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https://openalex.org/W4383706295	https://zenodo.org/records/8128871/files/IBAST%200710.pdf	English	null	COMPARATIVE EFFECTIVENESS OF TREATMENT OF ORAL MUCOSAL HEMANGIOMAS WITH IQ-LASERS	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	3,423	COMPARATIVE EFFECTIVENESS OF TREATMENT OF ORAL MUCOSAL HEMANGIOMAS WITH IQ-LASERS Karimov M.A.1 Sadikov R.R.2 Karimkulov N.A.3 Nurmatova X.X.4 1 Department of Medicine, Namangan State University, Namangan sh., Uzbekistan 2 Toshkent Medical Academy Tashkent City., Uzbekistan. 3 Andijan State Medical Institute, Andijan sh., Uzbekistan. 4 public health technician named after Kosonsoy Abu Ali ibn Sino https://doi.org/10.5281/zenodo.8128871 Karimov M.A.1 Sadikov R.R.2 Karimkulov N.A.3 Nurmatova X.X.4 1 Department of Medicine, Namangan State University, Namangan sh., Uzbekistan 2 Toshkent Medical Academy Tashkent City., Uzbekistan. 3 Andijan State Medical Institute, Andijan sh., Uzbekistan. 4 public health technician named after Kosonsoy Abu Ali ibn Sino https://doi.org/10.5281/zenodo.8128871 Resume. This article describes the results of treatment of oral mucosal hemangioma in the clinic, spread, modern diagnosis, stages of development, classification and modern methods of treatment with IQ-lasers, as well as medicamentous treatment, sclerotic treatment and joint treatment. The study analyzed the results of the treatment of 178 patients. Keywords. Oral mucosal hemangioma, IR-laser, sclerotic treatment, medicamentous treatment. Introduction. Hemangioma is a safe tumor that has developed from good quality blood vessels and is mainly observed in the first 18 months of a child's life, characterized by rapid growth and invasion of surrounding tissues. Hemangioma (ga) changes color, consistency, and tissue shape during growth, leading to varying degrees of cosmetic defects as well as functional impairment of the limbs. Located around the oral cavity to suppuration, can be observed with complications such as secondary infection as well as wound bleeding. The total incidence of Ga In newborn children is 10-15%, of which 40% is observed in the area of the area of the mouth circumference. In girls and children, the comparative frequency is observed in a ratio of 5:1. 83% of gas located in the head area call dysmorphophobia [4, 7, 15, 16]. Located in the deviation cavity, ga is often accompanied by complications such as scarring, bleeding and pain syndrome. IBAST \| Volume 3, Issue 7, June ECHNOLOGY The fetal organism becomes relatively sensitive due to the formation of the vascular system in the first 3 months of pregnancy. The process of vascular formation ends at the 7th month of pregnancy, then the endothelial lining of the vessels appears [1, 9, 17]. Skin and mucous membrane hemangiomas are manifested in the process of their development in 80% of cases in pink or bluish spotting at birth. Often during this period, a congenital rash or confusion with postpartum bleeding occurs. COMPARATIVE EFFECTIVENESS OF TREATMENT OF ORAL MUCOSAL HEMANGIOMAS WITH IQ-LASERS Later, during the first 2 weeks after childbirth, there is an increase in the size of the weaving and hemangioma. High-grade growth of GA is observed at 4 weeks (manifested in the appearance of a red spot rising above the level of the skin or mucous membrane) [2, 8]. Ga growth develops in 4 stages: onset, proliferation, maturation, and involution [3, 5, 10]. Hemangiomas of the oral mucosa (skin) usually grow rapidly within 18 months. In the Gada located on the skin, 15-20% absorption is observed, involution for mucous membrane hemangiomas is not considered characteristic. Obgs often cause a child's eating disorder due to the size or the presence of complications [9, 11]. According to data from many studies, the skin per 80% is observed in the head and neck of the body, of which 40% corresponds to the area of the oral cavity [7, 14, 17]. 44 44 INTERNATIONAL BULLETIN OF APPLIED SCIENCE AND TECHNOLOGY UIF = 8.2 \| SJIF = 5.955 Currently, there are more than 20 classifications of vascular tumors-hemangiomas worldwide according to their shape, character, histological structure, complications and other characteristics [13, 17]. Some of these are still used in the clinical practice of some clinics, such as the simplified ga classification proposed by Kondrashin (1963).) [1, 11, 16, 17].  Simple capillary  Simple hypertrophic  Simple hypertrophic  Cavernous  Mixed  Systemic hemangiomatosis International practice currently employs a classification adopted by the International Society for the study of Vascular Anamalies (ISSVA), the Society for the study of vascular anamalies, at the 1996 Xi International Society for the study of vascular anamalies symposium in Rome [1,4,5,6,13]. In 2007, 2016, and 2018, This accepted classification included further replenishment [1, 10, 11]. Research objective. Improving the results of complex treatment of oral hemangiomas using different wave range beams of the IR-laser device. Materials and inspection styles. Scientific work was carried out during 2015-2022 at the Department of "surgical diseases" of the Tashkent Medical Academy (City Hospital No. 1) and the Department of "Oncology and medical radiology" of the Andijan State Medical Institute (Andijan branch of the Republican specialized scientific practical medical center of oncology and radiology). In scientific research, Fdt with high energy lasers based on IR radiation (SO2, NDYAG, Pulse dye laser) has been used in the treatment of hemangiomas located in the oral cavity. In the research work, 178 patients were examined, of which 96 were treated by selecting treatment tactics depending on the stage of development of oral hemangioma. The comparative group is made up of 82 patients, the base of the Department of Oncology and medical radiology of the Tashkent Medical Academy and the Andijan State Medical Institute is the branch of the Republican specialized oncology and radiology scientific practical Medical Center Andijan and on the basis of various treatment institutions of our Republic used all known methods of treatment (1. and 2. listed in tables). IBAST \| Volume 3, Issue 7, June The age contingent of patients is from the age of newborn to 60 years. 78% were for girls and 22% for boys. Table 1. 45 ECHNOLOGY Distribution of patients with hemangioma in groups by age. Main group Comparative group Age group Number of patients Frequency (%) Number of patients Frequency (%) Breast ages 59 61,4±4.9* 5 6,1±2,6 Preschool age 18 18,8±3,9* 38 46,3±5,6 School prep and junior school age 11 11,4±3,2* 26 31,7±5,1 Senior school age, adolescence 8 8,4±2,8 13 15,9±4,0 Total: 96 100 82 100 Distribution of patients with hemangioma in groups by age. 45 INTERNATIONAL BULLETIN OF APPLIED SCIENCE AND TECHNOLOGY UIF = 8.2 \| SJIF = 5.955 ISSN: 2750-3402 IBAST INTERNATIONAL BULLETIN OF APPLIED SCIENCE AND TECHNOLOGY UIF = 8.2 \| SJIF = 5.955 * Р < 0,05 relative to the comparison group. Table 2. * Р < 0,05 relative to the comparison group. Table 2. Arrangement of hemangiomas in groups. Arrangement of hemangiomas in groups. Location Main group Comparative group Number % Number % Language 13 13,5±3,4 10 12,2±3,6 Upper and lower lip 44 45,8±5,1 53 64,6±5,2* Lunj 25 26,0±4,4* 16 19,6±4,3 Soft and hard palate 11 11,5±3,2 2 2,4±1,6 Milk 3 3,2±1,7 1 1,2±1,2 Total 96 100,0 82 100,0 * Р < 0,05 relative to the comparison group. All 178 patients with Obg si were treated with β-adrenoblocator (Propronalol). The main criterion for the appointment of a medicamentous treatment is the age of the child, which should not exceed 6 months, after this age, the effectiveness of the medicamentous treatment does not exceed 20%. Medicamentosis was often used in amounts of 1-2 mg/kg when undergoing treatment. The course of treatment was carried out for at least 2 months and continued for up to 6 months, when positive results were obtained. Before and during treatment, it is imperative to control the cardiovascular activity of the patient, and such examination methods as ECG, measurement of the number of heartbeats, blood pressure control and exocardiogram were performed. Patient children should be under the supervision of a cardiologist. Treatment was carried out in 90% outpatient, 10% inpatient conditions. A study of the long-term results of the use of β-adrenoblocators separately showed that some patients experienced Bradycardia When using the drug. This was manifested externally by the drowsiness and physical inactivity of the child (Table 3). IBAST \| Volume 3, Issue 7, June Table 3. Results of treatment of patients with Obg with β-adrenoblocator (propronalol). INTERNATIONAL BULLETIN OF APPLIED SCIENCE AND TECHNOLOGY UIF = 8.2 \| SJIF = 5.955 maserasia with scarring, in 18 patients bleeding due to frequent injury, while in 9 cases infection was recorded when ga was located in the oral cavity. 20 patients complained of pain due to Ga injury. The presence of GA has led to functional changes in the form of pain in 13 patients as well as difficulties in eating and swallowing food. For example, a 12-year-old patient with tongue cavernous Gass has been noted to have a change in taste perception. Gani caused deformities in 5 patients as a result of its growth into members. In all patients with small-sized ga (up to 1 cm) and weak blood flow, treatment with the Gaga so-2 laser ("scalpel-1" device) was performed during the first treatment session. The exposure was carried out in remote mode using focused beams, which leads to a minimal risk of bleeding. The output power of the so-2 laser radiation was 10-15 w, the Surface power density (Ps) was 40-150 W/cm2 (average 136.66±1 W/cm2), respectively, the surface energy density (Es) was achieved at mean values 6644.22±10 J/cm2 (1000 to 60000 J/cm2). In the process of visually controlled photocoagulation, the pathalogical tissue was selectively affected to the limit of healthy tissue. During the operation, ga decreased in size, flattened and changed its previous color. The operation process is completed by moistening the coagulation with a solution of potassium permanganate or brilliant green to the place where the Shell was formed. In order to protect the GAEs located in the lip area from mechanical damage to the coagulation scabbard in the natural folds after the procedure, only 6 patients had an aseptic ligament attached. In the post-laser application period, the laser does not require special attention to the affected areas, once every 1-2 days it is enough to treat with one of the antiseptic solutions (solution of briliant Green in 1% ethyl alcohol, 70% ethyl alcohol). In gas located in the area of the corners of the lips, maseration, secondary infection fall, strict adherence to hygiene measures and daily aseptic ligament renewal have been carried out to prevent damage to the coagulation curtain. In a number of patients, coagulation of the veil and punctate exudation along its circumference were recorded for 2-3 days. Table 1. Result Up to 6 months old After 6 months of age Good 47% 20% Satisfactory 42% 15% Unsatisfactory 11% 65% When choosing the type of laser in the treatment of Ga, we followed the following criteria: sults of treatment of patients with Obg with β-adrenoblocator (propronalol). IBAST \| Volume 3, Issue When choosing the type of laser in the treatment of Ga, we followed the following criteria: ECHNOLOGY A 10.6 µm wavelength so-2 laser allows for visually controlled coagulation and cut and radical removal of hemangioma tissue. The surrounding tissue damage area is minimal and does not exceed 300 µm. The main problem with the use of this type of laser in the treatment of gas is poor hemostatic exposure to venous vessels 1 mm in diameter and greater, as well as the presence of free blood at the site of exposure causing inefficiency of the treatment. Thus, this type of laser is applied to cut and remove the IGs in the stabilization growth phase, located in the area of the tip of the tongue and the lip. ECHNOLOGY Children receiving treatment treatments have been referred with basic complaints such as the presence of the derivative, cosmetic damage, functional disorders during meals. In some cases, objective changes have also been observed: in 15 cases ga 46 ges have also INTERNATIONAL BULLETIN OF APPLIED SCIENCE AND TECHNOLOGY UIF = 8.2 \| SJIF = 5.955 ISSN: 2750-3402 IBAST INTERNATIONAL BULLETIN OF APPLIED SCIENCE AND TECHNOLOGY UIF = 8.2 \| SJIF = 5.955 INTERNATIONAL BULLETIN OF APPLIED SCIENCE AND TECHNOLOGY UIF = 8.2 \| SJIF = 5.955 In one case, due to damage to the coagulation curtain in the oral mucosa, a 5-month-old child was observed to have a bleeding condition at a level that stopped with mechanical pressure. IBAST \| Volume 3, Issue 7, June It is noteworthy that only 35 patients showed local changes in the form of edema lasting up to 1-3 days. After one-time photocoagulation with so-2 Laser, good and satisfactory results were obtained in the treatment of 27 gas. In 3 patients observed to normal hypertrophic, flat red spots remained after a single so-2 laser treatment, in general, a reduction in Ga up to 1/3 of the initial size was observed. In 2 patients observed to cavernous so-2 Laser photocoagulation, Ga sites with a diameter of 0.5-0.7 cm were retained that rose slightly above the skin level. Repeated so-2 Laser photocaogulation treatments were carried out in addition to 3 of the patients with such types of relapsed and residual ga parts. ECHNOLOGY The best results in the treatment of kgs were obtained with satisfactory results from the use of so-2 lasers (83.9%) and only pulsed diode lasers in capillary ga forms. ECHNOLOGY The effectiveness of galari's treatment was assessed through visual and UTT data output. The next treatment sessions of laser destruction are carried out depending on whether the nourishing blood vessels of the GA are completely devascularized, as well as 47 ISSN: 2750-3402 IBAST INTERNATIONAL BULLETIN OF APPLIED SCIENCE AND TECHNOLOGY UIF = 8.2 \| SJIF = 5.955 the effectiveness of additional methods of treatment, such as sclerotherapy, general treatment, the use of local hormonal drugs. To devascularization level the rate of blood flow in the GA and the level of blood supply is determined through the UTT dopplerography examination. the effectiveness of additional methods of treatment, such as sclerotherapy, general treatment, the use of local hormonal drugs. To devascularization level the rate of blood flow in the GA and the level of blood supply is determined through the UTT dopplerography examination. Venous blood flow before treatment is 8.6±1.1 cm\SEC, 1.0±0.1 cm\SEC (R < 0.05) after treatment; 18.7±2.2 cm\SEC before treatment in mixed gas, 2.1±0.1 cm\SEC (R < 0.05) after treatment. Conclusion: Color dopplerography examination in cases of increased arterial, venous and mixed blood flow, against the background of medicamentous treatment, there will be an indication to undergo sclerotic therapy procedures aimed at reducing the blood flow rate to 5-10 cm/s and reduce the occurrence of complications during and after complex treatment with IQ lasers. In the presence of weak venous or partially accelerated blood flow in the deep tissues of the oral mucosa, the practice of laser coagulation is carried out up to 7-15 times using a distance Diode Laser (up to 5 W, in continuous exposure, energy 2-3 J) and interstitial radiation (3-5 W, energy up to 15 J). In the presence of residual blood flow and in the case of outward growth of the hemangioma, laser coagulation of so-2 in a moderately focused mode (power 7-10 W, Energy 20-30 J) will be indicated. In the so-2 Laser pathological tissue cutting mode (power 10 W, Energy 50 J), there will be an indication for deformation in the lip and lunge area and surgical correction of scars. References: 1.Гончарова Я.А. Гемангиомы раннего детского возраста. Тактика ведения // Пластическая хирургия и косметология. — 2012. — № 1. — С. 140-145. 2.Коноплицкий Д.В. Классификационный алгоритм лечения гемангиом наружной локализации у детей // Молодий вчений. 2015. 2—6, № 17. С. 618—622. 3.М. А. Каримов, 1Д. З. Мамарасулова, 2Р. Р. Садиков, 3Х. Х. Нурматова\РЕЗУЛЬТАТЫ ЛЕЧЕНИЯ ГЕМАНГИОМ РОТОВОЙ ПОЛОСТИ Доктор ахборотномаси № 4 (97)—2020 с. 162-165 Ў References: IBAST \| Volume 3, Issue 7, June 4.М.А.Каримов, Р.Р.Садиков, Х.Х.Нурматова. (2023). ОҒИЗ БЎШЛИҒИ ГЕМАНГИОМАЛАРИНИ КОМПЛЕКС ДАВОЛАШ НАТИЖАЛАРИ. EURASIAN JOURNAL OF ACADEMIC RESEARCH, 3(4), 102–107. https://doi.org/10.5281/zenodo.7805172 5.Нурмеев И.Н., Миролжубов Л.М., Осипов А.Ю. и др. Вожможности комбинированного лечения осложнённых гемангиом у детей. / Фундаментальные исследования. 2015. INTERNATIONAL BULLETIN OF APPLIED SCIENCE AND TECHNOLOGY UIF = 8.2 \| SJIF = 5.95 9.Castaneda S., S. Melendez-Lopez, E. Garcia [et al.] / The Role of the Pharmacist in the Treatment of Patients with Infantile Hemangioma Using Propranolol // Adv Ther. – 2016. Vol 33 №10 P 1831 1839 9.Castaneda S., S. Melendez-Lopez, E. Garcia [et al.] / The Role of the Pharmacist in the T t t f P ti t ith I f til H i U i P l l // Ad Th 2016 neda S., S. Melendez-Lopez, E. Garcia [et al.] / The Role of the Pharmacist in the 9.Castaneda S., S. Melendez Lopez, E. Garcia [et al.] / The Role of the Pharmacist in the Treatment of Patients with Infantile Hemangioma Using Propranolol // Adv Ther. – 2016. – Vol. 33, № 10. – P. 1831–1839. Treatment of Patients with Infantile Hemangioma Using Propranolol // Adv Ther. – 2016. – Vol. 33, № 10. – P. 1831–1839. 10.Dachlan, I., Wahdini, S. I., Putri, I. L., Seswandhana, M. R., Wicaksana, A., & Fauzi, A. R. (2020). Integrated propranolol, methylprednisolone, and surgery in managing a rare case of infantile hemangioma with concurrent cleft lip and palate. Ann Med Surg (Lond), 56, 91- 94.Retrieved from https://www. ncbi.nlm.nih.gov/pubmed/32612824. doi:10.1016/j.amsu.2020.06.015 11.Goto, K., Ozeki, M., Yasue, S., Endo, S., & Fukao, T. (2020). A retrospective study of 2 or 3 mg/kg/day propranolol for infantile hemangioma. Pediatr Int, 62(6), 751-753. 11.Goto, K., Ozeki, M., Yasue, S., Endo, S., & Fukao, T. (2020). A retrospective study of 2 or 3 mg/kg/day propranolol for infantile hemangioma. Pediatr Int, 62(6), 751-753. 12.Hartmann F., A. Lockmann, L. L. Grönemeyer [et al.] / Nd:YAG and pulsed dye laser therapy in infantile haemangiomas: a retrospective analysis of 271 treated haemangiomas in 149 children // J Eu.r Acad. Dermatol Venereol. – 2017. – Vol. 31, № 8. – P. 1372–1379. Doi: 10.1111/jdv.14074. Epub 2017 Jan 23. 13.Kagami, S. 1. Oral propranolol for infantile hemangiomas beyond the proliferative phase / S. 1. Kagami // J Dermatol. – 2018. – Vol. 45, № 10. – P. 1199–1202. Doi: 10.1111/1346- 8138.14581. 12.Hartmann F., A. Lockmann, L. L. Grönemeyer [et al.] / Nd:YAG and pulsed dye laser therapy in infantile haemangiomas: a retrospective analysis of 271 treated haemangiomas in 149 children // J Eu.r Acad. Dermatol Venereol. – 2017. – Vol. 31, № 8. – P. 1372–1379. Doi: 10.1111/jdv.14074. Epub 2017 Jan 23. 13.Kagami, S. 1. Oral propranolol for infantile hemangiomas beyond the proliferative phase / S. INTERNATIONAL BULLETIN OF APPLIED SCIENCE AND TECHNOLOGY UIF = 8.2 \| SJIF = 5.955 №1. стр.1208-1211. 6.Трапезникова Т.В., Хлебникова А.Н., Писклакова Т.П. Инфантильные гемангиомы: абсолютные показания к лечению. Человек. Спорт. Медицина 2017. Т. 17, № 3. С. 52–60 59 7.Чижевская, И.Д. Неинвазивный метод лечения врожденных гемангиом челюстнолицевой области у детей / И.Д. Чижевская // Педиатрия. Восточная Европа. – 2015. – № 3. – С. 160–166. ECHNOLOGY 8.Admani S., Feldstein S., Gonzalez E.M., Friedlander S.F. Beta blockers: an innovation in the treatment of infantile hemangiomas. J. Clin. Aesthet. Dermatol. 2014; 7(7): 37–45. 48 INTERNATIONAL BULLETIN OF APPLIED SCIENCE AND TECHNOLOGY UIF = 8.2 \| SJIF = 5.95 1. Kagami // J Dermatol. – 2018. – Vol. 45, № 10. – P. 1199–1202. Doi: 10.1111/1346- 8138.14581. 14.Karimov M.A. Sadykov R.R., Sadykov R.A., Mahmudov G. 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https://openalex.org/W3020062430	https://pubs.rsc.org/en/content/articlepdf/2020/na/d0na00153h	English	null	Size-dependent melting point depression of nickel nanoparticles	Nanoscale advances	2,020	cc-by	3,638	Introduction Metal and metal oxide nanoparticles exhibit uniquely size- dependent properties which normally follow an inverse surface area to volume proportionality.1 This has a depressive eﬀect on the melting point, and has been shown experimentally for many metals.2–9 This eﬀect was rst predicted by P. Pawlow in 190910 and demonstrated experimentally in 1954 by M. Takagi.11 Buﬀat et al. investigated the melting point depression of spherical gold nanoparticles using the same ED method as Takagi et al., and noted that this method could only determine the range of melting points for an ensemble of particles.4 This has long been a standard technology in determining nano- particle melting points.12 It has been purported, however, that measurements of melting points via ED becomes increasingly inaccurate as nanoparticle size decreases due to line-broad- ening.8 Determination of melting point depression has also been undertaken through the use of nano-DSC, Lai et al. investigated this property of tin nanoparticles, determining the melting point to be when there was an abrupt increase (“jump”) in heat ow required to sustain the increasing temperature.8 We apply a similar method as Zhang et al. in order to measure individual nanoparticles by direct observation of nanoparticles via TEM recognising record melting as an abrupt increase in observed diameter.13 In a typical synthesis (synthesis 1) a 5% (w/v) nickel nitrate solution was prepared by dissolving nickel nitrate hexahydrate (11.25 mg, 38.67 mmol) in water (0.225 mL). A 5% (w/v) alkaline NaBH4 solution was prepared in a similar fashion. Two reverse microemulsions (RME-1 and RME-2) were made up of cyclo- hexane (22.744 mL), 1-hexanol (0.3 mL) and TX-100 (1.731 mL, 2.50 mmol). To RME-1 under stirring 5% (w/v) nickel nitrate solution (0.225 mL) was added so that W0 was equal to 5 or 1 respectively. To RME-2 under stirring 5% (w/v) alkaline NaBH4 (0.225 mL) solution was added. Both reverse microemulsions were leto stir for 30 min to achieve homogeneity aer which RME-2 was added dropwise to RME-1 with continuous stirring under an inert (argon) atmosphere. The resulting reverse micro- emulsion was leto stir for 3 hours to allow for nanoparticle growth via Ostwald ripening. The nickel nanoparticles were separated via centrifugation and washed with ethanol and separately with toluene then dispersed in water (Fig. 2). aDepartment of Pure and Applied Chemistry, University of Strathclyde, 295 Cathedral Street, Glasgow, G1 1XL, UK bSchool of Chemistry, University of Bristol, Bristol, BS8 1TS, UK. E-mail: s.a.davis@ bristol.ac.uk; Simon.Hall@bristol.ac.uk † Electronic supplementary information (ESI) available. See DOI: 10.1039/d0na00153h Synthesis Nickel nitrate hexahydrate (Extra Pure SLR), cyclohexane ($99.5%) and sodium hydroxide ($97% NaOH) were purchased from Fisher Scientic. TX-100 (laboratory grade), 1-hexanol Open Access Article. Published on 21 April 2020. Downloaded on 10/24/2024 5: This article is licensed under a Creative Commons Attribution 3.0 U Received 24th February 2020 Accepted 21st April 2020 DOI: 10.1039/d0na00153h rsc.li/nanoscale-advances Received 24th February 2020 Accepted 21st April 2020 (98%) and NaBH4 ($96%) were purchased from Sigma-Aldrich. The reaction procedure was scaled down from the work of Kumar et al.14 Four syntheses were conducted with diﬀerent reactant concentrations (ESI Table 1†). The size of the nickel nanoparticles was controlled by varying the molar ratio of water to surfactant (W0). Each reaction does however produce a range of sizes (ESI Table 1 & ESI Fig. 1–4†), which we nd itself useful in this study. Introduction These reverse microemulsions continually collide and coalesce, mix- ing their internal constituents – this allows the sodium boro- hydride to reduce the nickel ions which then grow into a nanoparticle within the microemulsions. The synthesis has been visualized in Fig. 1. ss Article. Published on 21 April 2020. Downloaded on 10/24/2024 5:07:45 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Cite this: Nanoscale Adv., 2020, 2, 2347 Cite this: Nanoscale Adv., 2020, 2, 2347 Alexander van Teijlingen, a Sean A. Davis b and Simon R. Hall b We investigate the phase-transition behaviour of nickel nanoparticles (3–6 nm) via dynamic TEM. The nanoparticles were synthesized within a reverse microemulsion and then monitored via dynamic TEM simultaneously while undergoing controlled heating. The size-dependent melting point depression experimentally observed is compared with, and is in good agreement with existing thermodynamic and molecular dynamic predictions. Melting point characterization Phase-pure and oxide free nickel nanoparticles on carbon coated copper TEM grids were prepared by drop-casting of a colloidal suspension of nickel nanoparticles in toluene. Using a Gatan heating stage (model number 628) with applied water This journal is © The Royal Society of Chemistry 2020 Nanoscale Adv., 2020, 2, 2347–2351 \| 2347 View Article Online View Article Online Paper Nanoscale Advances Fig. 2 TEM images of (A) nickel nanoparticles encapsulated wit a reverse microemulsions of TX-100/1-hexanol (d ¼ 100 nm) and a single nickel nanoparticle (d ¼ 4.5 nm). 2 TEM images of (A) nickel nanoparticles encapsulat i l i f TX 100/1 h l (d 100 Fig. 1 Nickel nanoparticle synthesis procedure consisting of prepa ration of two reverse microemulsions (TX-100 with cosurfactant 1 hexanol stabilizing the spherical structure), one with nickel(II) ion within and another with sodium borohydride within. Open Access Article. Published on 21 April 2020. Downloaded on 10/24/2024 5:07:45 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 21 April 2020. Downloaded on 10/24/2024 5:07:45 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 2 TEM images of (A) nickel nanoparticles encapsulated within a reverse microemulsions of TX-100/1-hexanol (d ¼ 100 nm) and (B) a single nickel nanoparticle (d ¼ 4.5 nm). measurements. Recording the expansion of nanoparticles around the melting point allows the determination of this physical property unambiguously. Nanoparticles were deter- mined to have melted when three requirements had been met: 1. The average diameter of the spherical nanoparticle increased by at least 4.3% (this percentage was chosen as it is the diﬀerence in diameter for two equimolar spherical nickel nanoparticles, one a solid and one a liquid with perfect wetting on a substrate, densities 8.908 g cm3 and 7.81 g cm3 respectively). 1. The average diameter of the spherical nanoparticle increased by at least 4.3% (this percentage was chosen as it is the diﬀerence in diameter for two equimolar spherical nickel nanoparticles, one a solid and one a liquid with perfect wetting on a substrate, densities 8.908 g cm3 and 7.81 g cm3 respectively). Fig. 1 Nickel nanoparticle synthesis procedure consisting of prepa- ration of two reverse microemulsions (TX-100 with cosurfactant 1- hexanol stabilizing the spherical structure), one with nickel(II) ions within and another with sodium borohydride within. Fig. Melting point characterization The orange trendline and markers display the experimental data collected in this study, the vertical error bars show a 50 C uncertainty in temperature and the horizontal error bars show a 0.1 nm uncertainty in diameter. projected TEM image surface area of individual nanoparticles (Fig. 3) and the diameter calculated according to (1). d ¼ 2 ﬃﬃﬃﬃ A p r (1) (1) Fig. 3 shows the change in mean diameter as a function of projected nanoparticle surface area, for a single nickel nano- particle over increasing temperatures. There is an abrupt increase at the melting point (925 C 25 C). The melting point was taken as the temperature halfway between the upper and lower points of the increase. This method is similar to the nano-DSC method described by Lai et al.8 This process was repeated for seven diﬀerent nickel nanoparticles and the determined melting points plotted in Fig. 4. This experimental data is in good agreement with the MD studies and thermody- namic models also shown in Fig. 4. nanoparticles,2 it is generally believed that it is an incomplete model of solid–liquid phase transition in nanoparticles. More sophisticated models, such as the liquid nucleation and growth model (eqn (4) and (5)), which supposes that the liquid phase is initiated at a surface (a discontinuity) and propagates inwards towards the centre. At a critical radius (r) an unstable equi- librium is formed, where r ¼ r. The temperature at which this is formed marks the upper boundary of the melting range (green area, Fig. 4), while the lower boundary is given by eqn (5).17 Tm ¼ T0 1 1 d (2) (2) The empirical relationship between diameter and melting point can be described by eqn (2) where T0 denotes the bulk melting point, d the diameter, Tm the melting point of the nanoparticle and the melting point is inversely related to the diameter. r* ¼ 2gsvnmðsÞT0 DmHðT T0Þ (4) TmðdÞ ¼ T0 1 3 DmH gsvnmðsÞ r glvnðlÞ r (5) (4) (5) TmðdÞ ¼ T0 1 4gsl DmHrsd (3) (3) Thermodynamically, for the solid-to-liquid phase trans- formation to be energetically favourable, the sum of the ener- gies of the newly formed solid–liquid and liquid–vapor interfaces must be less than or equal to the energy of the solid– vapor interface (eqn (6)). Melting point characterization 1 Nickel nanoparticle synthesis procedure consisting of prepa- ration of two reverse microemulsions (TX-100 with cosurfactant 1- hexanol stabilizing the spherical structure), one with nickel(II) ions within and another with sodium borohydride within. 2. The melting point is clearly identied by a rapid and sustained increase in size as opposed to gradual expansion due to heating of a solid. cooling coupled to a JEOL-2100F TEM, we recorded the melting point of eight diﬀerent nickel nanoparticles over a temperature range of 700–1100 C at a rate of 5 C min1. Nanoparticle diameters were measured only when the video capture showed clearly de-ned edges. The heating stage independently measured the temperature of the sample and therefore heating eﬀects from the electron beam are accounted for in our 3. The diameter of the liquid phase nanoparticle used to deter-mine the melting point does not have overlapping error bars with the diameter measurements of the solid nanoparticle. Images at diﬀerent temperatures were analysed using the so-ware package ImageJ2, Fiji distribution15,16 by recording the 2348 \| Nanoscale Adv., 2020, 2, 2347–2351 This journal is © The Royal Society of Chemistry 2020 View Article Online Paper Nanoscale Advances Fig. 3 A line graph showing the diameter of a 4.3 nm nickel nano- sphere over a temperature range of 700–1050 C, at intervals of 50 C. The solid nanoparticle diameter was taken as an average of 5 measurements between 700 C and 900 C. The red X in the centre marks the melting point at 925 25 C, which was determined from the abrupt, sustained and signiﬁcant increase in nanoparticle diameter. Paper Fig. 4 A graph showing the results of evaluating the melting point depression of nickel nanoparticles by four diﬀerent methods. The red line denotes the melting point of nickel nanoparticles according to the empirical model (eqn (2)) from d ¼ 1 nm to d ¼ 6 nm with 1 nm increments. The blue trendline and markers plot the melting point results of the molecular dynamics study using the quantum-Sutton- Chen (QSC) force ﬁeld reported by Qi et al.3 The purple line denotes values predict by the liquid-drop model24 with a b value given by Hanszen et al.25 The black line and markers represent values for the melting point as a function of size predicted by the Gibbs–Thompson (eqn (3)). This journal is © The Royal Society of Chemistry 2020 Melting point characterization The green area shows the temperatures between the upper and lower bounds of the liquid nucleation and growth model (eqn (4) and (5)) – this is in very close agreement with the experimental data between 3.5 nm and 5.3 nm, where most of the experimental data points where collected. The orange trendline and markers display the experimental data collected in this study, the vertical error bars show a 50 C uncertainty in temperature and the horizontal error bars show a 0.1 nm uncertainty in diameter. Fig. 3 A line graph showing the diameter of a 4.3 nm nickel nano- sphere over a temperature range of 700–1050 C, at intervals of 50 C. The solid nanoparticle diameter was taken as an average of 5 measurements between 700 C and 900 C. The red X in the centre marks the melting point at 925 25 C, which was determined from the abrupt, sustained and signiﬁcant increase in nanoparticle diameter. Fig. 3 A line graph showing the diameter of a 4.3 nm nickel nano- sphere over a temperature range of 700–1050 C, at intervals of 50 C. The solid nanoparticle diameter was taken as an average of 5 measurements between 700 C and 900 C. The red X in the centre marks the melting point at 925 25 C, which was determined from the abrupt, sustained and signiﬁcant increase in nanoparticle diameter. Fig. 4 A graph showing the results of evaluating the melting point depression of nickel nanoparticles by four diﬀerent methods. The red line denotes the melting point of nickel nanoparticles according to the empirical model (eqn (2)) from d ¼ 1 nm to d ¼ 6 nm with 1 nm increments. The blue trendline and markers plot the melting point results of the molecular dynamics study using the quantum-Sutton- Chen (QSC) force ﬁeld reported by Qi et al.3 The purple line denotes values predict by the liquid-drop model24 with a b value given by Hanszen et al.25 The black line and markers represent values for the melting point as a function of size predicted by the Gibbs–Thompson (eqn (3)). The green area shows the temperatures between the upper and lower bounds of the liquid nucleation and growth model (eqn (4) and (5)) – this is in very close agreement with the experimental data between 3.5 nm and 5.3 nm, where most of the experimental data points where collected. Acknowledgements where: (8) (9) The authors would like to acknowledge the Bristol Centre for Functional NanoMaterials funded by the Engineering and Physical Sciences Research Council (EPRSC) UK (grantsEP/ G036780/1 and EP/L015544). TEM studies were carried out in the Chemistry Imaging Facility with equipment funded by UoB and EPSRC (EP/K035746/1 and EP/M028216/1). (8) (9) The authors would like to acknowledge the Bristol Centre for Functional NanoMaterials funded by the Engineering and Physical Sciences Research Council (EPRSC) UK (grantsEP/ G036780/1 and EP/L015544). TEM studies were carried out in the Chemistry Imaging Facility with equipment funded by UoB and EPSRC (EP/K035746/1 and EP/M028216/1). and: (9) Conﬂicts of interest There are no conicts to declare. There are no conicts to declare. Utot ¼ X i¼1 3 "X isj V rij C ﬃﬃﬃﬃri p # (7) VðrÞ ¼ a rij n (8) ri ¼ X a rij m (9) Tm ¼ Tþ þ T ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ Tþ þ T p (10) Utot ¼ X i¼1 3 "X isj V rij C ﬃﬃﬃﬃri p # (7) VðrÞ ¼ a rij n (8) ri ¼ X a rij m (9) Tm ¼ Tþ þ T ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ Tþ þ T p (10) (7) Nanoscale Advances Paper A key limitation of thermodynamic models is that melting is equivalent to stability such that changes in individual atoms in a system are not considered and therefore cannot reveal the mechanism of the melting process alone. In 2001 Qi et al. published a molecular dynamics study calculating the degree of melting point depression in nickel nanoparticles.3 By super- heating and super-cooling nickel nanoparticles under the QSC potential (eqn (7)), they found the equilibrium melting temperature according to eqn (10). The data presented in Fig. 4 sheds light onto the accuracy of some thermodynamic and molecular dynamic models that have been used to predict the melting point depression as a function of size in nickel, showing that the liquid nucleation and growth model (green area) to be the model that t best with the experimental data (orange line). experiment and modelling to be tested over a larger size range. Future experiments may also include both heating/cooling cycles where the properties of diﬀerent nanoparticles can be analysed at the 2nd, 3rd, 4th etc. cycle of heating as well as their behaviour upon cooling.23 With suﬃcient distribution on the grid, we speculate that this method would be appropriate to measure the properties of completely diﬀerent nanoparticles simultaneously. Just as we have used it to study diﬀerent sizes of nickel nanoparticles simultaneously, it could be used to deter- mine melting point depression in magnesium oxide and aluminium oxide simultaneously or the sintering of any two or more diﬀerent nanoparticles. Melting point characterization There exist several proposed mechanisms in which melting initialization and propagation in nanomaterials is described as a function of interface energies and the enthalpy of fusion (DmH). One of these models is the Gibbs–Thompson eqn (3), which assumes an isolated spherical nanoparticle situated within it's a liquid of its own type.7 While this model has some success in predicting melting point depression in our gsv $ gsl + glv (6) (6) Nanoscale Adv., 2020, 2, 2347–2351 \| 2349 This journal is © The Royal Society of Chemistry 2020 Paper View Article Online View Article Online View Article Online Paper Article Online Notes and references (10) Tm ¼ Tþ þ T ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ Tþ þ T p 1 D. K. Pattadar and F. P. Zamborini, J. Am. Chem. Soc., 2018, 140, 14126–14133. 1 D. K. Pattadar and F. P. Zamborini, J. Am. Chem. Soc., 2018, 140, 14126–14133. Experimental data presented herein (Fig. 4) have shown that MD studies using the QSC force eld, while generally in agreement with the experimental data,3,18,19 consistently over- predict the melting points of all sizes of nickel nanoparticles studied – a predictable outcome as their system was shown to determine the melting point of bulk nickel to be 1487 C (32 C higher than the real bulk melting point). This QSC force eld has previously been used to accurately predict melting point depression in gold,20 copper,6 platinum21 and iron.22 It is of note that in each system the bulk melting point of each element was overpredicted. Finally we compared the normalised (DT ¼ T/T0) and undercooled (DT ¼ T0 T) QSC MD study and nd that, while closer to the experimental data they over- and under- predict the individual melting points respectively (ESI Fig. 5 and 6†). 2 W. Luo, W. Hu and S. Xiao, J. Phys. Chem. C, 2008, 112, 2359– 2369. 3 Y. Qi, T. Çain, W. L. Johnson and W. A. Goddard, J. Chem. Phys., 2001, 115, 385–394. 4 P. Buﬀat and J.-P. Borel, Phys. Rev. A, 1976, 13, 2287–2298. 5 K. Dick, T. Dhanasekaran, Z. Zhang and D. Meisel, J. Am. Chem. Soc., 2002, 124, 2312–2317. 6 H. H. Kart, H. Yildirim, S. Ozdemir Kart and T. Çaˇgin, Mater. Chem. Phys., 2014, 147, 204–212. y 7 J. Sun and S. L. Simon, Thermochim. Acta, 2007, 463, 32–40. 8 S. L. Lai, J. Y. Guo, V. Petrova, G. Ramanath and L. H. Allen, Phys. Rev. Lett., 1996, 77, 99–102. 9 H. J. Kim, I. K. Beak, K. H. Kim and S. P. Jang, Trans. Korean Soc. Mech. Eng. B, 2014, 38, 695–700. 10 P. Pawlow, Z. phys. Chem., 1909, 65, 545–548. 11 M. Takagi, J. Phys. Soc. Jpn., 1954, 9, 359–363. 2350 \| Nanoscale Adv., 2020, 2, 2347–2351 Nanoscale Adv., 2020, 2, 2347–2351 \| 2351 This journal is © The Royal Society of Chemistry 2020 Conclusions 12 J. Zhang, Y. Zheng, D. Zhao, S. Yang, L. Yang, Z. Liu, R. Zhang, S. Wang, D. Zhang and L. Chen, J. Phys. Chem. C, 2016, 120, 10686–10690. In this study we have synthesized nickel nanoparticles of varying diameter via a reverse microemulsion synthesis and for the rst time we report experimental studies which complement and validate previous modelling of nickel nanoparticle melting point depression. We have used this method to report a detailed investigation of the melting point depression in nickel nano- particles. The method could be improved and expanded using alternative TEM grids, such as silicon nitride grids, as they are more stable than copper at elevated temperatures, allowing 13 M. Zhang, E. A. Olson, R. D. Twesten, J. G. Wen, L. H. Allen, I. M. Robertson and I. Petrov, J. Mater. Res., 2005, 20, 1802– 1807. 14 A. Kumar, A. Saxena, A. De, R. Shankar and S. Mozumdar, Adv. Nat. Sci.: Nanosci. Nanotechnol., 2013, 4, 25009. 15 J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, 15 J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J. Y. Tinevez, D. J. White, This journal is © The Royal Society of Chemistry 2020 2350 \| Nanoscale Adv., 2020, 2, 2347–2351 Paper Nanoscale Advances 20 Z. Qiao, H. Feng and J. Zhou, Phase Transitions, 2014, 87, 59– 70. V. Hartenstein, K. Eliceiri, P. Tomancak and A. Cardona, Nat. Methods, 2012, 9, 676–682. 16 C. T. Rueden, J. Schindelin, M. C. Hiner, B. E. DeZonia, 21 H. Akbarzadeh and G. A. Parsafar, Fluid Phase Equilib., 2009, 280, 16–21. A. E. Walter, E. T. Arena and K. W. Eliceiri, BMC Bioinf., 2017, 18, 1–26. 22 T. Shen, W. Meng, Y. Wu and X. Lu, Appl. Surf. Sci., 2013, 277, 7–14. 17 G. Guenther and O. Guillon, J. Mater. Sci., 2014, 49, 7915– 7932. 23 H. Chamati and K. Gaminchev, in J. Phys.: Conf. Ser., IOP Publishing, 2012, vol. 398, p. 012042. 18 T. D. Nguyen, C. C. Nguyen and V. H. Tran, RSC Adv., 2017, 7, 25406–25413. 24 K. K. Nanda, S. N. Sahu and S. N. Behera, Phys. Rev. A: At., Mol., Opt. Phys., 2002, 66, 132081–132088. 19 T. Çagin, Y. Qi, H. Li, Y. Kimura, H. 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https://openalex.org/W4249752919	https://www.jmir.org/2020/10/e21632/PDF	English	null	Physical Distancing Measures and Walking Activity in Middle-aged and Older Residents in Changsha, China, During the COVID-19 Epidemic Period: Longitudinal Observational Study (Preprint)	null	2,020	cc-by	9,195	JOURNAL OF MEDICAL INTERNET RESEARCH JOURNAL OF MEDICAL INTERNET RESEARCH Wang et al Original Paper Physical Distancing Measures and Walking Activity in Middle-aged and Older Residents in Changsha, China, During the COVID-19 Epidemic Period: Longitudinal Observational Study Yilun Wang1, MD, PhD; Yuqing Zhang2,3, DSC; Kim Bennell4, PhD; Daniel Kenta White5, PT, SCD; Jie Wei2,3,6, PhD; Ziying Wu1, MD; Hongyi He1, MD; Shaohui Liu6, MS; Xianghang Luo7,8, MD, PhD; Shuo Hu8,9, MD, PhD; Chao Zeng1,2,3, MD, PhD; Guanghua Lei1,8,10, MD, PhD 1Department of Orthopaedics, Xiangya Hospital, Central South University, Changsha, China 2Division of Rheumatology, Allergy, and Immunology, Department of Medicine, Massachusetts General Hospital, Boston, MA, United States 3The Mongan Institute, Massachusetts General Hospital, Harvard Medical School, Boston, MA, United States 4Centre for Health, Exercise and Sports Medicine, The University of Melbourne, Melbourne, Australia 5Department of Physical Therapy, University of Delaware, Newark, DE, United States 6Health Management Center, Xiangya Hospital, Central South University, Changsha, China 7Department of Endocrinology, Endocrinology Research Center, Xiangya Hospital, Central South University, Changsha, China 8National Clinical Research Center of Geriatric Disorders, Xiangya Hospital, Central South University, Changsha, China 9Department of Nuclear Medicine, Xiangya Hospital, Central South University, Changsha, China 10Hunan Key Laboratory of Joint Degeneration and Injury, Changsha, China these authors contributed equally Corresponding Author: Guanghua Lei, MD, PhD Department of Orthopaedics Xiangya Hospital Central South University Xiangya Road 87 Changsha, 410008 China Phone: 86 0731 89753006 Email: lei_guanghua@csu.edu.cn http://www.jmir.org/2020/10/e21632/ J Med Internet Res 2020 \| vol. 22 \| iss. 10 \| e21632 \| p. 1 (page number not for citation purposes) 1Department of Orthopaedics, Xiangya Hospital, Central South University, Changsha, China 2Division of Rheumatology, Allergy, and Immunology, Department of Medicine, Massachusetts General Hospital, Boston, MA, United States 3The Mongan Institute, Massachusetts General Hospital, Harvard Medical School, Boston, MA, United States 4Centre for Health, Exercise and Sports Medicine, The University of Melbourne, Melbourne, Australia 5Department of Physical Therapy, University of Delaware, Newark, DE, United States 6Health Management Center, Xiangya Hospital, Central South University, Changsha, China 7Department of Endocrinology, Endocrinology Research Center, Xiangya Hospital, Central South University, Changsha, China 8National Clinical Research Center of Geriatric Disorders, Xiangya Hospital, Central South University, Changsha, China 9Department of Nuclear Medicine, Xiangya Hospital, Central South University, Changsha, China 10Hunan Key Laboratory of Joint Degeneration and Injury, Changsha, China these authors contributed equally Study Outcome The outcome variable was daily step counts recorded by the accelerometer sensor in the smartphone and extracted by WeChat. To be eligible for the current analysis of daily step counts, we required participants to wear their smartphone for ≥10 hours on a given day, a standard convention for measuring daily walking [18,19]. The smartphone wear time was calculated as the difference between the times of the first recorded step count and the last recorded step count each day. This algorithm was used in the Activity Inequality Project to calculate daily step counts for more than 700,000 individuals across 111 countries [19]. Participants with no valid daily step count were excluded in the current analysis. We defined a low daily step count as ≤1500 steps/day [8,9]. If a participant had ≥14 days of low daily step counts over a 30-day period, we considered that person as having experienced frequent low daily steps [8,9]. Using data collected from the Step Study, a longitudinal study conducted among the urban residents of Changsha, China, we therefore described the trends of daily steps around the COVID-19 epidemic period in 2020 and compared them with that of a similar period one year earlier. We examined the relationships between several sociodemographic factors, anthropometric factors, and comorbidity and the prevalence of frequent low daily steps. We conducted two validation studies to assess the accuracy of daily step counts collected from WeChat. To determine the accuracy of steps measured at various walking speeds by iOS and Android devices, we visually counted steps from 14 subjects walking on a treadmill at 4.8, 6.4, 8.0, and 9.6 km/h while subjects held/wore smartphones in different positions (ie, pants pocket, hand, and arm). These methods are consistent with previous studies [20-22]. We found step count accuracy to be high, with intraclass correlation coefficients (ICCs) ranging from 0.64 to 0.99. Second, we assessed the accuracy of step counts extracted from WeChat in free-living conditions. Specifically, 36 participants from the Step Study were instructed to wear both a Fitbit Charge 3 (Fitbit Inc), as the criterion measure [23], and their personal smartphone for 7 consecutive days [24]. The results also demonstrated a moderate to high KEYWORDS COVID-19; pandemic; physical distancing; steps; walking activity account; and (4) be willing and able to give consent. Individuals with severe mental illness were ineligible. During each annual physical checkup, participants were queried about their sociodemographic and lifestyle factors, comorbidities, health-related symptoms and signs (eg, joint pain), and ability to perform daily activities. They also received clinical examinations and laboratory tests, including physical function tests (eg, lower limb muscle strength measurements). Abstract Background: Physical distancing measures taken to contain COVID-19 transmission may substantially reduce physical activity levels and cause individuals to adopt a more sedentary lifestyle. Background: Physical distancing measures taken to contain COVID-19 transmission may substantially reduce physical activity levels and cause individuals to adopt a more sedentary lifestyle. Objective: The objective of this study is to determine if there was any change in daily steps, an important component of daily physical activity, and examine risk factors for frequent low daily steps during the COVID-19 epidemic. Methods: We used data collected from the Step Study, a population-based longitudinal study of walking activity among residents aged ≥40 years in Changsha, China. Daily steps were collected via a smartphone linked to WeChat, a social networking platform. We plotted mean daily steps and the prevalence of low daily steps (≤1500 steps/day) 30 days before (reference period) and 30 days after (epidemic period) January 21, 2020 (date of the first COVID-19 case diagnosed in Changsha), and compared it with the same corresponding period from 2019. We examined the association of risk factors with the prevalence of frequent low daily steps (≤1500 steps/day for ≥14 days) using logistic regression. Results: Among 3544 participants (mean age 51.6 years; n=1226 females, 34.6%), mean daily steps dropped from 8097 to 5440 and the prevalence of low daily steps increased from 3% (2287/76,136 person-day) to 18.5% (12,951/70,183 person-day) during the reference and epidemic periods, respectively. No such phenomenon was observed during the corresponding period in 2019. Older age (P for interaction=.001) and female sex (P for interaction<.001) were both associated with a higher prevalence of frequent low daily steps and were more pronounced during the epidemic period. More education was associated with a lower prevalence of frequent low daily steps during the reference period but not the epidemic period (P for interaction=.34). Body mass index or comorbidity were not associated with frequent low daily steps during either period. J Med Internet Res 2020 \| vol. 22 \| iss. 10 \| e21632 \| p. 1 (page number not for citation purposes) J Med Internet Res 2020 \| vol. 22 \| iss. 10 \| e21632 \| p. 1 (page number not for citation purposes) XSL•FO RenderX XSL•FO RenderX XSL•FO RenderX Wang et al Introduction COVID-19 has caused morbidity and mortality worldwide [1]. To control this highly contagious infectious disease, many countries have implemented “physical distancing” and “shelter-in-place” measures to contain COVID-19 transmission [2,3]. However, such measures may substantially reduce physical activity levels and cause individuals to adopt a more sedentary lifestyle [4]. For each participant, walking activity measured as daily step counts was collected through a smartphone linked to WeChat. WeChat is a multipurpose social network platform (Tencent Inc) with approximately 1.1 billion monthly active users in China [17]. One of its apps can extract daily step count information from the accelerometer sensor in a smartphone. Thus, if a participant is a WeChat user and wears a smartphone, his/her step counts can be captured by WeChat’s app. There is ample evidence that regular physical activity is crucial for health, with physical inactivity characterized as the fourth leading cause of global mortality [5]. Results from the National Health and Nutrition Examination Survey (NHANES) and the Women’s Health Study both showed that a lower number of daily steps, which is an important component of daily physical activity, was significantly associated with higher all-cause mortality [6,7]. Studies also demonstrated that individuals who walked less than 1500 daily steps for 14 days were at a higher risk of muscle mass loss and low insulin sensitivity [8,9]. To date, several studies have reported changes in daily steps of residents in areas affected by COVID-19 [10-14]; however, none of these studies specifically described the prevalence of frequent low daily steps (≤1500 steps/day for ≥14 days over one month) [8,9], a strong predictor of poor health outcomes, and examined its risk factors. JOURNAL OF MEDICAL INTERNET RESEARCH Wang et al Conclusions: Daily steps of Changsha residents aged ≥40 years dropped significantly during the COVID-19 period, especially among older adults and females. Although successful physical distancing, measured by the rapid downward trend in daily step counts of residents, played a critical role in the containment of the COVID-19 epidemic, our findings of an increase in the prevalence of frequent low daily steps raise concerns about unintended effects on physical activity. (J Med Internet Res 2020;22(10):e21632) doi: 10.2196/21632 J Med Internet Res 2020 \| vol. 22 \| iss. 10 \| e21632 \| p. 2 (page number not for citation purposes) http://www.jmir.org/2020/10/e21632/ Statistical Analyses On January 22, 2020, one day after the first case of COVID-19 was diagnosed in Changsha (January 21, 2020, ie, the index day) the municipal government issued an emergency notice to implement physical distancing measures (ie, staying at home, closing schools, working from home if possible, travelling only when necessary, and cancelling mass gatherings) [26,27]. In this analysis, we defined the time interval from January 22 to February 20, 2020 (30 days after the index date), as the COVID-19 epidemic period, and the time interval from December 22, 2019, to January 20, 2020 (30 days before the index date), as the reference period. Since the COVID-19 epidemic occurred around the holiday season of Chinese New Year, for the purpose of comparison we also used data collected between January 2 and March 3, 2019, which corresponded to the same Chinese lunar calendar period. Details of the selection of study periods are shown in Multimedia Appendix 2. All P values for interaction were two-sided and P for interaction<.05 was considered statistically significant for all tests. All statistical analyses were conducted using SAS (Version 9.4). Study Exposures Information on age, sex, educational level, height, weight, and comorbidity were obtained from the annual physical checkup visit. Body mass index was calculated. The modified Charlson Comorbidity Index (CCI) was computed based on self-reported comorbidities [25]. Similarly, prevalence ratios (PR) were calculated for the prevalence of low daily steps (≤1500 steps/day) between the two comparative time periods in 2019 and 2020, respectively [30]. Finally, we estimated the prevalence of frequent low daily steps (≤1500 steps/day for ≥14 days over 30 days) during epidemic and reference periods and examined whether age, sex, BMI, educational level, and comorbidity were associated with the prevalence of frequent low daily steps using logistic regression. We tested the additive effect measure of modification of physical distancing with each of the risk factors mentioned above by adding an interaction term in the regression model [31]. Study Population The Step Study (registration number: ChiCTR1800017977) is a population-based longitudinal study initiated in September 2018 in Changsha, China. The aims of the Step Study were to describe patterns of walking activity, factors related to walking activity, and its sequelae among the community-living population. Participants in the Step Study comprised individuals who had their annual physical checkup at Xiangya Hospital of Central South University in Changsha, China [15,16]. To be eligible for the Step Study, participants had to meet the following criteria: (1) be a resident of Changsha; (2) be aged ≥40 years; (3) own a personal smartphone and have a WeChat http://www.jmir.org/2020/10/e21632/ XSL•FO RenderX Wang et al JOURNAL OF MEDICAL INTERNET RESEARCH Wang et al Wang et al in SAS (Version 9.4; SAS Institute) with identity links to calculate the MD and its 95% CI between the epidemic and reference periods. We added the REPEATED statement to account for correlation of the repeated measurements of individuals’ daily step counts [29]. agreement on step counts, with ICCs ranging from 0.67 to 0.81. Detailed information from these validation studies is shown in Multimedia Appendix 1. Ethical Considerations This study was approved by the Ethics Committee of Xiangya Hospital, Central South University (#201806910), and written informed consent was obtained from study participants. J Med Internet Res 2020 \| vol. 22 \| iss. 10 \| e21632 \| p. 3 (page number not for citation purposes) Results First, we plotted the mean daily steps from December 22, 2019, to February 20, 2020 (around COVID-19 epidemic period), and mean daily steps from January 2 to March 3, 2019 (historic comparison period), respectively, using a 3-day moving average smooth method [28]. Second, we used the same approach to plot the prevalence of low daily steps (≤1500 steps/day) for the corresponding periods. We calculated the mean difference (MD) and its 95% confidence interval for daily steps using Generalized Estimating Equations (GEE) between the epidemic period and reference period in 2020, and the corresponding periods in 2019, respectively. Specifically, we included each qualified daily step count into the GEE model using the PROC GENMOD procedure A total of 7262 Changsha residents aged ≥40 years had a physical checkup at the study center between September 2018 and January 2020, and 4145 of them (57.1%) had a WeChat account and agreed to participate in the Step Study. Of these, 3544 with at least one valid daily step count during the study period were included in the analysis (Table 1). The mean age of participants was 51.6 (SD 8.9) years, 1226 (34.6%) participants were females, and the mean BMI of participants was 24.0 (SD 4.3) kg/m2. Overall, 2616 (73.8%) participants were Android users, 765 (21.6%) were iOS users, and 163 (4.6%) participants’ phone types were unknown. J Med Internet Res 2020 \| vol. 22 \| iss. 10 \| e21632 \| p. 3 (page number not for citation purposes) http://www.jmir.org/2020/10/e21632/ XSL•FO RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Table 1. Baseline characteristics of included participants. Females (n=1226) Males (n=2318) Total sample (N=3544) Characteristics 51.5 (9.0) 51.6 (8.8) 51.6 (8.9) Age (years), mean (SD) Age (years), n (%) 616 (50.2) 1117 (48.2) 1733 (48.9) 40-49 372 (30.3) 818 (35.3) 1190 (33.6) 50-59 181 (14.8) 271 (11.7) 452 (12.7) 60-70 57 (4.7) 112 (4.8) 169 (4.8) ≥70 22.5 (4.3) 24.8 (4.2) 24.0 (4.3) BMI (kg/m2), mean (SD)a BMI (kg/m2), n (%)b 865 (80.0) 1043 (49.2) 1908 (59.6) <25 219 (20.0) 1075 (50.8) 1294 (40.4) ≥25 Education, n (%)b 236 (26.5) 328 (19.7) 564 (22.1) High school or below 249 (27.9) 372 (22.4) 621 (24.3) Junior college 407 (45.6) 962 (57.9) 1369 (53.6) University or above Charlson Comorbidity Index, n (%) 970 (79.1) 1789 (77.2) 2759 (77.8) 0 256 (20.9) 529 (22.8) 785 (22.2) ≥1 aN=3202. bN=2554. J Med Internet Res 2020 \| vol. 22 \| iss. 10 \| e21632 \| p. 4 (page number not for citation purposes) Results As shown in Figure 1 and Table 2, daily steps (mean 8097 steps/day, range 6942-9153 steps/day) during the reference period (30 days prior to the COVID-19 epidemic in 2020) were similar to that during the corresponding period in 2019 (mean 7872 steps/day, range 6649-8912 steps/day). However, the daily steps decreased substantially after implementing physical distancing measures, from 8624 steps/day on the day before the index day to 4121 steps/day on Day 4 after the index day; this trend continued during the rest of the epidemic period. Compared with the reference period, the mean daily steps dropped by 2678 steps (95% CI 2582-2763). However, this trend was not observed during the corresponding period in 2019. g Table 1. Baseline characteristics of included participants. distancing measures, from 8624 steps/day on the day before the index day to 4121 steps/day on Day 4 after the index day; this trend continued during the rest of the epidemic period. Compared with the reference period, the mean daily steps dropped by 2678 steps (95% CI 2582-2763). However, this trend was not observed during the corresponding period in 2019. As shown in Figure 1 and Table 2, daily steps (mean 8097 steps/day, range 6942-9153 steps/day) during the reference period (30 days prior to the COVID-19 epidemic in 2020) were similar to that during the corresponding period in 2019 (mean 7872 steps/day, range 6649-8912 steps/day). However, the daily steps decreased substantially after implementing physical J Med Internet Res 2020 \| vol. 22 \| iss. 10 \| e21632 \| p. 4 (page number not for citation purposes) J Med Internet Res 2020 \| vol. 22 \| iss. 10 \| e21632 \| p. 4 (page number not for citation purposes) http://www.jmir.org/2020/10/e21632/ XSL•FO RenderX Wang et al J Med Internet Res 2020 \| vol. 22 \| iss. 10 \| e21632 \| p. 5 (page number not for citation purposes) JOURNAL OF MEDICAL INTERNET RESEARCH Figure 1. Average daily step count around the Chinese Lunar New Year period among participants in 2019 and 2020. Day 0 represents the index date; in 2020, this represents the date of the first COVID-19 case diagnosed in Changsha. in 2020, this represents the date of the first COVID-19 case diagnosed in Changsha. Table 2. Associations of time period with daily step count. 2020 daily step count 2019 daily step count Time period Mean difference (95% CI)a Mean (SD) Mean difference (95% CI)a Mean (SD) 0 (reference) 8097 (4793) 0 (reference) 7872 (4842) Reference –2672 (–2763 to –2582) 5440 (4571) –413 (–501 to –325) 7472 (4979) Epidemic aMean differences were adjusted for age and sex. Figure 2 and Table 3 present the prevalence of low daily steps (≤1500 steps/day) in 2019 and 2020 The prevalence of low (53/2693) on the day prior to the index date to 25.5% (639/2505) on Day 4 after the index date; this trend continued during the Table 2. Associations of time period with daily step count. 2020 daily step count 2019 daily step count Time period Mean difference (95% CI)a Mean (SD) Mean difference (95% CI)a Mean (SD) 0 (reference) 8097 (4793) 0 (reference) 7872 (4842) Reference –2672 (–2763 to –2582) 5440 (4571) –413 (–501 to –325) 7472 (4979) Epidemic aMean differences were adjusted for age and sex. Table 2. Associations of time period with daily step count. aMean differences were adjusted for age and sex. aMean differences were adjusted for age and sex. (53/2693) on the day prior to the index date to 25.5% (639/2505) on Day 4 after the index date; this trend continued during the rest of the follow-up period. Compared with the reference period, the PR of low daily steps was 6.2 (95% CI 5.8-6.7). However, no such trend was observed during the entire historic comparison period in 2019. Figure 2 and Table 3 present the prevalence of low daily steps (≤1500 steps/day) in 2019 and 2020. The prevalence of low daily steps was similar during the reference period in 2020 (3.0%, 2287/76,136 person-day) and the corresponding period in 2019 (3.3%, 1006/30,647 person-day). In contrast, the prevalence of low daily steps increased substantially after implementing physical distancing measures, from 2.0% J Med Internet Res 2020 \| vol. 22 \| iss. 10 \| e21632 \| p. 5 (page number not for citation purposes) http://www.jmir.org/2020/10/e21632/ http://www.jmir.org/2020/10/e21632/ XSL•FO RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Wang et al Figure 2. Prevalence of low daily step count (≤1500 steps/day) around the Chinese Lunar New Year period among participants in 2019 and 2020. Day 0 represents the index date; in 2020, this represents the date of the first COVID-19 case diagnosed in Changsha. g y p ( p y) p g p p y 0 represents the index date; in 2020, this represents the date of the first COVID-19 case diagnosed in Changsha. Table 3. Associations of time period with prevalence of low daily steps (≤1500 steps/day). 2020 low daily steps 2019 low daily steps Time period Prevalence ratio (95% CI)a Person-day, n (%) Prevalence ratio (95% CI)a Person-day, n (%) 1.0 (reference) 2287 (3.0) 1.0 (reference) 1006 (3.3) Reference 6.2 (5.8-6.7) 12,951 (18.5) 1.6 (1.4-1.7) 1488 (5.1) Epidemic aPrevalence ratios were adjusted for age and sex. As shown in Table 4, only 12 (0.4%) of 2879 participants had walked less than ≤1500 steps/day for 14 days or more (frequent low daily steps) during the 2020 reference period; however, the prevalence of frequent low daily steps increased to 7.4% (196/2655) during the COVID-19 epidemic period, after physical distancing measures were implemented. Older age (P for interaction=.001) and female sex (P for interaction<.001) were both associated with a higher prevalence of frequent low daily steps than their counterparts during the reference and COVID-19 epidemic periods, and such associations were more pronounced during the epidemic period. Participants with a university education or more had a lower prevalence of frequent low daily steps than those with only high school or less education during the reference period but this was not observed during the COVID-19 epidemic period. During the physical distancing period, no significant interaction between education and prevalence of frequent low daily steps was observed (P for interaction=.34). There was no apparent association of either BMI or CCI with the prevalence of frequent low daily steps during either period. Table 3. Associations of time period with prevalence of low daily steps (≤1500 steps/day). 2020 low daily steps 2019 low daily steps Time period Prevalence ratio (95% CI)a Person-day, n (%) Prevalence ratio (95% CI)a Person-day, n (%) 1.0 (reference) 2287 (3.0) 1.0 (reference) 1006 (3.3) Reference 6.2 (5.8-6.7) 12,951 (18.5) 1.6 (1.4-1.7) 1488 (5.1) Epidemic aPrevalence ratios were adjusted for age and sex. Table 3. Associations of time period with prevalence of low daily steps (≤1500 steps/day). J Med Internet Res 2020 \| vol. 22 \| iss. 10 \| e21632 \| p. 6 (page number not for citation purposes) JOURNAL OF MEDICAL INTERNET RESEARCH aPrevalence ratios were adjusted for age and sex. As shown in Table 4, only 12 (0.4%) of 2879 participants had walked less than ≤1500 steps/day for 14 days or more (frequent low daily steps) during the 2020 reference period; however, the prevalence of frequent low daily steps increased to 7.4% (196/2655) during the COVID-19 epidemic period, after physical distancing measures were implemented. Older age (P for interaction=.001) and female sex (P for interaction<.001) were both associated with a higher prevalence of frequent low daily steps than their counterparts during the reference and COVID-19 epidemic periods, and such associations were more pronounced during the epidemic period. Participants with a university education or more had a lower prevalence of frequent low daily steps than those with only high school or less education during the reference period but this was not observed during the COVID-19 epidemic period. During the physical distancing period, no significant interaction between education and prevalence of frequent low daily steps was observed (P for interaction=.34). There was no apparent association of either BMI or CCI with the prevalence of frequent low daily steps during either period. J Med Internet Res 2020 \| vol. 22 \| iss. 10 \| e21632 \| p. 6 (page number not for citation purposes) http://www.jmir.org/2020/10/e21632/ http://www.jmir.org/2020/10/e21632/ XSL•FO RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Wang et al Wang et al Table 4. Association of prevalence of frequent low daily steps cases (≤1500 steps/day for ≥14 days) and basic characteristics. Principal Findings Using objective data collected from the longitudinal Step Study, we found that daily steps among middle-aged and older residents in Changsha dropped rapidly and substantially (2672 fewer daily steps on average) after implementing physical distancing measures during the COVID-19 epidemic period. In addition, more than 7% (196/2655) of residents had walked ≤1500 steps/day for ≥14 days over the one-month epidemic period compared with 0.4% (12/2879) of residents in the month prior to the epidemic. The reduction of steps/day during the COVID-19 epidemic was more pronounced among older adults and females. In addition, we found that the effect of implementation of physical distancing measures on frequent low daily steps was more pronounced among older adults and females. Previous studies have examined associations between walking activities and sociodemographic factors, anthropometric factors, and comorbidity. The results from the NHANES report indicated that those of advancing age (OR 1.95, per 16.7-year increments) and female sex (OR 1.86) both had higher odds of walking less than 5000 steps/day [32]. Another study found that education was associated with increased walking activity, with one additional year of education associated with a 560 daily steps JOURNAL OF MEDICAL INTERNET RESEARCH P values for interaction Epidemic period Reference period Characteristics PR (95% CI)b Cases, n (%) Participants, n PRa (95% CI)b Cases, n (%) Participants, n N/A N/A 196 (7.4) 2655 N/Ac 12 (0.4) 2879 Total .001 Age (years) N/A 1.0 (ref) 84 (5.7) 1470 1.0 (ref)d 1 (0.1) 1543 40-49 N/A 1.7 (1.2-2.3) 69 (7.9) 871 9.9 (1.2-0.0) 8 (0.8) 957 50-59 N/A 2.2 (1.5-3.4) 32 (12.5) 257 9.2 (0.8-102.6) 2 (0.7) 308 60-70 N/A 3.0 (1.6-5.7) 11 (19.3) 57 21.0 (1.3-331.8) 1 (1.4) 71 ≥70 <.001 Sex N/A 1.0 (ref) 75 (4.2) 1793 1.0 (ref) 7 (0.4) 1904 Males N/A 3.4 (2.5-4.6) 121 (14.0) 862 2.2 (0.6-7.9) 5 (0.5) 975 Females .250 Body mass index N/A 1.0 (ref) 109 (7.7) 1415 1.0 (ref) 7 (0.5) 1557 <25 N/A 1.2 (0.9-1.7) 61 (6.2) 269 0.8 (0.2-3.2) 3 (0.3) 1006 ≥25 .335 Education N/A 1.0 (ref) 45 (11.6) 389 1.0 (ref) 6 (1.4) 441 High school or be- low N/A 0.7 (0.4-1.0) 31 (6.7) 460 0.3 (0.1-1.7) 2 (0.4) 522 Junior college N/A 1.0 (0.7-1.4) 90 (7.9) 1134 0.2 (0.0-1.1) 2 (0.2) 1228 University or above .345 Charlson Comorbidity Index N/A 1.0 (ref) 150 (7.1) 2115 1.0 (ref) 9 (0.4) 2294 0 N/A 1.1 (0.7-1.5) 46 (8.5) 540 0.9 (0.2-3.8) 3 (0.5) 585 ≥1 aPR: prevalence ratio. bPrevalence ratios were adjusted for age and sex. on of prevalence of frequent low daily steps cases (≤1500 steps/day for ≥14 days) and basic characteristics. used a wristwatch with an embedded accelerometer (Withings) demonstrated a marked decrease in daily steps (from 25% to 54%) following the official dates of home confinement in countries adopting a total lockdown [11]. Similar findings were also reported in other countries [12-14]. Our results demonstrated that such a change also occurred in the Chinese population. Furthermore, we examined daily steps within the same period in the previous year and observed no such change during this period, which enabled us to minimize the potential impact of the holiday season of Chinese New Year on daily steps. J Med Internet Res 2020 \| vol. 22 \| iss. 10 \| e21632 \| p. 7 (page number not for citation purposes) http://www.jmir.org/2020/10/e21632/ Public Health Implications Physical distancing measures play a critical role in containing COVID-19 transmission and monitoring population mobility data can provide evidence as to whether people are complying with these measures [37]; however, the impact of physical distancing on other aspects of daily life should not be overlooked. Recently, Hall and colleagues [4] commented that “The world is experiencing an extraordinary, life-altering challenge due to the COVID-19 pandemic. Many countries have become accustomed to a new normal – ‘social distancing’ and ‘shelter-in-place’ are now a part of everyday vernacular and life.” The authors warned that this health crisis has the potential to further impact and accelerate the pandemic of physical inactivity and sedentary behavior. Potential limitations of our study also deserve comment. First, the WeChat app may underestimate daily step counts because some individuals may not always carry their smartphone with them [43], especially when participants were housebound. Thus, some light walking activities at home may not be captured. However, one previous study reported that the average wear time of smartphones among Chinese citizens was more than 13 hours during the day time, indicating that most walking activities should be captured by a smartphone [19]. Second, although our validation study demonstrated that daily step counts collected from iOS and Android devices both showed close agreement with actual step counts under controlled laboratory settings, previous studies found that iOS and Android devices have different accuracies in capturing the daily step count under free-living conditions [44]. This could have led to misclassification of daily step counts in this study. Third, participants in our study were slightly younger (51.6 versus 54.8 years) and more likely to be male (65.4% versus 51.2%) than those who are aged ≥40 years and live in Changsha, according to the latest census data in 2010. However, our results showed that the prevalence of frequent low daily steps increased more among women and older people, suggesting that the overall prevalence of frequent low steps among all residents in Changsha aged ≥40 years may be even higher than what was reported in our study. Fourth, participants in our study were urban residents in Changsha who came to Xiangya Hospital for their annual health checkup. The percentage of WeChat users among these individuals (57.1%) was higher than that of the population with the same age range in China (41.5%) [45]. Strengths and Limitations Several strengths of our study are noteworthy. First, we used data from a large population-based longitudinal study (Step Study), which allowed not only investigation of COVID-19–related changes in daily step counts from over 3500 residents, but also inclusion of a historic comparison period from the prior year to account for a secular trend in daily step counts. Second, the study took advantage of a social network platform (WeChat) to capture daily step count data via smartphone in real time among the community-living population. As a smartphone has become a daily necessity for most adults, this approach allowed long-term monitoring of daily step count trends. This contrasts to previous studies using wearable devices (accelerometers) that generally only collect step count data for a relatively short time (eg, a week or less) [42]. It can be challenging to extrapolate such short-term data to describe step count patterns over a long period owing to various potential confounders [42]. Comparison With Previous Studies To date, several studies have reported changes in daily steps during the COVID-19 epidemic [10-14]. One worldwide study based on a smartphone app (Argus) showed that mean daily steps in different regions decreased by 5.5% and 27.3% (287 and 1432 steps/day, respectively) within 10 and 30 days after the COVID-19 pandemic was declared [10]. Another study that http://www.jmir.org/2020/10/e21632/ XSL•FO RenderX XSL•FO RenderX Wang et al JOURNAL OF MEDICAL INTERNET RESEARCH health issue. It is also unknown whether the observed decline in daily walking is a temporary phenomenon that may revert back to baseline levels after the disease is under control; thus, further longitudinal studies are needed so that evidence-based strategies can be developed and implemented to encourage greater participation in regular physical activity. increase [33]. Our results corroborate these findings. However, after the implementation of physical distancing measures, the prevalence of frequent low daily steps among residents with university or above education was similar to those with high school or below education, suggesting both groups followed physical distancing measures and reduced their outdoor walking activities. Nevertheless, the magnitude of relative increase in the prevalence of frequent low daily steps during the COVID-19 epidemic appeared to be greater among residents with university or above education than among those with high school or below education, indicating the former are more likely to follow instructions and communicate effectively with health providers [33,34]. Previous studies also showed that both BMI and comorbidities (eg, hypertension and diabetes) were associated with fewer daily steps [6,7,35,36], but this was not the case in the present study, nor was any association modified by the implementation of physical distancing measures. J Med Internet Res 2020 \| vol. 22 \| iss. 10 \| e21632 \| p. 8 (page number not for citation purposes) http://www.jmir.org/2020/10/e21632/ Public Health Implications They also call for further studies to evaluate whether the low levels of walking activity observed following the implementation physical distancing measures will be maintained and whether they will have significant adverse impacts on health outcomes. to be an important predictor of mortality independent of step intensity [6]. Authors' Contributions GL and CZ had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. CZ and GL are joint corresponding authors. All authors have read the manuscript, provided critical feedback on intellectual content, and approved the final manuscript. CZ, GL, YW, and YZ contributed to the concept and design. All authors contibuted to the acquisition, analysis, or interpretation of data. YW, CZ, GL, and YZ drafted the manuscript. All authors critically revised the manuscript for important intellectual content. YZ and JW contributed to statistical analysis. CZ, GL, and JW obtained funding. YW, CZ, and GL provided administrative, technical, or material support. GL, YZ, and CZ provided supervision. Conclusions Using data collected from a large population-based longitudinal study, we demonstrated that walking activity, indicated by daily step count, decreased rapidly and substantially during the COVID-19 epidemic period among middle-aged and older adult Acknowledgments This work was supported by the National Natural Science Foundation of China (81772413, 81702207, 81702206, 81930071), the National Health and Medical Research Council Principal Research Fellowship, the National Key Research and Development Project (2018YFB1105705), and the Key Research and Development Program of Hunan Province (2018SK2070, 2018SK2071). No funding bodies had any role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors thank Xiaoxiao Li, Wei Li, Huizhong Long, Jiatian Li, Haochen Wang, Yuqing Wang, Ning Wang, Bei Xu, Zidan Yang, Jing Wu, Zhenglei Zhu, and Xin Huang (Xiangya Hospital) for their contribution to this study. Conflicts of Interest None declared. Multimedia Appendix 1 Validation study. [DOCX File , 55 KB-Multimedia Appendix 1] Multimedia Appendix 2 Diagram of the study periods. Chinese New Year fell on January 25 in 2020 and February 5 in 2019. [PNG File , 234 KB-Multimedia Appendix 2] Public Health Implications Thus, our findings may not be generalizable to residents living in other parts of China, especially those living in rural areas. Finally, we were unable to capture the intensity of steps (eg, slow versus fast steps); however, total steps/day has recently been shown Our data showed that average daily steps among residents of Changsha dropped by more than 30% (from 8097 to 5440) during the COVID-19 epidemic period after implementation of physical distancing measures. These data raise concerns about the potential adverse effects of such measures on health and well-being. Previous studies have showed that higher daily steps are associated with better cardiometabolic profiles and lower all-cause mortality [6,7,38]. Other studies have also found that a decrease in daily steps of 2000 steps, irrespective of previous habitual step counts, increases the risk of insulin sensitivity and higher cardiovascular events [39,40]. Thus, our findings have important implications for public health recommendations and the prevention of other health crises during the COVID-19 pandemic. Furthermore, our data showed that the reduction in daily steps is much more common among older adults and females. In general, older adults and females are more likely to develop sedentary behaviors, placing them at greater risk of various diseases related to inactivity [32,41]; thus, the worsening trend of physical inactivity during the COVID-19 epidemic period compounds the risk of sequelae related to a sedentary lifestyle. It is uncertain as to how long it will take to completely control the COVID-19 pandemic worldwide. If similar trends toward a sedentary lifestyle are seen in other countries, the avoidance of further sedentary lifestyle behaviors and promotion of regular physical activity during this time are an urgent global public XSL•FO RenderX XSL•FO RenderX Wang et al JOURNAL OF MEDICAL INTERNET RESEARCH Chinese residents living in urban areas. These results suggest that appropriate strategies need to be taken to encourage residents to actively engage in regular physical activity while maintaining personal hygiene and physical distancing. They also call for further studies to evaluate whether the low levels of walking activity observed following the implementation physical distancing measures will be maintained and whether they will have significant adverse impacts on health outcomes. Chinese residents living in urban areas. These results suggest that appropriate strategies need to be taken to encourage residents to actively engage in regular physical activity while maintaining personal hygiene and physical distancing. http://www.jmir.org/2020/10/e21632/ JOURNAL OF MEDICAL INTERNET RESEARCH Dose-response relationship between lower serum magnesium level and higher prevalence of knee chondrocalcinosis. Arthritis Res Ther 2017 Oct 24;19(1):236 [FREE Full text] [doi: 10.1186/s13075-017-1450-6] [Medline: 29065924] 16. 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[doi: 10.1111/sms.13653] [Medline: 32176397] J Med Internet Res 2020 \| vol. 22 \| iss. 10 \| e21632 \| p. 12 (page number not for citation purposes) Abbreviations Abbreviations CCI: Charlson Comorbidity Index GEE: Generalized Estimating Equations ICC: intraclass correlation coefficients MD: mean difference NHANES: National Health and Nutrition Examination Survey PR: prevalence ratio J Med Internet Res 2020 \| vol. 22 \| iss. 10 \| e21632 \| p. 11 (page number not for citation purposes) http://www.jmir.org/2020/10/e21632/ http://www.jmir.org/2020/10/e21632/ Wang et al JOURNAL OF MEDICAL INTERNET RESEARCH Edited by G Eysenbach; submitted 19.06.20; peer-reviewed by M Alharbi, H Sasai; comments to author 12.09.20; r received 01.10.20; accepted 02.10.20; published 26.10.20 Please cite as: Wang Y, Zhang Y, Bennell K, White DK, Wei J, Wu Z, He H, Liu S, Luo X, Hu S, Zeng C, Lei G Physical Distancing Measures and Walking Activity in Middle-aged and Older Residents in Changsha, China, During the COVID-19 Epidemic Period: Longitudinal Observational Study J Med Internet Res 2020;22(10):e21632 URL: http://www.jmir.org/2020/10/e21632/ doi: 10.2196/21632 PMID: 33027035 Please cite as: Wang Y, Zhang Y, Bennell K, White DK, Wei J, Wu Z, He H, Liu S, Luo X, Hu S, Zeng C, Lei G Physical Distancing Measures and Walking Activity in Middle-aged and Older Residents in Changsha, China, During the COVID-19 Epidemic Period: Longitudinal Observational Study J Med Internet Res 2020;22(10):e21632 URL: http://www.jmir.org/2020/10/e21632/ doi: 10.2196/21632 PMID: 33027035 ©Yilun Wang, Yuqing Zhang, Kim Bennell, Daniel Kenta White, Jie Wei, Ziying Wu, Hongyi He, Shaohui Liu, Xianghang Luo, Shuo Hu, Chao Zeng, Guanghua Lei. Originally published in the Journal of Medical Internet Research (http://www.jmir.org), 26.10.2020. This is an open-access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work, first published in the Journal of Medical Internet Research, is properly cited. The complete bibliographic information, a link to the original publication on http://www.jmir.org/, as well as this copyright and license information must be included. ©Yilun Wang, Yuqing Zhang, Kim Bennell, Daniel Kenta White, Jie Wei, Ziying Wu, Hongyi He, Shaohui Liu, Xianghang Luo, Shuo Hu, Chao Zeng, Guanghua Lei. Originally published in the Journal of Medical Internet Research (http://www.jmir.org), 26.10.2020. This is an open-access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work, first published in the Journal of Medical Internet Research, is properly cited. Please cite as: Wang Y, Zhang Y, Bennell K, White DK, Wei J, Wu Z, He H, Liu S, Luo X, Hu S, Zeng C, Lei G Physical Distancing Measures and Walking Activity in Middle-aged and Older Residents in Changsha, China, During the COVID-19 Epidemic Period: Longitudinal Observational Study J Med Internet Res 2020;22(10):e21632 URL: http://www.jmir.org/2020/10/e21632/ doi: 10.2196/21632 PMID: 33027035 Abbreviations The complete bibliographic information, a link to the original publication on http://www.jmir.org/, as well as this copyright and license information must be included. http://www.jmir.org/2020/10/e21632/ http://www.jmir.org/2020/10/e21632/ XSL•FO RenderX
https://openalex.org/W4280651990	https://www.iberpapers.org/jour/article/download/468/373	Kirghiz, Kyrgyz	null	A roadmap to make Peru regional power in Latin America	Iberoamerikanskie tetradi	2,022	cc-by	7,536	Aceptado: 24.12.2021 Para citar: Sánchez, Wilder Alejandro. “Una hoja de ruta para transformar a Perú en una potencia de Latinoamérica” [A roadmap to make Peru regional power in Latin America]. Cuadernos Iberoamericanos 9, no. 4 (2021): 146-159. https://doi.org/10.46272/2409-3416-2021-9-4-146-159. [In Spanish] № 4 № 4 Том IX 2021: 146-159 Ибероамериканские тетради Политика • Política Artículo de investigación https://doi.org/10.46272/2409-3416-2021-9-4-146-159 © Wilder Alejandro Sánchez, 2021 © Wilder Alejandro Sánchez, 2021 E-mail: wilder.a.sanchez@gmail.com Wilder Alejandro Sánchez, analista de temas de geopolítica y defensa Para la correspondencia: DC 20016, Washington, D.C., P.O. Box 9747 Wilder Alejandro Sánchez, analista de temas de geopolítica y defensa Para la correspondencia: DC 20016, Washington, D.C., P.O. Box 9747 E-mail: wilder.a.sanchez@gmail.com Recibido: 17.11.2021 Revisado: 08.12.2021 Aceptado: 24.12.2021 Accepted: 24.12.2021 For citation: Sánchez, Wilder Alejandro. “Una hoja de ruta para transformar a Perú en una potencia de Latinoamérica” [A roadmap to make Peru regional power in Latin America]. Cuadernos Iberoamericanos 9, no. 4 (2021): 146-159. https://doi.org/10.46272/2409-3416-2021-9-4-146-159. [In Spanish] Declaración de divulgación: El autor declara que no existe ningún potencial conflicto de interés. Declaración de divulgación: El autor declara que no existe ningún potencial conflicto de interés. Research article https://doi.org/10.46272/2409-3416-2021-9-4-146-159 → Resumen La República del Perú celebró su bicentenario el 28 julio de 2021 con inestabilidad política, problemas económicos, un movimiento narco-terrorista, alrededor de un millón de refugiados venezolanos, y la pandemia del COVID-19. Aunque la nación andina tiene muchos aspectos a su favor que deberían ayudar a que el país tenga una mayor imagen internacional, el peso geopolítico de Perú es limitado. En este ensayo, discutiremos los principales factores que son tomados en cuenta para catalogar a un país como una potencia regional. Asimismo, se ofrecerá una hoja de ruta que el Perú puede utilizar para incrementar su imagen internacional de cara al tricentenario. Estas recomendaciones incluyen que las Fuerzas Armadas peruanas tengan un rol de liderazgo en las misiones de paz de la Organización de las Naciones Unidas (ONU); que el Perú tome a la Comunidad Andina de Naciones (CAN) bajo su dirección para que el bloque obtenga más relevancia, además de sugerencias estilo “soft power.” Igualmente, es necesario que, en el Perú, no solo el Poder Ejecutivo, sino también los diferentes sectores estratégicos, tengan la ambición de lograr que el país llegue a ser una potencia regional. En el 2021, en una Latinoamérica fragmentada, sin claros líderes regionales, y con problemas como la pandemia y la situación en Venezuela, hay lugar para el surgimiento de una nueva potencia regional. El Perú podría alcanzar ese lugar, si es que tiene la ambición y visión estratégica a largo plazo para lograrlo. 146 Vol. IX 2021: 146-159 Cuadernos Iberoamericanos No. 4 © Wilder Alejandro Sánchez, 2021 E-mail: wilder.a.sanchez@gmail.com → Palabras clave Perú, política exterior, desarrollo, estrategia, Latinoamérica, diplomacia, influencia, pax Inca Introducción La República del Perú celebró su bicentenario de independencia el 28 de julio de 2021, a pesar de que ésta es una fecha histórica y motivo de celebración para la nación sudamericana, heredera del Imperio de los Incas, la situación del país no es la ideal. La pandemia del Coronavirus (COVID-19), ahora en sus variantes Delta, Lambda (descubierta por primera vez en el Perú), Mu y Omicrón, ha golpeado duramente a la sociedad y afectado la economía nacional; mientras tanto el país mantiene su larga tradición de inestabilidad política y de corrupción en los más altos niveles del gobierno. Una muestra de dicha inestabilidad son los cuatro presidentes que gobernaron el país durante el período presidencial del 2016-2021. A nivel internacional, el Perú tiene buenas relaciones con sus cinco países vecinos, no obstante, las históricas tensiones con Chile; también es sede de la Comunidad Andina de Naciones y país miembro de los principales bloques regionales como la Alianza del Pacífico, un bloque de economías emergentes en Latinoamérica. Históricamente, ha habido peruanos que han ocupado altos cargos en organizaciones internacionales como Javier Pérez de Cuéllar, quien fue secretario general de las Naciones Unidas en la década de los ochenta. En cuanto a la economía peruana, previa a la pandemia, ha permanecido en franco crecimiento gracias a su industria y producción minera de cobre, oro, plata, zinc, hierro, entre otros metales. El país también exporta petróleo, gas, y es reconocido mundialmente por su riqueza natural, es uno de los mayores exportadores de productos agrícolas tales como quinua, papa y arándanos, y por sus atracciones turísticas, como la ciudadela de Machu Picchu. Sin embargo, a pesar de estos logros y de tener una imagen positiva para atraer inversiones, Perú no es considerado como aún una potencia regional. Si aplicamos los índices creados para catalogar a los países como “potencias mundiales,” “potencias medianas,” o “potencias regionales,” a lo mucho, hoy en día el Perú sería una potencia secundaria en el hemisferio occidental. ¿Por qué es que, dos siglos después de lograr su independencia de la corona española, Perú no ha podido retomar el lugar protagónico a nivel internacional que ocupó durante el Imperio de los Incas?, y mirando al futuro, ¿cómo se podría cambiar esta situación para que, de cara al tricentenario de su independencia, la nación andina sea reconocida como una potencia sudamericana? Estas preguntas son el eje de este análisis. → Abstract The Republic of Peru marked its bicentennial on July 28, 2021 with political instability, economic problems, a narco-terrorist movement, around one million Venezuelan refugees, and the COVID-19 pandemic. Although the Andean nation has many resources to obtain a greater international role, Peru’s geopolitical weight is limited. The article discusses the main factors that are taken into account to classify a country as a regional power. Moreover, a roadmap is offered for Peru to improve its international image for the tercentennial. These recommendations include ensuring the leading role of the Peruvian armed forces in United Nations (UN) peacekeeping missions; The Andean Community of Nations under the leadership of Peru to ensure its greatest relevance, as well as “soft power” proposals. Likewise, it is necessary that in Peru, not only the executive power, but also the different governmental agencies and civilian sectors, strive towards making their homeland a regional power. In 2021, in a fragmented Latin America, with no clear regional leader, and with problems such as the pandemic and the situation in Venezuela, there is room for the new regional power to emerge. Peru could take the role, if it has the ambition and a long-term strategic vision. 147 Том IX 2021: 146-159 № 4 № 4 Ибероамериканские тетради → Keywords Peru, foreign policy, development, strategy, Latin America, diplomacy, influence, Pax Inca Disclosure statement: No potential conflict of interest was reported by the author. No. 4 Vol. IX 2021: 146-159 Vol. IX 2021: 146-159 Vol. IX 2021: 146-159 Es interesante notar que el término Pax no se ha aplicado a un país Latinoamericano. Este ensayo teoriza que, si Perú se vuelve una potencia regional, esto traería consecuencias positivas para el resto de la región, creando una Pax Inca en Latinoamérica. Desde 1438 hasta 1532, cuando ocurrió la llegada de los conquistadores españoles, el Imperio de los Incas conquistó un tercio de Sudamérica, gobernando desde lo que hoy en día es Ecuador en el norte, hasta regiones del norte de Argentina y Chile en el sur, abarcando gran parte de los actuales territorios de Perú y Bolivia. Hoy en día, lejos de ser equivalente a un imperio o una potencia mundial, la República del Perú tiene poca influencia a nivel internacional. Cambiar esta situación requerirá no solo crecimiento económico y desarrollo social, tiempo y esfuerzo, sino también ambición y una visión a largo plazo para elevar la imagen del país andino. Introducción Mucho se ha escrito sobre los aspectos que hacen que un país sea considerado como un poder mundial – como lo son la República Popular China, la Federación Rusa, y los Estados Unidos – o para que un país sea una potencia regional con aspiraciones mundiales – como Alemania, Corea del Sur, India, Japón y en el hemisferio occidental Brasil. Sin embargo, también es importante analizar qué es lo que necesita hacer un país para transformarse en una potencia regional y qué significa tener este título. 148 Cuadernos Iberoamericanos Construcción de una potencia regional Las características que definen a una potencia mundial y a una potencia regional es un tema que ha sido discutido por académicos y analistas, tomando a varios países como casos de estudio. Sin embargo, términos como “potencia “mundial,” y “potencia regional,” siguen siendo difíciles de definir, especialmente en el globalizado mundo de la postguerra fría. Como lo argumenta Defte Nolte, se puede decir que hay una “falta en general, de instrumentos analíticos para identificar y comparar a los países regionales, y para diferenciar a las potencias regionales de potenciales mundiales y medianas. Tal vez la diferencia involucra los términos, “potencia” y “región,” que son contextualizados de manera diferente en la teoría de relaciones internacionales, con “gran variación en sus significados,” explica Nolte.1 Como resultado, si se suman dos conceptos ambiguos y complejos, los problemas semánticos se van a acumular, dificultando un eficiente análisis o la creación de un apropiado catálogo de los países.2 Martha Ardila cataloga a los países de de la siguienta manera: “potencia (gran potencia), potencia media (PM), potencia regional (PR) y potencia regional secundaria (PRS).”3 Mientras tanto, Marcos Pablo Moloeznik clasifica a los países como: las potencias mundiales; las potencias medias, y las potencias regionales. “Ambas, potencias medias y regionales se ubican, estructuralmente, entre las potencias mundiales o centrales (rubro que incluye a las superpotencias) y los Estados menores o periféricos,” explica Moloeznik.4 El lector se dará cuenta que se utilizan adjetivos genéricos como “media,” “gran,” y “secundaria” para catalogar a los países. Algunas de las características usuales que son utilizadas para definir a una gran potencia. “El concepto de potencia es un referente geopolítico que incluye aspectos militares, políticos y económicos, el cual hace alusión a un conjunto de países y a su jerarquización en el sistema internacional.”5 Los países se diferencian en “la capacidad de proyectar su poder militar en diferentes regiones del mundo y en la posibilidad de ejercer su influencia política en forma global.”6 Por otro lado, Nolte propone una lista de temas a tomar en consideración para definir a un “gran poder regional”: – Un país que es geográficamente parte de una región específica – Un país que tiene mucha influencia en asuntos regionales – Un país que tiene mucha influencia en asuntos regionales – Un país que, a diferencia de un “poder medio” también puede volverse un poder mundial, además de ser una potencia regional.7 1 1 Nolte 2010, 883. № 4 Том IX 2021: 146-159 Том IX 2021: 146-159 De igual importancia es la intención de un gobierno de tener una mayor presencia en la geopolítica internacional. Es necesario tener “la pretensión de liderar; la influencia en la geopolítica e identidad, la disposición de recursos naturales, organizativos e ideológicos para la proyección de poder,” como otras características esenciales. También es importante la conexión económica, política y cultural con la región y la influencia en la estructura de gobernanza regional.1 También se han publicado varios análisis que discuten las potencias regionales emergentes, con un enfoque en Latinoamérica. Argentina es analizada como una potencia regional durante los gobiernos de Néstor Kirchner (2003–2007), Cristina Fernández de Kirchner (2007–2015), y Mauricio Macri (2015–2019), argumentando que “la geografía, el número de habitantes, el acceso a las tecnologías, las capacidades militares y económicas, normativas y diplomáticas comenzaron a ser consideradas algunas de las más relevantes a la hora de categorizar a un estado como ‘potencia regional’.”2 La política exterior de un país, incluyendo la membresía a organizaciones de elite regionales, es vital para volverse una potencia regional. Al analizar tres administraciones argentinas, se menciona como “la administración Macri asumió en relación con su posicionamiento en tanto potencia emergente: y el ingreso a Argentina a la Organización de Cooperación y Desarrollo Económico (OCDE).” También, “otra de las características que un poder regional debería detentar es aquella referida a la experiencia negociadora en la integración con los países vecinos y la capacidad de articular allí los instrumentos domésticos y regionales para mejorar las relaciones con los mismos.”3 La palabra liderazgo es de vital importancia para un país que quiere transformarse en potencia. Es decir, una potencia lidera “proyectos de integración, concertación y cooperación en términos militares, económicos y políticos. Dentro de ellos, los de seguridad adquieren un especial significado, bien sea como complejo o como comunidad de seguridad.”4 En este contexto una potencia es reconocida como tal no solo por su poder militar y económico, sino también porque toma la iniciativa y un rol protagónico en temas de interés regional, siendo seguido y apoyado por otros gobiernos regionales. Aquí hay un detalle críticamente importante y relevante para nuestro análisis: el reconocimiento por parte de los demás gobiernos. № 4 En otras palabras, no es suficiente que un país se autodefina, o actúe como una potencia; otros estados regionales deben comenzar a ver a dicho estado como un líder, apoyar sus iniciativas, y buscar aliarse a dicho estado en diferentes áreas; un país se vuelve una potencia cuando otros estados lo reconocen como tal. Finalmente, el simplemente hablar del “gobierno” o “estado” en alusión a un país no es suficiente, ya que usualmente se asume que se refiere solamente al poder ejecutivo y ministerios como cancillería, comercio exterior y defensa. Sin embargo, para estructurar la política exterior se incluyen otros niveles de gobierno como el poder legislativo, electoral y de organizaciones sociales, así como la dinámica público-privada. Construcción de una potencia regional 2 Ibid. 3 Ardila 2014, 89. 4 Moloeznik 2012, 197. 5 Ardila 2014, 89. 6 Ibid. 7 Nolte 2010, 889. 1 Nolte 2010, 883. 2 Ibid. 3 Ardila 2014, 89. 4 Moloeznik 2012, 197. 5 Ardila 2014, 89. 6 Ibid. 7 Nolte 2010, 889. 1 Nolte 2010, 883. 2 Ibid. 3 Ardila 2014, 89. 4 Moloeznik 2012, 197. 5 Ardila 2014, 89. 6 Ibid. 7 Nolte 2010, 889. 149 Ибероамериканские тетради № 4 1 Ardila 2012, 297. 2 González 2019, 307. 3 Ibid., 338 4 Ardila 2012, 297. No. 4 Vol. IX 2021: 146-159 Vol. IX 2021: 146-159 drásticamente y, la combinación de las políticas regionales de Brasil y Venezuela significaría el fin de la influencia histórica de Washington sobre Latinoamérica y el Caribe. drásticamente y, la combinación de las políticas regionales de Brasil y Venezuela significaría el fin de la influencia histórica de Washington sobre Latinoamérica y el Caribe. En ese entonces, Brasil era una potencia económica, y bajo la presidencia de Lula da Silva, lideraba la integración regional. Gracias a Brasil fueron creados dos bloques: la Unión de Naciones Sudamericanas (UNASUR) y la Comunidad de Estados Latinoamericanos y del Caribe (CELAC)1 Es más, Brasil era parte de un grupo de países que pretendía reformar el Consejo de Seguridad de las Naciones Unidas para expandir sus miembros permanentes y tener un representante por región. Brasil era el favorito para representar a Latinoamérica y el Caribe. Hay tres razones principales por las que Brasil era visto como actor global de Latinoamérica: “1) por su política exterior de largo plazo y el altamente profesionalizado servicio exterior que actúa con independencia del Ejecutivo; 2) por sus relaciones comerciales divididas en partes iguales entre Europa, EE. UU., América Latina y Asia, y 3) por su larga presencia y activa participación en foros y negociaciones internacionales.”2 Sin embargo, el análisis citado en el párrafo anterior fue escrito en el 2013, antes de la crisis política del 2015 que terminó con la vacancia de la entonces presidente Dilma Rousseff, y una nueva crisis financiera brasilera que ha sido exacerbada por la pandemia. También hay que agregar la presidencia de Jair Bolsonaro (electo el 2018) ha cambiado la orientación y objetivos de la política exterior del país, ya que Brasilia se enfoca más en Estados Unidos y Europa, y menos en sus vecinos geográficos, con excepción de Venezuela.3 Brasil ha sido elegido para representar a Latinoamérica y el Caribe como miembro no permanente del Consejo de la ONU para el periodo 2022–2023, y la economía brasilera sigue siendo fuerte, particularmente si es comparada con varios países vecinos. Similarmente, hace una década, Venezuela, con el ex-presidente Hugo Chávez aún en control del país, se había vuelto una potencia regional gracias al alto precio del petróleo, que permitió a Caracas financiar varios ambiciosos proyectos, como la compra de armamento y la creación de Petrocaribe. 1 Jorge Marirrodriga, “Brasil impulsa un organismo común de defensa suramericano,” El País, May 23, 2008, https://elpais.com/internacional/2008/05/24/actualidad/1211580001_850215.html. También ver: Martín Granovsky, “La Celac en diez claves,” Foro de Sao Paulo, December 3, 2011, https://forodesaopaulo.org/la-celac-en-diez-claves/. 2 Casado et al. 2013 25. 4 Estefania Pozzo, “Opinión: Crisis económica en Argentina: el problema no es solo la inflación, también los salarios,” Washington Post, August 9, 2021, https://www.washingtonpost.com/es/ post-opinion/2021/08/09/crisis-economica-argentina-sueldos-inflacion-dolar-fernandez- macri/. 3 Sánchez 2020. ¿Hay potencias en América Latina en la actualidad? En el 2011, la respuesta a esta pregunta hubiera sido relativamente fácil de responder. En ese entonces, se especulaba que la geopolítica de las Américas iba a cambiar 1 Ardila 2012, 297. 2 González 2019, 307. 3 Ibid., 338 4 Ardila 2012, 297. 1 Ardila 2012, 297. 2 González 2019, 307. 3 Ibid., 338 4 Ardila 2012, 297. 150 Cuadernos Iberoamericanos No. 4 p p 2 Casado et al. 2013, 25. No. 4 Con Chávez como líder, Venezuela creó la Alternativa Bolivariana para las Américas (ALBA), con varios países regionales como miembros. La situación es diferente hoy en día, Venezuela atraviesa por una crisis socioeconómica muy fuerte, que ha resultado en una crisis migratoria, algunos gobiernos han limitado sus relaciones con el gobierno de Nicolás Maduro. Aunque el gobierno de Maduro se mantiene en el poder, y tiene aliados regionales como Bolivia, Cuba y Nicaragua, y algunos países caribeños, su peso internacional es actualmente diferente al de hace algunos años. Entre tanto Argentina sigue siendo vista como un líder en MERCOSUR, pero la pandemia del COVID-19 y la actual presidencia del presidente Alberto Fernández comenzaron casi paralelamente, esta realidad afecta un análisis sobre la política exterior Argentina durante la era Fernández.4 Cabe recalcar que en los dos últimos años se han presentado sucesivas protestas en la región, que han estremecido a los gobiernos regionales. Tres ejemplos ocurrieron en 2019: Chile, que antes era visto como una isla de estabilidad, sufrió de violentas protestas 151 Ибероамериканские тетради Том IX 2021: 146-159 № 4 que forzaron al gobierno a convocar a un referéndum para re-escribir la constitución.1 Igualmente, violentas protestas en Ecuador obligaron al entonces-presidente Lenin Moreno a escapar de Quito y temporalmente reubicarse en Guayaquil.2 Mientras tanto, en Bolivia las protestas luego de las elecciones presidenciales del 2019 terminaron en la renuncia del entonces Presidente Evo Morales.3 que forzaron al gobierno a convocar a un referéndum para re-escribir la constitución.1 Igualmente, violentas protestas en Ecuador obligaron al entonces-presidente Lenin Moreno a escapar de Quito y temporalmente reubicarse en Guayaquil.2 Mientras tanto, en Bolivia las protestas luego de las elecciones presidenciales del 2019 terminaron en la renuncia del entonces Presidente Evo Morales.3 Existen otros candidatos, como Colombia y México para Latinoamérica, y Jamaica en el Caribe. Aunque estos países tienen una buena imagen regional, a pesar de sus problemas, el autor de este análisis argumentaría que no han aparecido nuevos gobernantes que tengan la intención de incrementar el peso de sus países a nivel regional. No. 4 La pandemia del COVID-19 y sus consecuencias socio-económicas son argumentos válidos para explicar por qué no hay un país líder en Latinoamérica hoy en día; por otro lado es precisamente en medio de una crisis cuando los gobernantes, no solo presidentes sino también ministros, jefes de institutos de salud y hasta jefes militares, con visiones amplias deben aparecer para guiar no solo al país sino también a la región a través de estos tiempos turbulentos, promoviendo la cooperación y políticas de salud unificadas y efectivas. En otras palabras, hasta ahora, la pandemia ha sido una oportunidad no aprovechada para promover la cooperación latinoamericana y ver si algún individuo, o gobierno, puede alzarse para convertirse en un líder regional durante esta tormenta. 1 “Protestas en Chile: la histórica marcha de más de un millón de personas que tomó las calles de Santiago,” BBC News Mundo, October 26, 2019, https://www.bbc.com/mundo/noticias- america-latina-50190029. 2 “Crisis en Ecuador: continúan las protestas mientras el gobierno y el movimiento indígena se preparan para dialogar este domingo,” BBC News Mundo, October 11, 2019, https://www.bbc.com/mundo/noticias-america-latina-50009459. 3 Ciara Nugent, “Bolivian President Evo Morales Has Resigned After Nearly 14 Years in Power. Here’s What to Know,” Time, November 11, 2019, https://time.com/5723753/bolivia-evo-morales- resigns/. 4 Jacqueline Fowks, “La economía en Perú cayó un 11% en 2020, el mayor retroceso en 30 años,” El País, February 15, 2021, https://elpais.com/economia/2021-02-16/la-economia-en-peru- cayo-un-11-en-2020-el-mayor-retroceso-en-30-anos.htm. 5 “Migración venezolana genera impacto fiscal de US$ 35 millones en Perú,” Gestión, July 27, 2021, https://gestion.pe/peru/migracion-venezolana-genera-impacto-fiscal-de-us-35-millones- en-peru-noticia/. 6 “La economía peruana registrará uno de los mayores crecimientos a nivel mundial entre el 2021 y 2022 y fortalecerá sus cuentas fiscales,” Ministerio de Economía y Finanzas (Perú), August 27, 2021, https://www.mef.gob.pe/index.php/?option=com_content&view=article&id=7134&Itemid=1 01108&lang=es. g 4 Jacqueline Fowks, “La economía en Perú cayó un 11% en 2020, el mayor retroceso en 30 años,” El País, February 15, 2021, https://elpais.com/economia/2021-02-16/la-economia-en-peru- cayo-un-11-en-2020-el-mayor-retroceso-en-30-anos.htm. p 6 “La economía peruana registrará uno de los mayores crecimientos a nivel mundial entre el 2021 y 2022 y fortalecerá sus cuentas fiscales,” Ministerio de Economía y Finanzas (Perú), August 27, 2021, https://www.mef.gob.pe/index.php/?option=com_content&view=article&id=7134&Itemid=1 01108&lang=es. 1 “Protestas en Chile: la histórica marcha de más de un millón de personas que tomó las calles de Santiago,” BBC News Mundo, October 26, 2019, https://www.bbc.com/mundo/noticias- america-latina-50190029. y y 5 “Migración venezolana genera impacto fiscal de US$ 35 millones en Perú,” Gestión, July 27, 2021, https://gestion.pe/peru/migracion-venezolana-genera-impacto-fiscal-de-us-35-millones- en-peru-noticia/. p 3 Ciara Nugent, “Bolivian President Evo Morales Has Resigned After Nearly 14 Years in Power. Here’s What to Know,” Time, November 11, 2019, https://time.com/5723753/bolivia-evo-morales- resigns/. 1 1 Alberto Vergara, “El fujimorismo ha sido un mal innecesario para Perú,” The New York Times, October 22, 2018, https://www.nytimes.com/es/2018/10/22/espanol/opinion/opinion- fujimorismo-innecesario-peru.html. También ver: “¿Realmente es Keiko Fujimori la mujer más poderosa de Perú?” France 24, September 12, 2019, https://www.france24.com/es/20190912- keiko-fujimori-mujer-poderosa-peru. 2 “Así fue el camino de Pedro Pablo Kuczynski hasta la renuncia: los escándalos que lo sacudieron,” CNN Español, March 21, 2018, https://cnnespanol.cnn.com/2018/03/21/ kuczynski-renuncia-escandalos-odebrecht-vacancia-indulto/. 3 “Peruvian Congress votes to impeach President Martín Vizcarra,” BBC News, November 10, 2020, https://www.bbc.com/news/world-latin-america-54872826. 4 Stefano Pozzebon et al., “Peru’s interim president resigns after just five days,” CNN, November 15, 2020, https://www.cnn.com/2020/11/15/americas/peru-interim-president-resigns-intl/index.html. 5 Dan Collyns, “Peru’s congress elects Francisco Sagasti as new interim president,” The Guardian, November 16, 2020, https://www.theguardian.com/world/2020/nov/16/perus-congress-elects- francisco-sagasti-as-new-interim-president. 6 Stefanie Eschenbacher, and Marco Aquino, “Peru leftist Castillo claims election win as Fujimori fights result,” Reuters, June 16, 2021, https://www.reuters.com/world/americas/perus-castillo- leads-election-with-501-votes-after-all-ballots-tallied-2021-06-15/. No. 4 Vol. IX 2021: 146-159 Vol. IX 2021: 146-159 Para el año 2022, la economía mantendrá su dinamismo y se proyecta que crecería 4,8%, impulsada por el incremento del gasto privado, el avance de las exportaciones (ante la mayor producción de Mina Justa y el inicio gradual de Quellaveco) y la mejora de la demanda externa, en un entorno de mayor control de la pandemia y vacunación masiva de la población. Por otro lado, Perú aún tiene un fuerte sector económico informal. A su vez, el país depende de su industria minera, agricultora y ganadera para mantener a la economía creciendo y aún más, el país andino no es productor y exportador de productos manufacturados a gran escala. A nivel político la inestabilidad sigue siendo un problema. Esto es una pena ya que se esperaba que luego de la dictadura de Alberto Fujimori (1990–2000), habría más estabilidad y buena gobernanza. En los últimos años la corrupción y los intereses de partidos políticos como Fuerza Popular liderado por Keiko Fujimori, política e hija del ex-dictador Fujimori, han impedido que los presidentes puedan gobernar.1 Por ejemplo, el presidente Pedro Pablo Kuczynski no pudo cumplir su mandato presidencial, gobernando solamente de 2016 a 2018. El jefe de estado sobrevivió dos procesos de vacancia, organizados por congresistas aliados de Fujimori, sin embargo tuvo que renunciar por acusaciones de sobornos y corrupción.2 Él fue reemplazado por su vicepresidente, el moqueguano Martin Vizcarra, quien tampoco pudo terminar el mandato, gobernando del 23 de marzo de 2018 al 9 de Noviembre del 2020.3 Debido a dos mociones de censura llevadas a cabo por los congresistas fujimoristas, Vizcarra renunció al cargo, siendo reemplazado por Manuel Merino, quien solamente gobernó por menos de una semana, del 10 al 15 de noviembre del 2020. Cuadernos Iberoamericanos Cuadernos Iberoamericanos Bicentenario del Perú La República del Perú recibió el bicentenario con celebraciones patrióticas, optimismo, problemas y desafíos. La famosa frase del país sudamericano “el Perú es más grande que sus problemas” es muy apropiada hoy en día. Ciertamente la pandemia del COVID-19, sigue siendo un grave problema. A pesar de tener uno de los programas de cuarentena más fuertes de la región, más de 200 mil peruanos han fallecido hasta la fecha. Además, se necesitará de más tiempo para que el programa nacional de vacunación cubra toda la población con las dos dosis, o hasta una tercera dosis. Es importante mencionar que la economía ha sido afectada por dos crisis, que ocurrieron muy cercanas una a la otra.4 La otra crisis comenzó antes de la pandemia: debido a la situación en Venezuela, más de un millón de refugiados venezolanos salieron de su patria en busca de una mejor vida y ahora viven en Perú.5 A pesar de todo, en temas económicos el Ministerio de Economía y Finanzas ha reportado que para el 2021 “se elevó la proyección de crecimiento del PBI a 10,5%, como resultado de la fuerte recuperación de la actividad económica impulsada tanto por factores externos como internos.”6 Asimismo, 152 1 Álvaro Cordero, “Canciller de Perú, Héctor Béjar, renuncia a menos de un mes en el cargo,” France24, August 18, 2021, https://www.france24.com/es/am%C3%A9rica-latina/20210818- peru-renuncia-canciller-bejar-marina. 2 “Maritime Dispute (Peru v. Chile),” International Court of Justice, accessed September 20, 2021, https://www.icj-cij.org/en/case/137. 3 Perú no tiene reelección presidencial directa, se tiene que esperar por lo menos un periodo presidencial. 4 “Declaración del Grupo de Lima,” Comunicado de Prensa, Ministerio de Relaciones Exteriores (Perú), September 15, 2018, https://www.gob.pe/institucion/rree/noticias/19021-declaracion- del-grupo-de-lima. No. 4 En temas de política exterior, ya ha ocurrido la primera baja, ya que el Ministro de Relaciones Exteriores Héctor Béjar renunció después de 19 días como Canciller (29 de julio al 17 de agosto del 2021), debido a controversiales comentarios que hizo, antes de asumir el cargo, sobre el rol de la Marina de Guerra durante el conflicto interno peruano.1 Su sucesor fue Óscar José Maúrtua de Romaña, quien asumió el cargo el 20 de Agosto 2021; Maúrtua es diplomático de carrera, fue Embajador de Perú en Canadá, Bolivia, Tailandia, Laos y Ecuador entre otros, además de ex Canciller de la República (2005–2006). En temas de seguridad y defensa, la situación no ha variado en años recientes. Perú mantiene buenas relaciones con sus cinco países vecinos (Bolivia, Brasil, Chile, Colombia y Ecuador) a pesar de que con Santiago existe el legado de la Guerra del Pacifico del Siglo XIX. En 2014, la Corte Internacional de Justicia emitió un veredicto que puso final a una disputa del límite marítimo entre los dos países.2 En cuestión de seguridad interna, se producen protestas con regularidad y la inseguridad ciudadana sigue siendo un problema grave. Además, el grupo Militarizado Partido Comunista del Perú, MPCP (otrora conocido como Sendero Luminoso), aún existe, aunque su área de operaciones es muy limitada, es decir comprende ciertas zonas andinas y amazónicas del país. Este autor argumenta que lo que falta en Perú es un análisis pausado previo a la toma de decisiones en política interna y externa. La inestabilidad política demuestra que los planes de un gobierno no duran más que cinco años, equivalentes a un periodo presidencial.3 La situación es peor aún para los cancilleres y otros ministros, que tienen una vida muy corta en el cargo, usualmente no más de dos años, y en casos extremos, solamente unas semanas. En esta realidad, es imposible crear planes a largo plazo, mucho menos una hoja de ruta para mejorar la imagen del país a nivel internacional. La excepción a la regla es la economía, ya que los presidentes peruanos del siglo XXI no han afectado la dependencia económica que el gobierno tiene en la industria minera, ni han modificado el modelo económico con el fin de conservar la atractiva imagen del Perú para atraer a inversionistas extranjeros. No. 4 Esto ocurrió debido a masivas protestas populares causadas por el repudio de la población a la corrupción de las clases gobernantes y la salida de Vizcarra; Merino renunció en desgracia.4 El congresista Francisco Sagasti fue elegido por el Congreso para terminar el mandato presidencial de Kuczynski, cuyo periodo fue de julio 2016 a julio 2021.5 Sagasti gobernó del 17 de noviembre del 2020 hasta el 28 de julio del 2021, cuando entregó el mando a Pedro Castillo, quien ganó las elecciones del 2021, derrotando a Keiko Fujimori en segunda vuelta.6 Algunas ideas de Castillo son vistas con preocupación por las clases altas, mientras que se espera que los partidos aliados a Fujimori continúen con su bien conocida estrategia de no dejar gobernar al Presidente de la República de turno e impedir que lleve a cabo a su plan de gobierno, y fomentar el repudio popular para intentar vacarlo, como ya ocurrió con otros ex-jefes de estado. A la fecha, el presidente Castillo, aunque tiene el apoyo de gran parte de la población peruana, ha tenido problemas para gobernar, ya que el Congreso es controlado por partidos 153 Ибероамериканские тетради № 4 Том IX 2021: 146-159 Том IX 2021: 146-159 de oposición. En temas de política exterior, ya ha ocurrido la primera baja, ya que el Ministro de Relaciones Exteriores Héctor Béjar renunció después de 19 días como Canciller (29 de julio al 17 de agosto del 2021), debido a controversiales comentarios que hizo, antes de asumir el cargo, sobre el rol de la Marina de Guerra durante el conflicto interno peruano.1 Su sucesor fue Óscar José Maúrtua de Romaña, quien asumió el cargo el 20 de Agosto 2021; Maúrtua es diplomático de carrera, fue Embajador de Perú en Canadá, Bolivia, Tailandia, Laos y Ecuador entre otros, además de ex Canciller de la República (2005–2006). de oposición. No. 4 Sin embargo, la protección del modelo económico no es suficiente para crear una visión internacional o planes de desarrollo a largo plazo. 1 “Jefe de Estado presidió ceremonia de Afirmado del Pabellón Nacional del Buque Multipropósito BAP Pisco,” Marina de Guerra del Perú, June 6, 2018, https://www.marina.mil.pe/ es/noticia/jefe-de-estado-presidio-ceremonia-de-afirmado-del-pabellon-nacional-del- buque-multiproposito-bap-pisco/; “Peru signs deal for 20 KAI KT-1 basic trainers,” Flight Global, November 8, 2012, https://www.flightglobal.com/peru-signs-deal-for-20-kai-kt-1-basic- trainers/107766.article; “China donará materiales militares a Perú valorizados en US$ 7 millones,” Gestión (Perú), June 8, 2019, https://gestion.pe/peru/politica/china-donara-materiales- militares-peru-valorizados-us-7-millones-nndc-269584-noticia/. 2 La Carta Democrática Interamericana fue adoptada por aclamación en una Asamblea General extraordinaria de la OEA celebrada en Lima el 11 de septiembre de 2001. Pax Inca 1.0 El Ensayo suscrito por el autor en 2013 para el Centro Argentino de Estudios Internacionales (CAEI), abordó sugerencias sobre como Perú podía mejorar su desempeño internacional de cara al bicentenario del 2021. Estas recomendaciones incluyeron: – La organización de conferencias sobre el desminado humanitario, una misión en la que las fuerzas peruanas están muy envueltas; adicionalmente el fortalecimiento de las operaciones de paz de la Organización de las Naciones Unidas. En este contexto Perú también promovió la creación del Grupo de Lima;4 – Destacar el rol de la nación en los bloques multinacionales: Perú es miembro de entidades como la Organización de Estados Americanos, la Comunidad 154 Cuadernos Iberoamericanos No. 4 Vol. IX 2021: 146-159 de Estados Latinoamericanos y Caribeños, la Comunidad Andina, la Alianza del Pacífico, y el Grupo de Lima, entre otros. de Estados Latinoamericanos y Caribeños, la Comunidad Andina, la Alianza del Pacífico, y el Grupo de Lima, entre otros. – Reestructuración y fortalecimiento del contingente militar: El astillero estatal SIMA está construyendo buques de transporte y buques patrulla para la Marina de Guerra del Perú (MGP).1 – Difusión de la riqueza cultural y recursos minerales. p 2 La Carta Democrática Interamericana fue adoptada por aclamación en una Asam General extraordinaria de la OEA celebrada en Lima el 11 de septiembre de 2001. Pax Inca v2.0: Perú rumbo al tricentenario Esta versión ofrece una proyección de largo alcance sobre análisis previo, cuyo fondo pueda ser consolidado y cuya forma pueda ser modificada dependiendo de las nuevas realidades del país, región y mundo. Tomando en consideración los escritos académicos revisados en la tercera sección, estas propuestas incluyen: académicos revisados en la tercera sección, estas propuestas incluyen: – Crear el Plan de Desarrollo de la República hacia el 2121: “Plan Inca 2121,” con objetivos de corto, mediano y largo plazo: el propósito es garantizar la estabilidad y permanencia ante las eventualidades políticas de los gobernantes de turno. Para un enfoque pragmático se han determinado cuatro etapas generales: Estudios, análisis y elaboración; difusión, ejecución y evaluación del Plan de desarrollo; posicionamiento regional y, finalmente, consolidación de logros y liderazgo regional. – Promover centros de formación diplomática y experticia internacional para que los ciudadanos peruanos accedan e integren Organizaciones Internacionales como la Organización de Estados Americanos, Naciones Unidas, Corte Internacional de Justicia, el Tribunal Penal Internacional, o el Banco Mundial. – Liderar la labor de la Comunidad Andina de Naciones para lograr roles regionales. La sede oficial debe ser aprovechada de mejor manera. – Fomentar a Lima como la sede de los tratados multinacionales tal como se lo ha realizado en anteriores ocasiones (Carta Democrática Interamericana, Creación del Grupo de Lima)2 Existen localidades amazónicas cuyas características intrínsecas pueden favorecer la firma de tratados internacionales en defensa de la flora y fauna silvestre. – La exportación de gas o minerales en la actualidad pueden ser complementadas con otros productos como: los arándanos, el cacao, quinua, papas y plátanos entre otros, así como con líneas de productos exóticos o productos manufacturados; – Modernizar e incrementar la infraestructura pública. Un buen ejemplo son los sistemas portuarios. Debido a la ubicación geográfica del Perú, el país puede ser un punto clave del tráfico marítimo por el Océano Pacifico en 155 Ибероамериканские тетради Том IX 2021: 146-159 Том IX 2021: 146-159 № 4 dirección al Canal de Panamá. El puerto del Callao y los terminales portuarios de Salaverry, Chancay y San Martín deben recibir mayor inversión para este fin.1 dirección al Canal de Panamá. Pax Inca v2.0: Perú rumbo al tricentenario El puerto del Callao y los terminales portuarios de Salaverry, Chancay y San Martín deben recibir mayor inversión para este fin.1 – Tener un rol más grande en operaciones de paz de la ONU: Esto no solo significa contribuir con personal de tropas sino también con personal que esté preparado para comandar y liderar las misiones de paz. Por ejemplo el ejército brasilero comandó las misiones de paz en Haití (MINUSTAH) y ahora en la República Democrática del Congo (MONUSCO).2 El Centro de Entrenamiento y Capacitación Para Operaciones de Paz (CECOPAZ) de Perú puede especializarse en el entrenamiento diferentes zonas geográficas y traer personal militar de países vecinos para entrenarlos, o enviar instructores al exterior; – Poseer una Base permanente en la Antártida. Esto ayudaría a que Perú sea considerado como un miembro más activo de la comunidad internacional en la Antártida. Además desde esta Base, los científicos y académicos peruanos podrían impulsar estudios científicos.3 – Crear el centro del estudio de la quinua, similar al Centro Internacional de la Papa (International Potato Center, ubicado en Lima), con la finalidad de potenciar el consumo de este importante grano. De igual manera, se deben promover los centros de idiomas indígenas como el quechua y el aimara. Ya existen varios centros de estudio de estas lenguas en Perú, sin embargo, se deben crear nuevas alianzas entre éstos y entidades educativas internacionales. Cast o 0 0, 5 2 El Teniente General Marcos De Sá Affonso Da Costa, de Brasil, fue nombrado como el comandante de MONUSCO en Abril del 2021. Da Costa es el sucesor del Teniente General Ricardo Augusto Ferreira Costa Neves, también de Brasil, que comando a la fuerza hasta el 31 de Marzo del 2021. “Lieutenant General Marcos De Sá Affonso Da Costa of Brazil - Force Commander for the United Nations Organization Stabilization Mission in the Democratic Republic of the Congo (MONUSCO),” United Nations – Secretary General, April 8, 2021. 3 Sánchez and Tielemans, 2015. 4 Chile Colombia México y Perú 1 Castillo 2020, 15. Conclusión Este análisis, una continuación de un ensayo escrito por el autor en el 2013, propone estrategias para que la República del Perú se convierta en una potencia regional media en Latinoamérica. Para que un país sea considerado como una potencia regional, hay muchos elementos a considerar. El poder económico, militar, cultural, el “soft power,” y la influencia que tiene un gobierno sobre otros. Para el autor de este análisis, todo comienza con la ambición de, no solo del gobierno en turno, sino también de otras autoridades locales, jefes militares, grupos empresariales y la población en general, que aspiran a lograr que su país sea reconocido como una potencia y a que se tracen los lineamientos y objetivos para tal fin. El Imperio de los Incas dominó gran parte de Sudamérica; sus construcciones y otros logros son estudiados y alabados hoy en día. Por otro lado, tras dos siglos de independencia, el Perú aún tiene pendiente posicionarse a nivel internacional. Los problemas políticos internos, las débiles relaciones diplomáticas, entre otros son algunos de los impedimentos para que el país andino gane el título de potencia regional latinoamericana. Sin embargo, este autor argumenta que el mayor impedimento es la falta de planificación estratégica de largo alcance. Para crear una Pax Inca en Latinoamérica, Perú tiene las herramientas necesarias, pero aún queda irresuelta la articulación estatal. No. 4 Vol. IX 2021: 146-159 Vol. IX 2021: 146-159 Para ello Perú goza de buenas relaciones con Brasil y Colombia, desde 1974, las tres armadas realizan ejercicios y operaciones conjuntas, la iniciativa se llama BRACOLPER, en la triple frontera amazónica.1 De igual manera, el gobierno peruano debe buscar a otros países en Sudamérica, y también en Centroamérica y el Caribe con los que puede crear alianzas estratégicas, aparte de sus países vecinos. Otros analistas han discutido la importancia del “poder blando” o “soft power,” es decir las acciones no-militares que un gobierno puede llevar a cabo para incidir en las acciones o decisiones de otros actores. Perú en ese sentido tiene mucho que ofrecer: la creación de un centro internacional de la quinua, y un centro internacional de lenguas como el quechua y el aimara son buenas iniciativas. Estos centros pueden firmar acuerdos académicos y de investigación con universidades y centros de estudio de la región, por ejemplo, en Bolivia, un país con el que Perú tiene relaciones históricas muy cercanas. Este tipo de proyectos educacionales y científicos incrementarían las relaciones con otros países. 1 Julieta Pelcastre, “Brazil, Colombia, Peru Sail Together in Bracolper 2021,” Diálogo, August 17, 2021, https://dialogo-americas.com/articles/brazil-colombia-peru-sail-together-in- bracolper-2021/#.YT-ni3spBH4. Discusión La segunda sección de este análisis enumeró los aspectos que son discutidos por académicos y analistas para determinar cómo se cataloga a un país. Por ende, la calificación de potencia regional secundaria para Perú es apropiada. El desempeño peruano en sus relaciones regionales tuvo su mejor momento en la conformación del Grupo de Lima, ya que el Perú demostró su liderazgo para, temporalmente, unir a la región de cara a la situación en Venezuela, sin embargo, esta iniciativa parece ya estar disolviéndose. A la vez, a pesar de tener una economía en crecimiento, especialmente antes de la pandemia, Lima no ha tomado un rol de líder de la Alianza del Pacífico, un bloque de cuatro países con economías similares.4 De cara al futuro, hay que recordar que, a pesar de sus problemas, el país Andino tiene varios elementos que lo pueden ayudar a tener un rol más influyente y significativo en la política regional. Sin embargo, es menester un accionar articulado y de amplio espectro. Es decir, no es un plan que únicamente esté anclado a un periodo presidencial, sino a un objetivo centenario. Más aún, el interés de obtener esta distinción no lo debe tener solamente el presidente y sus ministros, ésta es una tarea conjunta entre el estado, la sociedad civil y los sectores productivos privados en áreas de impulsar la marca “Made in Perú”. Otro punto crucial es el reconocimiento regional como en su momento lo tuvo Brasil, para ello es necesario impulsar una agenda en conjunto cuyas iniciativas sean de interés para otros estados. 156 Cuadernos Iberoamericanos Ardila Ardila, Martha. “Características de inserción internacional de potencias regionales latinoamericanas. A propósito de Colombia y Venezuela.” Oasis 19 (2014): 87–101. → Referencias / References Sánchez, Wilder Alejandro. “La política exterior de Brasil y la eterna búsqueda de la integración latinoamericana.” Cuadernos Iberoamericanos 8, no. 3 (2020): 10–23. Sánchez, Wilder Alejandro. “La política exterior de Brasil y la eterna búsqueda de la integración latinoamericana.” Cuadernos Iberoamericanos 8, no. 3 (2020): 10–23. Исследовательская статья Принята к публикации: 24.12.2021 Для цитирования: Sánchez, Wilder Alejandro. “Una hoja de ruta para transformar a Perú en una potencia de Latinoamérica” [A roadmap to make Peru regional power in Latin America]. Cuadernos Iberoamericanos 9, no. 4 (2021): 146-159. https://doi.org/10.46272/2409-3416-2021-9-4-146-159. [In Spanish] → Referencias / References 157 Ardila Ardila, Martha. “Características de inserción internacional de potencias regionales latinoamericanas. A propósito de Colombia y Venezuela.” Oasis 19 (2014): 87–101. Ardila Ardila, Martha. “Potencia regional secundaria en definición: Colombia entre Sur y Centroamérica.” Papel Político 17, no. 1 (2012): 293–319. González Bergez, Tomás. “La política exterior argentina hacia América Latina: ¿una potencia regional emergente?” Pensamiento Propio, no. 49–50 (2019): 303–350. Moloeznik, Marcos Pablo. “Potencias medias y potencias regionales en el sistema político internacional de Guerra Fría y Posguerra Fría (Propuesta de dos modelos teóricos).” Espiral, Estudios sobre Estado y Sociedad, vol. XIX, no. 55 (2012): 193–200. Nolte, Detlef. “How to compare regional powers: analytical concepts and research topics.” Review of International Studies 36 (2010): 881–901. 1 Julieta Pelcastre, “Brazil, Colombia, Peru Sail Together in Bracolper 2021,” Diálogo, August 17, 2021, https://dialogo-americas.com/articles/brazil-colombia-peru-sail-together-in- bracolper-2021/#.YT-ni3spBH4. Ardila Ardila, Martha. “Características de inserción internacional de potencias regionales latinoamericanas. A propósito de Colombia y Venezuela.” Oasis 19 (2014): 87–101. Ardila Ardila, Martha. “Potencia regional secundaria en definición: Colombia entre Sur y Centroamérica.” Papel Político 17, no. 1 (2012): 293–319. Ardila Ardila, Martha. “Potencia regional secundaria en definición: Colombia entre Sur y Centroamérica.” Papel Político 17, no. 1 (2012): 293–319. González Bergez, Tomás. “La política exterior argentina hacia América Latina: ¿una potencia regional emergente?” Pensamiento Propio, no. 49–50 (2019): 303–350. González Bergez, Tomás. “La política exterior argentina hacia América Latina: ¿una potencia regional emergente?” Pensamiento Propio, no. 49–50 (2019): 303–350. Moloeznik, Marcos Pablo. “Potencias medias y potencias regionales en el sistema político internacional de Guerra Fría y Posguerra Fría (Propuesta de dos modelos teóricos).” Espiral, Estudios sobre Estado y Sociedad, vol. XIX, no. 55 (2012): 193–200. Moloeznik, Marcos Pablo. “Potencias medias y potencias regionales en el sistema político internacional de Guerra Fría y Posguerra Fría (Propuesta de dos modelos teóricos).” Espiral, Estudios sobr Estado y Sociedad, vol. XIX, no. 55 (2012): 193–200. Nolte, Detlef. “How to compare regional powers: analytical concepts and research topics.” Review of International Studies 36 (2010): 881–901. Nolte, Detlef. “How to compare regional powers: analytical concepts and research topics.” Review of International Studies 36 (2010): 881–901. 157 № 4 Том IX 2021: 146-159 Ибероамериканские тетради Sánchez, Wilder Alejandro, and Tielemans, Otto Raul. “Reinvigorating Peru’s role in Antarctic geopolitics.” The Polar Journal, Opinion 5, no. 1 (2015): 101–112. Sánchez, Wilder Alejandro, and Tielemans, Otto Raul. “Reinvigorating Peru’s role in Antarctic geopolitics.” The Polar Journal, Opinion 5, no. 1 (2015): 101–112. © У. Алехандро Санчес, 2021 E-mail: wilder.a.sanchez@gmail.com Уайлдер Алехандро Санчес, аналитик по вопросам обороны и геополитики Для корреспонденции: DC 20016, США, Вашингтон, округ Колумбия, п/я 9747 Статья поступила в редакцию: 17.11.2021 Доработана после рецензирования: 08.12.2021 Доработана после рецензирования: 08.12.2021 Принята к публикации: 24.12.2021 Cuadernos Iberoamericanos Cuadernos Iberoamericanos Cuadernos Iberoamericanos No. 4 No. 4 региональных лидеров и с такими проблемами, как пандемия и си- туация в Венесуэле, есть место для появления новой региональной державы. Перу могло бы занять это место при наличии амбиций и долгосрочного стратегического видения для достижения этой цели. → Аннотация 28 июля 2021 года Республика Перу отметила свое двухсотлетие по- литической нестабильностью, экономическими проблемами, нар- ко-террористическим движением, наплывом около миллиона ве- несуэльских беженцев и пандемией COVID-19. Хотя андская нация обладает многими возможностями, способными улучшить междуна- родный имидж страны, геополитический вес Перу ограничен. В ста- тье рассматриваются основные факторы, которые учитываются при классификации страны как региональной державы. Кроме того, предлагается дорожная карта, которую Перу может использовать для улучшения своего международного имиджа к трехсотлетию. Среди рекомендаций можно отметить обеспечение ведущей роли перуанских вооруженных сил в миротворческих миссиях Органи- зации Объединенных Наций (ООН); Андское сообщество наций под руководством Перу для обеспечения его наибольшей значимости, а также предложения в духе «мягкой силы». Также необходимо, чтобы в Перу не только исполнительная власть, но и различные стратегиче- ские секторы стремились превратить страну в региональную дер- жаву. В 2021 году в раздробленной Латинской Америке, без четких 158 Vol. IX 2021: 146-159 Vol. IX 2021: 146-159 → Ключевые слова Перу, внешняя политика, развитие, стратегия, Латинская Америка, дипломатия, влияние, империя инков Конфликт интересов: Автор заявляет об отсутствии потенциально- го конфликта интересов. 159
https://openalex.org/W2073720459	https://www.scielo.br/j/jbn/a/3Pqnx7RDthsHxD9Ddy6QkZk/?lang=pt&format=pdf	Portuguese	null	Cistatina C sérica: uma alternativa prática para avaliação de função renal?	Brazilian Journal of Nephrology	2,011	cc-by	5,528	3 Setor de Glomerulopa tias, Disciplina de Nefro logia, Departamento de Medicina, UNIFESP. 2 Laboratório de Imu nopatologia Renal e Glomerulopatias, Setor de Glomerulopatias, Uni versidade Federal de São Paulo (UNIFESP). Resumo Data de submissão: 02/12/2010 Data de aprovação: 14/02/2011 Correspondência para: Gianna Mastroianni Kirsztajn Disciplina de Nefrologia da UNIFESP Rua Botucatu, 740, Vila Clementino São Paulo – SP – Brasil CEP: 04023-900 E-mail: gianna@nefro. epm.br Resumo A taxa de filtração glomerular é o principal indicador de função renal em indivíduos saudáveis e doentes. Apesar de todo o de senvolvimento da medicina em nossos dias, ainda há dificuldade para definir-se essa taxa com precisão na prática diária. Marcadores precoces de lesão renal são importantes, por que a taxa de filtração glomerular se reduz antes do aparecimento dos sintomas ou si nais de insuficiência renal. A cistatina C tem sido apontada como uma alternativa, mas ainda não foi testada em muitas condições. Vantagens e desvantagens desse marcador foram aqui discutidas. Embora a determina ção sérica da cistatina C comece a ser usada na prática clínica em todo o mundo, ainda não foram completamente esclarecidas suas limitações ou as situações em que está de fa to indicada sua aplicação; por outro lado, a creatinina sérica (e sua depuração) é um marcador laboratorial facilmente acessível, de baixo custo, cujas limitações são bem co nhecidas, que pode ser usado de forma roti neira para avaliação de função renal. Glomerular filtration rate is the main marker of renal function in healthy in dividuals and patients. Despite incon testable advances in medicine, it is still difficult to define precisely this test in clinical practice. Early markers of renal lesion are important, because glomerular filtration rate usually decreases before the first chronic renal failure symptoms or signs appear. Cystatin C has been pointed as an alternative, but it was not tested in many diseases. Advantages and disadvantages of this marker are dis cussed. Although serum cystatin C de termination is increasingly being used in clinical practice worldwide, its limi tations as well as the conditions its use is in fact indicated are not adequately established; on the other hand serum creatinine (and creatinine clearance) is an easily available and low cost labo ratory marker with well-known limita tions that can be used routinely in the assessment of renal function. Ivana Cláudia Gabriel1 Sonia Kiyomi Nishida2 Gianna Mastroianni Kirsztajn3 1Hospital do Servidor Público Estadual (HSPE). 2 Laboratório de Imu nopatologia Renal e Glomerulopatias, Setor de Glomerulopatias, Uni versidade Federal de São Paulo (UNIFESP). Palavras-chave: cistatina c, creatinina, taxa de filtração glomerular, insuficiência renal crônica. Keywords: cystatin c, creatinine, glom erular filtration rate, Chronic renal failure. Artigo de Atualização \| Update Article Artigo de Atualização \| Update Article Data de submissão: 02/12/2010 Data de aprovação: 14/02/2011 Os autores declaram a inexistência de conflitos de interesse. O referido estudo foi realizado no Setor de Glomerulopatias do Departamento de Medicina (Nefrologia) da Unifesp. Como determinar a TFG Até o momento, apenas substâncias exógenas têm si do consideradas como marcadores ideais para a de terminação da TFG, entre elas destacam-se a inulina, o ácido etilenodiaminotetracético (EDTA), o ácido etilenodiaminopentacético (DTPA), o iotalamato e, recentemente, o iohexol. Entretanto, o uso de tais substâncias é limitado a alguns protocolos ou situa ções em que a informação precisa sobre a função re nal é mandatória, uma vez que as técnicas são caras, requerem tempo prolongado para sua realização e não são práticas para uso rotineiro.2-5 Com o intuito de superar essas limitações, muitas fórmulas têm sido desenvolvidas para estimar a depu ração de creatinina, utilizando a concentração sérica de creatinina e dados antropométricos.11,12 Em 1973, Cockcroft e Gault publicaram uma fórmula que leva va em consideração a idade e o peso, mas não a raça. Tal equação foi desenvolvida primariamente em indi víduos do sexo masculino e a extensão de sua aplica bilidade para o sexo feminino foi feita por meio de um ajuste arbitrário de 85%.12 Os resultados não foram corrigidos para a área de superfície corpórea e foram expressos em mL/min. A fórmula de Cockcroft-Gault foi rapidamente difundida e aceita, em virtude da fa cilidade de seu cálculo e aplicabilidade clínica;7,12 en tretanto, sabe-se que tal fórmula superestima a TFG em virtude da secreção tubular de creatinina, particu larmente na presença de proteinúria.7,12 Marcadores endógenos são de determinação menos complexa e oferecem resultados mais rápi dos. Atualmente, a creatinina sérica é o marcador en dógeno mais comumente utilizado na prática clínica, seja por meio de sua determinação sérica isolada ou em conjunto com a coleta de urina de 24 horas para a determinação da depuração de creatinina. A con centração da creatinina plasmática, um derivado da creatina muscular, com massa molecular de 113 Da,1 está inversamente relacionada à TFG. Entretanto, muitos fatores limitam sua acurácia, uma vez que a concentração sérica da mesma é reflexo de sua pro dução, é proporcional à massa muscular e é influen ciada pela idade e sexo.1-4 Isso favorece uma consi derável variação intra e interindividual, justificando o nível plasmático mais elevado em adultos que em crianças e em homens que em mulheres, bem como um aumento significante com o crescimento linear em crianças.6,7 Ao contrário, indivíduos que perdem massa muscular evoluem com redução na produ ção de creatinina e, consequentemente, em seu nível plasmático. Introdução tais órgãos, resultando em complicações como hipertensão arterial, desnutrição, anemia, osteodistrofia, neuropatia e uma qualidade de vida insatisfatória.1-3 A avaliação precisa do nível de função re nal é a chave para o diagnóstico, a moni torização e o manejo das doenças renais, bem como para o cálculo adequado de doses das drogas que são excretadas pelos rins. É fato bem estabelecido que a função renal declina de maneira progressiva na maioria das enfermidades que acometem O referido estudo foi realizado no Setor de Glomerulopatias do Departamento de Medicina (Nefrologia) da Unifesp. A taxa de filtração glomerular (TFG), definida como clearance de uma subs tância presente no plasma, metabolizada exclusivamente pelos rins e filtrada livre mente pelos glomérulos,4 é o principal 261 Cistatina C sérica na avaliação de função renal A estimativa da TFG em indivíduos idosos é extre mamente difícil. É fato bem estabelecido que a função renal declina com o aumento da idade. Alguns estudos mostraram que o número de glomérulos se reduz de aproximadamente 1 milhão por rim para 600.000 ou menos em torno da 8a década, resultando em uma di minuição na área de filtração e na permeabilidade da membrana basal glomerular, que, por sua vez, não é refletida pelo aumento da creatinina, em virtude da redução da massa muscular que ocorre em idosos.8-10. indicador de função renal. Sabe-se também que a TFG se reduz antes do aparecimento dos sintomas de insuficiência renal. A substância ideal a ser utilizada para sua deter minação deve apresentar as seguintes características: ritmo de produção estável, manutenção constante do seu nível circulante que não deve ser influenciado por outras doenças, livre filtração pelos glomérulos e ausência de interferência tubular, como secreção ou reabsorção.2,4,5 Outro fator que interfere na utilização da crea tinina como marcador ideal da TFG é o fato de ser secretada pelos túbulos renais, superestimando, dessa forma, a TFG.4,5 Em condições normais, a depuração tubular de creatinina corresponde a aproximadamen te 10 a 20% da depuração renal dessa substância.2 Como o percentual de creatinina depurada do plas ma por secreção depende de seu nível plasmático e da massa de tecido tubular funcionante, em algumas si tuações a depuração tubular da creatinina pode atin gir 50 a 70% da depuração renal. Cistatina C A cistatina C é uma proteína catiônica não glicosilada, cujo ponto isoelétrico é 9,3.15 Sua massa molecular é de 13.359 Da, sendo um constituinte da superfamília das cistatinas, que, por sua vez, é composta por 12 proteí nas (Tabela 1).15,16 É um potente inibidor de proteases cisteínicas (como as catepsinas humanas B, H e L),17 composta de 120 aminoácidos dispostos em uma cadeia polipeptídica simples, cuja sequência foi determinada em 1981.6,7,15,17 É sintetizada como uma pré-proteína.17 Estudos subsequentes demonstraram que a cistatina C é produzida num ritmo constante por todas as células nucleadas e está presente nos líquidos biológicos.1-6,18 Na população pediátrica, tem-se demonstrado vantagem da cistatina C sobre a creatinina sérica, particularmente na detecção de mudanças pequenas e precoces na TFG, uma vez que, nessa população, a massa muscular reduzida, principalmente em crianças com idade inferior a 4 anos, resulta em um valor mui to baixo de creatinina sérica.32 A concentração sérica de cistatina C está elevada no primeiro dia de vida, evoluindo com uma rápida redução nas semanas se guintes. Por outro lado, a concentração de creatinina sérica aumenta com a idade, até o início da adoles cência, em virtude do ganho de massa muscular que ocorre com o crescimento.26 Ela é livremente filtrada pelos glomérulos (em virtude de seu baixo peso molecular em combinação com uma carga elétrica positiva)4,5,19 e, segundo es tudos iniciais, sua concentração sérica independe da idade, sexo, dieta, massa muscular e peso corporal.5,16- 20 Dessa forma, não foi relatada diferença relevante entre os valores de referência para o sexo feminino e o masculino. Em crianças saudáveis, a concentração de cistatina C se estabiliza no segundo ano de vida e o valor de referência é idêntico ao dos adultos.7,15 Mais recentemente, Bokenkamp et al.33 relataram que a cistatina C sérica é maior em crianças submeti das a transplante renal que em crianças portadoras de outras doenças renais, embora a TFG seja semelhante. Em função desse achado, levantou-se a hipótese de que a imunossupressão seria o principal fator com poten cial de influenciar tais resultados, uma vez que todos os pacientes receberam prednisona e ciclosporina A. A cistatina C é quase completamente catabolizada no túbulo proximal, assim como outras proteínas de baixo peso molecular. Como determinar a TFG Associado a isso, alguns fatores externos interferem em sua determinação analítica, entre os quais citam-se outras substâncias endógenas (glico se, bilirrubinas, ácido úrico, triglicerídeos, cetonas e proteínas plasmáticas) e alguns medicamentos (ce falosporinas, sulfas e cimetidina), que inibem sua secreção tubular, elevando o nível sérico sem afetar a TFG.1,2,6 Em 1999, uma equação matemática mais com plexa foi publicada, baseada na análise dos dados obtidos por meio do estudo Modification of Diet in Renal Disease (MDRD).7,12-14 A estimativa da TFG baseada no MDRD levava em consideração dados de idade, sexo, etnia, creatinina sérica, nitrogênio urei co e albumina sérica. Um ano mais tarde, uma ver são simplificada da equação do MDRD original foi apresentada, utilizando apenas dados de idade, sexo, etnia e creatinina sérica.7,12,14 Essa equação foi inclu ída como marcador da TFG nas Practice Guidelines for Chronic Kidney Disease, publicadas em 2002 pe lo Kidney Disease Outcomes Quality Initiative of the National Kidney Foundation (K/DOQI).7 J Bras Nefrol 2011;33(2):261-267 262 Cistatina C sérica na avaliação de função renal Nos últimos anos, diversas proteínas plasmáticas de baixo peso molecular vêm sendo estudadas com o intuito de se identificar um melhor marcador de TFG. Em 1985, demonstrou-se que a cistatina C é ao me nos equivalente à creatinina sérica, como marcador de função renal.5 O gene que codifica a cistatina C foi sequenciado e localiza-se no cromossomo 20.21,24 A estrutura do ge ne parece ser do tipo housekeeping, que é compatível com um ritmo de produção estável pela maioria das células nucleadas.7,15,22,24,25 Os aspectos aqui expostos introduziram a ideia de que os níveis séricos de cistatina C seriam indicado res melhores da TFG que a creatinina, fato esse que vem sendo confirmado por uma série de estudos.3,5,8- 10,15,18,26-29 Por outro lado, alguns poucos estudos não evidenciaram diferença significante entre as determi nações sanguíneas de cistatina C e de creatinina para esse fim.5,30,31 Cistatina C Porém, ao contrário dessas (co mo a ß2-microglobulina que tem 11,8 kDa),21 seu nível sérico parece não ser afetado por outras condições ex trarrenais, como inflamação e neoplasias.3,7,15,22 Por ser reabsorvida e metabolizada a nível tubular, 22,23 a cistati na C não retorna à circulação em sua forma intacta e sua concentração urinária é praticamente indetectável.3,24 Bjarnadóttir et al.,34 procurando desvendar a even tual contribuição de uma dessas medicações sobre os níveis de cistatina C, realizaram um estudo in vitro no qual células HeLa foram expostas a diferentes con centrações de dexametasona. Foi, então, observado um aumento dose-dependente na produção de cistati na C por células expostas ao corticoide. Família 1 Família 2 Família 3 Cistatinas intracelulares Cistatinas extracelulares Cistatinas intravasculares e/ou transcelulares Cistatina A Cistatinas C, D, E, F, G LMW-cininogênio Cistatina B Cistatina S HMW-cininogênio Cistatina SA Cistatina SN Tabela 1 Superfamília das cistatinas humanas Tabela 1 Superfamília das cistatinas humanas Em 2001, Risch et al.35 publicaram estudo pros pectivo realizado com o intuito de esclarecer a influ ência da imunossupressão com glicocorticoide sobre a concentração sérica da cistatina C em pacientes submetidos a transplante renal. Nesse trabalho, 20 pacientes recebendo baixa dose de corticoide foram comparados a 20 pacientes em uso de ciclosporina isolada e 20 pacientes tratados com ciclosporina em associação com azatioprina. Além disso, 13 pacientes receberam um curso rápido de altas doses de metil prednisolona. Esse estudo demonstrou que pacientes J Bras Nefrol 2011;33(2):261-267 263 Cistatina C sérica na avaliação de função renal recebendo corticoide apresentavam níveis séricos de cistatina C superiores aos dos grupos que não re ceberam tal imunossupressor. Além disso, os níveis de cistatina C foram significativamente maiores no grupo que recebeu altas doses de metilprednisolona quando comparado aos do grupo que recebeu baixa dose de prednisona. Esse achado demonstra uma re lação dose-dependente, embora tal aumento tenha sido transitório, uma vez que, em média, 8 dias após a suspensão da metilprednisolona, a concentração sérica de cistatina C havia retornado ao nível basal. na avaliação da função renal. Cistatina C Em estudo recente, en volvendo um grande número de pacientes com doença renal crônica moderada a severa, a cistatina C sérica, diferentemente da creatinina sérica, não foi afetada pe lo conteúdo proteico da dieta independente de mudan ças na TFG, indicando que a cistatina pode fornecer estimativas mais precisas da TFG que a creatinina em pacientes com ingestão reduzida de proteínas.43 Há in dícios de que os níveis de cistatina C não são afetados pela desnutrição, enquanto os de creatinina caem, fa zendo com que a TFG seja superestimada.44 Cistatina C parece ser também uma alternativa na avaliação da função renal em indivíduos com grande massa muscular, quando se suspeita de déficit de função renal discreto.45 Nesse último estudo, foi possível obser var que peso corporal e massa magra não se correlacio naram com os níveis séricos de cistatina. Também em indivíduos obesos, existem dificuldades para avaliar a função renal e a falta de consenso entre os estudos. Na avaliação de obesos, constatou-se que a adiposidade está associada aos níveis séricos de cistatina. Contrariando as expectativas de que seria um marcador adequado, contatou-se que, nesse grupo, fórmulas baseadas na cis tatina C superestimam a TFG nos níveis de índice de massa corporal (IMC) mais elevados.46 Alguns outros trabalhos evidenciaram elevação no nível sérico de cistatina C relacionada a altas doses de corticoide, em pacientes portadores de asma brônqui ca, hemorragia subaracnoidea e oftalmopatia severa secundária à Doença de Graves.36-39 Entretanto, os mecanismos envolvidos nessas mudanças ainda não estão bem esclarecidos. Por outro lado, em 2002, foram publicados dados de um estudo realizado com crianças portadoras de síndrome nefrótica idiopática, córtico-sensível, trata das de acordo com o protocolo do German Working Group for Pediatric Nephrology, segundo os quais a concentração sérica de cistatina C não foi afetada pe la administração de altas doses de corticoide.40 É fato conhecido que a função tireoideana pode interferir no nível sérico de creatinina. Foi demons trado que os pacientes com hipotireoidismo apresen tavam níveis de creatinina mais elevados, enquanto pacientes com hipertireoidismo apresentavam níveis menores. Após o tratamento e consequente estado de eutireoidismo, os níveis se reduziram e se elevaram, respectivamente. Diante disso, estudos foram rea lizados com o intuito de se identificar uma possível interferência dos hormônios tireoideanos sobre os ní veis de cistatina C. J Bras Nefrol 2011;33(2):261-267 Cistatina C Observou-se que, ao contrário do que ocorre com a creatinina, a concentração sérica de cistatina C é menor no estado de hipotireoidismo e maior no hipertireoidismo, quando comparada àque la observada no estado de eutireoidismo. Vale salientar que, também no caso da cistatina C, como vem acontecendo com a creatinina sérica, fo ram desenvolvidas fórmulas nos últimos anos com o objetivo de melhor avaliar a função renal, estimando a TFG,20,22,27,47-51 como listado Tabela 2. Tabela 2 Equações para a estimativa da taxa de filtração glomerular com base na cistatina C sérica (mg/L) isoladamente ou em combinação com a creatinina sérica (mg/dL) Autores Fórmulas propostas Hoek et al.20 TFG = -4,32 + 80,35 x 1/ cistatina Tan et al.22 TFG = 87,1 / cistatina - 6,87 Rule et al.27 TFG = 66,8 x cistatina -1,30 Grubb et al.47 TFG = 99,19 x cistatina -1,713 x 0,823 (se sexo feminino) Grubb et al.48 TFG = 87,62 x cistatina-1,693 x 0,94 (se sexo feminino) MacIsaac et al.49 TFG = 86,7 / cistatina - 4,2 Larsson et al.50 TFG = 77,239 x cistatina -1,2623 Stevens et al.51 TFG = 177,6 x creatinina-0,65x cistatina-0,57x idade-0,20x 0,82 (se sexo feminino) x 1,11 (se raça negra) Tabela 2 Equações para a estimativa da taxa de filtração glomerular com base na cistatina C sérica (mg/L) isoladamente ou em combinação com a creatinina sérica (mg/dL) Possíveis explicações para esses achados baseiam- se nos efeitos dos hormônios tireoideanos sobre a he modinâmica renal, a homeostase renal de sal e água e o transporte tubular ativo de sódio, potássio e íons hidrogênio. No que se refere à creatinina, é possível que sua secreção tubular esteja reduzida no hipoti reoidismo e aumentada no estado oposto. Referências 1. Stevens LA, Coresh J, Greene T, Levey AS. Assessing kidney function – measured and estimated glomerular filtration rate. N Engl J Med 2006; 354:2473-83. 1. Stevens LA, Coresh J, Greene T, Levey AS. Assessing kidney function – measured and estimated glomerular filtration rate. N Engl J Med 2006; 354:2473-83. Os idosos representam hoje uma grande preocu pação no que se refere à determinação da TFG. Em revisão sistemática recente da literatura,59 concluiu-se que não existe ainda um método preciso para avaliar a função renal nesse grupo de indivíduos, mas a cis tatina C, assim como as fórmulas de Cockcroft-Gault e MDRD são parâmetros úteis – embora as evidên cias favoráveis a um ou outro marcador ainda sejam insuficientes. 2. Hojs R, Bevc S, Ekart R, Gorenjak M, Puklavec L. Serum cystatin C as an endogenous marker of renal function in patients with mild to moderate impairment of kidney function. Nephrol Dial Transplant 2006; 21:1855-62. 2. Hojs R, Bevc S, Ekart R, Gorenjak M, Puklavec L. Serum cystatin C as an endogenous marker of renal function in patients with mild to moderate impairment of kidney function. Nephrol Dial Transplant 2006; 21:1855-62. 3. Martinez IKH, Simón DJ. Utilidad clínica de la cistatina C como marcador de función renal. An Med Asoc Med Hosp ABC 2003; 48:216-22. 3. Martinez IKH, Simón DJ. Utilidad clínica de la cistatina C como marcador de función renal. An Med Asoc Med Hosp ABC 2003; 48:216-22. p 4. Deinum J, Derk FHM. Cystatin for estimation of glomerular filtration rate? Lancet 2000; 6:1624-5. 4. Deinum J, Derk FHM. Cystatin for estimation of glomerular filtration rate? Lancet 2000; 6:1624-5. 5. Dharnidharka VR, Kwon C, Stevens G. Serum cystatin C is superior to serum creatinine as a marker of kidney function: A meta-analysis. Am J Kidney Dis 2002; 40:221-6. Por fim, uma condição em que a utilização da cis tatina C parece particularmente promissora é a lesão renal aguda, na qual tem-se revelado um biomarcador preciso para detecção precoce e, em populações sele cionadas, mostrou-se superior à creatinina em alguns estudos; de qualquer forma, os resultados ainda são inconsistentes. Os investigadores questionam ainda se ela é custo-efetiva em relação à creatinina e se os dois testes teriam papéis complementares.60 6. Bökenkamp A, Domanetzki M, Zinck R, Schumann G, Byrd D, Brodenl J. Cystatin C – A new marker of glomerular filtration rate in children independent of age and height. Cistatina C Já no que tange à cistatina C, como o estado tireoideano in fluencia o metabolismo geral, ele pode influenciar a sua produção.41,42 Outro aspecto que exige atenção é a interferência da ingestão de proteínas e mesmo do estado nutricional J Bras Nefrol 2011;33(2):261-267 264 Cistatina C sérica na avaliação de função renal Algumas dessas fórmulas que envolvem a cistati na C apresentaram, segundo os investigadores que as utilizaram, melhor desempenho que equações que uti lizam a creatinina20,47,48 ou foram similares.27,49 Para alguns, a combinação das dosagens séricas de crea tinina e cistatina C em fórmulas foi a melhor opção, particularmente quando foram levados em conta da dos demográficos.51-54 Porém, não há consenso ainda quanto à superioridade das fórmulas que envolvem a cistatina, nem mesmo a combinação dos dois marca dores, considerando-se que, possivelmente, elas não são adequadas para uso em diferentes populações, como testado por Urbaniak et al.55 de referência variam conforme o kit e cada laborató rio deve fazer a verificação dos valores de referência. No Brasil, esse exame não está disponível na maioria dos serviços e seu custo ainda é elevado; em alguns laboratórios de qualidade reconhecida no país que realizam tal exame, o custo é de aproximadamente oito vezes o da creatinina. Considerações finais A determinação sérica da cistatina C começa a ser usa da na prática clínica em todo o mundo, mas ainda não foram completamente esclarecidas suas limitações ou as situações em que está de fato indicada sua aplica ção. Aqui procuramos mostrar as vantagens e desvan tagens já conhecidas desse teste laboratorial, que tem sido visto como promissor na avaliação da TFG. Deve-se ter em mente, entretanto, que foram rea lizados ou estão em andamento numerosos estudos, para se definir melhor o papel da cistatina C. Um desses demonstrou, recentemente, que ela identifi cou com maior precisão que a creatinina sérica quais indivíduos evoluiriam com complicações cardiovas culares numa grande população com doença renal crônica, divisando-se assim que a cistatina C pode ser um importante marcador de risco nesse grupo de indivíduos.56,57 Eriksen et al. argumentam com base nos resultados que encontraram, avaliando um grupo de indivíduos bastante representativo, que estimativas da TFG baseadas na cistatina C não são superiores às da creatinina quando se fala da população geral, e que o melhor desempenho anteriormente descrito na identificação de risco cardiovascular pode dever-se a outros fatores que não a TFG em si.58 Vale salientar que sua determinação ainda é de al to custo em nosso meio, o que se constitui em mais um motivo para estar atento à sua real contribuição no acompanhamento de diferentes doenças/distúrbios renais. Enquanto tais aspectos não estiverem bem estabelecidos, aconselhamos não desprezar outros testes, como a dosagem sérica de creatinina, cujas li mitações são bem conhecidas e podem, ao menos em parte, ser contornadas. Referências Clin Chem 2002; 48:2297-8. 15. Filler G, Bökenkamp A, Hofmann W, Le Bricon T, Martínez-Brú C, Grugg A. Cystatin C as a marker of GFR—history, indications, and future research. Clin Biochem 2005; 38:1-8. 31. Daniel JP, Chantrel F, Offner M, Moulin B, Hannedouche T. Comparison of cystatin C, creatinine and creatinine clearance vs. GFR for detection of renal failure in renal transplant patients. Ren Fail 2004; 26:253-7. 32. Laterza OF, Price CP, Scott MG. Cystatin C. An improved estimator of glomerular filtration rate? Clin Chem 2002; 48:699-707. 16. Shimizu-Tokiwa A, Kobata M, Io H, Kobayashi N, Shou I, Funabiki K, et al. Serum cystatin C is a more sensitive marker of glomerular function than serum creatinine. Nephron 2002; 92:224-6. 33. Bokenkamp A, Domanetzki M, Zinck R, Schumann G, Byrd D, Brodehl J. Cystatin C serum concentrations underestimate glomerular filtration rate in renal transplant recipients. Clin Chem 1999; 45:18668. 7. Abrahamson M, Mason RW, Hansson H, Buttle DJ, Grubb A, Ohlsson K. Human cystatin C – Role of the N-terminal segment in the inhibition of human cysteine proteinases and in its inactivation by leucocyte elastase. Biochem J 1991; 273:621-6. 34. Bjarnadóttir M, Grubb A, Ólafsson I. Promoter- mediated, dexamethasone-induced increase in cystatin C production by HeLa cells. Scand J Clin Lab Invest 1995; 55:617-23. 18. Jung K, Jung M. Cystatin C. A promising marker of glomerular filtration rate to replace creatinine. Nephron 1995; 70:370-1. 35. Risch L, Herklotz R, Blumberg A, Huber A. Effects of glucocorticoid immunosupression on serum cystatin C concentrations in renal transplant patients. Clin Chem 2001; 47:2055-9. 19. Curhan G. Cystatin C. A marker of renal function or something more? Clin Chem 2005; 51:294. 20. Hoek FJ, Kempermen FAW, Krediet RT. A comparison between cystatic C, plasma creatinine and the Cockcroft and Gault for the estimation of glomerular filtration rate. Nephrol Dial Transplant 2003; 18:2024-31. 36. Risch L, Huber AR. Glucocorticoids and increased serum cystatin C concentrations. Clin Chim Acta 2002; 320:113-34. 37. Risch L, Saely C, Reist U, Reist K, Hefti M, Huber A. Course of glomerular filtration rate markers in patients receiving high-dose glucocorticoids following subarachnoidal hemorrhage. Clin Chim Acta 2005; 360:205-7. 21. Bökenkamp A, Grabensee A, Stoffel-Wagner B, Hasan C, Henne T, Offner G, et al. The ß2-microblobulin/ cystatin C ratio – a potential marker of post-transplant lymphoproliferative disease. Clin Nephrol 2001; 58:417-22. 38. Referências Pediatrics 1998; 101(5):875-81. 7. Rosenthal SH, Bökenkamp A, Hofmann W. How to estimate GFR-serum creatinine, serum cystatin C or equations? Clin Biochem 2007; 4:153-61. 8. Finney H, Bates CJ, Price CP. Plasma cystatin C determinations in a healthy elderly population. Arch Gerontol Geriatr 1999; 29:75-94. Do ponto de vista prático, vale lembrar que a de terminação da cistatina C, como marcador direto de função renal, pode ser feita em soro ou plasma, nas mesmas condições de coleta da creatinina. Os valores 9. Ognibene A, Mannucci E, Caldini A, Terreni A, Brogi M, Bardini G, et al. Cystatin C reference values and aging. Clin Biochem 2006; 39:658-61. J Bras Nefrol 2011;33(2):261-267 265 Cistatina C sérica na avaliação de função renal concentration in children. Pediatr Nephrol 1998; 12:125-9. 10. Burkhardt H, Bojarsky G, Gladisch R. Diagnostic efficiency of cystatin C and creatinine as markers of reduced glomerular filtration rate in the elderly. Clin Chem Lab Med 2002; 40:1135-8. 27. Rule AD, Bergstralh EJ, Slezak JM, Bergert J, Larson TS. Glomerular filtration rate estimated by cystatin C among different clinical presentations. 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Contribution of the MDRD equation and of cystatin C for renal function estimates in cancer patients. Med Oncol 2006; 23:63-73. 30. Willems HL, Hilbrands LB, Van De Calseyde JF, Monnens LA, Swink DW. Is serum cystatin C the marker of choice to predict glomerular filtration rate in paediatric patients? Ann Clin Biochem 2003; 40(Pt 1):60-4. 14. Lupovitch A. More accurate alternatives to serum creatinine for evaluating glomerular filtration rate. Referências Manetti L, Genovesi M, Grasso L, Lupi I, Morselli LL, Martino E. Early effects of methylprednisolone infusion on serum cystatin C in patients with severe Graves’ophthalmopathy. Clin Chim Acta 2005; 356:227-8. 22. Tan GD, LEWIS AV, James TJ, Altmann P, Taylor RP, Levy JC. Clinical usefulness of cystatin C for the estimation of glomerular filtration rate in type 1 diabetes: reproducibility and accuracy compared with standard measures and iohexol clearance. Diabetes Care 2002; 25:2004-9. 39. Cimerman N, Brguljan PM, Krasovel M, Suskovic S, Kos J. Serum cystatin C, a potent inhibitor of cysteine proteinases, is elevated in asthmatic patients. Clin Chim Acta 2000; 300:83-95. 23. Seco ML, Rus A, Sierra M, Caballero M, Borque L. Determination of serum cystatin C in patients with essential hypertension. Nephron 1999; 81:446-7. yp p ; 24. Uchida K, Gotoh A. Measurement of cystatin C and creatinine in urine. Clin Chim Acta 2002; 323:121-8. 40. Bokenkamp A, van Wijk JAE, Lentze MJ, Stoffel- Wagner B. Effect of corticosteroid therapy on serum cystatin C and ß2-microglobulin concentrations. Clin Chem 2002; 48:1123-6. 25. Demirtas S, Akan O, Can M, Elmali E, Akan H. Cystatin C can be affected by nonrenal factors: A preliminary study on leukemia. Clin Biochem 2006; 39:115-8. 41. Hollander JG, Wulkan RW, Mantel MJ, Berghout A. Is cystatin C a marker of glomerular filtration rate in thyroid dysfunction? Clin Chem 2003; 49:1558-9. 26. Bökenkamp A, Domanetzki M, Zinck R, Schumann G, Brodehl J. Reference values for cystatin C serum J Bras Nefrol 2011;33(2):261-267 266 Cistatina C sérica na avaliação de função renal 42. Manetti L, Pardini E, Genovesi M, Campomori A, Grasso L, Morselli LL, et al. Thyroid function differently affects serum cystatin C and creatinine concentrations. J Endocrinol Invest 2005; 28:346-9. 51. Stevens LA, Coresh J, Schmid CH, Feldman HI, Froissart M, Kusek J, et al. Estimating GFR using serum cystatin C alone and in combination with serum creatinine: a pooled analysis of 3,418 individuals with CKD. Am J Kidney Dis 2008; 51:395-406. 43. Tangri N, Stevens LA, Schmid CH, Zhang YL, Beck GJ, Greene T, Coresh J, Levey AS. Changes in dietary protein intake has no effect on serum cystatin C levels independent of the glomerular filtration rate. Kidney Int 2011; 79:471-7. 52. Rigalleau V, Beauvieux MC, Lasseur C, Chauveau P, Raffaitin C, Perlemoine C, et al. Referências The combination of cystatin C and serum creatinine improves the monitoring of kidney function in patients with diabetes and chronic kidney disease. Clin Chem 2007; 53:1988-9. 44. Hari P, Bagga A, Mahajan P, Lakshmy R. Effect of malnutrition on serum creatinine and cystatin C levels. Pediatr Nephrol 2007; 22:1757-61. 53. Ma YC, Zuo L, Chen JH, Luo Q, Yu XQ, Li Y, et al. Improved GFR estimation by combined creatinine and cystatin C measurements. Kidney Int 2007; 72:1535-42. p 45. Baxmann AC, Ahmed MS, Marques NC, Menon VB, Pereira AB, Kirsztajn GM, et al. Influence of muscle mass and physical activity on serum and urinary creatinine and serum cystatin C. Clin J Am Soc Nephrol 2008; 3:348-54. 54. Tidman M, Sjostrom P, Jones I. A comparison of GFR estimating formulae based upon s-cystatin C and s-creatinine and a combination of the two. Nephrol Dial Transplant 2008; 23:154-60. 46. Vupputuri S, Fox CS, Coresh J, Woodward M, Muntner P. Differential estimation of CKD using creatinine- versus cystatin C-based estimating equations by category of body mass index. Am J Kidney Dis 2009; 53:993-1001 55. Urbaniak J, Weyde W, Smolska D, Zagocka E, Klak R, Kusztal M, et al. S-cystatin C formulae or combination of scystatin C and s-creatinine formulae do not improve prediction of GFR. Nephrol Dial Transplant 2008; 23:2425-6. 47. Grubb A, Bjork J, Lindstrom V, Sterner G, Bondesson P, Nyman U. A cystatin C-based formula without anthropometric variables estimates glomerular filtration rate better than creatinine clearance using the Cockcroft-Gault formula. Scand J Clin Lab Invest 2005; 65:153-62. 56. Peralta CA, Katz R, Sarnak MJ, Ix J, Fried LF, De Boer I, Palmas W, et al. Cystatin C identifies chronic kidney disease patients at higher risk for complications. J Am Soc Nephrol 2011; 22:147-55. 57. Wu CK, Lin JW, Caffrey JL, Chang MH, Hwang JJ, Lin YS. Cystatin C and long-term mortality among subjects with normal creatinine-based estimated glomerular filtration rates: NHANES III (Third National Health and Nutrition Examination Survey). J Am Coll Cardiol 2010; 56:1930-6. 48. Grubb A, Nyman U, Bjork J, Lindstrom V, Rippe B, Sterner G, et al. Simple cystatin C-based prediction equations for glomerular filtration rate compared with the modification of diet in renal disease prediction equation for adults and the Schwartz and the Counahan- Barratt prediction equations for children. Clin Chem 2005; 51:1420-31. 58. Referências Eriksen BO, Mathisen UD, Melsom T, Ingebretsen OC, Jenssen TG, Njølstad I, et al. Cystatin C is not a better estimator of GFR than plasma creatinine in the general population. Kidney Int 2010; 78:1305-11. 49. MacIsaac RJ, Tsalamandris C, Thomas MC, Premaratne E, Panagiotopoulos S, Smith TJ, et al. Estimating glomerular filtration rate in diabetes: a comparison of cystatin-C- and creatinine-based methods. 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https://openalex.org/W4391476378	https://bmcnurs.biomedcentral.com/counter/pdf/10.1186/s12912-024-01744-z	English	null	Nursing students’experience of flipped classroom combined with problem-based learning in a paediatric nursing course: a qualitative study	BMC nursing	2,024	cc-by	7,668	Abstract Background Problem-based learning (PBL) is a student-centred approach that triggers learning by presenting problems cenarios early in the learning process.Flipped classrooms have been used in various disciplines using various models.Pre-class e- learning in aflipped classrooms can enrich knowledge acquisition in PBL teaching. This study was conducted to explore nursing students’experience of flipped classroom combined with problem-based learning in a paediatric nursing course. Method This descriptive qualitative study was conducted between January and June 2022.Semi-structured interviews were conducted with nursing students who were participated in flipped classrooms combined with PBL teaching in a paediatric nursing course at Soochow university in China. Nursing students were selected using a purposive sampling method until no new data were generated (n = 16). Results We identified ten sub-themes and four higher-order themes based on these sub-themes: (1)stimulating interest in learning and enhancing autonomous learning,(2)improving independent thinking and problem-solving skills,(3)cultivating team work spirit, and (4) gaining knowledge and improving skills.The findings of our research contribute to show the effectiveness of the flipped classroom combined with PBL in a paediatric nursing course. Conclusion The flipped classroom combined with PBL in a paediatric nursing course can enhances communication and cooperation abilities among nursing students, promoting common progress and the comprehensive development of nursing students. Keywords Nursing students, Flipped classroom, Problem-based learning, Qualitative study †Zhi Hong Ni and Jie Huang contributed equally to this work. BMC Nursing BMC Nursing BMC Nursing Ni et al. BMC Nursing (2024) 23:88 https://doi.org/10.1186/s12912-024-01744-z Open Access Nursing students’experience of flipped classroom combined with problem-based learning in a paediatric nursing course: a qualitative study Zhi Hong Ni1†, Jie Huang1†, Dao Ping Yang1 and Jing Wang1 Zhi Hong Ni1†, Jie Huang1†, Dao Ping Yang1 and Jing Wang1 †Zhi Hong Ni and Jie Huang contributed equally to this work. Correspondence: Zhi Hong Ni nizhihong8888@163.com 1Children’s Hospital of Soochow University, No. 92, Zhong nan St, 215025 Suzhou, China Correspondence: Zhi Hong Ni nizhihong8888@163.com 1Children’s Hospital of Soochow University, No. 92, Zhong nan St, 215025 Suzhou, China *Correspondence: Zhi Hong Ni nizhihong8888@163.com 1Children’s Hospital of Soochow University, No. 92, Zhong nan St, 215025 Suzhou, China © The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Introduction Flipped classrooms have been used in various disci plines using various models. In nursing education, one of the most important teaching methods with the advan tage of improving nursing students’ clinical performance and higher-level thinking abilities, PBLcan be used as an in-class activity in flipped classrooms. However, to some extent, Pre-class e- learning in a flipped classrooms can enrich knowledge acquisition in PBL teaching [16]. Few studies have investigated the combination of flipped classrooms and PBL in paediatric nursing education. [17–18]. Introduction Problem-based learning (PBL) is a student-centred approach that triggers learning by presenting prob lems cenarios early in the learning process [1]. Barrows defined PBL as “learning that results from the process of working toward the understanding or resolution of a problem” [2], Four critical conditions for a deep approach to learning are encompassed within the PBL approach: a well-structured knowledge base, active learning, col laborative learner interaction, and a context designed to promote internal motivation through the provision of pragmatic goals (Margetson, 1994).Each student group addresses this problem in the presence of a facilitator [3]. The role of the problems cenario is to encourage stu dents to activate their prior knowledge and stimulate their interest in the subject matter [4], thereby engaging the students in active discussions and creating a posi tive learning environment [5]. Several factors affect the quality of PBL scenario and its effectiveness in stimulat ing discussions. These factors include the reality of the scenarios, variety of experiences, degree of challenge, supporting group work, and ability to activate prior knowledge [6].PBL aims to help students define their new learning needs. Problem-based learning (PBL) is a student-centred approach that triggers learning by presenting prob lems cenarios early in the learning process [1]. Barrows defined PBL as “learning that results from the process of working toward the understanding or resolution of a problem” [2], Four critical conditions for a deep approach to learning are encompassed within the PBL approach: a well-structured knowledge base, active learning, col laborative learner interaction, and a context designed to promote internal motivation through the provision of pragmatic goals (Margetson, 1994).Each student group addresses this problem in the presence of a facilitator [3].h Paediatric Nursing course is one of nursing programs’ most important core courses. The course aims to build students’ knowledge systems for caring for paediatric patients and cultivate their professional skills to assess, analyse, andsolve clinical problems. Introduction Paediatric Nursing aims to study the laws of children’s growth and develop ment and improve children’s physical and mental health and disease care measures. Individual gender and age differences among the children are significant. There fore, the knowledge points of paediatric disease nursing are relatively complex, and nursing students experience certain learning difficulties.Traditional in-class lectures are not conducive to the cultivation of students’ thinking abilities and are unable to promote the connection of the oretical knowledge and clinical practice skills. In view of the advantages and benefits of PBL and the flipped class room, these methods may be suitable for changing the traditional classroom to meet the needs of the students, and a combination of these methods may produce better outcomes in the teaching of Paediatric Nursing.h The role of the problems cenario is to encourage stu dents to activate their prior knowledge and stimulate their interest in the subject matter [4], thereby engaging the students in active discussions and creating a posi tive learning environment [5]. Several factors affect the quality of PBL scenario and its effectiveness in stimulat ing discussions. These factors include the reality of the scenarios, variety of experiences, degree of challenge, supporting group work, and ability to activate prior knowledge [6].PBL aims to help students define their new learning needs. PBL is widely used in health professionals’ education [7]. where real-world scenarios provide the stimulus for a deeper understanding of both the theoretical and practical concepts in focus. Thus, PBL is a promising pedagogical model for nursing education [8]. Through PBL, students practice and develop problem-solving, self-directed learning, and collaborative skills, which are important professional skills for registered nurses. Thus, teamwork and collaborative skills are taught as part of PBL-based education. The aim of this study was to explore nursing students’ experiences of applyingl flipped classrooms combined with PBL in a paediatric nursing course.We conducted a flipped classroom com bined with problem-based learning in a paediatric nurs ing course, and conducted interviews to understand students’experiences after taking the course to provide a reference for further improvement of the paediatric nurs ing course. PBL has been provento improve nursing students’ application of theory lessons in clinical practice, learn ing motivation, critical thinking, self-learning capabili ties, and satisfaction with teaching [9–10]. As a result of recent developments in the fields of Internet technolo gies, social networks, and learning management systems, educators are increasingly using flipped classrooms. Ni et al. BMC Nursing (2024) 23:88 Page 2 of 9 Ni et al. BMC Nursing Introduction The flipped classroom is a blended learning methodol ogy that combines e-learning and face-to-face classroom techniques [11]. It is intended to improve the efficacy of classroom learning by allowing students to control the timing and pace of their online learning and maxi mise their opportunities for active learning by engaging in class discussions and collaborative exercises in the company of peers and instructors [12]. Previous studies have demonstrated that, compared to traditional lecture- based classrooms, flipped classrooms are more effective in improving academic performance and appraisal of the course, as well as in developing engagement and higher- level thinking skills in nursing students [13–15]. Online pre-class teaching session To capture nursing students’ experiences of the pae diatric flipped classroom combined with PBLcoursesin real-time,we conducted one-on-one interviews with nursing students after they completed the paediatric problem-based learning programme.Only the nurs ing students and interviewers were present during the interviews. No other participants were allowed to enter the interview room.Each interview lasted 30–45 min. Data were collected continuously until no new events occurred, thereby achieving data saturation [22].Audio recordings were used to collect data and field notes were taken after each interview. Each student entered the Paediatric Nursing for autono mous learning through an online course. Students must watch videos, complete homework, and take online tests independently before class. Students can participate in the platform’s interactive forum,an online communica tion platform for consultation if they encounter problems duringonline learning. Data collection Th The interviews were conducted in a quiet classroom at Soochow University. The qualitative data collec tion method included semi-structured, face-to-face interviews.A senior researcher (NZH) performed the interviews and trained less-experienced co-workers. NZH is an experienced Ph.D.teacher. Moreover, all researchers in this study were experienced in performing qualitative research. Initially, preliminary interviews were conducted with five students. The data from the prelimi nary interviews were not included in this study but were used to modify the interview structure according to the preliminary outcomes. The final interviews used in this study included the following items: Teaching Steps [21]:Flipped Classroom includes online pre-class teaching session and offline in-Class PBL teach ing sessions. Teachers created teaching videos, determined teach ing materials, prepared teaching tasks based on course knowledge points, arranged for the students to com plete learning tasks with the help of the online learning platform, and conducted PBL group in-class teaching in the PBL classroom so that the students can carry out learning activities such as discussion and sharing in the classroom. The nursing studentswere asked:(1)What are your thoughts and reflections after attending the paediatric flipped classroom combined with the PBL course?(2) What do you think is the impact on learning after attend ing the paediatric flipped classroom combined with PBL course? Methods Designh The study used a qualitative descriptive design employing interviews [19]. The interview data were analysed using qualitative content analysis [20].Qualitative research as a systematic and subjective approach leads to increased insight, understanding and awareness of human experi ences. Therefore, to discover and explain dimensions of the phenomenon in question and reveala deep under standing of the social world of the participants, an induc tive qualitative research approach is most suitable. Ni et al. BMC Nursing (2024) 23:88 Page 3 of 9 Page 3 of 9 Ni et al. BMC Nursing (2024) 23:88 Ni et al. BMC Nursing Offline in-class PBL teaching sessions (1)Building a PBL case base. Before the class began, teachers carefully selected and adapted real clinical cases together with medical and nursing staff, according to the requirements of the teach ing objectives, to form a PBL teaching case library. (2) Formation of PBL student groups. Setting Thi This study used a qualitative design to analyse nurs ing students’experiences of flipped classrooms com bined with PBL teaching in a paediatric nursing course. All interviews were conducted between September and December 2022 in a classroom at Soochow university. Purposive sampling was used to enrol nursing students who attended a flipped classroom combined with PBL in a paediatric nursing course. Participants and procedure Each group hadeightto ten pupils. Students with different characteristics were reasonably matched based on gen der, learning capacity, personality, and other factors. Each group hadeightto ten pupils. Students with different characteristics were reasonably matched based on gen der, learning capacity, personality, and other factors. (3) Conducting PBL teaching.Students worked in small groups, each with an instructor, and were provided with cases in acts and sections. Students presented the prob lems to be solved in thecase scenario and discussed them in small groups to propose ways to solve the problems. (4) Students reported, the teacher summarised, and students reported the results of the case discussions in groups. The teacher remarked on and summarised the reports from each group, briefly summarised the knowl edge in correlation with the problems encountered by the students in their autonomous learning, and explained and answered questions about the students’ error-prone and confusing areas as well as common difficult problems. As previously mentioned, the purposive sampling method was used to select participants. All the partici pants were undergraduate nursing students in their third year who attended a flipped classroom combined with PBL in a paediatric nursing course and met the follow ing inclusion criteria: the participants were (1) under graduate nursing students enrolled in 2020, (2) who participated in a flipped classroom combined with a PBL paediatric nursing course, and (3) who voluntarily par ticipated in this study. After applying the selection crite ria, a total of 16 nursing students(12 female students and 10 male students) aged 20−21 years were enrolled in this study. (3) Conducting PBL teaching.Students worked in small groups, each with an instructor, and were provided with cases in acts and sections. Students presented the prob lems to be solved in thecase scenario and discussed them in small groups to propose ways to solve the problems. (4) Students reported, the teacher summarised, and students reported the results of the case discussions in groups. The teacher remarked on and summarised the reports from each group, briefly summarised the knowl edge in correlation with the problems encountered by the students in their autonomous learning, and explained and answered questions about the students’ error-prone and confusing areas as well as common difficult problems. Resultsh Through data analysis, we identified the following four themes:(1)stimulating interest in learning and enhancin gautonomous learning; (2)improving independent think ing and problem-solving skills; (3)cultivatinga teamwork spirit; and (4) gaining knowledge and improving skills (Table 1). Each theme is described below with supporting quotes from the participants. Proactively learning Before class, the nursing students searched for materi als and discussed them with group members through the teaching content and cases posted by the teacher, thereby creating a strong learning atmosphere. ‘Before class, I try my best to understand the learning content and search for knowledge according to the tasks assigned by the teacher.Because I need to report on the class,I hope to vividly explain what I understand and learn. I will search for extended knowledge online and practice many times beforeclass.’—Participant 16. ‘Before class, the teacher assigned paediatric cases for us to think about, to answer the questions, I searched a lot of literature and actively thought’.—Participant 6. ‘I liked the combination of flipped classroom and PBL course teaching. In the course, we organised discussions and speeches; therefore, we must carefully study the rel evant knowledge before class.—Participant 3.hl Ethical considerationsh This study was performed in compliance with the estab lished ethical guidelines expressed in the Declaration of Helsinki.This study was approved by the Ethics Com mittee of the Children’s Hospital of Soochow University (approval no. #2021cs196).At the time of the study,the authors had no student–teacher relationship with involved participants. Verbal and written informed con sent was obtained fromall participants before the inter view took place. All participants were informed of their right to withdraw from the study at any time, without giving any reason for withdrawal. All participants were provideda guarantee of confidentiality by coding the transcripts and assigning each quotation a code. The combination of the flipped classroom and PBL course teaching can improve students’interest in learn ing. During pre-class preparation and learning in the class, students are able to proactively identify problems and actively discuss and solve them. ‘I think this teaching method will encourage us to learn the paediatric course ourselves, actively collect various knowledge related to the paediatric nursing course, and make learning more proactive’.—Participant 1. I think the flipped classroom combined with PBL course teaching is very interesting, which can better motivate students and exercise our ability to learn inde pendently.—Participant 8. Data analysis h l For the qualitative content analysis [20]the interviews were first transcribed word-by-word, and then the inter view notes were compiled. Data analysis was conducted Ni et al. BMC Nursing (2024) 23:88 Page 4 of 9 Page 4 of 9 Ni et al. BMC Nursing Stimulate learning interest and enhance autonomous learning Stimulate learning interest and enhance autonomous learning using the NVIVO software (QST International, Cam bridge, MA, USA).The investigators read the tran scripts to familiarise themselves with the data. Then they extracted the most relevant words and phrases to describe the nursing students’experiences in paediat ric flipped classrooms combined with PBL course. The investigators read all transcripts and extracted sentences that conveyed the most meaningful information regard ing the nursing students’experiences. This was followed by the preparation of coding sheets, grouping of the data, and creation and abstraction of categories. Data categori sation was performed multiple times by investigators who worked closely together until the four main catego ries were identified. As a confirmatory test, the four cate gories were shown to the caregivers, who agreed that the results accurately represented their experiences [20]. Improve independent thinking and problem-solving skills Independently think about problems Improve independent thinking and problem-solving skills Independently think about problems understand what they do not understand in the course of classroom discussion. Improve independent thinking and problem-solving skills Independently think about problems A flipped classroom combined with PBL course teaching is no longer a teacher’s lecture but student-centred teach ing, which can enable students to develop the habit of independent thinking and analysis of problems. ‘In PBL class, everyone can answer questions and solve problems with each other. If there are any problems, they can be solved through group discussions. If they cannot be solved within the group, they can work together with the teacher or with the entire class to solve them. This is very good.’—Participant 10. ‘Discovering their strengths and weaknesses while reported from other groups is also an improvement for oneself’.—Participant 2. During normal classes, it is necessary to follow teach ers’ ideas, and areas that are not understood are easily overlooked. ‘We will preview before class based on the learning materialsreleased by the teacher, and check the materials for any questions we cannot understand’.—Participant 5. ‘Because PBL discussions require everyone in the group to raise questions, it is necessary to read thepae diatric nursing textbooks and identify the areas of your doubts’.—Participant 13. ‘In group discussions and speeches, everyone has their own opinions, which has helped me develop the habit of diligent thinking’.—Participant 11. ‘Combining the case study and analysis provided by the teacher expanded our thinking’.—Participant 7. ‘I can learn from this classroom feedback what I need to strengthen and do. I won’t feel defeated; just treat it as a constructive thing.—Participant 16. Students believe that it is very important to be able to think about problems actively rather than passively lis tening to teachers’ indoctrination when combining PBL teaching with a flipped classroom. PBL coursescan broaden students’knowledge horizon tally and deepen their understanding of disease knowl edge vertically. Most students believe that PBL course methods are novel and diverseand can stimulate interest and deepen their understanding. l ‘In the paediatric nursing course learning,it gave me the feeling I had before, which was to think proactively’.— Participant 4. ‘For example, there is PBL teaching for children with congenital heart disease. The teacher will notify us in advance and let us find pictures of congenital heart dis ease, such as cardiac vascular anatomy, angiography, etc., and share them in class. Active learning atmosphere Flipped classrooms combined with PBL course teaching adopta group teaching mode. Under the teacher’s guid ance, students freely expressed their opinions. Most stu dents expressed that an active teaching atmosphere made them more willing to participate in learning activities.h ‘The teacher’s humourous and witty language, appro priate guidance, and positive responses from team mem bers made the entire classroom atmosphere more lively. Different people have different thinking patterns, and their emphasis on answering questions varies. Therefore, I have a more comprehensive understanding of the learn ing pointsafter class’.—Participant 12. Table 1 Themes and Sub-themes Themes Sub-themes (1) Stimulate learning interest and enhance autonomous learning Proactively learning Active learning atmosphere (2) Improve independent think ing and problem-solving skills Independently think about problems Positive thinking (3) Cultivate teamwork spirit Thinking collision and supplement missing knowledge Proactively and enhance communication Gurop collaboration and team growth (4) Gain knowledge and im prove skills Knowledge Extension Improvement of cognitive skills Table 1 Themes and Sub-themes Themes Sub-themes (1) Stimulate learning interest and enhance autonomous learning Proactively learning Active learning atmosphere (2) Improve independent think ing and problem-solving skills Independently think about problems Positive thinking (3) Cultivate teamwork spirit Thinking collision and supplement missing knowledge Proactively and enhance communication Gurop collaboration and team growth (4) Gain knowledge and im prove skills Knowledge Extension Improvement of cognitive skills Table 1 Themes and Sub-themes ‘We gathered with the teachertogether to discuss the case, which brought us closer to the teacher. The entire format and atmosphere are also relatively relaxed; just like a few friends gather to discuss and learn, the teacher can have a deeper understanding of each student.—Par ticipant 15. Page 5 of 9 Page 5 of 9 Ni et al. BMC Nursing (2024) 23:88 (2024) 23:88 Ni et al. BMC Nursing Improve independent thinking and problem-solving skills Independently think about problems This teachingmethod can help us understand the formation of congenital heart disease and other knowledge.—Participant 2. ‘During the problem discussion section,the teacher asked a question, and she asked us to review the materi als ourselves and think for ourselves. We would check the materials ourselves in and out of class, which would feel better.—Participant 14. p Thinking collision and supplement missing knowledge ‘In PBL class, my attention is very focused. We will identify problems together and then thinkabout and discuss them with classmates. Compared with regular classes, this can improve thinking ability.—Participant 11. During the PBL discussion process, it is possible to iden tify and fill in gaps, supplement overlooked knowledge, and comprehensively grasp the course content. ‘Sometimes, I may not notice the questions raised by other classmates, so I tend to pay more attention in the study. Sometimes, I feel that other classmates’ideas are not correct, and I also bring them up to them.’—Partici pant 4. ‘When we learned the PBL course about children’s respiratory disease and bronchopneumonia, the teacher asked us how to observe the changes of hypoxia in chil dren with pneumonia according to the on-site case study. We all expressed our own opinions, and the teacher also encouraged us to share our ideas so that we could have our own ideas.—Participant 9. ‘During the discussion, if some students have differ ent opinions, they will also raise them, and everyone will have a collision of ideas so that the answer will be more comprehensive’.—Participant 15. Positive thinking ‘I feel that after attending the PBL course, I have gained a deeper understanding of paediatricdiseases, and a clearer understanding and observation of clinical mani festations’.—Participant 6. Most students realised that teachers are not simply imparting knowledge but rather combining practi cal cases and specific clinical nursing problems, asking students questions, and guiding them to find answers, thereby triggering positive thinking among students. Proactively and enhance communicationh Students proposed that, compared with traditional teaching, flipped classrooms combined with PBL cur riculum teaching are more willing to ask questions and Through PBL discussions, students can get along harmo niously and better understand each other. Ni et al. BMC Nursing (2024) 23:88 Ni et al. BMC Nursing (2024) 23:88 Page 6 of 9 Ni et al. BMC Nursing (202 Ni et al. BMC Nursing more effective but also promote everyone to develop good learning habits’.—Participant 15. ‘When we discussed the question, we raised the question and then solved the question, which leads to dialogue and gradually becomes familiar with them’.— Participant 7. Gain knowledge and improve skills Knowledge extensionhl ‘I tend to be distracted in my study. There will be a question-answering session in the flipped classroom that will improve my interest in learning. It’s very good’.—Par ticipant 9. The flipped classroom combines PBL course teaching, which encourages students to find diversified teach ing resources and prepare fully before class. During the teaching process, the teacher helps students compre hensively learn the course from multiple perspectives, such as theoretical knowledge, clinical applications, and patient needs, thereby helping them expand their knowledge.h Gurop collaboration and team growthl In a flipped classroom that combines PBL course learn ing, team members conduct preclass previews accord ing to a course planorganised by the team leader. This requires independent access to relevant materials, prob lem-solving, and, more importantly, a reasonable division of labour among team members to share learning meth ods with each other. The teacher also guided us on the concept of neona tal developmental care, which is not mentioned in our paediatric nursing book but has important clinical sig nificance. The flipped classroom combined with the PBL course not only allows us to focus on books but also broadens our horizons..’—Participant 14. ‘Classmates study together to divide and collaborate, check materials, write reports, etc., and then several classmates discuss learning outcomes together, which is better than learning alone’.—Participant 3. ‘Flipped classrooms that combine PBL courses with paediatric cases usually involve knowledge from multiple disciplines. By discussing and learning about this case, we can not only learn the knowledge within the textbook but also expand it to other knowledge outside the text book, which helps us integrate knowledge from various disciplines’.—Participant 3. ‘We work together as a group of classmates, each of whom has their strengths and can learn from them to make up for my weaknesses. As a member of the group, I must also conscientiously complete the relevant con tent.—Participant 1. ‘Although we have all assigned learning tasks, each stage requires everyone to discuss and learn together, communicate with each other, and reach an agreement’.— Participant 12. Discussion Th d This study explored nursing students’experiences of flipped classrooms combined with PBL in a paediat ric nursing course and found that nursing students stimulated learning interest and enhanced autonomous learning,improved independent thinking and problem- solving skills,cultivated teamwork spirit, and gained knowledge and improved skills after attending a flipped classroom combined with PBL in a paediatric nursing course [23–24]. f The flipped classroom combines PBL course-teach ing methods to develop students’skills in various areas, such as problem-solving, time management, teamwork, higher-level thinking, and the ability to obtain and evalu ate information and use it to construct knowledge flex ibly [36–37].However, a drawback of the PBL teaching method is the lack of systematically acquired informa tion [38–39], and the lack of effective teaching resources can significantly affect PBL teaching. This study used an online course platform to fully meet students’self-study needs before and after classes. It was combined with offline PBL teaching to fully integrate the optimal online and offline teaching resources for the flipped classroom model, which improved students’ problem-solving and clinical thinking abilities while promoting the systemati sation and integrity of knowledge acquisition andachiev ing good teaching results. Compared to the traditional teaching mode, through a flipped classroom combined with PBL course learning, students can complete learning within a certain range according to their own schedule, freely choose learning places, and ensure that they can complete the course in a better state [25]. Hanet al proposed that in a flipped classroom, improving the learning effect is conducive for students who are good at managing time [26]. Therefore, to ensure the effectiveness of students’self-directed learn ing, teachers must strengthen the supervision and guid ance of students’ self-directed learning and implement personalised self-directed learning. The traditional teaching model advocates the leading role of teachers, neglecting students’initiative and creativ ity [13]. Before entering the flipped classroom,mastery of the content increases students’ intuitive learning skills and confidence to perform classroom activities [27]. Studies have also shown that flipped classrooms improve students’ self-directed learning readiness and increase their satisfaction [28]. There is evidence suggesting that the flipped classroom approach positively affects stu dents’ motivation and significantly improves self directed readiness skills [29–30].The flipped classroom teaching model combined with PBL can develop nursing students’ interest in learning and meet learners’ needs. Improvement of cognitive skills Flipped classrooms combined with PBL teaching can shorten the process of disease knowledge development from theoretical learning to practical applications. Some students mentioned that disease knowledge and nursing measures learned in flipped classrooms combined PBL courses could help them achieve better results in clinical health education and nursing practice. When I encounter problems that I don’t understand, I will consult with my group members and actively learn to improve my learning outcomes.’—Participant 14. In the class, team members collaborate and answer questions in an interactive manner. This shared learning method can promote the overall growth of teams. They realised that listening to the opinions of other team mem bers can help them achieve alternative ways of thinking and enhance their thinking processes. ‘By studying cases of various paediatric diseases, we can connect knowledge with practical applications, so that we can directly apply the medical knowledge we have learned to clinical cases’.—Participant 12.hl ‘By adopting this teaching method, we can have a more comprehensive understanding of our own shortcomings and blind knowledge spots, which can help us think more comprehensively and effectively’.—Participant 8.hfl ‘Through flipped classroom combined with PBL course teaching, I think that when nursing patients with such diseases, I can find their nursing problems quickly, which is more targeted. Patients also think that I am a profes sional.—Participant 2. f ‘The biggest difference between flipped classroom com bined with PBL teaching and regular teaching is that it requires our active cooperation. From the preparation of materials, we need to combine the outline and case studies to review relevant materials and actively com municate with other students so that everyone can work together to reach the best conclusion.—Participant 13. Most students have a strong sense of benefit from the combination of flipped classrooms and PBL teaching and can gain useful knowledge and experiences from the curriculum. ‘For the flipped classroom teaching combined with PBL course teaching, I think I have mastered a lot of knowl edge in the classroom. During the internship, when I performed a physical examination for children, I felt that my professional knowledge was very comprehensive and ‘Engaging in self-learning, discussion, and raising one’s own questions or opinions on a certain issue among group participants can not only make group learning Ni et al. BMC Nursing (2024) 23:88 (2024) 23:88 Page 7 of 9 Page 7 of 9 Ni et al. BMC Nursing more confident. Improvement of cognitive skills I can answer all the questions asked by the children’s families.—Participant 5. combined with PBL improved students’ learning out comes to some extent by actively discussing issues and reducing classroom-learning loopholes. ‘I think that compared with ordinary teaching, Flipped classroom combined with PBL course teaching course is very helpful to me. This enables me to better understand the clinical manifestations, nursing problems, and nurs ing measures of paediatric diseases.—Participant 8. In this study, students raised questions in classroom discussions and engaged in brainstorming through group discussions, which was beneficial for promoting mutual learning and progress among students as well as enhancing communication between teachers and class mates. Research has shown that group discussions can help students experience a spirit of teamwork and culti vate team qualities [33–34]. Schlairetetal [35]. proposed that flipped classrooms allow teachers sufficient time to observe students’learning and provide targeted guidance. Therefore, teachers, in addition to preparing well before class, should not ignore effective guidance for students.hl Data availability Th d h The data that support the findings of this study are available from the corresponding author upon reasonable request. Acknowledgements The authors wish to express our profound gratitude to all the nursing students who volunteered to participate in the study. 12. Sangestani G, Khatiban M. Comparison of problem-based learning and lecture-based learning in midwifery. Nurse Educ Today. 2013;33(8):791–5. https://doi.org/10.1016/j.nedt.2012.03.010. 12. Sangestani G, Khatiban M. Comparison of problem-based learning and lecture-based learning in midwifery. Nurse Educ Today. 2013;33(8):791–5. https://doi.org/10.1016/j.nedt.2012.03.010. Funding As an important core educational course in the nurs ing profession, paediatric nursing not only emphasises the mastery of basic knowledge and concepts but also focuses on the cultivation of students’ clinical thinking and practical problem-solving abilities. Based on real clinicalsituations, the flipped classroom combined with PBL encourages students to find, analyse, and solve prob lems in the process of independent exploration, which isconducive to promoting the cultivation of students’ clinical thinking ability and shortening the distance between school and the clinic [43]. We found that stu dents can obtain a greatsense of self-satisfaction in the process of independent analysis and problem-solving, whichis beneficial for promoting their self-improvement and increasing their interest inlearning. Moreover, the university stage is a key period of life development, and having good communication and cooperation skills is the basis for nursing students to improve their comprehen sive quality, clinical nurse-patient communication skills, and health careteam cooperation skills [44]. The authors thank the Suzhou Medical CollegeUndergraduate Teaching Projectfor funding this study (No.202106). References This study has both strengths and limitations. One strength is that the setting and the different stages of the research process, namely,data collection and analysis, were clearly described so that readers could follow the process, enhancing the credibility and transferability of this study. Quotations directly lifted from the interviews were included in the results section to illustrate that each category originated from the student’s perspective, thus creating transparency in the content analysis. 1. Norman GR, Schmidt HG. 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Does problem- based learning in nursing education Empower Learning? Nurse Educ Pract. 2020;44:102752. https://doi.org/10.1016/j.nepr.2020.102752. This study used the purposive sampling method, which can limit the transferability of the results. However,researchers attempted to use maximum varia tion by considering the participants’ characteristics regarding age,gender, and a proportional number of par ticipants for qualitative studies were used. 6. Azer SA, Peterson R, Guerrero AP, Edgren G. Twelve tips forconstructing problem-based learning cases. Med Teach. 2012;34(5):361–7. https://doi.org/ 10.3109/0142159X.2011.613500. 7. Servant-Miklos VFC, Woods NN, Dolmans DHJM. Celebrating 50 years of problem-b.ased learning: progress, pitfalls and possibilities. Adv Health Sci Educ Theory Pract. 2019;24(5):849–51. https://doi.org/10.1007/ s10459-019-09947-9. 8. Bodagh N, Bloomfield J, Birch P, Ricketts W. Problem-based learning: a review. 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Competing interests Competing interests The authors declare no competing interests. Competing interests The authors declare no competing interests. p g The authors declare no competing interests. Received: 16 September 2023 / Accepted: 18 January 2024 Received: 16 September 2023 / Accepted: 18 January 2024 Discussion Th d nursing students can choose the most proper ways to obtain new knowledge [31].h The flipped classroom teaching model combined with PBL was recognised by the students, and the tra ditional teaching model based on a teacher’s lecture could give full play to the teacher’s organizational role in classroom teaching. However, it ignores students’ initiative,creativity, and cognitive subject roles, which is not conducive to improving their interest in learning and autonomous learning abilities. Furthermore, there is insufficient teacher-student and student-student commu nication, and the classroom atmosphere is not active in the traditional teaching model [40].hl The flipped classroom combined with PBL not only promotes students’ autonomous learning of basic concepts and knowledge beforehand by using online resources, but, especially in the process of finding answers andsolving problems based on PBL cases, stu dents also need to retrieve andlearn relevant knowledge independently. This is conducive to promoting students’ subjective initiatives and improving their autonomous learning ability, in line with the results of Zeng andZhao [41–42]. This study transforms the role of teachers into that of initiators and coordinators of the curriculum and trans forms teacher-centred education into putting students at the centre creates an environment and atmosphere for active learning. Mortensen and Nicholson [32]proposed that active learning could promote students’independent think ing and increase their creativity. The results of this study showed that the flipped classroom teaching model Ni et al. BMC Nursing (2024) 23:88 Ni et al. BMC Nursing (2024) 23:88 Page 8 of 9 Consent for publication Not applicable. Consent for publication Not applicable. g The authors wish to express our profound gratitude to all the nursing students who volunteered to participate in the study. Ethics approval and consent to participate This study was performed in compliance with the established ethical guidelines expressed in the Declaration of Helsinki.This study was approved by the Ethics Committee of the Children’s Hospital of Soochow University (approval no. #2021cs196).Informed consent was signed by each participant before being interviewed and was coded to maintain anonymity. Author contributions 13. Shiau S, Kahn LG, Platt J, Li C, Guzman JT, Kornhauser ZG, Keyes KM, Martins SS. 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https://openalex.org/W139724090	https://hal.inria.fr/hal-01540894/document	English	null	Feature Selection on Handwriting Biometrics: Security Aspects of Artificial Forgeries	Lecture notes in computer science	2,012	cc-by	4,997	To cite this version: Karl Kümmel, Tobias Scheidat, Claus Vielhauer, Jana Dittmann. Feature Selection on Handwriting Biometrics: Security Aspects of Artificial Forgeries. 13th International Conference on Communications and Multimedia Security (CMS), Sep 2012, Canterbury, United Kingdom. pp.16-25, 10.1007/978-3- 642-32805-3_2. hal-01540894 Distributed under a Creative Commons Attribution 4.0 International License HAL Id: hal-01540894 https://inria.hal.science/hal-01540894v1 Submitted on 16 Jun 2017 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. 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Distributed under a Creative Commons Attribution 4.0 International License Feature Selection on Handwriting Biometrics: Security Aspects of Artificial Forgeries Feature Selection on Handwriting Biometrics: Security Aspects of Artificial Forgeries Karl Kümmel1,2, Tobias Scheidat1,2, Claus Vielhauer1 and Jana Dittmann2 1Brandenburg University of Applied Sciences, Germany {karl.kuemmel,tobias.scheidat,claus.vielhauer}@fh-brandenburg.de 2Otto-von-Guericke University Magdeburg, Germany {tobias.scheidat,jana.dittmann}@iti.cs.uni-magdeburg.de Karl Kümmel1,2, Tobias Scheidat1,2, Claus Vielhauer1 and Jana Dittmann2 1Brandenburg University of Applied Sciences, Germany karl.kuemmel,tobias.scheidat,claus.vielhauer}@fh-brandenburg.de 2Otto-von-Guericke University Magdeburg, Germany {tobias.scheidat,jana.dittmann}@iti.cs.uni-magdeburg.de 1Brandenburg University of Applied Sciences, Germany {karl.kuemmel,tobias.scheidat,claus.vielhauer}@fh-brandenburg.de 2Otto-von-Guericke University Magdeburg, Germany {tobias.scheidat,jana.dittmann}@iti.cs.uni-magdeburg.de Abstract. A lot of improvements were introduced lately in order to increase the verification performance of biometric user authentication systems. One method, besides many others, is the selection of specific features for each user during the verification process. In this paper we present a security analysis of a user specific bit mask vector, which was originally introduced to improve verifica- tion performance on a Biometric Hash algorithm for dynamic handwriting. Therefore, we use a reverse engineering attack method to generate artificial handwriting data and calculate error rates to examine the impact on the verifica- tion performance. Our goal is to study the effect of a feature selection by a mask vector on artificial data in comparison to genuine handwriting data. Our first experimental results show an average decrease of the equal error rate, gen- erate by the artificial data, by approx. 64%. In comparison, equal error rates of random attacks, using verification data of another user, decreases by an average of approx. 27%. Keywords: Biometrics, dynamic handwriting, feature selection, security analy- sis, reverse engineering 1 Introduction and Motivation Biometric user authentication is an important field in IT security today. It relies on personal physiological or behavioral characteristics of a person. The purpose of a generic biometric system is to determine and/or verify a person’s identity based on at least one biometric modality (i.e. fingerprint, iris, voice etc.). Like in every other au- thentication system, i.e. knowledge based (password or PIN), it is crucial to protect the reference data (templates) from being misused. A variety of biometric template protection methods were introduced during the last years to prevent the misuse of biometric data. Jain et al. categorize in [1] a selection of template protection schemes for several biometric modalities and describe their advantages and disadvantages. Besides security issues, authentication performance is also a key requirement to bio- metric authentication systems. During the last years a lot of research in almost every biometric authentication algorithm and modality is done to improve user authentica- tion performance. Many of which rely on the concept of feature selection. For exam- ple, Fratric et al. propose in [2] a novel method of feature extraction from face images to improve recognition accuracy. They use a so-called local binary linear discriminant analysis (LBLDA), which combines the good characteristics of both methods LDA and local feature extraction. Hollingsworth et al. introduce in [3] a method where potential fragile iris code bits are masked to increase the separation between the match and non-match distributions in iris based authentication systems. A further technique to improve user authentication performance is biometric fu- sion. Rathgeb et al. describe in [4] a generic fusion technique for iris recognition at bit-level (called Selective Bit Fusion) to improve accuracy and processing time. Nageshkumar et al. propose in [5] an authentication method for a multimodal biomet- ric identification system using the two traits face and palmprint. Specific feature selection is, besides many others, another method to improve au- thentication performance, whereby useful features are determined during a feature selection process. In this context, useful features are those which positively affect the user authentication and biometric hash generation performance. In [6] Kumar et al. show that an evaluation and selection of useful biometric features can improve the recognition accuracy. They used a correlation based feature selection (CFS) for bi- modal biometric systems and analyzed the classification performance. Makrushin et al. compare in [7] different feature selection strategies to determine sophisticated features. 2 Biometric Hash Algorithm The Biometric Hash algorithm for dynamic handwriting (hereafter BioHash) is ini- tially introduced by Vielhauer et al. in [9] and enhanced in [10] in order to generate stable individual biometric hash values as well as to perform biometric verification based on the hashes. Generally, the raw data of each dynamic handwriting sample consists of a time dependent sequence of physical values derived from a digitizer device (e.g. Tablet PC, signature tablet). The data consist of five values per sample point: pen tip positions x(t) and y(t), pen tip pressure p(t) and pen orientation angles altitude Φ(t) and azimuth Θ(t). A so-called Interval Matrix IM is determined by the BioHash algorithm during the enrollment process for each user separately. The IM calculation is based on raw data of the writer and the parameters Tolerance Factor and Tolerance Vector. From each raw data sample derived from each person during the enrollment process, a statistical feature vector (static and dynamic features) is calculated with a dimensionality of k (k=131 in the reference implementation used in this paper). The IM consists of a vec- tor containing the length of a mapping interval for each feature and an offset value vector. Both vectors are calculated based on an analysis of intra-class variability of the user using its statistical feature vectors acquired during enrollment session. The Biometric Hash algorithm provides two possibilities to parameterize the hash generation by scaling the mapping intervals stored in the IM: Tolerance Factor TF and Tolerance Vector TV. The Tolerance Factor TF is a global hash generation parameter, which is a scalar value. Using the TF, it is possible to scale the mapping intervals for all features by one global factor. In contrast to the TF, the aim of the Tolerance Vec- tor TV is to provide an individual scaling of the mapping interval of each statistical feature separately. Thus, the dimensionality of TV is also k. TV can be calculated indi- vidually for each user or globally by a group of users, e.g. either based on a disjoint group of users, but also on all or a selection of enrolled persons. Based on one statistical feature vector derived from the enrollment data and the us- ers’ individual IM the so-called interval mapping function determines the reference hash vector bref of a user. 2 Biometric Hash Algorithm Therefore, the feature dependent interval lengths and offsets provided by IM are used to map each of the k statistical features to the corresponding hash value. Each further biometric hash is calculated in the same manner, independ- ently if it is used for biometric verification or hash generation application. For verifi- cation, the hash vector b derived from the currently presented handwriting sample is compared against the reference hash vector bref by Hamming distance measurement. For more details of the single calculation steps, the interested reader is referred to reference [10]. 1 Introduction and Motivation It has been shown that forward and backward selection algorithms have al- ways better results than considered heuristics. We introduced in [8] a much simpler feature selection method which leads to simi- lar findings compared to [7]. A user specific bit mask is generated during the enroll- ment process to enable/disable certain features within the verification process. In this work we focus on the security perspective of this user specific bit mask ap- plied on a Biometric Hash algorithm for dynamic handwriting [10] with respect to a specific attack scenario. We analyze whether a potential attack gains any advantages or disadvantages when a user mask vector is used during the verification process. In order to perform a security analysis, we use a reverse engineering attack method in- troduced in [11] and [12] to check the security affects of a user specific bit mask vec- tor. Since we already observed in [11] and [12] that computer generated handwriting samples of this attack method are not as good as human forgeries, we like to examine if a selective feature approach may affect the false acceptance characteristics of syn- thetically generated data. Secondly, we like to compare the behavior of the system’s verification performance using artificial verification data similar to genuine verifica- tion data under feature selection approach. The structure of the paper is as follows. In section 2 the Biometric Hash algorithm for dynamic handwriting is shortly presented. The user specific feature mask and its generation are described in section 3. A reverse engineering attack method based on a spline interpolation technique is introduced in section 4. Experimental results are shown in section 5 and a conclusion and possible future prospects are given in the last section. 3 Feature Mask Vector In addition to the reference BioHash vector bref and the corresponding Interval Matrix IM, we generate a k dimensional (k=131) feature mask vector MV for each user. MV is created during the feature selection process after the enrollment. The main idea of creating a feature mask vector is to select or deselect specific features. If a bit is set to 1, the represented feature is considered during the verification process and if it is set to 0, it is not taken into account. To create a user specific feature mask vector MV raw data samples s0, s1, …, sn, which are not used during the enrollment process, are required. The identifier n indi- cates the maximum number of used samples. Three steps have to be executed to gen- erate MV. Firstly, feature vectors fv0, fv1, …, fvn are determined based on the raw data samples s0, s1, …, sn. Secondly, feature vector fv0, fv1, …, fvn of each user are mapped to BioHash vectors b0, b1, …, bn using the corresponding Interval Matrix IM of the user. Within the last step, one feature mask vector MV for each user are determined by an element-wise comparison of each BioHash vector b0, b1, …, bn and reference Bio- Hash bref. If a certain number of values at position i is equal, the corresponding i-th bit of MV is set to 1; otherwise it is set to 0. The result is a k-dimensional feature mask vector MV. This vector is a new part of the reference data of each user and therefore stored together with corresponding Interval Matrix IM and BioHash bref, for example in a database or on a Smart Card. During the verification process only selected fea- tures, which are marked by “ones” within MV, are considered. This method allows a simplistic user specific enabling or disabling of used fea- tures. We come in [8] to the first conclusion that the application of feature mask vec- tor MV leads to improved recognition accuracy. In our tests, the equal error rates (EER see section 5.1) decreases noticeable by approximately three percentage points. Furthermore, the reproducibility of generated biometric hashes increases in all tests considerable. The average increase of the reproduction rate (RR see section 5.1) is approx. 26%. 3 Feature Mask Vector These results show that a simple feature selection strategy is able to substantial increase the biometric hash generation as well as the user authentication performance. 4 Reverse Engineering based Attack Method In previous work ([11] and [12]) we introduced a method for constructing biometric raw data from given reference data. This method is based on the following conditions. A potential attacker has compromised a biometric based verification system and has access and knowledge to username, reference BioHash bRef, and Interval Matrix (IM) for each registered individual. The operating principle of the BioHash algorithm is openly published and is accessible for everyone who is interested in (Kerkhoff princi- ples). The attacker’s aim is to generate synthetic raw data that produces a BioHash batt, which is almost identical to the reference BioHash bref. He determines the differ- ences by calculating the Hamming Distance between them. Consequently, he tries to provoke a false-acceptance using his artificially generated raw data. In [11] we determined the following vulnerability of the Biometric Hash algorithm. When BioHash bref and corresponding Interval Matrix IM are given, a reverse map- ping to create a feature vector fvcalc can be performed. Due to the fact that fvcalc is determined from bref and corresponding IM, it can be mapped with help of IM to bref again and therefore be used to reconstruct raw data, based on it. If an attacker takes advantage of this vulnerability (reverse mapping) he can reduce his work on recon- structing raw data based on that calculated feature vector fvcalc, i.e. in feature space rather than on the BioHash. Feature Vector ncalc Basic Raw Data Construction Feature Vector Calculation Basic Raw Data Structure BioHash bref Interval Matrix (IM) Additional Feature Integration Reconstructed Raw Data Step 1 Step 2 Step 3 PRNG Seed Fig. 1. Illustration of steps 1 to 3 of the raw data generation approach Fig. 1. Illustration of steps 1 to 3 of the raw data generation approach Figure 1 illustrates the three main steps of the attack method to generate raw data. Step 1 implies the calculation of fvcalc using reference BioHash bref and corresponding IM. Within step 2 a spline interpolation function generates a basic raw data structure. This is done based on specific feature elements of fvcalc, which contains the amount of maxima and minima for horizontal pen movement signal X respectively vertical pen movement signal Y. All splines are set randomly using a pseudo random number generator (PRNG), which requires a seed value. The implementation of additional feature into the basic raw data structure is done in step 3. 4 Reverse Engineering based Attack Method These additional features represent minimum, maximum and average of pressure and angle values. The algo- rithm simply sets an arbitrary chosen sample point and marks it with the maximum or minimum value. All other sample points are set in a way that the sum matches the average value. This procedure can be done for pressure and both angle values. The result of all three steps is a set of synthetic raw data of an artificial handwriting signal. Due to the reverse engineering algorithm the shape of genuine handwriting signals and artificial handwriting signals do not look similar at all (see figure 2). A detailed description on the algorithm is described in [11] and [12]. Fig. 2. Original genuine handwriting signal (left) and artificial handwriting signal (right) Fig. 2. Original genuine handwriting signal (left) and artificial handwriting signal (right) 5.1 Methodology In order to demonstrate the improved verification performance of an applied user specific feature mask vector MV on the BioHash algorithm, we compare the verifica- tion performance with and without applied MV. Afterwards we use the raw data, which is generated by the attack method described in section 4, to test the security impact of an applied MV. Therefore, biometric error rates FRR, FAR and EER are calculated for both settings. The ratio between the number of false rejections of au- thentic persons and the total number of tests is described by the false rejection rate (FRR). The FAR (false acceptance rate) is the ratio between number of false accep- tances of non-authentic persons and the entire number of authentication attempts. The equal error rate (EER) is a common measurement in biometrics for a comparative analysis of the verification performance. EER denotes the point in error characteris- tics, where FRR and FAR yield identical value. Furthermore, we calculate the false acceptance rate caused by the reverse engineering attack method and corresponding equal error rate (EERre). The FARre is the ratio between number of false acceptance of artificially generated imposter data (attack raw data) and the entire number of authen- tication attempts. Consequently, EERre donates the point in error characteristics where FRR and FARre yield identical value. We also evaluate the reproducibility rate (RR) and collision rate (CR) for both set- tings including the attack data; these values are related sums of identical reproduced hashes in genuine and both imposter trials (see [12]). Because of the reciprocal effect of RR and CR, a tuning of the system to improve RR leads to a degradation of CR and vice versa. Therefore, the collision reproduction rate (CRR, [12]) is selected as a hash generation quality criterion. The CRR is defined in the following equation, whereas CR and RR are equally weighted. )) 1( ( 2 1 RR CR CRR − + = (1) )) 1( ( 2 1 RR CR CRR − + = (1) CRre and CRRre describe collision rate and collision reproduction rate of the attack data, which is generated by the reverse engineering attack method. 5 Experimental Evaluation In this section we describe our methodology and setup for the experimental evaluation and show first results on the verification and security performance of the user specific feature mask vector MV in context of reverse engineering based attacks. 5.2 Experimental Settings The biometric database of our initial tests consists of 39 subjects, which have donated 30 handwriting samples in three sessions with an interval of at least one month be- tween two sessions. Within a session a user provides 10 handwritten samples for five different semantics (5850 test samples overall). These semantics are “Free chosen Pseudonym” (pseudonym), “Free chosen Symbol” (symbol), “Answer to the Question: Where are you from?” (place), “Fixed 5 digit PIN: 77993” (public PIN) and “Free chosen 5 digit PIN” (secret PIN). It has been observed in [10] that semantics produce similar recognition accuracy compared to handwriting signatures, without disclosing the true identity of the writer. All samples were captured under similar laboratory conditions using a Toshiba M200 Portege tablet PC. The handwriting samples ac- quired during the first session are used as enrollment data in order to determine the reference BioHash bref as well as to generate the Interval Matrix IM. The samples of the second session are used for tuning of the Tolerance Factor TF and feature selec- tion in terms of feature mask vector calculation. Finally, the data collected within the third session are used for evaluation. Ten raw data samples are generated by the attack method for each user based on its reference data BioHash bref and IM (39 users times 10 test samples). These test samples are used to calculate EERre, CRre, and CRRre. In addition, an attempt of one user to be verified as another one is considered as an imposter trial (random attack). Each test implies 390 genuine trials, where reference data of a user is matched against its own verification data (39 user times 10 test sam- ples) and 14,820 imposter trials (random attacks), where reference data of a user is matched against all other verification data except its own (38 user claims times 39 actual users times 10 test samples). Within the feature extraction process of the Bio- Hash algorithm 131 features are calculated based on the handwritten samples. Table 1. Tolerance factor (TF) values used during the evaluation Semantic TF in CRR mode TF in EER mode Public PIN 1.50 1.00 Secret PIN 1.75 1.00 Pseudonym 2.50 1.25 Symbol 3.50 1.50 Place 2.50 1.25 Table 1. Tolerance factor (TF) values used during the evaluation Since all features are considered equally, the tolerance vector TV is set to (1, …, 1). 5.3 Experimental Results Table 2 shows equal error rates (EER and EERre) of all semantics with and without applied MV. By comparing the first columns of each section, where the EER is pre- sented for all semantics, a decrease of all EER is clearly noticeable. The highest drop of 4.67 percentage points (relative drop of 42.61%) is caused by the semantic pseudo- nym. Similar results are recorded by the EERre. The highest drop of 2.2 percentage points (relative drop of 46.8%) is achieved by semantic pseudonym. Semantic public PIN even reaches an EERre of 0%. In one case a slightly increase of the EERre of 0.2 percentage points occurs (semantic place). Table 3 shows reproduction rates, collision rates and collision reproduction rates of all semantic classes with and without applied specific feature mask vector MV. If a MV is used all reproduction rates increases significantly by an average of approxi- mately 19%, whereas the collision rates also increase by an average of 31%. Table 2. Equal error rates (EER) of all semantic classes (in %) with and without applied MV. No MV MV Semantic EER EERre EER EERre Public PIN 17.46 2.56 13.25 0.00 Secret PIN 12.71 1.41 11.54 0.25 Pseudonym 10.96 4.70 6.29 2.50 Symbol 9.45 1.86 6.44 1.35 Place 9.79 2.30 7.09 2.50 Table 2. Equal error rates (EER) of all semantic classes (in %) with and without applied MV. The largest reproduction rate increase is obtained by the semantic public PIN (51.79% up to 72.54%) and the highest reproduction rate was achieved by semantic symbol (94.35%). Collision rates of associated attack test samples are in almost every semantic zero. Within semantic symbol a slightly collision rate of 0.76% is recorded during the experimental tests. Table 3. Collision reproduction rates (CRR/CRRre), reproduction rates (RR) and collision rates (CR/CRre) of all semantic classes (in %) with and without user specific feature mask vector MV. 5.2 Experimental Settings Thus, the tolerance factor (TF) is the main parameter for controlling hash generation as well as verification performance. In previous work [7] we already determined tol- erance factor values of the same evaluation data for two scenarios, lowest EER (EER mode) and highest RR (CRR mode), in all semantics. According to these results of the previous test, based on all 131 features, the TF values are set as shown in table 1. Feature mask vectors are generated for each user in all semantic classes separately, as described in section 3, using the evaluation data of the second session. During the MV generation, only if all values at a specific position i of all BioHash vectors are equal, then MVi is set to 1. The minimal, average and maximal amounts of selected features are determined to show how many features are actually used during the veri- fication or hash generation process. Note that the evaluation protocol leads to a realis- tic scenario since the reference data has already undergone an aging of at least 2 month compared to the evaluation data. 5.3 Experimental Results No MV MV Semantic RR CR CRre CRR CRRre RR CR CRre CRR CRRre Public PIN 51.79 5.10 0.00 26.65 24.10 72.54 8.34 0.00 17.88 13.71 Secret PIN 60.00 4.85 0.00 22.42 20.00 78.20 9.17 0.00 15.48 10.89 Pseudonym 71.02 4.33 0.00 16.65 14.48 84.61 5.84 0.00 10.61 7.69 Symbol 86.92 5.40 0.00 9.24 6.53 94.35 6.72 0.76 6.18 3.20 Place 71.02 4.06 0.00 16.52 14.48 87.17 5.57 0.00 9.19 6.41 Table 4 shows the minimal, average and maximal amount of selected features rep- resented by the feature mask vector in each semantic class for both scenarios (verifi- Table 4 shows the minimal, average and maximal amount of selected features rep- resented by the feature mask vector in each semantic class for both scenarios (verifi- cation and hash generation mode). The minimal amount (87) of features used during a verification process is obtained by semantic public PIN within the EER mode. In CRR mode the number of used features is always higher than in EER mode. The av- erage amount of selected features over all semantics in EER mode is 122 and in CRR mode 128. Table 4. Minimal, average and maximal amount of selected features for each semantic in both scenarios (verification and hash generation mode) Table 4. Minimal, average and maximal amount of selected features for each semantic in both scenarios (verification and hash generation mode) Public PIN Secret PIN Pseudonym Symbol Place Mode EER CRR EER CRR EER CRR EER CRR EER CRR Min. 87 96 89 107 103 120 113 126 107 123 Avg. 120 126 120 127 123 128 125 130 122 128 Max. 129 131 130 131 131 131 131 131 131 131 Table 4. Minimal, average and maximal amount of selected features for each semantic in both scenarios (verification and hash generation mode) References 1. Jain, A. K., Nandakumar, K., Nagar, A.: Biometric Template Security. In EURASIP Jour- nal on Advances in Signal Processing, Article ID 579416 (2008) 2. Fratric, I., Ribaric S.: Local Binary LDA for Face Recognition. In Proceedings of the 3rd European Workshop on Biometrics and Identity Management (BioID2011), pp. 144-155, Germany, Brandenburg (2011) 3. Hollingsworth, K.P., Bowyer, K.W., Flynn, P.J.: The Best Bits in an Iris Code. IEEE Transactions on Pattern Analysis and Machine Intelligence Volume 31 Issue 6, 964-973 (2009) 4. Rathgeb, C., Uhl, A., Wild, P.: Combining Selective Best Bits of Iris-Codes. In Proceed- ings of the 3rd European Workshop on Biometrics and Identity Management (BioID2011), pp. 127-137, Germany, Brandenburg (2011) 5. Nageshkumar, M.; Mahesh, P., Swamy, M. S.: An Efficient Secure Multimodal Biometric Fusion Using Palmprint and Face Image. International Journal of Computer Science Is- sues, IJCSI, Volume 2, pp. 49-53 (2009) 6. Kumar, A., Zhang, D.: Biometric Recognition using Feature Selection and Combination. In: Audio- and Video-Based Biometric Person Authentication, LNCS 3546, pp. 813-822 (2005) 7. Makrushin, A., Scheidat, T., Vielhauer, C.: Handwriting Biometrics: Feature Selection based Improvements in Authentication and Hash Generation Accuracy. In Proceedings of the 3rd European Workshop on Biometrics and Identity Management (BioID2011), pp. 37- 48, Germany, Brandenburg (2011) 8. Kümmel, K.; Scheidat, T.; Arndt, C., Vielhauer, C.: Feature Selection by User Specific Feature Mask on a Biometric Hash Algorithm for Dynamic Handwriting. In Proceedings of the 12th Joint IFIP TC6 and TC11 Conference on Communications and Multimedia Se- curity, pp. 85-93 (2011) 9. Vielhauer, C., Steinmetz, R., Mayerhöfer, A., “Biometric Hash based on Statistical Fea- tures of Online Signature”. In Proceedings of the International Conference on Pattern Rec- ognition (ICPR), Quebec City, Canada, Vol. 1 (2002) 10. Vielhauer, C.: Biometric User Authentication for IT Security: From Fundamentals to Handwriting, Springer, New York (2006) 11. Kümmel, K., Vielhauer, C.: Potentielle Rückführbarkeit eines biometrischen Hashes für Handschriften. In Proceedings of the D-A-CH Security Conference, pp. 66-77, Klagenfurt, Germany (2010) 12. Kümmel, K., Vielhauer, C., Scheidat, T., Franke, D., Dittmann, J.: Handwriting Biometric Hash Attack: A Genetic Algorithm with User Interaction for Raw Data Reconstruction. In Proceedings of the 11th Joint IFIP TC6/TC11 International Conference on Communica- tions and Multimedia Security, pp. 178-190, Austria (2010) 13. Scheidat, T., Vielhauer, C., Dittmann, J.: Advanced Studies on Reproducibility of Biomet- ric Hashes. In Proceedings of the First Workshop on Biometrics and Identity Management (BIOID 2008), pp. 6 Conclusion and Future Work The content of this document is under the sole responsibility of the authors. We also like to thank the StepOver GmbH for supporting the project “OptiBio- HashEmbedded”. Acknowledgements. This work is supported by the German Federal Ministry of Edu- cation and Research (BMBF), project “OptiBioHashEmbedded” under grant number 17N3109. The content of this document is under the sole responsibility of the authors. p y We also like to thank the StepOver GmbH for supporting the project “OptiBio- HashEmbedded”. 6 Conclusion and Future Work 6 In this work we study the security impact of an applied user specific feature mask vector MV introduced in [8] on a Biometric Hash algorithm for dynamic handwriting [10]. Therefore, we use a reverse engineering attack method introduced in [11] and [12] to generate attack test samples for each user. Our goal was to see if the feature mask vector MV has any affect on artificial data and if so, are the results similar to genuine verification data. Within the experimental tests, equal error rates for both settings, with and without applied MV, were evaluated. First results indicate that an applied feature mask vector reduces the false acceptance rates caused by the attack test samples significantly. Consequently, this leads to a decrease of EERre in almost every semantic class by an average of approx. 64%. Compared to the decrease caused by the MV on the EER of random attacks (average drop by 27%), artificial data are more effected. It seems that an applied feature mask vector has even a greater security impact on artificially generated data then on genuine data and random attacks. Colli- sion rates caused by the artificial generated data (in almost every semantic 0%) sup- port this assumption. These results point out, that an applied specific feature mask vector improves not only the verification but also the security performance of the Biometric Hash algorithm by reducing error rates imposed by artificial samples. Nev- ertheless, a slightly increase of an EERre of 0.2 percentage points and a minor colli- sion rate of 0.76% during an applied MV needs to be studied further. In order to sub- stantiate the first experimental results further tests, using more individuals and a greater amount of attack test samples, have to be carried out. Also the reverse engi- neering method can be improved to generate more efficient imposter data in order to execute a more sophisticated security performance test. One possible research direc- tion here could be a composition of handwriting signals from sets of base letter struc- ture components with the pseudorandom spline function. This method may also lead to more realistic looking artificial handwriting data then those which were generated by the actual reverse engineering method. Acknowledgements. This work is supported by the German Federal Ministry of Edu- cation and Research (BMBF), project “OptiBioHashEmbedded” under grant number 17N3109. 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https://openalex.org/W2151437159	https://europepmc.org/articles/pmc3212939?pdf=render	English	null	A qualitative exploration of the human resource policy implications of voluntary counselling and testing scale-up in Kenya: applying a model for policy analysis	BMC public health	2,011	cc-by	8,428	Abstract Background: Kenya experienced rapid scale up of HIV testing and counselling services in government health services from 2001. We set out to examine the human resource policy implications of scaling up HIV testing and counselling in Kenya and to analyse the resultant policy against a recognised theoretical framework of health policy reform (policy analysis triangle). Methods: Qualitative methods were used to gain in-depth insights from policy makers who shaped scale up. This included 22 in-depth interviews with Voluntary Counselling and Testing (VCT) task force members, critical analysis of 53 sets of minutes and diary notes. We explore points of consensus and conflict amongst policymakers in Kenya and analyse this content to assess who favoured and resisted new policies, how scale up was achieved and the importance of the local context in which scale up occurred. Results: The scale up of VCT in Kenya had a number of human resource policy implications resulting from the introduction of lay counsellors and their authorisation to conduct rapid HIV testing using newly introduced rapid testing technologies. Our findings indicate that three key groups of actors were critical: laboratory professionals, counselling associations and the Ministry of Health. Strategic alliances between donors, NGOs and these three key groups underpinned the process. The process of reaching consensus required compromise and time commitment but was critical to a unified nationwide approach. Policies around quality assurance were integral in ensuring standardisation of content and approach. Conclusion: The introduction and scale up of new health service initiatives such as HIV voluntary counselling and testing necessitates changes to existing health systems and modification of entrenched interests around professional counselling and laboratory testing. Our methodological approach enabled exploration of complexities of scale up of HIV testing and counselling in Kenya. We argue that a better understanding of the diverse actors, the context and the process, is required to mitigate risks and maximise impact. to the HIV epidemic [1,2]. 2008 saw the launch of new task shifting guidelines [3] with a number of countries reporting the success of this approach in HIV services generally [4,5] and in HIV counselling and testing speci- fically [6,7]. However, as new cadres of staff emerge, concerns remain over the professional recognition of lay staff, their training and supervisory support as well as quality assurance mechanisms [8,9]. Taegtmeyer et al. BMC Public Health 2011, 11:812 http://www.biomedcentral.com/1471-2458/11/812 Taegtmeyer et al. BMC Public Health 2011, 11:812 http://www.biomedcentral.com/1471-2458/11/812 A qualitative exploration of the human resource policy implications of voluntary counselling and testing scale-up in Kenya: applying a model for policy analysis Miriam Taegtmeyer1, Tim Martineau2, Jane H Namwebya3, Annrita Ikahu4, Carol W Ngare5, James Sakwa6, David G Lalloo1 and Sally Theobald2 © 2011 Taegtmeyer et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. RESEARCH ARTICLE Open Access © 2011 Taegtmeyer et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Correspondence: M.Taegtmeyer@liverpool.ac.uk 1Clinical Group, Liverpool School of Tropical Medicine, Liverpool, UK Full list of author information is available at the end of the article Background h While the authors’ involvement in the process may have led to subjectivity, the close ‘insider’ involvement allowed unique insights, grounded in a reflexive approach. Awareness of the difficulties, dilemmas and potential biases of the methodology meant that steps were taken to mitigate them. Open-ended questions followed by probes were used in interviews to enable participants’ own views to be clearly expressed. The roles of indivi- duals within the group are examined reflexively in the analytical process to assess their influence on policy and in shaping strategies for VCT scale up. In 2000, when this work commenced, an estimated 2.2 million adult Kenyans were infected with HIV [15]. Pre- valence rates showed significant regional and rural/ urban variations, average urban prevalence was (10%) nearly twice that in rural areas (5-6%) and some districts in Nyanza province had prevalence rates in excess of 30% [16]. Kenya is an example of a country where task shifting has been employed to facilitate the rapid scale up of client-initiated counselling and testing services in government health facilities [17,18] and high prevalence areas were targeted in the initial phase of scale up. The introduction in 2000 of rapid HIV testing technology that enabled whole blood from a finger prick to be used for testing made it possible for lay counsellors to con- duct HIV testing with rapid and reliable results [19]. Kenya then saw a rapid increase in the availability of VCT from 3 sites in 2000 to over 400 registered sites in early 2004 [17]. Purposive sampling was employed to recruit intervie- wees with rich and relevant experience [24]. Intervie- wees ranged from those with first hand experiences establishing VCT programmes, to technical advisors, donors and Ministry of Health (MoH) officials. Not all were permanent members of the taskforce but all were key political drivers of the scale up process. A total of 22 interviews were conducted, 14 of whom were task- force members. Interviews were conducted in English by MT between March and May 2004 and were audio- taped with concomitant note-taking. Informed consent was obtained and participants’ details were anonymised and coded. However, the gender and position of partici- pants means that many would be able to recognise not only their own inputs but also those of fellow members of the VCT taskforce. Wherever possible (given the time delay) results were sent to participants for feedback and ‘participant checking’, thus ensuring quality control. Background h The expansion of voluntary counselling and testing for HIV (VCT) has implications for human resources. There has been increasing global recognition of the need for task shifting (whereby medical tasks are dele- gated to less specialised health care workers) in response Taegtmeyer et al. BMC Public Health 2011, 11:812 http://www.biomedcentral.com/1471-2458/11/812 Taegtmeyer et al. BMC Public Health 2011, 11:812 http://www.biomedcentral.com/1471-2458/11/812 Page 2 of 10 Kenya, like many resource-poor countries, has a chronically under-resourced health care system and a high prevalence of poverty [10]. In the public sector in 2004, there were 3 doctors and 49 nurses per 100 000 population and more than half of all health personnel were based in urban areas [11]. The average health cen- tre has no laboratory staff and a district hospital having an average of only 4 technicians and technologists. Kenya continues to lose large numbers of qualified health care workers, including high numbers of labora- tory staff every year [12]. On the one hand the Govern- ment of Kenya is committed to scale up of access to HIV services, on the other hand donors are unwilling to fund salaries and a recent International Monetary Fund moratorium on hiring of civil servants means that despite increasing workloads, the government is not able to hire additional health professionals [13,14]. attitudes of human resources for VCT scale up [21,22]. Methods which were carried out simultaneously included participant observation, review of minutes of taskforce meetings and individual in-depth interviews with key informants all conducted by the principal investigator (MT). Scale-up occurred across the country and so the whole of Kenya may therefore be seen as the study area. With the exception of a one week retreat in 2001, all taskforce meetings were held in Nairobi and this is where the policymaker interviews were conducted. A total of 53 sets of minutes, representing all of the taskforce meetings held between Sept 2000 and July 2004 were reviewed. Close to 80% (42/53) of these had matching diary notes. Consideration was given to the interaction of the individuals as well as the group pro- cesses and decisions in analysis of diary notes. MT was thus both a participant and an observer [23], and ‘insi- ders’ (co-authors who were members of the VCT task- force - AI, JHN, CN, JS) as well as ‘outsiders’ were included in the qualitative analysis process. Background h Feedback was received from 15 of 22 interviewees (some were no longer contactable), who indicated they were happy with the way their views were identified and por- trayed. For these participants, details of gender and The Kenyan scenario represents the kind of challenges that health policymakers are faced with in many resource poor countries: the need to increase the flex- ibility of health services provision balanced with the need for quality and professionalism. New HIV services are provided in a fast-changing context with wide reach- ing implications for human resources. Kenya has a chronically under-resourced health care system [20]. Staffing implications need to be understood in the con- text of serious shortages, mal-distribution of staff and financing constraints [1]. We set out to explore the complex interactions between the key actors, the con- text they operated in and the processes that evolved as human resources policies for the scale up of HIV testing and counselling (HTC) were developed. Methods Qualitative research methodologies were employed, appropriate to exploring policymakers’ perceptions and Taegtmeyer et al. BMC Public Health 2011, 11:812 http://www.biomedcentral.com/1471-2458/11/812 Page 3 of 10 location are provided with illustrative quotations; for others these details are not included to maintain confidentiality. the actors. This model, or variants of it, continues to be used in resource poor contexts for analysis of health policy [30-32]. In our analysis, too, particular attention was paid to the complex inter-relationships between the actors (forming the centre of the triangle) and the con- text, processes and content. All 22 interviews were included in the analysis. Data analysis commenced during the data collection period allowing emerging issues to be included in subsequent interviews and issues from participant observation to be included in the interview guide. Once complete, the data were analysed using a thematic framework [25], integrating themes from interviews with those from par- ticipant observations and diary notes enabling cross con- nections to be made. Our analysis was informed by conceptual and empirical work by Walt and Gilson [26,27] and Buse [28], who developed a simplified analy- tical model, called the ‘health policy triangle’ (see Figure 1). The model was developed to address the problem that the focus of most policy analysis at the time - in this case related to health reform - was on the content of the policy. Like Reich [29] they believed that policy change is a highly political process and therefore actors were at the centre of the process. In addition, attention should be paid to the processes of developing policy including how the policy issue arises (agenda setting), how decisions are made in the development and finally how the policy is implemented. Finally, all this takes place with a particular context (for example, the particu- lar role of the state, within a specific cultures and the current economic situation), which along with the policy content formed the three apices of the triangle around All study participants provided written informed con- sent and were informed of the study by the VCT task- force at the Kenyan National AIDS and STD Control Programme, which gave explicit permission for meeting minutes to be reviewed. Ethical approval was obtained from the Kenya Medical Research Institute for opera- tional research on the scale up of VCT. Methods This specific study also received institutional approval from the Liver- pool School of Tropical Medicine Research Ethics Com- mittee (ref 03.62). Results Data was synthesized from all the different qualitative methods employed, the results shed light on the difficul- ties faced in instigating task shifting policy. The health policy triangle (see Figure 1) is used to graphically illus- trate and structure the findings. The findings are thus presented against the four areas in the triangle: ‘context’, ‘actors’, ‘process’ and ‘content’. Key actors and their interests in task shifting The VCT taskforce was established with donor fund- ing for initial meetings under MoH leadership in late 2000 and was convened by NASCOP. While the back- bone of membership was MoH officials, donors and their implementing partners, a number of technical Table 1 contains a summary of the key actors and their interests in task shifting. The strategic alliance between donors (who did not support salaries for government staff) and the central Ministry of Health (who were not Table 1 Key actors and their potential impact on task shifting policy Actor Engagement with taskforce Interest regarding scale up and task shifting policy Potential support for lay counsellor testing* Evidence of support or opposition to task shifting Ministry of Health (central) Limited but aware Use existing salary lines: encourage task shifting +++ Agrees to meetings with all players Ministry of Health (NACC) Limited but aware No significant engagement. Supportive if CBOs and NGOs using lay counsellors + National AIDS Strategy sets targets for scale up No significant involvement in the processes Ministry of Health (NASCOP) Convened taskforce in 2001 Host meetings Open as many sites as possible with wide geographic coverage ++ Provides enabling policy environment Open wording to appease all players Accepts variety of financial contributions Ministry of Health (provincial and district level) Occasionally invited to meetings Ensure quality of sites in districts Pragmatic: work within existing resources Maintain good relationships and a motivated workforce ++++ Provides services and input practical advice into policy development Seeks additional donor support Laboratory associations Individuals attend meetings on behalf of other actors (e.g. if working for a donor but also an association member). Context of VCT scale up in Kenya At this point it is impossible for a major redirection of health personnel into VCT.” (Male donor, key informant interview.) “We have had a non-replacement policy within the hospitals and within most of the MoH now for years. At this point it is impossible for a major redirection of health personnel into VCT.” (Male donor, key informant interview.) Context of VCT scale up in Kenya In late 2000 a consultative meeting [19] on VCT set the agenda for scale up, outlined government strategy and reviewed the results of pilot studies conducted in Kenya [33] as well as experience with rapid testing from else- where. Key to the discussion was the availability of new rapid testing technologies. These were easy to conduct by lay people, meaning that specialised laboratory train- ing was not necessary, did not require electricity or external reagents and gave results in 15 minutes, thus enabling the giving of results immediately after testing. In 2001 a national taskforce convened to oversee VCT services in Kenya. From the outset it intended to actively promote task shifting such that the burden of extra work created by HIV counselling and testing was distributed throughout the different categories of staff and maximum benefit was gained from the use of lay counsellors. With this in mind a new combined role, designated ‘VCT counsellor’, was conceived and a uni- fied curriculum for training was published by the National AIDS and STD Control Programme (NAS- COP). This covered counselling skills, rapid HIV testing using whole blood and quality assurance [34]. It was open to health care professionals as well as lay counsellors. Figure 1 Policy analysis of VCT scale up (adapted from Walt and Gilson 1994). Over 2500 VCT counsellors were trained and certified and 900 new sites opened in the following six years. The majority (80%) of these VCT sites were located in Figure 1 Policy analysis of VCT scale up (adapted from Walt and Gilson 1994). Taegtmeyer et al. BMC Public Health 2011, 11:812 http://www.biomedcentral.com/1471-2458/11/812 Page 4 of 10 in a position to hire new staff) was instrumental in encouraging task shifting. government health facilities. At the same time HIV test- ing in clinical settings was increasing in Kenya with antenatal clinics and hospitals incorporating provider- initiated HIV testing and counselling (PITC) as a rou- tine standard of care. Health facilities trying to find ways to provide HIV counselling and testing in addition to their existing clinical services were experiencing sig- nificant new human resource challenges. “We have had a non-replacement policy within the hospitals and within most of the MoH now for years. Key actors and their interests in task shifting Not formally engaged in taskforce until 2003 Maintain quality of tests Maintain professionalism in testing Create jobs for unemployed laboratory technicians and technologists - Engages senior Ministry of Health officials and at times national newspapers to try to prevent task shifting Counselling associations Only one (of two possible) counselling associations engaged in taskforce Recognition for counselling as a profession Maintain quality of counselling Maintain a monopoly on recognised training institutions Continue current curricula on theories of counselling + Disagree with length and focus of training curriculum Approach central Ministry of Health directly to request counselling recognised as a cadre in the Ministry Donors and their implementing partners Provided driving force for taskforce establishment, including funds for secretariat No funding for government salaries: encourage task shifting but varying approaches to incentives for lay counsellors Report high client flow Report high numbers of sites opened +++ Funding to government directly to convene taskforce, collect data etc. Influence through support to implementing partners whose remit is policy change Influence through support to NGO/CBOs +indicates support of lay counsellor testing and - indicates lack of support Table 1 Key actors and their potential impact on task shifting policy +indicates support of lay counsellor testing and - indicates lack of support Taegtmeyer et al. BMC Public Health 2011, 11:812 http://www.biomedcentral.com/1471-2458/11/812 Page 5 of 10 trainees as ‘VCT counsellors’ as distinct from ‘profes- sional counsellors’ the associations shared training curri- cula with the national committee, took part in the process of developing a unified curriculum and encour- aged as many as possible to attend their own or other recognised VCT counsellor training courses, whilst try- ing to maintain prime position in this market. advisors, laboratory and counselling experts with experi- ence in HIV testing and other key stakeholders were invited to attend either from the onset or at various stages over the course of the next few years (seen in the first column of table 1). The inclusion of individuals with experience at the district level allowed human resource lessons, pilot studies and operational research to shape policy development. A number of large international non-governmental organisations and smaller local organizations, funded by donors, were involved in the provision of VCT services or technical support including procurement, logistics, counselling and testing and social marketing. Some were tasked by their donors to work with government staff, whilst others provided independent services. Key actors and their interests in task shifting Donor poli- cies generally restricted direct salary support to govern- ment staff but organisations were able to give technical and quality support. A number of the smaller organisa- tions established mechanisms for donor funds to be rou- ted through NGOs most of whom also had a vested interest in sustaining their own funds. The relationship between donors as taskforce drivers who promoted the scale up of lay counsellors and concerns around sustain- ability were openly debated in Kenya, where donors had high expectations of the speed of scale up and pushed the lay counsellor agenda from early on without formal assessment of longer-term sustainability (see table 1). p p y p Professional associations were also influential, but had a potential to undermine moves towards task shifting. Traditionally, both laboratory and counselling associa- tions have been involved in HIV testing and counselling. Both expressed concerns that scale up and task shifting might compromise quality. The two associations looked at quality from two different angles. Whereas the coun- selling association did not mind lay counsellors conduct- ing the testing, they were concerned over the length and content of the counselling training. On the other hand, the laboratory association were less concerned with this and more opposed to lay counsellors conducting testing. Laboratory operations are regulated by the Kenya Medi- cal Laboratory Technicians and Technologists Board under an Act of Parliament and personnel may join the Association of Kenya Medical Laboratory Scientific Offi- cers [35] that acted as advocates for its members, rather like a union [36]. Initial taskforce meetings agreeing the VCT guidelines included a number of laboratory profes- sionals who were members, but not official representa- tives, of the board and association. Like many cadres of health professionals in Kenya there are a significant number of unemployed laboratory staff, as well as an employment embargo, which according to some was imposed by the International Monetary Fund [37,38]. The association did not see the use of task shifting as being in the best interests of it members, the majority of whom saw HIV testing as the prerogative of laboratory technicians and technologists. They referred to the VCT guidelines, which state that they should conduct testing where available, to reinforce their arguments. Processes in task shifting Th f hi The process of reaching consensus on task shifting suffi- cient for the publication of unified national guidelines and curriculum involved lengthy discussions. The task- force took a number of pragmatic early decisions based on the results of a pilot study [39]. They were: to use health professionals selected from all cadres already on the government pay roll, to come up with a uniform training curriculum and to allow non-laboratory staff to conduct testing. The development of the training manual for VCT counsellors was a contentious issue in the VCT task- force and between counselling organisations, the MoH, international agencies and technical advisors (subcom- mittee minutes 2001). The development of the manual to the satisfaction of all parties with different views took almost two years to complete. There was disagreement on the length of training, counselling organizations wanted longer training, government agreed to a two- week training and some technical and donor agencies pushed for two-day training on testing alone. The last two groups were concerned that time away from service provision for training would exacerbate existing shortages. There was also disagreement on the content of counselling. The professional counsellors were push- ing for a training that was grounded in counselling the- ories leaving them able to conduct fully client-centred “it rightly belongs to the lab people to test.” (Labora- tory technologist, key informant interview.) “it rightly belongs to the lab people to test.” (Labora- tory technologist, key informant interview.) “it rightly belongs to the lab people to test.” (Labora- tory technologist, key informant interview.) The government does not currently recognize the counselling profession as a cadre in the MoH. As well as theory-based university courses in counselling psy- chology there are many registered counselling organisa- tions. Two large associations of professional counsellors undertake generic training to certificate, higher diploma and Master’s level. These organisations saw HIV testing as an opportunity to promote counselling as a profes- sion. They underscored its central role in HIV services and encouraged members to learn how to conduct rapid testing. Once happy with the move to rename the new Taegtmeyer et al. BMC Public Health 2011, 11:812 http://www.biomedcentral.com/1471-2458/11/812 Page 6 of 10 country, the issue was brought to the attention of the central MoH who tried to placate both sides, changing the wording of the guidelines to add ‘or any other per- son authorised by the Minister of Health’. therapeutic sessions. Others argued for an approach that was still client-centred but focused on exploring indivi- dual HIV risk issues and others still wanted a simplified training that taught counselling through providing trai- nees with lists of statements and questions on pre- printed cards that were to be used to guide the sessions. The disparate elements, content, curricula and theories of adult education were brought together in a participa- tory process and a manual was published in 2002. “because the government has supported VCT scale up knowing that we don’t have enough lab people to cope.” (Female programme officer, Nairobi, KII.) At the Annual Conference of Laboratory Scientific Officers in Kakamega in October 2002, data from imple- menting partners VCT sites were presented that con- firmed that lay counsellors who conducted testing were getting a 99.6% level of accuracy according to rigorous external quality assurance [40]. Despite this and due in part to a lack of clear statement from leadership, the dispute continued to rage. Variously described in inter- views as a ’turf war’ a ’threat to professionalism’ and ’a concern over quality standards’, the deliberations over who should conduct testing almost brought VCT scale up to a halt and reached the press in August 2003 with articles raising quality concerns about non-laboratory personnel testing. “it rightly belongs to the lab people to test.” (Labora- tory technologist, key informant interview.) Popular daily national papers includ- ing the Nation, Standard and East African carried almost daily articles on the debate with headings such as ‘Sincerity Lacking in VCTs’, ‘The Big Issue: Panic over HIV testing’; ‘Fix it now, VCT debate getting out of control’ and ‘Government defends VCTs’ (Figure 2). The debate was intended to air the concerns of the Association about the professional erosion of the labora- tory staff but actually gave a platform for donors to sponsor articles explaining quality assurance procedures and stimulated public interest in VCT sites. Acrimonious debates in the taskforce (recurring theme in taskforce minutes Nov 2000 - Sept 2003) dominated the decisions on whether health workers (primarily nurses) or counsellors should provide the counselling and testing. Some committee members argued that it was wrong to divert health workers from clinical duties; others argued that medical diagnoses should remain the prerogative of clinicians and HIV testing the prerogative of laboratory technicians and technologists (diary notes, March 7-9th, 2001; Guidelines Retreat). There has been an evolution over time in response to these concerns. Initially health workers provided VCT services delivered in health facilities, and lay or non-medical counsellors provided services in stand-alone or community sites. By 2005 lay counsellors were employed to work as VCT counsellors in health facilities but government funding for these finished in 2009. By 2011, few remained in place. Instead health workers provide HIV testing and counselling as part of ‘provider-initiated testing and counselling’ (PITC) in health facilities and lay counsel- lors are employed in stand-alone VCT sites, mobile, out- reach and door-to-door HIV testing and counselling programmes. Senior laboratory staff who have been on a training course train and certify VCT counsellors in HIV rapid testing, supervise the VCT counsellors doing testing and support the district medical laboratory in- charges to provide quality assurance mechanisms in VCT sites. In the end, the agreed wording for the guide- lines was: Despite this the conflict was barely noticed outside Nairobi. In fact some of the district and provincial inter- viewees thought the bad publicity may have stimulated Figure 2 Sample VCT newspaper headlines August and September 2003. “All HIV testing for VCT should be done by labora- tory technologists or technicians. However, in some locations and settings this is not possible. “it rightly belongs to the lab people to test.” (Labora- tory technologist, key informant interview.) If they have successfully completed an accredited training in test- ing procedures and are supervised by a laboratory technologist/technician, other health workers may perform simple rapid tests for VCT purposes.” Practically however, only a proportion of testing was conducted by laboratory personnel. A few large stand- alone sites were able to hire one technician or technolo- gist, but most sites, including health facilities, were con- ducting in-room testing by health care workers (participant observation, diary notes Nov 21st 2001). Some time after VCT was well established in the Figure 2 Sample VCT newspaper headlines August and September 2003. Figure 2 Sample VCT newspaper headlines August and September 2003. Taegtmeyer et al. BMC Public Health 2011, 11:812 http://www.biomedcentral.com/1471-2458/11/812 Page 7 of 10 interest in VCT. Most had good working relationships between the routine laboratory staff and VCT. understand why someone would serve only 8 or 15 cli- ents. Those who tried to perform both roles were fru- strated and overworked. This resulted in internal conflict or frustrated clients as VCT service opening times became irregular in order to fit round other work expectations. “At national level there has been a lot of politics but in our area so far no problem.” (District Medical Officer of Health, KII.) Subsequently the lack of promotional structures and of recognition for counselling as a cadre threatened the task shifting policies as far as the use of other health professionals was concerned and increasingly opened the door for lay counsellors to provide services in health facilities. The laboratory members interviewed would have wel- comed their inclusion in planning, in study design to evaluate quality, and in early stages of roll-out. The omission of the official representatives of the profes- sional association and senior laboratory personnel work- ing with the government was felt to be an error. However, there were still those who felt that an early official lab presence may have inhibited scale up in the public sector. On the other hand, policies around quality assurance were integral to the scale-up strategy in Kenya [18] and were central in guiding how to task shift to lay counsel- lors, many of whom were volunteers. The guidelines and training curriculum became a key element of the subsequent site registration and quality assurance sys- tems. “VCT are manned from all directions. Some are pub- lic health technicians, some are nurses etc.” (Male, programme officer, KII.) There was an awareness that similar HIV counselling programmes had failed in the past due to the impact of dual roles (doing counselling as well as nursing) on pro- vider stress, high attrition rates and inconsistent service delivery. This led to deliberate policies on the preven- tion of counsellor burn out through a policy of manda- tory attendance at support supervision sessions. Furthermore lay personnel and ‘volunteer counsellors’ were encouraged to apply for training positions and to provide services with NGO support. Despite efforts to mitigate the impact of task shifting on strained resources, concerns arose not only about the lack of deliberate policy but also about the lack of clear line management structures that ensued. Staff reported to - and were evaluated by - their former bosses in their main discipline, many of whom were not versed in the expectations or job descriptions for VCT counsellors. This created tensions. For example, counsellors were told to see a max of 8-15 clients per day. A departmen- tal head who had over 50 patients waiting to be served in another department by one nurse/midwife could not The registration system then formed the basis of a comprehensive quality assurance programme that included supervision of counselling and testing, continu- ing education, laboratory-based testing using dried blood spots and inspection of sites. In late 2003 a National Quality Assurance Team was established, including laboratory supervisory staff, who visited all sites annually. The team found high standards of testing in registered sites [40] and reported high client confi- dence in the approach used. “It is important for the client and the counsellor to do their thing together and for the client to watch. Then clients have absolute confidence in their results.” (Male, laboratory, KII.) “it rightly belongs to the lab people to test.” (Labora- tory technologist, key informant interview.) Within a year of the establishment of the task- force, the guidelines had been disseminated throughout Kenya and an enforced registration system meant that all VCT sites should be operating under the same stan- dards (Report on piloting of registration system VCT taskforce minutes Nov 7th 2001). Only registered sites could access the free HIV test kits funded by a World Bank loan [41] in exchange for data on client uptake. A national data form was developed and registered sites completed a form for each individual. “If they were on the taskforce earlier it might have affected scale up. Depending on how strong they were or how forceful of their point of view it could have blocked expansion.” (Female, donor, KII.) Content of task shifting The initial strategies for VCT scale up did not make task shifting policies for VCT explicit. Assumptions were made that health care workers, taken from a num- ber of different cadres, would perform dual roles. “The government took a loan from the World Bank for the purchase of test kits. It helped us. Free kits probably accelerated the process and allowed us to control it.” (Male MoH taskforce member, KII.) “VCT are manned from all directions. Some are pub- lic health technicians, some are nurses etc.” (Male, programme officer, KII.) Discussion Our findings indicate that programmers have to deal with more than the content (the technical components) Taegtmeyer et al. BMC Public Health 2011, 11:812 http://www.biomedcentral.com/1471-2458/11/812 Page 8 of 10 of a policy in order to get it successfully implemented. A qualitative approach has revealed multiple complex layers as individuals and groups with their own personal and political histories and interactions all play a role. Our methodology and our policy analysis, that was not restricted to the policy content, but included all four components of the policy triangle, have enabled an in- depth understanding of the human resource implica- tions of VCT scale up from the perspective of the full breadth of task force members, including representative, experienced service providers. First we need to be clear about the actors and their interests (which may or may not be supportive). Next we need to understand the context as this might influence the interests of the actors (for example an employment embargo heighten- ing the opposition of the lab staff to task-shifting). Finally, with a better understanding of the actors and the context and the potential risks involved, the content and the process can be better planned. These findings have resonance for the provision of new services, such as new rapid diagnostic tests or new care pathways in many settings. indicated that the success of task shifting in Kenya was in part due to the quality assurance systems for HIV testing. While the introduction of task shifting for HIV counselling and testing in Kenya has enabled rapid scale up of services, it was the flexibility around who conducts testing that allowed expansion into non-medical areas, outreach services and non-government facilities. This flexibility was possible through the scale up of one uni- fied regulatory and quality assurance system [18]. Qual- ity assurance systems for VCT scale up in Kenya are thus both part of the context (as they were planned as part of scale up from the outset) and the content as policies evolved and new systems piloted as scale up progressed. p g In 2005 Kenyan unified guidelines broadened the scope of HIV testing further to include all health cadres with minimal training [43]. Here, even more so than in VCT, rapid expansion has threatened the quality of ser- vices, both in Kenya and globally [44,45]. Discussion External qual- ity assurance systems for testing and counselling are not maintained in provider-initiated HIV testing and coun- selling to the same standard as those in VCT sites. Test- ing sites do not conduct proficiency panels and do not send dried blood spots from rapid tests to reference laboratories (personal communication, NASCOP, 2008). Indeed there is no national picture of where testing is conducted, who is doing the testing and what the qual- ity of ward and clinic-based testing is. It has also become the expectation that every health care worker will do counselling in every clinical setting. While this has increased the rates of HIV testing and counselling, it has also led to missed opportunities for prevention messages and created a coercive atmosphere around testing in some settings [44,46]. Involving lay counsel- lors in busy clinics conducting HTC has been a success- ful approach in Botswana [47] and may yet have a role to play in PITC scale up with a renewed focus on uni- versal access to HTC. Issues of sustainability, quality assurance of testing and the use of lay counsellors are still debated and local actors, processes and content are vital components in understanding how these opportu- nities can be maximised in different contexts. y g Despite staffing shortages, medical training colleges in many resource-poor countries continue to produce health care workers, without significant overlapping skills, who strive to maintain high standards. These health care workers are understandably protective of professional boundaries and this protectionism has the potential to threaten or even deliberately sabotage the move to task shifting. The advent of a new testing tech- nology which could be performed without extensive laboratory training clearly raised concerns among laboratory workers Kenya. The VCT taskforce com- pounded this by failing to engage adequately with all the officially recognized groups (Board and Association) of laboratory seniors. A better understanding of the pre- ceding political conflicts between the Association and the Board as well as of the positions and legitimate con- cerns of the members would have allowed a smoother path to scale up. Some members’ concerns may have been rooted in their training. Discussion As a result, despite docu- mented high standards of lay counsellors conducting testing [40], the professional associations diverted time and attention away from supervision and the establish- ment of comprehensive quality assurance systems for HIV testing in all settings (including provider-initiated HIV testing and counselling) to do battle with VCT. Time has shown that their concerns over quality of test- ing are justified in the absence of comprehensive and funded systems of training, certification, data recording and external retesting of samples. Author details 1Cli i l G 15. National Council for Population and Development: Demographic Health Survey Maryland: Marco International Inc 1999; 1998. 1Clinical Group, Liverpool School of Tropical Medicine, Liverpool, UK. 2International Health Group, Liverpool School of Tropical Medicine, Liverpool, UK. 3Family Health International, Nairobi, Kenya. 4Counselling Department, Liverpool VCT, Treatment & Care, Nairobi, Kenya. 5Quality Assurance Coordinator, National AIDS and STD Control Programme, Nairobi, Kenya. 6Elected Chair, Association of Kenya Medical Laboratory Scientific Officers, Nairobi, Kenya. y y 16. Kenya: Ministry of Planning and National Development: Demographic Health Survey 2003 Nairobi Central Bureau of Statistics; 2004. Survey 2003 Nairobi Central Bureau of Statistics; 2004. 17. Kenya Ministry of Health: AIDS in Kenya: trends, interventions 17. Kenya Ministry of Health: AIDS in Kenya: trends, interventions and impact Nairobi: National AIDS and STD Control Programme; 2005. Nairobi: National AIDS and STD Control Programme; 2005. 18. Marum E, Taegtmeyer M, Chebet K: Scale-up of v and testing in Kenya. JAMA 2006, 296:859-62. 18. Marum E, Taegtmeyer M, Chebet K: Scale-up of voluntary HIV counselin and testing in Kenya. JAMA 2006, 296:859-62. 19. Proceedings of the Consultative Technical Meeting on HIV Voluntary Counselling and Testing VCT. [http://www.fhi.org/en/hivaids/pub/archive/ confrpts/pro_consult_tech_meeting_hiv_vct.htm], accessed on July 4th 2011. Acknowledgements Th h ld lik 10. World Health Organisation: Country profile: Kenya [document on the Internet]. Geneva: WHO; 2006 [http://www.who.int/hac/crises/ken/ Kenya_may06.pdf], [cited 2011 Jan 12].. The authors would like to thank all interview participants as well as all members of the VCT taskforce, the Kenya Medical Laboratory Technicians and Technologists Board, the Association of Kenya Medical Laboratory Scientific Officers, the Kenyan Association of Professional Counsellors and the staff of Liverpool VCT Treatment and Care, Kenya. All gave of their time and insights whether directly or indirectly. Important feedback from interview participants was incorporated in the paper and many read and critically appraised earlier drafts of this document. The authors wish to thank them all for very helpful comments. While this study did not receive direct donor funding the programmes it describes were funded by numerous donors including USAID, The US Centers for Diseases Control and the UK’s Department for International Development (DfID). Kenya_may06.pdf], [cited 2011 Jan 12].. 11. Republic of Kenya Ministry of Health: Report on Human Resources mapping and Verification Exercise. Nairobi; 2005. 12. Chankova S, Muchiri S, Kombe G: Health workforce attrition in the public sector in Kenya: a look at the reasons. Human Resources for Health 2009, 7:58. 13. International Monetary Fund: Country report: Kenya poverty reduction strategy paper [document on the Internet] Washington DC: IMF; 2005 [http://www. imf.org/external/pubs/ft/scr/2005/cr0511.pdf], [cited 2011 April 5].. 14. Ambrose S: Preserving disorder: IMF policies and Kenya’s health care crisis Pambazuka 2006, 257[http://pambazuka.org/en/category/features/34800], News[cited 8th April 2011]. Conclusion Our findings are still relevant today as new approaches to HTC are scaled up within health systems and staff struggle to balance the human resources requirements with the need to maintain quality of service and labora- tory and counselling professionalism. Our analysis shows that the use of lay counsellors and health care workers from a number of cadres to conduct counsel- ling as well as testing in Kenya served to strengthen rather than undermine primary care without diverting significant resources from laboratories or depleting any In keeping with findings from Zambia and Cameroon we highlight the importance of clearly re-defining roles and task-shifting procedures [6,42]. Our data also Taegtmeyer et al. BMC Public Health 2011, 11:812 http://www.biomedcentral.com/1471-2458/11/812 Page 9 of 10 access to antiretroviral therapy in Thyolo, Malawi through task shifting and decentralization of HIV/AIDS care. Trop Med Int Health 2010, 12:1413-20. one cadre. Concentrating only on the content or techni- cal side of programme scale up is likely to fail, whatever the approach to HTC scale up that is being used. A cri- tical and informed analysis of context and actors and the prioritisation of quality assurance are central to the successful scale up of HIV services. 8. Brentlinger PE, Assan A, Mudender F, Ghee AE, Vallejo Torres J, Martínez Martínez P, Bacon O, Bastos R, Manuel R, Ramirez Li L, McKinney C, Nelson LJ: Task shifting in Mozambique: cross-sectional evaluation of non-physician clinicians’ performance in HIV/AIDS care. Human Resources for Health 2010, 8:23. 9. Fulton BD, Scheffler RM, Sparkes SP, Auh EY, Vujicic M, Soucat A: Health workforce skill mix and task shifting in low income countries: a review of recent evidence. Human Resources for Health 2011, 9(1):1. Authors’ contributions MT conceived and designed the study, conducted the interviews, analysed the data and wrote the paper. ST designed the study, reviewed data analysis and critically appraised the document. DGL contributed to the analysis and read and critically reviewed the document. AI, JHN, CW and J were intimately involved in the scale up of VCT that the paper describes and read and critically appraised the document. TM contributed to the writing of the paper and read and critically appraised later versions. All authors have read and approved the final version of this manuscript. 20. World Health Organisation: Country profile: Kenya [document on the Internet] Geneva: WHO; 2006 [http://www.who.int/hac/crises/ken/Kenya_may06.pdf], [cited 2007 Jan 12].. 21. Brewer J, Hunter A: Foundations of multimethod research. Thousand Oaks: Sage Publications; 2006. 22. Silverman D: Interpreting qualitative data. London: Sage Publications; 2006. Conflict of interests Th h d l h 23. Elliott J: Using narrative in social research: qualitative and quantitative approaches. London: Sage Publications; 2005. The authors declare that they have no competing interests. The authors declare that they have no competing interests. 24. Ritchie J, Lewis J, Elam G: Designing and selecting samples. In Qualitative research practice: a guide for social science students and researchers. Edited by: Ritchie J, Lewis J. London: Sage Publications; 2003:77-108. Received: 15 April 2011 Accepted: 18 October 2011 Published: 18 October 2011 Received: 15 April 2011 Accepted: 18 October 2011 Published: 18 October 2011 Received: 15 April 2011 Accepted: 18 October 2011 Published: 18 October 2011 25. Pope C, Ziebland S, Mays N: Qualitative research in health care. Analysing qualitative data. BMJ 2000, 320:114-6. References 1. Callaghan M, Ford N, Schneider H: A systematic review of task-shifting for HIV treatment and care in Africa. Human Resources for Health 2010, 8(1):8. 1. Callaghan M, Ford N, Schneider H: A systematic review of task-shifting for HIV treatment and care in Africa. Human Resources for Health 2010, 8(1):8. 26. Walt G, Gilson L: Reforming the health sector in developing countries: the central role of policy analysis. 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Working Paper 10 Harvard School of Public Health, Boston; 1994. 5. Makombe SD, Jahn A, Tweya H, Chuka S, Yu JK, Hochgesang M, Aberle- Grasse J, Pasulani O, Schouten EJ, Kamoto K, Harries AD: A national survey of the impact of rapid scale-up of antiretroviral therapy on health-care workers in Malawi: effects on human resources and survival. Bull World Health Organ 2007, 85:851-7. 30. Pearson M, Zwi AB, Buckley N: Prospective policy analysis: how an epistemic community informed policymaking on intentional self poisoning in Sri Lanka Health Research Policy and Systems. 2010, 8:19. 31. Buse K, Lalji N, Mayhew SH, Imran M, Hawkes SJ: Political feasibility of scaling-up five evidence-informed HIV interventions in Pakistan: a policy analysis. Sex Transm Infect 2009, 85(Suppl_2):ii37-ii42. 6. Sanjana P, Torpey K, Schwarzwalder A, Simumba C, Kasonde P, Nyirenda L, Kapanda P, Kakungu-Simpungwe M, Kabaso M, Thompson C: Task-shifting HIV counselling and testing services in Zambia: the role of lay counsellors. Hum Resour Health 2009, 7:44. 6. Taegtmeyer et al. BMC Public Health 2011, 11:812 http://www.biomedcentral.com/1471-2458/11/812 Njeru MK, Blystad A, Shayo EH, Nyamongo IK, Fylkesnes K: Practicing provider-initiated HIV testing in high prevalence settings: consent concerns and missed preventive opportunities. BMC Health Serv Res 2011, 11:87. 45. Evans C, Ndirangu E: The nursing implications of routine provider- initiated HIV testing and counselling in sub-Saharan Africa: a critical review of new policy guidance from WHO/UNAIDS. Int J Nurs Stud 2009, 46(5):723-31. 46. Hardon A, Kageha E, Kinsman J, Kyaddondo D, Wanyenze R, Obermeyer CM: Dynamics of care, situations of choice: HIV tests in times of ART. Med Anthropol 2011, 30(2):183-201. 46. 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https://openalex.org/W4313426616	https://journals.ajsrp.com/index.php/jctm/article/download/6029/5751	Arabic	null	أثر استخدام استراتيجية التعليم المتمايز في تنمية المهارات النحوية في مقرر اللغة العربية لدى طلبة الأقسام العلمية بكلية التربية – صنعاء	Maǧallaẗ al-manāhiǧ wa-ṭurūq al-tadrīs	2,022	cc-by	20,835	Journal of Curriculum and Teaching Methodology Volume (1), Issue (16): 30 Dec 2022 P: 91 - 121 ISSN: 2790-7333 مجلة املناهج وطرق التدريس ( املجلد1 ،) ( العدد16 ) : 30 ديسمبر2022م ص : 91 - 121 Journal of Curriculum and Teaching Methodology Volume (1), Issue (16): 30 Dec 2022 P: 91 - 121 ISSN: 2790-7333 مجلة املناهج وطرق التدريس ( املجلد1 ،) ( العدد16 ) : 30 ديسمبر2022م ص : 91 - 121 ISSN: 2790-7333 مجلة املناهج وطرق التدريس ( املجلد1 ،) ( العدد16 ) : 30 ديسمبر2022م ص : 91 - 121 Journal of Curriculum and Teaching Methodology Volume (1), Issue (16): 30 Dec 2022 P: 91 - 121 Journal of Curriculum and Teaching Methodology Volume (1), Issue (16): 30 Dec 2022 P: 91 - 121 The effect of using the differentiated education strategy in developing grammatical skills in the Arabic language course among students of the scientific departments of the College of Education- Sana’a إبراهيم إسماعيل عباس الجعدي كلية التربية\|\| جامعة صنعاء\|\| اليمن املستخلص :هدفت الدراسة إلى معرفة أثر استخدام استراتيجية التعليم املتمايز في تنمية املهارات النحوية في مقرر اللغة العربية لدى طلبة األقسام العلمية بكلي ة التربية– صنعاء، معتمدة على املنهجين (الوصفي وشبه التجريبي)، ولتحقيق هدف الدراسة، أعدّ الباحث قائمة باملهارات النحوية، وبرنامجً ا تعليميًا مصممً ا وفق استراتيجية التعليم املتمايز، واختبارًا تحصيليًا، وتكونت عينة الدراسة ا مليدانية ( من41) طالبًا وطالبة من ط( لبة األقسام العلمية مستوى أول املسجلين بقسمي (الرياضيات وعلوم الحياة) في العام الجامعي2021 م– 2022 ) ، مثلوا مجموعة تجريبية واحدة، تم تدريسهم املهارات النحوية املقررة عليهم في كتاب (اللغة العربية102 ) باستخدام استراتيجية التعليم املتمايز، و أسفرت نتائج الدر( اسة عن وجود فروق دالة إحصائيًا عند مستوى الداللة0.05 ) بين متوسطي درجات املجموعة التجريبية في التطبيقين (القبلي والبعدي) الختبار املهارات النحوية على مستوى االختبار ككل وعلى مستوى كل مهارة مستقلة لصالح التطبيق البعدي، يُعزى إلى تدريس املهارات النحوية باست راتيجية التعليم املتمايز ، وأوص ى الباحث بتضمين استراتيجية التعليم املتمايز .ضمن طرائق التدريس الحديثة لتدريسها في كليات التربية الكلمات املفتاحية:استراتيجية–التعليم املتمايز–املهارات النحوية //www.ajsrp.com : https : Available at //www.ajsrp.com : https : Available at DOI: https://doi.org/10.26389/AJSRP.D150822 ( 91 ) //www.ajsrp.com : https : Available at Faculty of Education \|\| Sana'a University \|\| Yemen Abstract: The study aimed to know the effect of using the differentiated education strategy in developing grammatical skills in the Arabic language course among students of the scientific departments of the College of Education- Sana’a، based on the two approaches (descriptive and quasi-experimental). differentiated education، achievement test، The sample of the field study consisted of (41) male and female students from the first-level scientific departments who were registered in the two departments (Mathematics and Life Sciences) in the academic year (2021 AD- 2022 AD)، they represented one experimental group who were taught the grammatical skills prescribed to them in the book (Arabic Language 102) using the strategy differentiated education، The results of the study revealed that there were statistically significant differences at the significance level (0.05) between the mean scores of the experimental group in the two applications (pre and post) to test the grammatical skills at the test level as a whole and at the level of each independent skill in favor of the post application، attributed to the teaching of grammatical skills using the differentiated education strategy. The researcher recommended the inclusion of a differentiated education strategy within modern teaching methods to be taught in colleges of education. Keywords: Strategy- differentiated education- grammatical skills. أثر استخدام استراتيجية التعليم املتمايز في تنمية املهارات النحوية في مقرر اللغة العربية لدى طل بة األقسام العلمية بكلية التربية– صنعاء املقدمة. م إن هللا شرف اللغة العربية على سائر اللغات بأن جعلها لغة القرآن الكريم املُعجز املُتَعبَّد بتالوته قال تعالى : ِ{وَإِنَّهُ لَتَنْزِيلُ رَبِّ الْعَاملَِينَ ، نَزَلَ بِهِ الرُّوحُ األْ َمِينُ ، عَلَ ى قَلْبِكَ لِتَكُونَ مِنَ املْ ُنْذِ رِينَ ، ب} ٍلِسَ انٍ عَرَبِيٍّ مُ بِين الشعراء [ 192 - 195 ]، ف اللغة العربية من أهم الدعا ُئم األساسية التي تقوم عليها الحياة العربية اإلسالمية؛ فهي ت ميز اإلنسان عن ،غيره من سائر املخلوقات و هي قوام الحياة، ومطلب أساس ي للفرد، فضالً عن أنها سجل لتراث الشعوب وثقافتهم .وحضارتهم ولهذا فإن تعليم اللغة العربية منذ بداية املراحل الدراسية يهدف إلى تمكي ن الدارس من مهاراتها، عن طريق تزويده باملهارات األساسية ومساعدته على اكتساب ممارساتها الصحيحة، والتدرج في تنميتها على امتداد املراحل التعليمية، بحيث يصل الطالب في نهاية هذه املرحلة إلى مستوى لُغوي يمكنه من استخدام اللغة بشكل يساعده على مواصلة الدراسة في امل،راحل التعليمية التالية (الناقة، شي العيد2009 ،م4 .) ويُعد النحو أهم فروع اللغة العربية؛ الرتباطه بتقويم اللسان من الخطأ عند الحديث والقراءة، وتقويم القلم من الخطأ عند الكتابة، فهو مرتبط بصحة الجملة التي تُعد األساس لسالمة اللغة العربية، ومن شأنه الحفاظ عل يها من االضمحالل، ويُعِدُّ دارسِ يه على أُسس علمية تمكنهم من الحفاظ على لغتهم نطقً ا وكتابة، وتظهر أهمية النحو بصورة أوضح عندما نعرف األسباب التي وُضع من أجلها كعلم له قواعد والتي من أهمها الباعث الديني الذي يهدف إلى أداء نصوص القرآن الكريم والحديث أداءً سلي مًا بعد شيوع اللحن على ألسنة الناس، وملّا كان للنحو هذه األهمية بين فروع اللغة العربية تأتي أهمية دراسته في املناهج التعليمية؛ ملا له من أثر كبير في تصحيح أخطاء اللسان، فهو يعين الدارسين على الحديث والكتابة بلغة صحيحة وسليمة، ويمكنهم من استخدام اللغة استخد امًا ،سليمً ا في يُسر ومهارة في املواقف اللغوية املختلفة (الحبيش ي2008 ،م1 .) وتُعد منزلة النحو من العلوم األخرى بمثابة الدستور من القوانين الحديثة، فهو مَ ثَلها الذي تعود إليه في جميع مسائلها، فال نجد علمً ا من هذه العلوم مستقالً عن النحو، أو يسترشد بغير نوره و هداه، والنحو علم يُعرف به .حقائق املعاني إن القواعد النحوية تنمي العديد من مهارات اللغة وتزداد أهميتها لتقاطعها مع املهارات اللغوية األربع؛ لذا ُي عدُّ مقرر اللغة العربية عنصرًا رئيسً ا في منظومة االتِّصال اللغَويّ الفاعل، فبدون إتقان مهاراته ال يمكن أن يكون ٌهناك فهْمٌ وإفهامٌ سليم ، فبوّابة النّجاح اللغويّ تمرُّ عبر مراعاةِ قوانين املهارات النحوية األساسية، وذلك من خالل إدراكِ أوجه العالقات بين الجُ مَ ل، والقدرةِ على ضبْط الكلمات وإعرابها بصورةٍ تقود إلى سالمة اللغة، وصحّ ة األساليب املستخدمة، وتقويم اللسان من ٍالزّلل، األمر الذي يؤدّي إلى فهْم املعاني، وإفهامها لآلخرين بكل كفاءة .ٍواقتدار لذا احتلّت املهارات النحوية مكانةً متميزةً لدى القائمين على برامج اإلعداد اللغوي في املرحلة الجامعيّة؛ حيثُ جاء على رأس أولويَّات أهداف تعليم النَّحو :تدريب الطلبة على مهارات التّفكير النَّحويّ بما تشتمل عليه من تمييز، وفهم، وتحليل، وتركيب، وإصدار أحكام، والقدرة على استخدام مهاراته بتمكّ نٍ وفاعليّة و اقتدار (عبد الباري : 212، ص5). DOI: https://doi.org/10.26389/AJSRP.D150822 جلة العربية للعلوم ونشر األبحاث ــ مجلة المناهج وطرق التدريس ــ المجلد األول ــ العدد ال سادس عشر ــ ديسمبر 2022م المجلة العربية للعلوم ونشر األبحاث ــ مجلة المناهج وطرق التدريس ــ المجلد األول ــ العدد ال سادس عشر ــ ديسمبر 2022 م املقدمة. و لقد أشار العديد من التربويين إلى أن السبب الرئيس في الضعف ال نحوي يعود إلى فشل طرائق التدريس التقليدية في معالجة ضعف الطلبة في النحو وشيوع األخطاء بينهم، إذ أخفقت في تحقيق أهدافها، وغرست روح كراهية القواعد النحوية الجامدة في نفوس الطلبة؛ ملعاناتهم في استظهارها، مما أدى إلى ظهور اتجاهات جديدة تدعو إلى تطبيق طرائق واس تراتيجيات تدريس حديثة (العربي، والعقيلي : 1986 ،م24 .) و لقد أشار العديد من التربويين إلى أن السبب الرئيس في الضعف ال نحوي يعود إلى فشل طرائق التدريس التقليدية في معالجة ضعف الطلبة في النحو وشيوع األخطاء بينهم، إذ أخفقت في تحقيق أهدافها، وغرست روح كراهية القواعد النحوية الجامدة في نفوس الطلبة؛ ملعاناتهم في استظهارها، مما أدى إلى ظهور اتجاهات جديدة تدعو إلى تطبيق طرائق واس تراتيجيات تدريس حديثة (العربي، والعقيلي : 1986 ،م24 .) والتعليم الجامعي هو مرحلة التخصص واإلعداد العملي في كافة مستويات حاجات املجتمع املختلفة حاضره ومستقبله بما يساير التطور في العصر الحديث (كامل : 2005م، ص136 ) ، ويتطلب ضمان نوعية التعليم الجامعي بناء برامج ت ًعليمية في ضوء احتياجات الطلبة وقدراتهم وإمكانياتهم العقلية والنفسية سواء كانوا متفوقين وموهوبين أم بطيئ ي .التعلم والتعليم الجامعي هو مرحلة التخصص واإلعداد العملي في كافة مستويات حاجات املجتمع املختلفة حاضره ومستقبله بما يساير التطور في العصر الحديث (كامل : 2005م، ص136 ) ، ويتطلب ضمان نوعية التعليم الجامعي بناء برامج ت ًعليمية في ضوء احتياجات الطلبة وقدراتهم وإمكانياتهم العقلية والنفسية سواء كانوا متفوقين وموهوبين أم بطيئ ي .التعلم وبناءً على ّما سبق فال بد من تنوع استراتيجيات التدريس الحديثة تبعًا لتغي ر النظرة إلى طبيعة عملية التعليم، فبعد أن كانت تعتمد على الحفظ والتسمي ع اتسعت لتشمل املستويات اإلدراكية املعرفية، مما يتطلب إيجابية املتعلم في التعليم؛ بهدف إظهار قدرات الطلبة الكامنة واالرتقاء بها، ولم تعد األساليب التقليدية في التدريس ،تالئم الحياة املعاصرة وبالتالي ظهرت طرائق حديثة عملت على تغيير النظرة من املتعلم إلى علم النحو وتحبيبه إليه ،وإكسابه مهاراته ومن ها استراتيجية التعليم املتمايز التي تساعد على اكتساب العديد من املهارات العقلية واالجتماعية والحركية، وتتمثل مهمة املعلم في إتاحة الفرصة للمتعلمين لتحصيل املعرفة بأنفسهم، واملشاركة بفاعلية في كافة أنشطة التعليم، واإل.قبال على ذلك برغبة ونشاط حتى يعتادوا االستقالل في الفكر والعمل واالعتماد على الذات و ًالتدريس املتمايز يأخذ أشكاال ً عدة، فقد يكون التدريس وفق ا لنظرية الذكاءات املتعددة، ًوقد يكون وفق ا ألنماط املتعلمين، فمنهم : السمعي، والبصري، والحركي، والحس ي، واللفظي، وقد يأخذ شكل التعلم التعاوني إذا ارتأى املعلم تنظيم املهام وتوزيعها وفق اهتمامات املتعلمين. املقدمة. َ وتزدادُ أهمِّيَّة املهارات النحوية لدى طلبة املرحلة الجامعيّة، الذين وصلوا إلى مستوى عقليّ يسمح لهم ٍبإتقان هذه املهارات وتوظيفها ألولئكَ امللتحقين بكليات جامعة صنعاء، الذين يُؤمَّل منهم أن يضطلعوا بأدوار مستقبليةٍ رئيسةٍ في تيسير تعليمها وتعلّمها (عبده : 2009م، ص5) . ( 92 ) جلة العربية للعلوم ونشر األبحاث ــ مجلة المناهج وطرق التدريس ــ المجلد األول ــ العدد ال سادس عشر ــ ديسمبر 2022م المجلة العربية للعلوم ونشر األبحاث ــ مجلة المناهج وطرق التدريس ــ المجلد األول ــ العدد ال سادس عشر ــ ديسمبر 2022 م وعلى الطلبة أن يكرسوا جهودهم لتحقيق أعلى مستويات اإلنجاز األكاديمي، ويبذلوا جهودهم وإبد اعاتهم ّوصوالً إلى مستوى التمك ،ن واإلتقان، شريطة أن يتوفر في كل نظام تعليمي خبرات وقيادات تعليمية رفيعة ومخلصة وإداريون أكفاء، وإمكانات مادية مناسبة، وبيئة ثرية للعمل يسودها روح الفريق املتعاون واملتسامح، واملصلحة العامة "فوق كل اعتبار (شحاتة، والنجار : 2003م، ص5) . وعلى الرغم من تطوير مناهج النحو في الوطن العربي بالتركيز على املفاهيم النحوية الوظيفية لتخليص القواعد النحوية من األغالل والقيود والعلل والخالفات وتعدد اآلراء، إالّ أن مالمحَ الواقع الذي أثبتته الدراسات ُوالبحوث السابقة ت شير إلى أنّ ثمةّ ضعفً ا وا ضحً ا في املهارات النحوية وال سيما لدى طلبة الجامعات، ويدلُّ على هذا ًالضَّعْفِ كثرة أخطائهم النحوية تحدُّ ث .ا وكتابةً، بصورة تقلّلُ من قدرتهم على فهم املعاني وإفهامها لآلخرين (عافش ي : 1997م، ص9). املقدمة. وال يقتصر التمايز في التدريس على مجال واحد، فقد يتمايز التدريس في األهداف، واألساليب واالستراتيجيات، ومصادر التعلم، واملخرجات، كأن يكتفي بمخرجات محدودة يحققها بعض الطلب ة، ًفي حين يطلب من آخرين مخرجات أكثر عمق.ا (عبيدات وأبو السميد : 2007 ) و أكدت العديد من الدراسات على أهمية توظيف استراتيجية التعليم املتمايز في العملية التعليمية كدراسة (أحمد : 2019 ) التي هدفت إلى معرفة فاعلية برنامج قائم على مدخل التعليم املتمايز لتنمية مهار ات القراءة، ودراسة (أبو شباب : 2019 ) التي هدفت إلى التعرف على فاعلية برنامج تدريبي مستند إلى التعليم املتمايز في تحسين مستوى التحصيل في مادة الرياضيات ،وأظهرت دراسة (عمار حسن : 2016 )، ودراسة (النبهان والكنعاني : 2016 ) ، ودراسة (األحمد والجهيمي : 2015) فاعلية ال تعليم املتمايز في العملية التعليمية ، وهذا ما دفع الباحث إلى بناء برنامج تعليمي .مصمّ م وفق استراتيجية التعليم املتمايز في تنمية املهارات النحوية لدى طلبة األقسام العلمية بكلية التربية صنعاء أثر استخدا م استراتيجية التعليم المتمايز في تنمية المهارات النحوية في مقرر اللغة العربية لدى طلبة األقسام العلمية ( 93 ) الجعدي جلة العربية للعلوم ونشر األبحاث ــ مجلة المناهج وطرق التدريس ــ المجلد األول ــ العدد ال سادس عشر ــ ديسمبر 2022م مشكلة الدراسة : وفي ضوء ما أشارت إليه نتائج مجمل الدِّ راسات والبحوث إلى َّأن ثمَّ ة ًا ضعْف ًا بيِّن في مهارات النحو لدى طلبة الجامعات؛ كالضعف في تحديد العالقة بين مكوِّنات التركيب ،اللغوي وتعليل ضبط بعض ،الكلمات والتمييز بين املوقع اإلعرابي والحكم ،اإلعرابي وتوظيف الكلمة داخل التركيب ّالل غوي ٍبشكل ، ٍصحيح وتحليل التّركيب الل غوي إلى عناصره ،الرئيسة وإعادة ،بنائه وتصويب األخطاء .النَّحوية وفي ضوء ذلك يمكن صياغة مشكلة الدراسة في السؤال الرئيس اآلتي: ما أثر استخدام استراتيجية التعليم املتمايز في تنمية املهارات النحوية في مقرر اللغة العربية لدى طلبة األقسام العلمية بكلية التربية– صنعاء؟. ويتفرع منه األسئلة اآلتية : 1- امل ما هارات النحوية الالزمة ل طلبة األقسام العلمية بكلية التربية– صنعاء؟ 2- ما صورة برنامج قائم على استراتيجية التعليم املتمايز في تنمية املهارات النحوية لدى طلبة األقسام العلمية بكلية التربية– صنعاء؟ 3- ما أثر تدريس البرنا مج القائم على استراتيجية التعليم املتمايز في تنمية املهارات النحوية لدى طلبة األقسام العلمية بكلية التربية– صنعاء؟ العلمية بكلية التربية– صنعاء؟ فرضيات الدراسة : ولإلجابة عن أسئلة الدراسة تم صياغة فرضي ة الدراسة اآلتية : - توجد فروق دالة إحصائيًا عند مستوى الداللة ( 0.05 )بين متوسطي درجات املجموعة التجريبية في التطبيقين (القبلي والبعدي )الختبار املهارات النحوية على مستوى االختبار ككل وعلى مستوى كل مهارة مستقلة لصالح التطبيق ،البعدي يُعزى إلى تدريس املهارات النحوية باستراتيجية التعليم املتمايز. فرضيات الدراسة : ولإلجابة عن أسئلة الدراسة تم صياغة فرضي ة الدراسة اآلتية : - توجد فروق دالة إحصائيًا عند مستوى الداللة ( 0.05 )بين متوسطي درجات املجموعة التجريبية في التطبيقين (القبلي والبعدي )الختبار املهارات النحوية على مستوى االختبار ككل وعلى مستوى كل مهارة مستقلة لصالح التطبيق ،البعدي يُعزى إلى تدريس املهارات النحوية باستراتيجية التعليم املتمايز. أهداف الدراسة : ت هدف الدراسة إلى : 1- تحديد املها رات النحوية ( في مقرر اللغة العربية102 ) الالزمة لطلبة األقسام العلمية بكلية التربية– .صنعاء 2- بناء برنامج تعليمي قائم على استراتيجية التعليم املتمايز لتنمية املهارات النحوية لدى طلبة األقسام العلمية بكلية التربية– .صنعاء ت هدف الدراسة إلى : 1- تحديد املها رات النحوية ( في مقرر اللغة العربية102 ) الالزمة لطلبة األقسام العلمية بكلية التربية– .صنعاء 2- بناء برنامج تعليمي قائم على استراتيجية التعليم املتمايز لتنمية املهارات النحوية لدى طلبة األقسام العلمية بكلية التربية– .صنعاء 3- التعرف على أثر تدريس البرنامج التعل يمي القائم على استراتيجية التعليم املتمايز في تنمية املهارات النحوية لدى طلبة األقسام العلمية بكلية التربية– .صنعاء 3- التعرف على أثر تدريس البرنامج التعل يمي القائم على استراتيجية التعليم املتمايز في تنمية املهارات النحوية لدى طلبة األقسام العلمية بكلية التربية– .صنعاء تنبع أهمية الدراسة من اآلتي : - ُت عد هذه الدراسة– حسب علم الباحث– الدراسة األولى في جامعة صنعاء، التي تناول ت تدريس املهارات النحوية في مقرر ( اللغة العربية102 ) على طلبة األقسام العلمية بكلية التربية– صنعاء باستراتيجية التعليم .املتمايز آ - ُت عد هذه الدراسة– حسب علم الباحث– الدراسة األولى في جامعة صنعاء، التي تناول ت تدريس املهارات النحوية في مقرر ( اللغة العربية102 ) على طلبة األقسام العلمية بكلية التربية– صنعاء باستراتيجية التعليم .املتمايز م - تناولت الدراسة جانبًا مهمًا من جوانب املعرفة التطبيقية في اللغة العربية وهو "املهارات النحوية." - تناولت الدراسة جانبًا مهمًا من جوانب املعرفة التطبيقية في اللغة العربية وهو "املهارات النحوية." أثر استخدا م استراتيجية التعليم المتمايز في تنمية المهارات النحوية في مقرر اللغة العربية لدى طلبة األقسام العلمية( 94 ) الجعدي ( 94 ) ( 94 ) جلة العربية للعلوم ونشر األبحاث ــ مجلة المناهج وطرق التدريس ــ المجلد األول ــ العدد ال سادس عشر ــ ديسمبر 2022م المجلة العربية للعلوم ونشر األبحاث ــ مجلة المناهج وطرق التدريس ــ المجلد األول ــ العدد ال سادس عشر ــ ديسمبر 2022 م - ُت فيد واضعي مقررات اللغة العربية في الجامعات ا ليمنية ومطوّريها في األقسام اللغوية املتخصّصة : ُحيث ت قدم لهم الدراسة الحالية عددًا من املوجهات التي يمكن اإلفادة منها في توجيه مقررات اللغة العربية؛ عبر تحديد املهارات النحوية ( في مقرر اللغة العربية102 ) لدى طلبة األقسام العلمية بكلية التربية– صنعاء باستر اتيجية ُالتعليم املتمايز، وإعداد أداة لقياسها، وتبني املعالجات التَّعليميَّة، التي تتَّسِ ق مع طبيعتها، وت.سهم في تنميتها - ُت فيد واضعي مقررات اللغة العربية في الجامعات ا ليمنية ومطوّريها في األقسام اللغوية املتخصّصة : ُحيث ت قدم لهم الدراسة الحالية عددًا من املوجهات التي يمكن اإلفادة منها في توجيه مقررات اللغة العربية؛ عبر تحديد املهارات النحوية ( في مقرر اللغة العربية102 ) لدى طلبة األقسام العلمية بكلية التربية– صنعاء باستر اتيجية ُالتعليم املتمايز، وإعداد أداة لقياسها، وتبني املعالجات التَّعليميَّة، التي تتَّسِ ق مع طبيعتها، وت.سهم في تنميتها - ُت فيد القائمين على تدريس مقرر اللغة العربية من أعضاء هيئة التدريس : ُحيث ت ًقدِّ مُ لهم الدراسة دليال إجرائيًا، يوضّ ح كيفيَّة استخدام البر.نامج القائم على استراتيجية التعليم املتمايز - ُت فيد القائمين على تدريس مقرر اللغة العربية من أعضاء هيئة التدريس : ُحيث ت ًقدِّ مُ لهم الدراسة دليال إجرائيًا، يوضّ ح كيفيَّة استخدام البر.نامج القائم على استراتيجية التعليم املتمايز - ُي.ؤّمّ ل أنْ تسهمَ الدراسة الحالية في إِثراء البحوث والدراسات املرتبطة بتعليم مهارات اللغة العربية - ُي.ؤّمّ ل أنْ تسهمَ الدراسة الحالية في إِثراء البحوث والدراسات املرتبطة بتعليم مهارات اللغة العربية م - .تفتح املجال للباحثين، إلجراء العديد من الدراسات املشابهة في متغيرات مختلفة - .تفتح املجال للباحثين، إلجراء العديد من الدراسات املشابهة في متغيرات مختلفة حدود الدراسة : اقتصرت الدراسة على ال حدود اآلتية : اقتصرت الدراسة على ال حدود اآلتية :  الحدود املوضوعية :املوضوعات النحوية املقررة في كتاب ( اللغة العربية102 ) املقرر على طلبة األقسام العلمية املستوى األول في كلية التربية– صنعاء.  الحدود املوضوعية :املوضوعات النحوية املقررة في كتاب ( اللغة العربية102 ) املقرر على طلبة األقسام العلمية املستوى األول في كلية التربية– صنعاء.  الحدود البشرية :عينة من طلبة املستوى األول في األقسام العلمية: (رياضيات – علوم .)حياة  الحدود املكان ية : كلية التربية– صنعاء.  الحدود البشرية :عينة من طلبة املستوى األول في األقسام العلمية: (رياضيات – علوم .)حياة  الحدود املكان ية : كلية التربية– صنعاء. م  الحدود الزمانية :الفصل الدراس ي الثاني من العام الجامعي ( 2021م – 2022 ). مصطلحات الدراسة : - األثر :األثر لغة : ّبقية الش يء، وجمعها آثار، وأثر الجرح أثر يبقى بعدما يبرأ، وأث ًر في الش يء ترك فيه أثر ا. (ابن منظور، ص ص49 - 53 ). o األثـر اصطال ًاح :"هو نتيج ة ،تترتب على حادث أو ظاهرة في عالقة سببية أو أثر حالة من اإلشباع، أو عدم اإلشباع ُكما يقال عن األثر أنه التأث ُر بصفةِ شخص أو التأث ر بفكرةٍ عامة" ،. (العاليلي1974م، ص8 .) أا o ويُعرّف األثر إجرائيًا بأنه :قياس الفرق بين ما تتقنه مجموعة الدراسة من طلبة األقسام العلمية بكلية التربية صنعاء من املهارات النحوية قبل تطبيق التجربة، وبين ما تتقنه بعد تطبيق التجربة، وتدريسهم املهارات النحوية .باستخدام استراتيجية التعليم املتمايز - ال ستراتيجية : ّعر ف (عطية) االستراتيجية بأنها :خط السير املوصل إلى الهدف، وتشمل جميع الخطوات ،األساسية التي يصفها املدرس من أجل تحقيق أهداف املنهج، فيدخل فيها كل فعل أو إجراء له غاية أو غرض ُلذلك فإن االستراتيجية بمعناها العام ت ّمث.ل كل ما يفعله املعلم لتحقيق أهداف املنهج ،(عطية2009 ،م ص138 ) - ال ستراتيجية : ّعر ف (عطية) االستراتيجية بأنها :خط السير املوصل إلى الهدف، وتشمل جميع الخطوات ،األساسية التي يصفها املدرس من أجل تحقيق أهداف املنهج، فيدخل فيها كل فعل أو إجراء له غاية أو غرض ُلذلك فإن االستراتيجية بمعناها العام ت ّمث.ل كل ما يفعله املعلم لتحقيق أهداف املنهج ،(عطية2009 ،م ص138 ) o ُوت ّعر ف االستراتيجية إجرائيًا بأنها :خطوات وإجرا ءات محددة ومخططة مسبقً ا لتدريس طلبة عينة الدراسة املهارات النحوية ب.استراتيجية التعليم املتمايز o ُوت ّعر ف االستراتيجية إجرائيًا بأنها :خطوات وإجرا ءات محددة ومخططة مسبقً ا لتدريس طلبة عينة الدراسة املهارات النحوية ب.استراتيجية التعليم املتمايز - التعليم املتمايز :عرفه (اللقاني والجمل) بأنه : أسلوب يعتمد على التنوع، حيث توجد الفروق الفردية بين تالميذ الص ف الواحد، األمر الذي يعني أن اعتماد املعلم على طريقة واحدة ال يؤدي بالضرورة إلى تعلم الجميع بالقدر ًوالنوع نفسيهما. ومن هنا فاملعلم مطالب بأن يستخدم عديد ا من الطرق، من أجل توفير مواقف تعليمية .متنوعة، ومناسبة ألكبر عدد ممكن من التالميذ ،(اللقاني، والجمل2003م، ص92 ) - التعليم املتمايز :عرفه (اللقاني والجمل) بأنه : أسلوب يعتمد على التنوع، حيث توجد الفروق الفردية بين تالميذ الص ف الواحد، األمر الذي يعني أن اعتماد املعلم على طريقة واحدة ال يؤدي بالضرورة إلى تعلم الجميع بالقدر ًوالنوع نفسيهما. مصطلحات الدراسة : ومن هنا فاملعلم مطالب بأن يستخدم عديد ا من الطرق، من أجل توفير مواقف تعليمية .متنوعة، ومناسبة ألكبر عدد ممكن من التالميذ ،(اللقاني، والجمل2003م، ص92 ) أثر استخدا م استراتيجية التعليم المتمايز في تنمية المهارات النحوية في مقرر اللغة العربية لدى طلبة األقسام العلمية ( 95 ) المجلة العربية للعلوم ونشر األبحاث ــ مجلة المناهج وطرق التدريس ــ المجلد األول ــ العدد ال سادس عشر ــ ديسمبر 2022 م جلة العربية للعلوم ونشر األبحاث ــ مجلة المناهج وطرق التدريس ــ المجلد األول ــ العدد ال سادس عشر ــ ديسمبر 2022م o ُوي ّعر ف التعليم املتمايز إجرائيًا بأنه :است راتيجية تعليمية حديثة تهدف إلى توفير بيئة تعليمية مناسبة لجميع الطلبة، وتراعي االختالفات بين طلبة عينة الدراسة كل حسب مستواه في جميع النواحي من حيث ما لدى كل ّواحد منهم من خبرات ومهارات واهتمامات، ونقطة البداية لكل .على حدة - التنمية :لغة :مشتقة من الفعل نما ً، ومنه نما املال يعني نماء. ،(الجوهري1987م، ص1691 ) o التنمية اصطالحً ا : ُي ّف عر( ها شحاتة والنجار2003م، ص157 ) بأنها : رفع مستوى أداء الطالب في مواقف تعليمية تعلمية مختلفة، وتتحدد التنمية بزيادة متوسط الدرجات التي يحصلون عليها بعد تدريبهم على برنامج محدد. o ُوت ّعر ف التنمية إجرائيًا بأنها : التغيير املطلوب الوصول إليه لدى عينة الدراسة من طلبة األقسام العلمية بكلية التربية صنعاء بعد تدريسهم املهارات النحوية ب.استراتيجية التعليم املتمايز - التنمية :لغة :مشتقة من الفعل نما ً، ومنه نما املال يعني نماء. ،(الجوهري1987م، ص1691 ) o التنمية اصطالحً ا : ُي ّف عر( ها شحاتة والنجار2003م، ص157 ) بأنها : رفع مستوى أداء الطالب في مواقف تعليمية تعلمية مختلفة، وتتحدد التنمية بزيادة متوسط الدرجات التي يحصلون عليها بعد تدريبهم على برنامج محدد. o ُوت ّعر ف التنمية إجرائيًا بأنها : التغيير املطلوب الوصول إليه لدى عينة الدراسة من طلبة األقسام العلمية بكلية التربية صنعاء بعد تدريسهم املهارات النحوية ب.استراتيجية التعليم املتمايز o ُوت ّعر ف التنمية إجرائيًا بأنها : التغيير املطلوب الوصول إليه لدى عينة الدراسة من طلبة األقسام العلمية بكلية التربية صنعاء بعد تدريسهم املهارات النحوية ب.استراتيجية التعليم املتمايز - املهارات النحوية : تُعرّف املهارة ًلغة بأنها :جمع مهارة وتعني : الحذق في الش يء واإل جادة فيه (الفيروز أبادي : 1993 ،م137 ) . - املهارات النحوية : تُعرّف املهارة ًلغة بأنها :جمع مهارة وتعني : الحذق في الش يء واإل جادة فيه (الفيروز أبادي : 1993 ،م137 ) . مصطلحات الدراسة : o وتُعرّف املهارة ًاصطالحا بأنها : ٌأداءٌ سهل ٌدقيق ٌقائم ًعلى ما يتعلمه اإلنسان حركي ًا وعقلي ا، مع توفير الوقت والجهد والتكاليف (اللقاني، والجمل : 1996م، ص187 ) . o وتُعرّف املهارة ًاصطالحا بأنها : ٌأداءٌ سهل ٌدقيق ٌقائم ًعلى ما يتعلمه اإلنسان حركي ًا وعقلي ا، مع توفير الوقت والجهد والتكاليف (اللقاني، والجمل : 1996م، ص187 ) . o ُوت ّعر ًف املهارة إجرائي ا بأنها : قدرة عينة الدراسة من طلبة األ قسام العلمية بكلية التربية- صنعاء على أداء عمل .معين بدقة وإتقان مع االقتصاد في الوقت والجهد o ُوت ّعر ًف املهارة إجرائي ا بأنها : قدرة عينة الدراسة من طلبة األ قسام العلمية بكلية التربية- صنعاء على أداء عمل .معين بدقة وإتقان مع االقتصاد في الوقت والجهد ا - النحو : ًلغة :هو "القصد والطريق، ُي قال (نحَ ا نَحوَه) أي قَصَدَ قَصدَ ه، وَنَحَ ا بَصَرَهُ إليه صَرَفَ ، وبابُها عَدَ ا (وانحَ نَى َبَصَره عَدَ لَهُ) ونَحاهُ عن موضعه فَت."نحَ ى (الرازي : 2005م، ص320 ) - النحو : ًلغة :هو "القصد والطريق، ُي قال (نحَ ا نَحوَه) أي قَصَدَ قَصدَ ه، وَنَحَ ا بَصَرَهُ إليه صَرَفَ ، وبابُها عَدَ ا (وانحَ نَى َبَصَره عَدَ لَهُ) ونَحاهُ عن موضعه فَت."نحَ ى (الرازي : 2005م، ص320 ) o ّوفي االصطالح عر فه ابن جني (ت : 392هـ) بأنه : ّ"انْتِحَ اءُ سَ مْ ت كالم العرب، في تصرفه من إعراب وغيره، كالت ثنية ّوالجمع والت ّحقير والت ّكسير واإلضافة والن ّسب والت ركيب وغير ذلك، ليلحق من ليس من أهل اللغة العربية بأهلها في الفصاحة، ف ّينطق بها، وإن لم يكن منهم، أو إن شذ بعضهم عنها رد به إليها، وهو في األصل مصدر شائع، أي ."نحوت نحواً، كقولك قصدت قصداً، ثم خص به انتحاء هذا القبيل من العلم (ابن جني : 1913م، ص34 ) o وتُعرَّف املهارات النحوية إجرائياً بأنها : مجموعة املؤشرات السلوكية الدالة على قدرة عينة الدراسة من طلبة األقسام العلمية بكلية التربية– صنعاء على األداء اللغوي الصحيح للنصوص املكتوبة من خالل تطبيقه ملهارات ُالنحو املحددة بي ُسرٍ وس .هولة 2- اإلطار النظري والدراسات السابقة. 2- اإلطار النظري والدراسات السابقة. 1- اإلطار النظري : أهمية استراتيجية التعليم املتمايز : م هناك جوانب عدة تؤكد أهمية التدريس املتمايز، وهي كاآلتي ،(الحليس ي2012م، ص54 ) : - ُي)عزز التدريس املتمايز مبدأ (التعليم حق للجميع، وعبارة (املقاس الوا.)حد ال يصلح للجميع - ُي راعي األنماط املختلفة للتعلم (سمعي- بصري– منطقي– اجتماعي- .)حس ي - ُي شبع ميول واتجاهات الطلبة ؛ مما يعزز الدافعية، ويرفع مستوى التحدي لديهم، وينمي االبتكار، .واإلبداع - .يقوم على التكامل بين االستراتيجيات التعليمية املختلفة - ُي حقق شروط ا ّلتعل ّم الفع.ال ًويرى الباحث أن استراتيجية التعليم املتمايز أصبحت حاجة ًملحة ؛ لتحقيق أهداف املناهج الدراسية؛ حيث تعمل على تحقيق مبدأ تكافؤ الفرص التعليمية بين الطلب ة في عملية التعليم، وتزيد من دافعيتهم نحو ه ، وتزيد من ُفعالية وجودة عملية التعليم، وت لبي احتي اجات هم .في املهارات واالهتمامات، والقدرات والذكاءات واالتجاهات وامليول مفهوم التعليم املتمايز : مم عرفه (الحليس ي) بأنه : تحديد احتياجات املتعلمين املختلفة ومعلوماتهم السابقة واستعداداتهم للتعلم ُومستواهم اللغوي وميولهم وأنماط تعلمهم امل ّفض لة، ثم االستجابة لذلك في عملية التدريس، أي عملية تنويع للتدريس، وهو عملية تعليم وتعلم طلبة بينهم اختالفات كثيرة في ص ف .دراس ي واحد (الحلي،س ي2012م، ص47 ) وعَرَّفته (كوجك وآخرون) بأنه :ابتكار طرق متعددة تُوفر للتالميذ على اختالف قدراتهم، وميولهم واهتماماتهم، ،واحتياجاتهم التعليمية فرصًا متكافئة لفهم واستيعاب املفاهيم واستخدامها في مواقف الحياة اليومية كما تسمح للتالميذ بتحمل مسئولية تعلمهم من.خالل تعليم وتعلم األقران والتعلم التعاوني (كوجك وآخرون : 2008م، ص24 ) الجعدي ( 96 ) المجلة العربية للعلوم ونشر األبحاث ــ مجلة المناهج وطرق التدريس ــ المجلد األول ــ العدد ال سادس عشر ــ ديسمبر 2022 م جلة العربية للعلوم ونشر األبحاث ــ مجلة المناهج وطرق التدريس ــ المجلد األول ــ العدد ال سادس عشر ــ ديسمبر 2022م و عرفت (توملينسون، 2001م، ص5) التعليم املتمايز بأنه : إعادة تنظيم ما يجري في غرفة الصف؛ لكي تتوفر للمتعلمين خيارات متعددة للوصول إلى املعلومة، وتكوين األفكار، والتعبير عما تعلموه، وبمعنى آخر يوفر التعليم ًاملتمايز سبال مختلفة للتمكن من املحتوى ومعالجة تكوين األفكار، وتطوير منتجات تمكن كل متعلم من التعلم .بفعالية ًويتبين من التعاريف السابقة أن التعليم املتمايز يهدف إلى رفع مستوى جميع الطلبة وصوال لتحقيق مخرجات تعليمية واحدة ؛ ب استخدام أساليب مت نوعة، مع األخذ بعين االعتبار اختالف الطلبة في القدرات .واالهتمامات وغيرها أهداف التعليم املتمايز : م م من أهداف التعليم املتمايز، اآلتي Heacox ، D. (2002).p11) ) : 1- .تطوير مهمات تتسم بالتحدي واالحتواء لكل متعلم 2- تطوير أنشطة تعليمية تعتمد على املوضوعات واملفاهيم الجو هرية والعمليات واملهارات املهمة، وكذلك تطوير .طرق متعددة لعرض عملية التعلم 3- .توفير مداخل تتسم باملرونة لكل من املحتوى والتدريس واملخرجات 4- االستجابة ملستويات االستعداد لدى الطلب ة ، واالحتياجات التدريسية واالهتمامات .في عملية التعلم 5- التوافق مع معايير ومتطلبات ا.ملنهج لكل متعلم 6- توفير الفرص للطلب ة .للعمل وفق طرق تدريس مختلفة ويرى الباحث أن استراتيجية التعليم املتمايز تهدف إلى رفع مستوى التحصيل العلمي عند الطلب ة ، وزرع الثقة بأنفسهم، .واالبتعاد عن اإلحباط والشعور باليأس مميزات استراتيجية التعليم املتمايز : من مميزات استراتيجية التعليم املتمايز اآلتي) Hall (2009)..p460 ) : - .تُمَ كن املعلمين من فتح فرص تعلم لجميع الطالب من خالل تقديم خبرات متنوعة - ُت.ساعد املعلمين على فهم واستخدام التقييم بصورة جيدة كما ينبغي أثر استخدا م استراتيجية التعليم المتمايز في تنمية المهارات النحوية في مقرر اللغة العربية لدى طلبة األقسام العلمية ( 97 ) الجعدي ( 97 ) المجلة العربية للعلوم ونشر األبحاث ــ مجلة المناهج وطرق التدريس ــ المجلد األول ــ العدد ال سادس عشر ــ ديسمبر 2022 م جلة العربية للعلوم ونشر األبحاث ــ مجلة المناهج وطرق التدريس ــ المجلد األول ــ العدد ال سادس عشر ــ ديسمبر 2022م - ُت ضيف استراتيجيات تعليمية جديدة للمعلمين، وتستحدث تقنيات ت ساعده م على التركيز على أساسيات .املنهج - ُت ضيف استراتيجيات تعليمية جديدة للمعلمين، وتستحدث تقنيات ت ساعده م على التركيز على أساسيات .املنهج - ُت طابق متطلبات املناهج بطريقة ذات معنى لتحقيق نجاح الطلب ة. ويذكر (أمجد الراعي، 2014م، ص22) بعض مميزات استراتيجية التعليم املتمايز، منها : - ُت .ساعد املعلمين على فهم واستخدام التقييم بصورة أفضل - ُت طابق متطلبات املناهج بطريقة ذات معنى لتحقيق نجاح الطلب ة. ويذكر (أمجد الراعي، 2014م، ص22) بعض مميزات استراتيجية التعليم املتمايز، منها : - ُت .ساعد املعلمين على فهم واستخدام التقييم بصورة أفضل - تنال رضا كل من املتعلمين و املعل.مين ويضيف (محسن عطية، 2009م، ص460) مميزات أخرى الستراتيجية التعليم املتمايز، وهي كاآلتي : - ُت وفر لك ل .طالب أو مجموعة طالب متطلبات التعليم التي تالئمهم - ُت وفر لك ل .طالب أو مجموعة طالب متطلبات التعليم التي تالئمهم - .تزيد من فعالية املتعلمين في التعليم استراتيجيات التعليم املتمايز : 5- استراتيجيات معالجة املعلومات :وتقوم على تعليم الطلب ة كيف ،ية تنظيم املعلومات، وتخزينها، واسترجاعها .وتطبيقها حسب قدراتهم 5- استراتيجيات معالجة املعلومات :وتقوم على تعليم الطلب ة كيف ،ية تنظيم املعلومات، وتخزينها، واسترجاعها .وتطبيقها حسب قدراتهم 6- استراتيجية التعلم بالتعاقد :حيث يتم عقد اتفاق بين املعلم والطلب ة قبل البدء في عملية التعلم يوضح فيه الغرض من التعلم، واملصادر التعليمية التي سوف يحتاجونها، وطبيعة األنشطة التي سيمارسونها، وأسل وب .التقييم وتوقيتاته 6- استراتيجية التعلم بالتعاقد :حيث يتم عقد اتفاق بين املعلم والطلب ة قبل البدء في عملية التعلم يوضح فيه الغرض من التعلم، واملصادر التعليمية التي سوف يحتاجونها، وطبيعة األنشطة التي سيمارسونها، وأسل وب .التقييم وتوقيتاته 7- استراتيجية حل املشكالت : تعتمد هذه االستراتيجية على وجود موقف تعليمي يمثل مشكلة حقيقية تواجه الطلب ة ، وتستثيرهم للوصول إلى أنسب الحلول املمكنة، كما تتنوع املشكالت لتتوافق مع تنوع الطلب ة . ودور املعلم املساعدة في تقديم حلول يختبرها الطلب ة الختيار أفضلها، وتزيد دافعية الطلب ة وتكون حلولهم أكثر ًنضج.ا كلما توافقت املشكالت مع ميولهم وأنماط تعلمهم وذكاءاتهم ومعرفتهم 7- استراتيجية حل املشكالت : تعتمد هذه االستراتيجية على وجود موقف تعليمي يمثل مشكلة حقيقية تواجه الطلب ة ، وتستثيرهم للوصول إلى أنسب الحلول املمكنة، كما تتنوع املشكالت لتتوافق مع تنوع الطلب ة . ودور املعلم املساعدة في تقديم حلول يختبرها الطلب ة الختيار أفضلها، وتزيد دافعية الطلب ة وتكون حلولهم أكثر ًنضج.ا كلما توافقت املشكالت مع ميولهم وأنماط تعلمهم وذكاءاتهم ومعرفتهم 8- استراتيجية (فكر- زاوج- )شارك : ٍتُعد إحدى االستراتيجيات التي تؤيد تنويع التدريس والتعلم النشط في آن واحدٍ ، وتعتمد على استثارة التال ؛ ميذ كي يفكروا كل على حدة، ثم يشترك كل تلميذين في مناقشة أفكار كل منهما، وذلك من خالل توجيه سؤال يستدعي تفكير التالميذ، وإعطائهم الفرصة ؛ كي يفكروا على مستويات .مختلفة 9- استراتيجية املجموعات املرنة :هي استراتيجية قريبة إلى التعلم التعاوني إال أنها تستند إلى ساس أ مهم هو أن كل تلميذ في الصف هو عضو في مجموعات مختلفة متعددة يشكلها املعلم في ضوء أهداف ،التعليم والتعلم ًوأيضا في ضوء خصائص التالميذ، ويسمح في ها بانتقال التلميذ من مجموعة إلى ،أخرى تبعًا الحتياجاته التعليمية. 9- استراتيجية املجموعات املرنة :هي استراتيجية قريبة إلى التعلم التعاوني إال أنها تستند إلى ساس أ مهم هو أن كل تلميذ في الصف هو عضو في مجموعات مختلفة متعددة يشكلها املعلم في ضوء أهداف ،التعليم والتعلم ًوأيضا في ضوء خصائص التالميذ، ويسمح في ها بانتقال التلميذ من مجموعة إلى ،أخرى تبعًا الحتياجاته التعليمية. خطوات استراتيجية التعليم املتمايز : م من الخطوات التي يجب ا تباعها أثناء تطبيق التعليم املتمايز، اآلتي (عطية : 2009م، ص328 ) : 1- التقويم القبلي : إن أول خطوة من خطوات التعليم املتمايز هو إجراء عملية تقويم تستهدف تحديد املعارف ،السابقة و ،تحديد القدرات واملواهب و ،تحديد امليول والخصائص الشخصية و تحديد أسلوب التعلم املالئم، وتحديد الخلف.يات الثقافية من الخطوات التي يجب ا تباعها أثناء تطبيق التعليم املتمايز، اآلتي (عطية : 2009م، ص328 ) : 1- التقويم القبلي : إن أول خطوة من خطوات التعليم املتمايز هو إجراء عملية تقويم تستهدف تحديد املعارف ،السابقة و ،تحديد القدرات واملواهب و ،تحديد امليول والخصائص الشخصية و تحديد أسلوب التعلم املالئم، وتحديد الخلف.يات الثقافية 2- تصنيف الطلبة في مجموعات في ضوء نتائج التقويم القبلي وفق ما بين أعضاء كل مجموعة من قواسم .مشتركة 2- تصنيف الطلبة في مجموعات في ضوء نتائج التقويم القبلي وفق ما بين أعضاء كل مجموعة من قواسم .مشتركة 3- .تحديد أهداف التعلم 4- .اختيار املواد واألنشطة التعليمية ومصادر التعلم وأدوات التعليم 5- .تنظيم البيئة التعليمية بطريقة تستجيب لجميع املجموعات 6- اختيار اس.تراتيجيات التدريس املالئمة للطلبة أو املجموعات 7- ُتحديد األنشطة التي ت َّكل.ف بها كل مجموعة 8- .إجراء عملية التقويم بعد التنفيذ لقياس مخرجات التعلم ويذكر ذوقان، وأبو السميد ( 2007م، ص229 ) ،عدة خطوات يجب مراعاتها عند تطبيق التدريس املتمايز وهذه الخطوات هي : 1- تحديد .)املعلم ملهارات وقدرات كل طالب (ماذا يعرف كل طالب؟ وماذا يحتاج؟ 2- .اختيار استراتيجية التدريس املالئمة 3- .تحديد املهام التي سيقوم بها الطلبة لتحقيق أهداف التعلم وقد استفاد الباحث من الخطوات السابقة في بناء البرنامج التعليمي، ووضع تصور عام لتدريس املهارات النح وية باستخدام استراتيجية التعليم املتمايز، وكذلك في تطبيق البرنامج التعليمي على املجموعة التجريبية حيث .اتبع الباحث الخطوات السابقة 6ي ر س.و ملجمو ب دريس ملال م ر يجي 7- ُتحديد األنشطة التي ت َّكل.ف بها كل مجموعة 8- .إجراء عملية التقويم بعد التنفيذ لقياس مخرجات التعلم ويذكر ذوقان، وأبو السميد ( 2007م، ص229 ) ،عدة خطوات يجب مراعاتها عند تطبيق التدريس املتمايز وهذه الخطوات هي : 1- تحديد .)املعلم ملهارات وقدرات كل طالب (ماذا يعرف كل طالب؟ وماذا يحتاج؟ 2- .اختيار استراتيجية التدريس املالئمة 3- .تحديد املهام التي سيقوم بها الطلبة لتحقيق أهداف التعلم وقد استفاد الباحث من الخطوات السابقة في بناء البرنامج التعليمي، ووضع تصور عام لتدريس املهارات ام 3- .تحديد املهام التي سيقوم بها الطلبة لتحقيق أهداف التعلم وقد استفاد الباحث من الخطوات السابقة في بناء البرنامج التعليمي، ووضع تصور عام لتدريس املهارات النح وية باستخدام استراتيجية التعليم املتمايز، وكذلك في تطبيق البرنامج التعليمي على املجموعة التجريبية حيث .اتبع الباحث الخطوات السابقة الجعدي ( 98 ) المجلة العربية للعلوم ونشر األبحاث ــ مجلة المناهج وطرق التدريس ــ المجلد األول ــ العدد ال سادس عشر ــ ديسمبر 2022 م جلة العربية للعلوم ونشر األبحاث ــ مجلة المناهج وطرق التدريس ــ المجلد األول ــ العدد ال سادس عشر ــ ديسمبر 2022م استراتيجيات التعليم املتمايز : م يرتكز التدريس املتمايز على مجموعة من االستراتيجيات التدريسية التي تتيح اختيارات تعل ي مية متنوعة تتناسب مع قدرات واحتياجات واهتمامات الطلبة، وفيما يلي بيان لبعض ما ورد في األدبيات التي اطلع عليها الباحث، والتي تناولت التدريس املتمايز، من أهمها اآلتي : 1- استراتيجية التعليم املباشر : ٍّيستخدمها املعلم لعرض كم ٍكبير من املعلومات في وقت محدد، وتوضيح كل ما ي حتاجه الطلب ة .للتعلم يرتكز التدريس املتمايز على مجموعة من االستراتيجيات التدريسية التي تتيح اختيارات تعل ي مية متنوعة تتناسب مع قدرات واحتياجات واهتمامات الطلبة، وفيما يلي بيان لبعض ما ورد في األدبيات التي اطلع عليها الباحث، والتي تناولت التدريس املتمايز، من أهمها اآلتي : آم 1- استراتيجية التعليم املباشر : ٍّيستخدمها املعلم لعرض كم ٍكبير من املعلومات في وقت محدد، وتوضيح كل ما ي حتاجه الطلب ة .للتعلم 1- استراتيجية التعليم املباشر : ٍّيستخدمها املعلم لعرض كم ٍكبير من املعلومات في وقت محدد، وتوضيح كل ما ي حتاجه الطلب ة .للتعلم 2- استراتيجية التعلم التعاوني : ًوتقوم على تجميع فرق صغيرة من التالميذ بطريقة غير متجانسة وفق ،ا لقدراتهم .واهتماماتهم، وخلفياتهم، ويتم توزيع مهام التعلم عليهم 2- استراتيجية التعلم التعاوني : ًوتقوم على تجميع فرق صغيرة من التالميذ بطريقة غير متجانسة وفق ،ا لقدراتهم .واهتماماتهم، وخلفياتهم، ويتم توزيع مهام التعلم عليهم 3- استراتيجية التعلم املرتكز على املهام : وفيها يقوم املعلم بتوزيع مجموعة من املهام على كل طالب داخل الصف، وتختلف هذه املهام حسب قدرات الطلب ة. 4- استراتيجية طرح األسئلة :وفيها يقوم املعلم بطرح مجموعة من األسئلة املتدرجة في الصعوبة على الطلب ة ، ويطلب منهم التفكير في إجاباتها حسب قدرات هم. 4- استراتيجية طرح األسئلة :وفيها يقوم املعلم بطرح مجموعة من األسئلة املتدرجة في الصعوبة على الطلب ة ، ويطلب منهم التفكير في إجاباتها حسب قدرات هم. استراتيجيات التعليم املتمايز : 10 - استراتيجية األنشطة املتدرجة : ُت ستخدم هذه اال ستراتيجية عند وجود ط لبة مختلفين في املستويات املعرفية أو املهارية، ويدرسون نفس املفاهيم واملهارات، وفيه يمكن أن يبدأ كل طالب من النشاط املالئم ملستواه املعرفي أو املهاري ويتقدم للوصول إلى مستوى متميز، ويمكن للمعلم تضمين ثالثة مستويات من النشاط تتوافق مع مس توى كل طالب، كما ينبغي توفر املرونة لدى املعلم في حال وضع ا لطالب في نشاط أعلى أو أقل من مستواه .الحقيقي ( 99 ) المجلة العربية للعلوم ونشر األبحاث ــ مجلة المناهج وطرق التدريس ــ المجلد األول ــ العدد ال سادس عشر ــ ديسمبر 2022 م جلة العربية للعلوم ونشر األبحاث ــ مجلة المناهج وطرق التدريس ــ المجلد األول ــ العدد ال سادس عشر ــ ديسمبر 2022م 11 - املحطات التعليمية :وهي أماكن مختلفة في غرفة الصف يعمل الطلبة فيها على مهمات مختلفة في وقت ،واحد ويمكن استخدام هذه املحطات مع الطلبة من جميع األعمار وفي جميع املوضوعات الدراسية، ويمكن تمييزها باإلشارات أو الرموز أو األلوان، أو يمكن للمعلم أن يطلب من مجموعات من الطلبة أن تتحرك إلى أجزاء معينة .من غرفة الصف 12 - استراتيجية الكتشاف : هي مجموعة من األنشطة تساعد املتعلم على أن يتوصل إلى املعرفة بنفسه من جهة ويتعرف لى ع أسلوب التعلم ،وعملياته ويكتسب مهارات البحث املتضمنة فيه واالتجاهات العلمية من جهة أخرى. 13 - األلعاب التعليمية : وهي مجموعة األلعاب التي تساعد املتعلم على التوصل إلى املعرفة بطريقة شائقة، حيث .تُدخل املرح إلى نفس املتعلم بأ ري رم إ ى و ى مم يأ و و ي .تُدخل املرح إلى نفس املتعلم 14 - األنشطة الثابتة :هي نوع من األنشطة الت عليمية التعلمية التي يصممها املعلم في ضوء أهداف ومحتوى املنهج ،املقرر ولكل نشاط من هذه األنشطة أهداف واضحة ،ومحددة ويُراعى في تصميمها أن تتنوع في أنواعها ومستواها؛ لتناسب احتياجات التالميذ .املختلفة 14 - األنشطة الثابتة :هي نوع من األنشطة الت عليمية التعلمية التي يصممها املعلم في ضوء أهداف ومحتوى املنهج ،املقرر ولكل نشاط من هذه األنشطة أهداف واضحة ،ومحددة ويُراعى في تصميمها أن تتنوع في أنواعها ومستواها؛ لتناسب احتياجات التالميذ .املختلفة ام ولقد قام الباحث بتوظيف جُ ل هذه االستراتيجيات في بناء وتنف يذ البرنامج التعليمي القائم على التعليم املتمايز في تدريس املهارات،النحوية وكان لها األثر الكبير في زيادة التحصيل لدى الطلب ة، وهي (املجموعات املرنة – األنشطة املتدرجة- املحطات التعليمية- فكر ،زاوج ،شارك – العصف الذهني– األلعاب التعليمية– األنشطة الثابتة – االكتشاف- .)حل املشكالت نشأة النحو العربي : نشأة النحو العربي : ملا أشرقت شمس البعثة النبو ية على ربوع الجزيرة العربية أرشدت العقول وهدت القلوب واستثمرت عناصر ُالخير، وأصول الفضل في هذه األمة، كما ت ستنبت البذور الصالحة في األرض الطيبة، وكان من ذلك لسان هذه األمة الذي ظهر فضله، وتميزه حين وعى ما في القرآن الكريم من حكم وتشريعات ونظم، ومواعظ ونصائ ح وقصص وأخبار (الطويل : 1991م، ص178 ) . ،وعندما انتشر اإلسالم ودخل الناس في دين هللا أفواجً ا، وامتزجت الثقافات، وتبادلت العادات والتقاليد ُّمما أثر على اللغة العربية، فضعفت السليقة الل ًغوية، وأخذ اللحن في الظهور شيئًا فشيئ ا إلى أن كثُر وشاع، وأصبح ًبالءً عام.ا وشرًا مستطيرًا (زقوت : 1999م، ص165 ) حتى إذا حاولت العجمة أن تختال الفصاحة في اللسان العربي هبّ أبناؤه ا لوضع الضوابط، وتقعيد القواعد التي تصونه ا من عائلة االستعجام وتحفظه ا .من الضياع وفي الحقيقة انبثق الخوف على العربية من تسرب اللحن إلى بنياتها من معين حب ،اإلسالم وخاف عليه وبدأه املربي األول محمد- صلى هللا عليه وسلم- حيث قال للمسلمين عندما لحن أحدهم في كالمه : "أرشدوا أخاكم فقد ضل". ثم من بعده قال أبو بكر– رض ي هللا عنه- ( .""لئن أقرأ فأسقط أحب إليّ من أن أقرأ فألحن السيد ، ب ،ت143 ) ،وعندما انتشر اإلسالم ودخل الناس في دين هللا أفواجً ا، وامتزجت الثقافات، وتبادلت العادات والتقاليد ُّمما أثر على اللغة العربية، فضعفت السليقة الل ًغوية، وأخذ اللحن في الظهور شيئًا فشيئ ا إلى أن كثُر وشاع، وأصبح ًبالءً عام.ا وشرًا مستطيرًا (زقوت : 1999م، ص165 ) وزاد اإلحساس بالخطر من ب عدهم فقال عبد امللك بن مروان : ،"اللحن في الكالم أقبح من التفتيق في الثوب والجدري في الوجه". وفي موضع آخر قيل له :"لقد عجل عليك الشيب يا أمير املؤمنين، فقال : شيبني ارتقاء املنابر وتوقيع اللحن (األندلس ي ب. ت، ص308 ) . الفرق بين التعليم املتمايز والتعليم التقليدي : ُإن من الفروقات التي ت ميز التعليم املتمايز عن التعليم التقليدي اآلتي ،(الحليس ي2012م، ص82 ) : آم 1- إن التعليم التقليدي يعامل الطلب ة وفق طريقة واحدة وبمستوى واحد، أما التعليم املتمايز يلبي ا حتياجات .الطالب املختلفة واملتنوعة 1- إن التعليم التقليدي يعامل الطلب ة وفق طريقة واحدة وبمستوى واحد، أما التعليم املتمايز يلبي ا حتياجات .الطالب املختلفة واملتنوعة مم 2- ،إن عملية التقييم في التعليم التقليدي تتم في نهاية الوحدة أو ،األسبوع أو السنة، أما التقييم في التعليم .املتمايز فإنه عملية متفاعلة ومستمرة تحدث في كل األوقات واألشكال 2- ،إن عملية التقييم في التعليم التقليدي تتم في نهاية الوحدة أو ،األسبوع أو السنة، أما التقييم في التعليم .املتمايز فإنه عملية متفاعلة ومستمرة تحدث في كل األوقات واألشكال 3- ًإن أنماط التعلم واهتمامات الطلبة نادر ا ما تأخذ في أي ،حيز في إعداد الدروس بالنسبة للتعليم التقليدي .أما في التعليم املتمايز فإنه يتم أخذ أساليب التعلم املتنوعة واهتمامات الطلبة بعين االعتبار 3- ًإن أنماط التعلم واهتمامات الطلبة نادر ا ما تأخذ في أي ،حيز في إعداد الدروس بالنسبة للتعليم التقليدي .أما في التعليم املتمايز فإنه يتم أخذ أساليب التعلم املتنوعة واهتمامات الطلبة بعين االعتبار امم 4- ُنوع الواجبات واألعمال التي ي كلف بها الطالب في الصف التقليدي، ُفإنه ي ّكل ،ف بواجب واحد لجميع الصف أما في التعليم املتم.ايز فإن الخيارات متعددة للطالب 4- ُنوع الواجبات واألعمال التي ي كلف بها الطالب في الصف التقليدي، ُفإنه ي ّكل ،ف بواجب واحد لجميع الصف أما في التعليم املتم.ايز فإن الخيارات متعددة للطالب 5- أما فيما يخص العوامل املوجهة للتعليم ففي الصف التقليدي يوجد منهج واحد ومواد تعليمية واحدة وكتاب مدرس ي واحد، ًأما في الصف املتمايز فإنه يتم اعتماد معايير تعليم أساسية لكنه يأخذ أنواعاً وأشكاال .حسب احتياجات الطلبة 5- أما فيما يخص العوامل املوجهة للتعليم ففي الصف التقليدي يوجد منهج واحد ومواد تعليمية واحدة وكتاب مدرس ي واحد، ًأما في الصف املتمايز فإنه يتم اعتماد معايير تعليم أساسية لكنه يأخذ أنواعاً وأشكاال .حسب احتياجات الطلبة يتضح مما سب ق أن التعليم املتمايز أفضل من التعليم التقليدي؛ ألنه ينسجم مع أسس بناء املناهج ًالحديثة، والتي تولي اهتمام ًا كبير ل ا ًلمتعلم واهتماماته وحاجاته وميوله وقدراته، وتجعله محور.ا للعملية التعليمية يتضح مما سب ق أن التعليم املتمايز أفضل من التعليم التقليدي؛ ألنه ينسجم مع أسس بناء املناهج ًالحديثة، والتي تولي اهتمام ًا كبير ل ا ًلمتعلم واهتماماته وحاجاته وميوله وقدراته، وتجعله محور.ا للعملية التعليمية أثر استخدا م استراتيجية التعليم المتمايز في تنمية المهارات النحوية في مقرر اللغة العربية لدى طلبة األقسام العلمية ( 100 ) الجعدي أثر استخدا م استراتيجية التعليم المتمايز النحوية في مقرر اللغة العربية لدى ط( 100 ) الجعدي ( 100 ) المجلة العربية للعلوم ونشر األبحاث ــ مجلة المناهج وطرق التدريس ــ المجلد األول ــ العدد ال سادس عشر ــ ديسمبر 2022 م جلة العربية للعلوم ونشر األبحاث ــ مجلة المناهج وطرق التدريس ــ المجلد األول ــ العدد ال سادس عشر ــ ديسمبر 2022م املهارات النحوية : املهارات النحوية : نشأة النحو العربي : ومن هنا هب علماء اللغة العربية إلى التفكير في إنقاذها، ودرء الخطر .املحدق بها، فكان تأليفهم لقواعد النحو، ووضع الضوابط لها مكانة النحو في اللغة العربية : ُتتجلى مكانة النحو بأنها ت ُعتبر العمود الفقري في جسم اللغة العربية؛ وذلك ملا ي بنى عليها في فهم املعاني والكلمات والجمل، ومعرفة قواعدها، وحركاتها .اإلعرابية التي ال نستطيع التحدث بشكل سليم إال باتباعها ُوي عد النحو العربي العصب النابض للغة وقطب رحاها، والحصن املنيع الذي ال غنى عنه في صون اللسان ُمن الهَنات، وإقالته من العثرات، وهو اإلطار التنظيمي الذي يحكم قوانين اللغة وفق نظام ل غوي موحد، وله قصب ال.سبق بين فنون اللغة (الجوجو : 2011م، ص1372 ) ُوي عد النحو الركيزة األ ُساسية للغة، فهو النظام الذي به يتم نظم اللغة، وهو باإلضافة إلى ذلك ي عد معيار ُالصالحية والدقة عند استخدام اللغة. وال ي ُعد ما ي ُقال أو ي كتب صحيحً ا ما لم يتم االلتزام به على نحو معلوم، وتبدو أهمية النحو في كل اللغات لكنه أكثر أهمية في تعلم العربية؛ أل ن علماء العربية يولون اهتمامًا خاصًا للصالحية ًوالدقة، إضافة إلى ذلك فإن دارس التراث العربي تحديد ا يجد صعوبة كبيرة في الفهم مالم يكن على معرفة جيدة بنحو العربية وصرفها. ولألسف الشديد فإن النحو هو ُالعلم املظلوم بين علوم العربية، حيث ي عده الكثيرون صعبًا العتبارات كثيرة، ولم يكن أسالفنا من العامة والخاصة يجدون تلك الصعوبة؛ كونهم كانوا يتحدثون العربية السليمة .سليقة يتلقونها في بيوتهم ومدارسهم ويتحدثون بها في أسواقهم ومراكز تعلمهم (املصري : 2016م، ص32 ) مكانة النحو في اللغة العربية : ُتتجلى مكانة النحو بأنها ت ُعتبر العمود الفقري في جسم اللغة العربية؛ وذلك ملا ي بنى عليها في فهم املعاني والكلمات والجمل، ومعرفة قواعدها، وحركاتها .اإلعرابية التي ال نستطيع التحدث بشكل سليم إال باتباعها ُوي عد النحو العربي العصب النابض للغة وقطب رحاها، والحصن املنيع الذي ال غنى عنه في صون اللسان ُمن الهَنات، وإقالته من العثرات، وهو اإلطار التنظيمي الذي يحكم قوانين اللغة وفق نظام ل غوي موحد، وله قصب ال.سبق بين فنون اللغة (الجوجو : 2011م، ص1372 ) ُوي عد النحو الركيزة األ ُساسية للغة، فهو النظام الذي به يتم نظم اللغة، وهو باإلضافة إلى ذلك ي عد معيار ُالصالحية والدقة عند استخدام اللغة. أهمية تعليم النحو العربي : أهمية تعليم النحو العربي : ًإن تعليم القواعد ما هي إال وسيلة تؤدي إلى سالمة التعبير، حديث ًا وكتابة ، وإلى فهم األفكار وإدراك املعاني ُبيسر، ولذلك فإن كثرة التدريب واملران، ومالحظة طرائق استعمال اللغة في نصوص ومواقف ل غوية حية تمكن التالميذ من فهم القواعد وا.إلحساس بها، واالنطالق في الكالم والكتابة بسهولة وسليقة (أبو مغلي : 2009م، ص63 ) ،فتبرز أهمية اللغة العربية من كونها ذات قدرة كبيرة على تذليل الصعاب، وقوة واضحة في مجابهة الحياة واستيعاب كل جديد من العلم والحكمة والفلسفة، وأنواع املعارف األخرى، وهي تتمتع برس وخ في األصول، وحيوية في .الفروع (الدليمي، والوائلي : 2003م، ص60 ) وتأتي أهمية النحو من أهمية اللغة العربية ذاتها، إذ أ نه يعمل على تقويم األلسنة، وتجنب الخطأ في الكالم والكتابة، ويعوٍّد على صحة الحكم، ودقة املالحظة، ونقد التراكيب، ويعمل على شحذ العقول، والتدر يب على التفكير ِّاملتواصل املنظم، ويمك.ن من فهم التراكيب املعقدة والغامضة (الدليمي : 2004م، ص25 ) ُوي شير السيد ( 1969 ) ُإلى أهمية النحو وأنه ذو فائدة تربوية عظيمة؛ حيث ي ،سهم في بناء شخصية الطالب ًوينمي لديه الجوانب اللغوية، والثقافية، فضال عما يكتسبه الطالب م ن مهارات رئيسة مثل : ،املالحظة، والتحليل .واملقارنة، والربط، واالستنباط، والتجريب، هذا إلى جانب ما تغرسه هذه املادة من اتجاهات إيجابية لدى الطالب ،فتدريس النحو للنشء ضرورة محتمة؛ ألنه وسيلة لحفظ اللغة العربية، ومنع اللحن من التسلل إليها ويساعد على عصمة اللس ،ان من الخطأ، والقلم من الزلل، ويساعد على التفكير العلمي املنظم؛ ألنه رياضة للعقل .ويحارب تفش ي العامية، ويساعد على االتصال الصحيح السليم من خالل نقل املعنى املقصود فيسهل فهمه (مجاور : 2000م، ص363 ) ،فتدريس النحو للنشء ضرورة محتمة؛ ألنه وسيلة لحفظ اللغة العربية، ومنع اللحن من التسلل إليها ويساعد على عصمة اللس ،ان من الخطأ، والقلم من الزلل، ويساعد على التفكير العلمي املنظم؛ ألنه رياضة للعقل .ويحارب تفش ي العامية، ويساعد على االتصال الصحيح السليم من خالل نقل املعنى املقصود فيسهل فهمه (مجاور : 2000م، ص363 ) ومما سبق يمكن تلخيص أهمية تعليم النحو العربي في اآلتي : - يعمل .على تقويم األلسنة وتجنب الخطأ في الكالم والكتابة - .يعمل على حماية اللغة العربية من اللحن - .يعمل على تنمية الجوانب اللغوية، والثقافية لدى الطالب ومما سبق يمكن تلخيص أهمية تعليم النحو العربي في اآلتي : - يعمل .على تقويم األلسنة وتجنب الخطأ في الكالم والكتابة - .يعمل على حماية اللغة العربية من اللحن -يعمل عل تنمية الجوانب اللغوية، والثقافية لدى الطالب آ - يعمل .على تقويم األلسنة وتجنب الخطأ في الكالم والكتابة - .يعمل على حماية اللغة العربية من اللحن - .يعمل على تنمية الجوانب اللغوية، والثقافية لدى الطالب - .يساعد املتعلم على صحة الحكم ودقة املالحظة ونقد التراكيب - .يساعد املتعلم على صحة الحكم ودقة املالحظة ونقد التراكيب - ّشحذ العقول، والتدريب على التفكير املتواصل املنظم، ويمك.ن من فهم التراكيب املعقدة والغامضة - ّشحذ العقول، والتدريب على التفكير املتواصل املنظم، ويمك.ن من فهم التراكيب املعقدة والغامضة نشأة النحو العربي : وال ي ُعد ما ي ُقال أو ي كتب صحيحً ا ما لم يتم االلتزام به على نحو معلوم، وتبدو ل ال ف أكث أ لك ف كل الل ا ال أ لأ ا ا لل ال ا ا ا ل ل ا ال مكانة النحو في اللغة العربية : ُتتجلى مكانة النحو بأنها ت ُعتبر العمود الفقري في جسم اللغة العربية؛ وذلك ملا ي بنى عليها في فهم املعاني والكلمات والجمل، ومعرفة قواعدها، وحركاتها .اإلعرابية التي ال نستطيع التحدث بشكل سليم إال باتباعها ُوي عد النحو العربي العصب النابض للغة وقطب رحاها، والحصن املنيع الذي ال غنى عنه في صون اللسان ُمن الهَنات، وإقالته من العثرات، وهو اإلطار التنظيمي الذي يحكم قوانين اللغة وفق نظام ل غوي موحد، وله قصب ال.سبق بين فنون اللغة (الجوجو : 2011م، ص1372 ) ُوي عد النحو الركيزة األ ُساسية للغة، فهو النظام الذي به يتم نظم اللغة، وهو باإلضافة إلى ذلك ي عد معيار ُالصالحية والدقة عند استخدام اللغة. وال ي ُعد ما ي ُقال أو ي كتب صحيحً ا ما لم يتم االلتزام به على نحو معلوم، وتبدو أهمية النحو في كل اللغات لكنه أكثر أهمية في تعلم العربية؛ أل ن علماء العربية يولون اهتمامًا خاصًا للصالحية ًوالدقة، إضافة إلى ذلك فإن دارس التراث العربي تحديد ا يجد صعوبة كبيرة في الفهم مالم يكن على معرفة جيدة بنحو العربية وصرفها. ولألسف الشديد فإن النحو هو ُالعلم املظلوم بين علوم العربية، حيث ي عده الكثيرون صعبًا العتبارات كثيرة، ولم يكن أسالفنا من العامة والخاصة يجدون تلك الصعوبة؛ كونهم كانوا يتحدثون العربية السليمة .سليقة يتلقونها في بيوتهم ومدارسهم ويتحدثون بها في أسواقهم ومراكز تعلمهم (املصري : 2016م، ص32 ) أثر استخدا م استراتيجية التعليم المتمايز في تنمية المهارات النحوية في مقرر اللغة العربية لدى طلبة األقسام العلمية ( 101 ) جلة العربية للعلوم ونشر األبحاث ــ مجلة المناهج وطرق التدريس ــ المجلد األول ــ العدد ال سادس عشر ــ ديسمبر 2022م المجلة العربية للعلوم ونشر األبحاث ــ مجلة المناهج وطرق التدريس ــ المجلد األول ــ العدد ال سادس عشر ــ ديسمبر 2022 م أهداف تدريس النحو العربي : لتدريس النحو العربي هدفان : نظري يتعلق باستيعاب القواعد النحوية، ووظيفي يتعلق بتطبيقها، ويندرج تحت هذين الهدفين األهداف اآلتية : آأ ه 1- تيسير إدراك الطلبة للمعاني املسموعة واملقروءة، والتعبير عنها بوضو ،ح ال يدخله غموض أو إبهام أو خطأ .وترتيب أجزاء الكالم على أساس ترابط املعاني ومواقع األلفاظ في التراكيب (عامر : 2000م، ص124 ) 2- .تقويم اعوجاج اللسان من الزلل في النطق، والقلم من الزلل في الكتابة، وتصحيح املعاني واملفاهيم 3- تمكين الطلب ة من القراءة والكتابة والحدي.ث بصورة خالية من األخطاء اللغوية 4- وق و ف الطلب ة على خصائص اللغة العربية وثراء صيغها؛ ألن هذه القواعد تكشف عن أوضاع اللغة املختلفة .بصيغها املتنوعة، والتغيرات التي تحدث في ألفاظها وتراكيبها 5- ًتدريب الطلبة على استعمال األلفاظ والتراكيب استعماال صحيحً ا؛ بإدراك ا لخصائص الفنية السهلة للجملة العربية، كمكونات ال ج.ملة ومكمالتها (شحاتة : 1993م، ص202 ) 1- تيسير إدراك الطلبة للمعاني املسموعة واملقروءة، والتعبير عنها بوضو ،ح ال يدخله غموض أو إبهام أو خطأ .وترتيب أجزاء الكالم على أساس ترابط املعاني ومواقع األلفاظ في التراكيب (عامر : 2000م، ص124 ) 2- .تقويم اعوجاج اللسان من الزلل في النطق، والقلم من الزلل في الكتابة، وتصحيح املعاني واملفاهيم 3- تمكين الطلب ة من القراءة والكتابة والحدي.ث بصورة خالية من األخطاء اللغوية أ 4- وق و ف الطلب ة على خصائص اللغة العربية وثراء صيغها؛ ألن هذه القواعد تكشف عن أوضاع اللغة املختلفة .بصيغها املتنوعة، والتغيرات التي تحدث في ألفاظها وتراكيبها 5- ًتدريب الطلبة على استعمال األلفاظ والتراكيب استعماال صحيحً ا؛ بإدراك ا لخصائص الفنية السهلة للجملة العربية، كمكونات ال ج.ملة ومكمالتها (شحاتة : 1993م، ص202 ) أثر استخدا م استراتيجية التعليم المتمايز في تنمية المهارات النحوية في مقرر اللغة العربية لدى طلبة األقسام العلمية الجعدي ( 102 ) المجلة العربية للعلوم ونشر األبحاث ــ مجلة المناهج وطرق التدريس ــ المجلد األول ــ العدد ال سادس عشر ــ ديسمبر 2022 م جلة العربية للعلوم ونشر األبحاث ــ مجلة المناهج وطرق التدريس ــ المجلد األول ــ العدد ال سادس عشر ــ ديسمبر 2022م 6- إن الطلبة الذين يدرسون لغة أجنبية إلى جانب لغتهم القومية يجدون في دراسة قواعد لغتهم ما يساعدهم ًعلى فهم اللغة األجنبية؛ ألن بين اللغات قدر ًا مشترك ا من القواعد العامة (عاشور والحوامدة : 2007 ،م ص106 ). أهداف تدريس النحو العربي : ومن خالل ما سبق يتضح أن أهم أهداف النحو ه ي ،إعانة الطلبة على التحدث والكتابة دون أخطاء والقدرة على فهم التراكيب الغامضة وإعادة الصياغة لها من خالل االستخدام السليم لقواعد اللغة، والتمكن من فهم مدلوالت كل الكلمات، ومعانيها الوظيفية .التي تخدم النص القرائي، وإتقان مهارات االتصال اللغوي ربي و ي ب ب ب يُعد فهم القواعد النحوية من أصعب مشكالت الطلبة وأعقدها، ولذا فقد أصبحت عندهم مصدر نفور منها ومن اللغة، ومن ثم تعالت صيحات الشكوى من إخفاق الطلبة في تعلمها، وضعفهم في اكتسابها في شتى مراحل التعليم، ولعل من األسباب التي أدت إلى ضعف الطلبة في النحو العربي اآلتي : 1- طبيعة املادة النحوية : العتمادها على قوانين مجردة، واحتياجها إلى عمليات عقلية عليا، وتعدد األوجه ُاإلعرابية، وما إلى ذلك من أمور ت.شعر الطالب بصعوبة املادة إم 2- املنهج النحوي :اشتمال املنهج أحيانًا على موضوعات غير مرتبطة بحاجات الطلب ة ومشاعرهم، وتفتت .وتبعثر بعض موضوعاتها 2- املنهج النحوي :اشتمال املنهج أحيانًا على موضوعات غير مرتبطة بحاجات الطلب ة ومشاعرهم، وتفتت .وتبعثر بعض موضوعاتها 3- كتاب النحو املقرر : غلبة الطابع املنفر من طبع وتغليف وتتابع موضوعات، وطريقة عرض املادة، وخلوها من أساليب اإليضاح، وطريقة عرض املادة، وخلقها نوعًا من امل لل والضجر للطلب ة. 3- كتاب النحو املقرر : غلبة الطابع املنفر من طبع وتغليف وتتابع موضوعات، وطريقة عرض املادة، وخلوها من أساليب اإليضاح، وطريقة عرض املادة، وخلقها نوعًا من امل لل والضجر للطلب ة. 4- طريقة التدريس : مهما بلغت صعوبة املادة وجفافها فإن للطريقة الجيدة دورً ا مهمًا في إيصال املعلومة ،وتبسيطها وإ ن ما نجده على أرض الواقع من طر ائ.ق تقليدية تجعل من النحو رموزً ا مجردة، وقوالب صماء 5- معلم اللغة العربية : معاناة كثير من املعلمين من انخفاض في املستوى اللغوي، وقلة التجديد والتنويع في ائ طر ق تدريسهم للمادة، انعكس سلبًا على مستوى الطلب ة. أهداف تدريس النحو العربي : 6- طبيعة املتعلم :انصراف الطلب ة عن تعلم اللغة العربية، النعدام الدافع، وعدم إدراكهم للهدف الصحيح من .وراء تعلم النحو 6- طبيعة املتعلم :انصراف الطلب ة عن تعلم اللغة العربية، النعدام الدافع، وعدم إدراكهم للهدف الصحيح من .وراء تعلم النحو 7- البيئة املحيطة باملتعلم : سيطرة العامية في مختلف مناحي الحياة، حرم الطلب ة من االستماع إلى األساليب .الصحيحة، والتراكيب السليمة 7- البيئة املحيطة باملتعلم : سيطرة العامية في مختلف مناحي الحياة، حرم الطلب ة من االستماع إلى األساليب .الصحيحة، والتراكيب السليمة 8- وسائل اإلعالم : ًشيوع اللحن والتحريف على ألسنة العاملين في وسائل اإلعالم سواء ًكانت مسموعة أو ًمكتوبة ًأو مرئية.، كان له دور في إبراز تلك املشكلة (زقوت : 1999م، ص ص178 - 185 ) 8- وسائل اإلعالم : ًشيوع اللحن والتحريف على ألسنة العاملين في وسائل اإلعالم سواء ًكانت مسموعة أو ًمكتوبة ًأو مرئية.، كان له دور في إبراز تلك املشكلة (زقوت : 1999م، ص ص178 - 185 ) ويرى الباحث أن من أسباب ضعف الطلبة في النحو، ه ي قلة تلبية األمثلة والنماذج النحوية لرغبات هم ، فال تثير دوافعهم وميولهم لتعلم القواعد النحوية، فبعض املعلمين يسرد األمثلة بطريقة تخلق نوعًا من النفور واالبتعاد لدى الطلبة عن املادة، فأكثر هم يحفظون هذه األمثلة من.أجل االختبار دون استيعاب عالج ضعف الطلبة في النحو العربي : ( ذكر عطية2007م، ص187 ( ) والضبعات2007م، ص201 ) أن ُهناك عدة وسائل ت ساعد على عالج ضعف الطلبة في القواعد النحوية، منها : 1- أن تتصل دروس النحو بالحياة، وأن تعالج املواقف التي تواجه املتعلمين في حيات.هم اليومية 2- .استثارة دوافع املتعلمين واستغاللها في تدريس القواعد النحوية عالج ضعف الطلبة في النحو العربي : ( ذكر عطية2007م، ص187 ( ) والضبعات2007م، ص201 ) أن ُهناك عدة وسائل ت ساعد على عالج ضعف الطلبة في القواعد النحوية، منها : 1- أن تتصل دروس النحو بالحياة، وأن تعالج املواقف التي تواجه املتعلمين في حيات.هم اليومية 2- .استثارة دوافع املتعلمين واستغاللها في تدريس القواعد النحوية ( 103 ) الجعدي الجعدي المجلة العربية للعلوم ونشر األبحاث ــ مجلة المناهج وطرق التدريس ــ المجلد األول ــ العدد ال سادس عشر ــ ديسمبر 2022 م جلة العربية للعلوم ونشر األبحاث ــ مجلة المناهج وطرق التدريس ــ المجلد األول ــ العدد ال سادس عشر ــ ديسمبر 2022م 3- االبتعاد عن األساليب املعقدة في عرض املادة النحوية وقواعد ها. أهداف تدريس النحو العربي : 4- .االستفادة من الوسائل التعليمية واملواقف التي يمكن أن ينطلق منها املدرس في تدريس مادة النحو العربي 5- زيادة تدريب املتعلمين ع.لى تحليل الجمل والتراكيب اللغوية، وتحديد املواقع اإلعرابية ملفرداتها 6- ُجعل فروع اللغة العربية مواد تطبيقية ملادة النحو، وعدم التهاون في أي تقصير ل غوي من جانب الطلب ة. 7- تعويد الطلب ة على سماع األساليب العربية الصحيحة وترديدها باستمرار، واإل ،تيان بأمثلة مشابهة بحيث .تكون حصة اللغة العربية تطبيقً ا لقواعد النحو العربي عن طريق التدريس والتقليد واملمارسة 8- ُضرورة ترتيب أبواب النحو بحيث ت.جمع املوضوعات ذات العالقة في أبواب مستقلة 9- أ ن تركز االختبارات وطرق التقويم على قياس مدى تحقق األهداف، فتتجنب االختبارات التركيز على ا.ملصطلحات أو تسميع القواعد 10 - ُاستخدام الوسائل التعليمية الحديثة في مواقف تعلم القواعد النحوية بال تكلف؛ ألن استخدامها ي فض ي .على الدرس فاعلية وتشويقً ا، ويحبب املتعلمين في استخدام هذه القواعد ويرى الباحث أن من وسائل عالج ضعف الطلبة في القواعد النحوية التنويع ف ي طر ائ ق التدريس، وتوظيف وسائل تعليمية متعددة، وربط املادة بالواقع، وهذا يتطلب من املعلم اطالعًا موسعًا على أهم طر ائ ق التدريس .الحديثة، والعمل على إثراء املنهج، وتطوير نفسه، وعدم االكتفاء بما هو تقليدي وممل ُضرورة ترتيب أبواب النحو بحيث ت.جمع املوضوعات ذات العالقة في أبواب مستقلة 9- أ ن تركز االختبارات وطرق التقويم على قياس مدى تحقق األهداف، فتتجنب االختبارات التركيز على ا.ملصطلحات أو تسميع القواعد 10 - ُاستخدام الوسائل التعليمية الحديثة في مواقف تعلم القواعد النحوية بال تكلف؛ ألن استخدامها ي فض ي .على الدرس فاعلية وتشويقً ا، ويحبب املتعلمين في استخدام هذه القواعد ويرى الباحث أن من وسائل عالج ضعف الطلبة في القواعد النحوية التنويع ف ي طر ائ ق التدريس، وتوظيف وسائل تعليمية متعددة، وربط املادة بالواقع، وهذا يتطلب من املعلم اطالعًا موسعًا على أهم طر ائ ق التدريس .الحديثة، والعمل على إثراء املنهج، وتطوير نفسه، وعدم االكتفاء بما هو تقليدي وممل 2- الدراسات السابقة : - ( دراسة إيمان أبو شباب2019 :) هدفت الدراسة إلى التعرف على فاعلية برنامج تدريبي مستند إلى التعليم املتمايز في تحسين مستوى التحصيل في مادة الرياضيات واالتجاهات نحوها لدى طلبة الصف الثالث األساس ي .ذوي صعوبات التعلم في املدارس الحكومية في األردن ولتحقيق هدف الدراسة اعتمد ت الباحث ة املنهج ا ،لتجريبي وأعد ت ًاختبار ًا تحصيلي.ا في الرياضيات، وبطاقة املالحظة؛ ملالحظة اتجاهات الطلبة نحوها وتكونت عينة ( الدراسة من70 ً) طالب ا وطالبة من طلبة الصف الثالث في ست مدارس حكومية تابعة ملديرية التربية والتعليم في محافظة جرش في األردن، وتم تقسيمهم بالتساوي إلى.)مجموعتين (تجريبية، وضابطة وقد أظهرت الدراسة ًعدد ا من النتائج أهمها : يوجد فروق ذات داللة في الدرجة الكلية لالختبار التحصيلي بين أفراد املجموعتين ُلصالح املجموعة التجريبية ت.عزى للبرنامج املستخدم في الدراسة - ( دراسة جواهر الشهراني2019 :) هدفت الدراسة إلى معر فة أثر تدريس العلوم باستخدام استراتيجية التعليم املتمايز القائم على األنشطة في تنمية االستيعاب املفاهيمي وتنمية عادات العقل لدى طالبات الصف الثاني .املتوسط ًولتحقيق هدف الدراسة استخدمت الباحثة املنهج التجريبي، وأعدت اختبارًا تحصيلي ًا، ودليال لل.معلم وتكونت ( عينة الدراسة من40 ُ) طالبة، اختيرت بالطريقة العشوائية من طالبات الصف الثاني املتوسط، وو ِّز َع ْت بالتساوي إلى مجموعتين )(تجريبية، وضابطة ،وأظهرت النتائج ًوجود فرق دال إحصائي ا بين متوسطي درجات طالبات املجموعة التجريبية ودرجات طالبات املجموعة الضابطة في االخ.تبار البعدي لصالح املجموعة التجريبي - ( دراسة خالد الشقران2019 :) هدفت الدراسة إلى تقص ي أثر التدريس املتمايز في اكتساب املفاهيم العلمية .ومهارات عمليات العلم واالتجاه نحو العلوم لدى طالب الصف السابع األساس ي ولتحقيق هدف الدراسة استخدم الباحث املنهج التجريبي ، ًوأعد اختبار ا؛ ( لقياس املفاهيم العلمية مكوناً من27 ) فقرة، واختبارًا لقياس ( عمليات العلم مكوناً من24 ً) فقرة، ومقياس االتجاه نحو العلوم مكون( ا من33 .) فقرة وتكونت عينة الدراسة ( من65 ً) طالب ا اختيروا بالطريق ة ًاملتيسرة، حيث اختيرت إحدى الشعب عشوائي ا كمجموعة تجريبية بلغ عددها ( 104 ) الجعدي المجلة العربية للعلوم ونشر األبحاث ــ مجلة المناهج وطرق التدريس ــ المجلد األول ــ العدد ال سادس عشر ــ ديسمبر 2022 م جلة العربية للعلوم ونشر األبحاث ــ مجلة المناهج وطرق التدريس ــ المجلد األول ــ العدد ال سادس عشر ــ ديسمبر 2022م ( 31 ً) طالب( ا درست بطريقة التدريس املتمايز، والشعبة األخرى مثلت املجموعة الضابطة بلغ عددها34 ً) طالب ا .درست بالطريقة االعتيادية ًوأظهرت الدراسة عدد ا من النتائج أهمها : ًوجود فروق دالة إحصائي ا بين متوسطي درجات املجموعتين التجريبية والضابطة.لصالح املجموعة التجريبية - ( دراسة عفاف يوسف2019 :) هدفت ل لكشف عن أثر استراتيجية خرائط املفاهيم في تحسين مهارات القواعد .النحويّة لدى طالب الصف السابع األساس ي ،ولتحقيق هدف الدراسة استخدمت الباحثة املنهج التجريبي ًوأعدت اختبارًا تحصيلي.ا للقواعد النحوية وتكون( ت عينة الدراسة من60 ً) طالب ا، بواقع شعبتين، جرى اختيارهما عشوائيًّا من بين شعب الصف السابع األساس ي في مدرسة امللك عبد هللا الثاني للتميز، في منطقة إربد ( للعام الدراس ي2016م/ 2017 )، واختيرت إحدى الشعب عشوائيًا ؛ لتمثل املجموعة التجريبية التي تمّ تدريسها وفق استراتيجية خرائط املفاهيم في حين عدّ ت الشعبة الثانية هي املجموعة الضابطة التي درست القواعد .النحويّة باستخدام الطريقة التقليدية ًوأظهرت الدراسة عدد ا من النتائج أهمها : وجود فر وق ذات داللة إحصائية بين متوسطي درجات املجموعتين التجريبية والضابطة على أدائهنّ في ،اختبار القواعد النحويّة البعدي .لصالح املجموعة التجريبية - ( دراسة رائد حميد2019 :) هدفت إلى تعرف أثر أنموذجي (كمب وبارمان) في اكتساب املفاهيم النحوية في مادة .قواعد اللغة العربية لدى طالبات الصف الرابع العلمي ولتحقيق هدف الدراسة استخدم الباحث املنهج التجري ًبي، وأعد اختبارًا تحصيلي ا، والخطط التدريسية املتعلقة بتدريس املجموعة التجريبية األولى باستعمال أ نموذج كمب، والخطط التدريسية املتعلقة بتدريس املجموعة التجريبية الثانية باستعمال أ ،نموذج بارمان ًوالخطط التدريسية املتعلقة بتدريس املجموعة الضابطة وفق ا للطريقة.االعتيادية وتكونت عينة الدراسة من ( 75 ِّ) طالبة، ووُ ز عَتْ إلى ثالث مجموعات، حيث تم اختيار إعدادية شط العرب للبنات املجموعة التجريبية األ ولى التي ستتعرض طالباتها إ لى املتغير املستقل األ ول (أ،)نموذج كمب وثانوية الزرقاء للبنات املجموعة التجريبية الثانية التي ستتعرض طالباتها إ( لى املتغير املستقل الثاني أ نموذج بارمان)، وثانوية التحرير للبنات املجموعة الضابطة التي ستتعرض طالباتها إلى املتغير املستقل .)(الطريقة االعتيادية ًوأظهرت الدراسة عدد ا من النتائج أهمها : أن أ )نموذج (كمب أ فاد طالبات املجموعة األولى، وذلك بارت فاع درجاتهن في اختبار اكتساب املفاهيم، وتفوقهن على املجموع ة الثانية واملجموعة الضابطة. 2- الدراسات السابقة : كذلك تفوق املجموعة التجريبية الثانية التي استعمل معها أ.نموذج (بارمان) على املجموعة الضابطة التعليق على الدراسات السابقة : هدفت بعض الدراسات السابقة إلى استخدام استراتيج ية التعليم املتمايز مع متغيرات تربوية مختلفة، كما هدفت بعض الدراسات إلى تنمية املهارات النحوية باستخدام طرائق تدريسية متنوعة، واتفقت الدراسة الحالية في (املتغير املستقل) مع بعض الدراسات السابقة التي استخدمت استراتيجية التعليم املتمايز واختلفت معها في املتغ ير ( التابع، كما اتفقت مع دراستي عفاف يوسف2019 ) ( ورائد حميد2019 ) في املتغير التابع وهو تنمية املهارات النحوية، واختلفت معها في املتغير املستقل، وتميزت هذه الدراسة بأنها استهدفت تدريس املهارات النحوية باستخدام .استراتيجية التعليم املتمايز م و اختلفت الدراسة ا لحالية زمنيًا عن جميع الدراسات السابقة؛ إذ أُجْ رِيَت في الفصل الدراس ي الثاني من العام الجامعي ( 2021م- 2022 ). ( 105 ) ( 105 ) الجعدي المجلة العربية للعلوم ونشر األبحاث ــ مجلة المناهج وطرق التدريس ــ المجلد األول ــ العدد ال سادس عشر ــ ديسمبر 2022 م جلة العربية للعلوم ونشر األبحاث ــ مجلة المناهج وطرق التدريس ــ المجلد األول ــ العدد ال سادس عشر ــ ديسمبر 2022م وتنوعت أداوت الدراسات السابقة بين البرامج واالختبارات واالستبانات، واتفقت الدراسة الحالية مع ،الدراسات السابقة التي استخدمت اختبارًا وقائمة مهارات واختلفت عنها في نوع البرنامج التعليمي، وتميزت هذه .الدراسة بأنها استخدمت برنامجً ا تعليمً ا قائمًا على استراتيجية التعليم املتمايز في تنمية املهارات النحوية واقتصرت أغلب الدراسات السابقة على منهج واحد، وما يميز هذه الدراسة أنها استخدمت منهجين، هما : (املنهج الوصفي، واملنهج شبه التجريبي)، واتفقت الدراسة الحالية مع الدراسات التي اختارت عينتها من املرحلة الجامعية، واختلفت عنها في العينة حيث اقتصرت الحالية على عينة من طلبة األقسام العلمية بكلية التربية- .صنعاء أوجه الستفادة من الدراسات السابقة : استفادت هذه الدر اسة من الدراسات السابقة من عدة وجوه، أهمها : استفادت هذه الدر اسة من الدراسات السابقة من عدة وجوه، أهمها : - ،تحديد مشكلة الدراسة وبلورة أسئلتها، وتعرف موقعها وأصالتها بين الدراسات السابقة وإثراء فصولها .النظرية والتطبيقية - ،تحديد مشكلة الدراسة وبلورة أسئلتها، وتعرف موقعها وأصالتها بين الدراسات السابقة وإثراء فصولها .النظرية والتطبيقية - تحديد منهج الدراسة املناسب، وأدوات جمع البيانات املناسبة، وكيفية التحقق من صدقها وثباتها ، وكيفية تتابع إجراءات دراسة املشكلة، واختيار األساليب اإلحصائية املناسبة ملعالجة البيانات، وكيفية استخالص .النتائج وعرضها ومناقشتها وتفسيرها - تحديد منهج الدراسة املناسب، وأدوات جمع البيانات املناسبة، وكيفية التحقق من صدقها وثباتها ، وكيفية تتابع إجراءات دراسة املشكلة، واختيار األساليب اإلحصائية املناسبة ملعالجة البيانات، وكيفية استخالص .النتائج وعرضها ومناقشتها وتفسيرها - تحديد امل هارات النحوية الالزمة لطلبة األقسام العلمية بكلية التربية صنعاء التي يمكن تنميتها عن طريق تدريسها باستراتيجية التعليم املتمايز. - تحديد امل هارات النحوية الالزمة لطلبة األقسام العلمية بكلية التربية صنعاء التي يمكن تنميتها عن طريق تدريسها باستراتيجية التعليم املتمايز. - كيفية إعداد برنامج تعليمي مصمم وفق استراتيجية التعليم املتمايز ، وإعداد اختبار امل هارات النحوية. م - .اإلفادة من بعض املراجع التي أشارت إليها الدراسات السابقة - .اإلفادة من بعض املراجع التي أشارت إليها الدراسات السابقة - .اإلفادة من بعض املراجع التي أشارت إليها الدراسات السابقة 3- منهجية الدراسة و إجراءات.ها 1. منهج الدراسة وتصميمها التجريبي : اعتمدت الدراسة منهجي ن هما : (املنهج الوصفي) في جمع املعلومات والبيانات وتحليلها وتفسيرها، و(املنهج التجريبي) الذي تم من خالله اختيار أسلوب تصميم املجموعة التجريبية الواحدة؛ لبيان أثر املتغير التجريبي املستقل .)(التعليم املتمايز) في تنمية املتغير التابع (املهارات النحوية 1. منهج الدراسة وتصميمها التجريبي : 2. مجتمع الدراسة وعينتها : تمثل مجتمع الدراسة بجميع طلبة األقسام العلمية بكلية التربية– صنعاء في املستوى األول للعام الجامعي ( 2021م- 2022 ) ( ، وتكونت عينة الدراسة امليدانية من41 ً) طالب ا وطالبة من طلبة األقسام العلمية مستوى أول املسجلين بقسمي (الرياضيات وعلوم الحياة ) في العام الجامعي ( 2021 م– 2022 ) ، تم اختيارهم بطريقة قصدية ليمثلوا مجموعة تجريبية واحدة تم تدريسهم املهارات النحوية املقررة عليهم في كتاب (اللغة العربية102 ) باستخدام .استراتيجية التعليم املتمايز وقد اقتصر الباحث على عينة تجريبية واحدة للمبررات اآلتية : لط أ ل ك ل لأ وقد اقتصر الباحث على عينة تجريبية واحدة للمبررات اآلتية :  أن عدد الطلبة في .األقسام العلمية بكلية التربية صنعاء قليلون  أن قسم الفيزياء لم يلتحق به أي طالب للمستوى األول في العام الجامعي ( 2021 م– 2022 ). كما أنه تم .اختيار العينة االستطالعية من قسم الكيمياء ب ن ي ء ي ون ربي ص مي ب ي مأ  أن قسم الفيزياء لم يلتحق به أي طالب للمستوى األول في العام الجامعي ( 2021 م– 2022 ). كما أنه تم .اختيار العينة االستطالعية من قسم الكيمياء أثر استخدا م استراتيجية التعليم المتمايز في تنمية المهارات النحوية في مقرر اللغة العربية لدى طلبة األقسام العلمية ( 106 ) ( 106 ) الجعدي المجلة العربية للعلوم ونشر األبحاث ــ مجلة المناهج وطرق التدريس ــ المجلد األول ــ العدد ال سادس عشر ــ ديسمبر 2022 م جلة العربية للعلوم ونشر األبحاث ــ مجلة المناهج وطرق التدريس ــ المجلد األول ــ العدد ال سادس عشر ــ ديسمبر 2022م  أن قسمي الرياضيات وعلوم الحياة يدرسون مقرر (اللغة العربية 102 ّ) في وقت مت حد وقاعة واحدة أي )(مدمجين ، مما يجعل من املتوقع تشابه خصائصهم االجتماعية والثقافية واالقتصادية وتشابه لهجة .كالمهم  أن قسمي الرياضيات وعلوم الحياة يدرسون مقرر (اللغة العربية 102 ّ) في وقت مت حد وقاعة واحدة أي )(مدمجين ، مما يجعل من املتوقع تشابه خصائصهم االجتماعية والثقافية واالقتصادية وتشابه لهجة .كالمهم  استعداد دكتور .املادة للتعاون مع الباحث  قدرة الباحث على بذل الجهد الذي يتطلبه تنفيذ التجربة مع هذه العينة املحدودة، ومناسبة ا لزمن لجمع .البيانات ومعالجتها إحصائيًا، ودقة نتائج التجارب ذات العينات املعتدلة  قدرة الباحث على بذل الجهد الذي يتطلبه تنفيذ التجربة مع هذه العينة املحدودة، ومناسبة ا لزمن لجمع .البيانات ومعالجتها إحصائيًا، ودقة نتائج التجارب ذات العينات املعتدلة 3. أداة الدراسة : اُستخدم في هذه الدراسة ٌاختبار ؛ لقياس امل هارات النحوية ، ويهدف االختبار إلى قياس أثر استخدام استراتيجية التعليم املتمايز في تنمية امل هارات النحوية املستهدفة لدى طلبة األقسام العلمية بكلية التربية– صنعاء .)من خالل مقارنة متوسطي التطبيقين (القبلي والبعدي ا وتكوّن اختبار امل هارات النحوية ( من32 ) ًسؤاال تسلسل حسب تسلسل قائمة امل هارات النحوية التي تم تحكيمها ،بنصاب سؤال واحد لكل مهارة و تمت صياغة مفردات االختبار باالعتم ًاد على األسئلة املوضوعية؛ ممثال في أحد أنواعها، وهو االختيار من متعدد، وجاء اختيار هذا النوع من االختبارات؛ ملا يحققه من إيجابيات، تتمثل في أنه يحتاج إلى مجهود ووقـت قليـل، والتصـافه بمواصفات االختبار الجيد؛ حيث إ نه يتصف بإعطاء نفس النتائج مهما اختلف املص ًححون؛ مما يقلل نسبة التقدير الذاتي، كما أن األسئلة فيه محـددة، واإلجابات محددة أيض ا، ويقل فيه االلتباس ، ًوقد تم وضع مجموعة من البدائل للسؤال، يختار منها الطالب بديالً واحـد ا. كما تم صياغة االختبار حسب جدول املواصفات للموازنة املوضوعية بين أهمية املهارات ا لنحوية ومستويات األهداف املعرفية من املجال املعرفي لتصنيف (بلو)، حيث تم التركيز على املهارات العقلية العليا :(الفهم، والتطبيق، والتحليل، والتقوي م). وبعد ذلك تم التأكد من الصدق الظاهري لالختبار بعرضه على عدد من املتخصصين في مناهج اللغة العربية وطرائق تدريسه ا وفي القياس والتقويم. كما صُدِّ ر االختبار بخطاب للمحكمين إلبداء آرائهم في صالحيته للتطبيق امليداني ألغراض البحث العلمي. بَيِّن لهم هدف االختبار وطريقة إبداء آرائهم فيه، وقد أجمع كل املحكمين على صالحية اختبار امل هارات النحوية وملحقاته ألغراض البحث العلمي ب صورته الراهنة عدا صياغة بعض الفقرات ( وترتيبها، وبذلك أصبح االختبار في صورته النهائية مكوناً من32 ) ًسؤاال. وبعد االنتهاء من إيجاد الصدق الظاهري لالختبار َجُ رِّب على عينة ( استطالعية من20 ) طالبًا ب قسم الكيمياء بكلية التربية- صنعاء ، من خارج عينة الدراسة؛ اس قي ل خصائص صدق االختبار وثباته ومعامالت صعوبته وتمييز فقراته. 3. أداة الدراسة : صدق التساق الداخلي : ًللتأكد من صدق فقرات االختبار، أوجد الباحث االتساق بين الفقرات والدرجة الكلية لالختبار مستخدم ا في ذلك البرنامج اإل( حصائيspss ) إليجاد ذلك، كما هو موضح في الجدول اآلتي : جدول(1) معامالت الرتباط بين كل فقرة من فقرات الختبار مع الدرجة الكلية لالختبار جدول(1) معامالت الرتباط بين كل فقرة من فقرات الختبار مع الدرجة الكلية لالختبار رقم الفقرة معامل الرتباط مستوى الدللة رقم الفقرة معامل الرتباط مستوى الدللة رقم الفقرة معامل الرتباط مستوى الدللة 1 .387* دال عند0.05 12 .380* دال عند0.05 23 .514* دال عن د0.05 2 .684 دال عند0.01 13 .577 دال عند0.01 24 .554* دال عند0.05 ج و(اب ر ي رج ب ر ع ر ر ن بين ل ارب ) رقم الفقرة معامل الرتباط مستوى الدللة رقم الفقرة معامل الرتباط مستوى الدللة رقم الفقرة معامل الرتباط مستوى الدللة 1 .387* دال عند0.05 12 .380* دال عند0.05 23 .514* دال عن د0.05 2 .684 دال عند0.01 13 .577 دال عند0.01 24 .554* دال عند0.05 ( 107 ) المجلة العربية للعلوم ونشر األبحاث ــ مجلة المناهج وطرق التدريس ــ المجلد األول ــ العدد ال سادس عشر ــ ديسمبر 022 رقم الفقرة معامل الرتباط مستوى الدللة رقم الفقرة معامل الرتباط مستوى الدللة رقم الفقرة معامل الرتباط مستوى الدللة 3 .524 دال عند0.01 14 .504 دال عند0.05 25 .622 دال عند0.01 4 .438 دال عند0.01 15 .460 دال عند0.01 26 .433 دال عند0.01 5 .702 دال عند0.01 16 .499 دال عند0.01 27 .358 دال عند0.01 6 .432 دال عند0.01 17 .434 دال عند0.01 28 .566 دال عند0.01 7 .662 دال عند0.01 18 .509 دال عند0.01 29 .391 دال عند0.01 8 .388 دال عند0.01 19 .652 دال عند0.01 30 .715 دال عند0.01 9 .734 دال عند0.01 20 .625 دال عند0.01 31 .584 دال عند0.01 10 .470 دال عند0.01 21 .482 دال عند0.01 32 .469 دال عند0.01 11 .524* دال عند0.05 22 .530 دال عند0.01 اا 6 .432 دال عند0.01 17 .434 دال عند0.01 28 .566 دال عند0.01 7 .662 دال عند0.01 18 .509 دال عند0.01 29 .391 دال عند0.01 8 .388 دال عند0.01 19 .652 دال عند0.01 30 .715 دال عند0.01 9 .734 دال عند0.01 20 .625 دال عند0.01 31 .584 دال عند0.01 10 .470 دال عند0.01 21 .482 دال عند0.01 32 .469 دال عند0.01 11 .524* دال عند0.05 22 .530 دال عند0.01 - دال احصائياً عند مستوى داللة ( 0.01 ) - دال احصائياً عند مستوى داللة ( 0.05 ) يتضح من ال جدول السابق أن جميع ًفقرات االختبار ترتبط بمتوسط الدرجة الكلية لالختبار ارتباط ا ا ذ داللة إ حصائية عند مستوى داللة ( 0.01) لبعض الفقرات، وعند مستوى داللة ( 0.05 ) لبعض الفقرات األخرى، وهذا يؤكد اتساق فقرات االختبار، ُأي صدق االختبار ملا أ.عد له معامل الصعوبة : إليجاد معامل صعوبة كل فقرة ،قام الباحث يرت بت ب نتائج االختبار االستطالعي ًترتيب ًا تنازلي ا (من األكبر إ لى األصغر )، وقسم العينة إ لى مجموعتين (عليا- دنيا)، وتم أخذ27 ، من املجموعة العليا% 27 من الدنيا. 3. أداة الدراسة : ثم استخدم% الباحث املعادلة اآلتية لحساب معامل الصعوبة وهي: وكانت النتائج كما هي موضحة في الجدول .اآلتي جدول (2) يوضح معامل الصعوبة لفقرات الختبار رقم الفقرة معامل الصعوبة رقم الفقرة معامل الصعوبة رقم الفقرة معامل الصعوبة 1 0.37 12 0.55 23 0.52 2 0.48 13 0.46 24 0.42 3 0.60 14 0.49 25 0.43 4 0.40 15 0.43 26 0.60 5 0.47 16 0.51 27 0.58 6 0.50 17 0.40 28 0.46 7 0.52 18 0.46 29 0.59 8 0.42 19 0.59 30 0.36 9 0.43 20 0.36 31 0.39 10 0.60 21 0.39 32 0.52 11 0.58 22 0.52 يتضح من الجدول (2 ( ) أن معامل الصعوبة تراوحت بين0.36 - 0.60) وهي معامالت صعوبة مق.بولة = معامل الصعوبة عدد الطلبة الذين أجابوا عن الفقرة إجابة صحيحة إجمالي عدد الطلبة × 100 - * دال احصائياً عند مستوى داللة ( 0.01 ) - *دال احصائياً عند مستوى داللة ( 0.05 ) يتضح من ال جدول السابق أن جميع ًفقرات االختبار ترتبط بمتوسط الدرجة الكلية لالختبار ارتباط ا ا ذ داللة إ حصائية عند مستوى داللة ( 0.01) لبعض الفقرات، وعند مستوى داللة ( 0.05 ) لبعض الفقرات األخرى، وهذا يؤكد اتساق فقرات االختبار، ُأي صدق االختبار ملا أ.عد له يتضح من ال جدول السابق أن جميع ًفقرات االختبار ترتبط بمتوسط الدرجة الكلية لالختبار ارتباط ا ا ذ داللة إ حصائية عند مستوى داللة ( 0.01) لبعض الفقرات، وعند مستوى داللة ( 0.05 ) لبعض الفقرات األخرى، وهذا يؤكد اتساق فقرات االختبار، ُأي صدق االختبار ملا أ.عد له معامل الصعوبة : إليجاد معامل صعوبة كل فقرة ،قام الباحث يرت بت ب نتائج االختبار االستطالعي ًترتيب ًا تنازلي ا (من األكبر إ لى األصغر )، وقسم العينة إ لى مجموعتين (عليا- دنيا)، وتم أخذ27 ، من املجموعة العليا% 27 من الدنيا. 3. أداة الدراسة : ثم استخدم% الباحث املعادلة اآلتية لحساب معامل الصعوبة وهي: = معامل الصعوبة عدد الطلبة الذين أجابوا عن الفقرة إجابة صحيحة إجمالي عدد الطلبة × 100 وكانت النتائج كما هي موضحة في الجدول .اآلتي جدول (2) يوضح معامل الصعوبة لفقرات الختبار رقم الفقرة معامل الصعوبة رقم الفقرة معامل الصعوبة رقم الفقرة معامل الصعوبة 1 0.37 12 0.55 23 0.52 2 0.48 13 0.46 24 0.42 3 0.60 14 0.49 25 0.43 4 0.40 15 0.43 26 0.60 5 0.47 16 0.51 27 0.58 6 0.50 17 0.40 28 0.46 7 0.52 18 0.46 29 0.59 8 0.42 19 0.59 30 0.36 9 0.43 20 0.36 31 0.39 10 0.60 21 0.39 32 0.52 11 0.58 22 0.52 يتضح من الجدول (2 ( ) أن معامل الصعوبة تراوحت بين0.36 - 0.60) وهي معامالت صعوبة مق.بولة ( 108 ) الجعدي المجلة العربية للعلوم ونشر األبحاث ــ مجلة المناهج وطرق التدريس ــ المجلد األول ــ العدد ال سادس عشر ــ ديسمبر 2022 م معامل التمييز : وإليجاد معامل التمييز استخدم الباحث املعادلة اآلتية : معامل التمييز = عدد اإلجابات الصحيحة للفئة العليا- عدد اإلج ابات الصحيحة للفئة الدنيا عدد أفراد إحدى املجموعتين × 100 معامل التمييز : وإليجاد معامل التمييز استخدم الباحث املعادلة اآلتية : وكانت النتائج كما هي موضحة في الجدول .اآلتي جدول (3) يوضح معامل التمييز للفقرات رقم الفقرة معامل الصعوبة رقم الفقرة معامل الصعوبة رقم الفقرة معامل الصعوبة 1 0.48 12 0.60 23 0.64 2 0.55 13 0.48 24 0.53 3 0.72 14 0.55 25 0.65 4 0.49 15 0.65 26 0.49 5 0.64 16 0.62 27 0.61 6 0.52 17 0.57 28 0.40 7 0.50 18 0.55 29 0.35 8 0.63 19 0.59 30 0.50 9 0.44 20 0.61 31 0.56 10 0.43 21 0.45 32 0.48 11 0.65 22 0.63 يتضح من الجدول (3) أن معامل التمييز لفقرات االختبار تراوحت بين ( 0.35 - 0.72) وهي معامالت مقبولة. 3. أداة الدراسة : معامل التمييز = عدد اإلجابات الصحيحة للفئة العليا- عدد اإلج ابات الصحيحة للفئة الدنيا عدد أفراد إحدى املجموعتين رقم الفقرة معامل التمييز رقم الفقرة معامل التمييز 1 0.30 17 0.50 2 0.40 18 0.30 3 0.40 19 0.40 4 0.50 20 0.50 5 0.30 21 0.70 6 0.50 22 0.40 7 0.30 23 0.40 8 0.40 24 0.50 9 0.60 25 0.60 10 0.70 26 0.60 11 0.50 27 0.70 12 0.40 28 0.70 13 0.60 29 0.60 14 0.50 30 0.60 15 0.40 31 0.70 16 0 70 32 0 50 × 100 ثبات الختبار : للتأكد من ثبات االختبار، استخدم الباحث طريقة )(ألفا كرونباخ : ( حيث حصل ثبات االختبار على0.918 .) وبهذا يكون الباحث قد تأكد من صدق وثبات االختبار، وأصبح االختبا ر يطمئن الباحث لتطبيقه على عينة الدراسة. ييز ل ي ي اج ب ي ي اج ب عدد أفراد إحدى المجموعتين ب ر ب للتأكد من ثبات االختبار، استخدم الباحث طريقة )(ألفا كرونباخ : ( حيث حصل ثبات االختبار على0.918 .) وبهذا يكون الباحث قد تأكد من صدق وثبات االختبار، وأصبح االختبا ر يطمئن الباحث لتطبيقه على عينة الدراسة. اا عدد أفراد إحدى المجموعتين تنفيذ التطبيق : ( قام الباحث بتطبيق اختبار املهارات النحوية القبلي على عينة الدراسة بتاري28 /2/ 2022 ) ، ثم بدأ الباحث بتنفيذ التجربة امليدانية بتدريس املهارات النحوية باستراتيجية التعليم املتمايز على املجموعة التجريبي ة تاري (7/3/ 2022 )؛ حيث استغرق تنفيذ التجربة امليدانية مع االختبارين القبلي والبعدي (تسعة أسابيع) بواقع محاضرة أسبوعيًا، تحددت بيوم (اإل ثنين) من كل أسبوع من الساعة العاشرة صباحً ا وحتى الساعة الثانية عشرة ظهرًا حسب ( الجدول املخصص ملحاضرة مقرر اللغة العربية102) بكلية التربية- صنعاء، ابتداءً من يوم اإل ثنين بتاري 7) /3/ 2022 )، وانتهاءً يوم اإل( ثنين بتاري23 /5/ 2022 ) ، ،تخلل الفترة إجازة شهر رمضان املبارك وبذلك تكون البرنامج التعليمي من تسع محاضرات، زمن املحاضرة (ساعت ا،ن) حسب املحاضرات الجامعية لشهادة البكالوريوس كما .خصص الباحث فقرة ختامية ملراجعة وتقييم البرنامج التعليمي م و طُبِقَ االختبار البعدي ل لمهارات النحوية يوم اإلثنين بتاري) 30 /5/ 2022 ) ، بإشراف الباحث في ظروف متشابهة لالختبار القبلي . تنفيذ التطبيق : والجدول اآلتي يوضح الخطة الزمنية لتنفيذ البرنامج التعليمي القائم على استرا تيجية .التعليم املتمايز ( 109 ) الجعدي ( 109 ) المجلة العربية للعلوم ونشر األبحاث ــ مجلة المناهج وطرق التدريس ــ المجلد األول ــ العدد ال سادس عشر ــ ديسمبر 2022 م (جدول4) الخطة الزمنية لتدريس البرنامج التعليمي للعينة التجريبية املحاضرة الدروس مدة املحاضرة التاريخ األولى االختبار القبلي 28 /2/ 2022م الثانية الفاعل– .نائب الفاعل ساعتان 7/3/ 2022م الثالثة املفعول به– املفعول املطلق ساعتان 14 /3/ 2022 .م الرابعة املفعول ألجله– املفعول فيه ساعتان 21 /3/ 2022 .م الخامسة املفعول معه– االستثناء ساعتان 28 /3/ 2022 .م السادسة الحال– التمييز ساعتان 9/5/ 2022 .م السابعة الفعل املضارع ساعتان 16 /5/ 2022 .م الثامنة العدد ساعتان 23 /5/ 2022 .م التاسعة االختبار الب عدي 30 /5/ 2022 .م (جدول4) الخطة الزمنية لتدريس البرنامج التعليمي للعينة التجريبية املحاضرة الدروس مدة املحاضرة التاريخ األولى االختبار القبلي 28 /2/ 2022م الثانية الفاعل– .نائب الفاعل ساعتان 7/3/ 2022م الثالثة املفعول به– املفعول املطلق ساعتان 14 /3/ 2022 .م الرابعة املفعول ألجله– املفعول فيه ساعتان 21 /3/ 2022 .م الخامسة املفعول معه– االستثناء ساعتان 28 /3/ 2022 .م السادسة الحال– التمييز ساعتان 9/5/ 2022 .م السابعة الفعل املضارع ساعتان 16 /5/ 2022 .م الثامنة العدد ساعتان 23 /5/ 2022 .م التاسعة االختبار الب عدي 30 /5/ 2022 .م األساليب اإلحصائية ملعالجة بيانات الدراسة : عُوْلِ جَ تْ بيانات الدراسة بواسطة الحاسب اآللي، باستعمال الرزمة اإلحصائية في العلوم االجتماعية ( واإلنسانيةSPSS ،)، واستُعْمِلَ كلٌّ من اإلحصاء الوصفي واالستداللي في معالجة بيانات الدراسة وتحليلها وهي : (املتوسطات الحسابية– االنحرافات املعيارية- معامل ارتباط (بيرسونpearson )- ( معامل كرونباخ ألفا)- ( اختبارT- test .) (جدول4) الخطة الزمنية لتدريس البرنامج التعليمي للعينة التجريبية األساليب اإلحصائية ملعالجة بيانات الدراسة : عُوْلِ جَ تْ بيانات الدراسة بواسطة الحاسب اآللي، باستعمال الرزمة اإلحصائية في العلوم االجتماعية ( واإلنسانيةSPSS ،)، واستُعْمِلَ كلٌّ من اإلحصاء الوصفي واالستداللي في معالجة بيانات الدراسة وتحليلها وهي : (املتوسطات الحسابية– االنحرافات املعيارية- معامل ارتباط (بيرسونpearson )- ( معامل كرونباخ ألفا)- ( اختبارT- test .) 4- عرض النتائج ومناقشتها. 4- عرض النتائج ومناقشتها.  نتائج اإلجابة عن السؤال األول :" ما املهارات النحوية الالزمة ل طلبة األقسام العلمية بكلية التربية– صنعاء ؟" ول إلجابة عن هذا السؤال، تم الرجوع إلى كتاب (اللغة العربية102 ) املقرر على طلبة األقسام العلمية بكلية التربية– ،صنعاء كمتطلب جامعي ومراجعة األدب النظري والدراسات السابقة ذات الصلة ب امل هارات النحوية ، وآراء ،بعض الخبراء في مناهج اللغة العربية وطرائق تدريسها ثم أُعِدَّ ت قائمة ب امل هارات النحوية التي يمكن تنميتها لدى طلبة األقسام العلمية بكلية التربية صنعاء من املوضوعات املقررة عليهم، وعرضها على املحكمين في أدوات الدراسة للتأكد من صدقها وإبداء آرائهم حول مدى مناسبة هذه املهارات ،لطلبة األقسام العلمية وتكونت القائم ة بعد ( تحكيمها من32) مهارة ، ويبين الجدول اآلتي املهارات النحوية في .صورتها النهائية ي ي يم يآ ( جدول5 )يبين قائمة املهارات النحوية الالزمة لطلبة األقسام العلمية بكلية التربية صنعاء في صورتها و ه و ه ي رب ب مأ زا وي مه ر ي م املهارات النحوية م املهارات النحوية 1 يستخرج الفاعل من أمثلة.معطاة 17 .يميز بين الظرف املتصرف وغير املتصرف من خالل أمثلة معطاة 2 .يعرب الفاعل في أمثلة معطاة 18 .يستخرج املفعول معه من أمثلة معطاة 3 .يوضح حكم إعراب الفاعل 19 .يعرب املفعول معه إعرابًا صحيحً ا 4 .يوضح نوع الفاعل في أمثلة معطاة 20 يستخرج أركان الستثن.اء من أمثلة معطاة 5 .يستخرج نائب الفاعل من أمثلة معطاة 21 .يقارن بين أنواع الستثناء من حيث األداة واإلعراب 6 .يعرب نائب الفاعل في أمثلة معطاة 22 .يعرب الحال في أمثلة معطاة 7 .يستخرج املفعول به من أمثلة معطاة 23 .يوضح نوع الحال من خالل أمثلة معطاة ( 110 ) المجلة العربية للعلوم ونشر األبحاث ــ مجلة المناهج وطرق التدريس ــ المجلد األول ــ العدد ال سادس عشر ــ ديسمبر 2022 م م املهارات النحوية م املهارات النحوية 8 ي.عرب املفعول به في أمثلة معطاة 24 .يبين صاحب الحال في أمثلة معطاة 9 .يوضح نوع املفعول به في أمثلة معطاة 25 .يوضح نوع التمييز في أمثلة معطاة 10 .يستخرج املفعول املطلق من أمثلة معطاة 26 .يعرب التمييز إعرابًا صحيحً ا 11 يوضح نوع املفعول املطلق في أمثلة معطا.ة 27 .يبين أدوات نصب الفعل املضارع 12 يستخرج النائب عن املفعول املطلق من أمثلة .معطاة 28 .يوضح سبب نصب الفعل املضارع بأن مضمرة وجوبًا أو جوازًا 13 .يستخرج املفعول ألجله من أمثلة معطاة 29 .يميز بين األدوات التي تجزم فعالً مضارعًا واحدً ا والتي تجزم فعلين 14 .يعرب املفعول ألجله في أمثلة معطاة 30 .يعرب الفعل املضارع املجزوم إعرابًا صحيحً ا 15 يستخرج ظرفي الزمان واملكان من أمثلة معطاة 31 .يبين سبب اقتران جواب الشرط بالفاء من خالل أمثلة معطاة 16 .يميز بين الظرف املحدد واملبهم في أمثلة معطاة 32 أن يكتب األعدا.د كتابة صحيحة  نتائج اإلجابة على السؤال الثاني : "ما صورة برنامج تعليمي قائم على استراتيجية التعليم املتمايز في تنمية املهارات النحوية لدى طلبة األقسام العلمية بكلية التربية – ."صنعاء؟ و لإلجابة عن هذا السؤال قام الباحث باالطالع على الدراسات السابقة ذات الص لة باستراتيجية التعليم املتمايز وتطبيقاته التربوية، وكيفية بناء البرامج التعليمية وفق استراتيجية التعليم املتمايز، كما قام الباحث ،باستشارة بعض الخبراء واملختصين في مناهج اللغة العربية وطرائق تدريسها واسترشد بآرائهم َّثم أُعد برنامجً ا تعليميًا قائمً ا على ا ستراتيجية التعليم املتمايز لتنمية املهارات النحوية ، وعرضه على املحكمين في أدوات الدراسة ؛ للتحقق من صدقه ومناسبته لطلبة األقسام العلمية ب الكلية ، ثم عرضه على املجموعة التجريبية بغرض تنمية امل هارات.النحوية وبدأ البرنامج التعليمي القائم على استراتيجية التعليم املتمايز بالهدف العام، وتحديد املستهدفين في البرنامج ،من طلبة األقسام العلمية بكلية التربية صنعاء، ومصادر بناء البرنامج ومبرراته، ثم أهدافه الخاصة املتمثلة امل ب هارات النحوية املحددة في قائمة املهارات، وتكوّن البرنامج التعليمي في صورته النهائية بعد تحكيمه( من7 ) محاضرات كل محاضرة احتوت على درسين من كتاب (اللغة العربية102 ) املقرر على طلبة األقسام العلمية بكلية التربية– ،صنعاء كمتطلب جامعي وكل درس فيه عدد من األنشطة القائمة على التعليم املتمايز املناسبة لكل درس .نحوي ُور وعي عند اختيار املحتوى مناسبته لحاجات ،الطلبة ،وميولهم وقدراتهم، والفروق الفردية بينهم، وربطه باألهداف العامة .للبرنامج أما استراتيجيات التدريس املقترحة لتنفيذ البرنامج التعليمي فقد تم االستناد إلى توليفية مجتمعة من استراتيجيات التدريس املتمايز املتمثلة في :(املجموعات املرنة – األنشطة املتدرج ة- املحطات التعليمية- فكر ،زاوج ، شارك – العصف الذهني– األلعاب التعليمية– األنشطة الثابتة– االكتشاف- حل املشكالت)، بحيث تمتزج هذ ه االستراتيجيات جميعًا مع بعضها.، وبحيث تناسب قدرات الطلبة ومستوياتهم، بهدف تمكينهم من املهارات النحوية وتم تحديد واختيار األن شطة التعليمية وفقً ا الهتمامات الطلبة وقدراتهم، وقد تضمن البرنامج عددًا من األنشطة، ورُ وعي في إعدادها واختيارها مجموعة من املعايير التي يجب أن تتصف بها األنشطة. 4- عرض النتائج ومناقشتها. 4- عرض النتائج ومناقشتها. 4- عرض النتائج ومناقشتها. كما تم استخدام العديد من الوسائل التعليمية التي تساعد على تنفيذ البرنامج التعليمي القائم على است راتيجية التعليم املتمايز، ومنها : ،(بطاقات ملونة تساعد على تنمية املهارات النحوية، ولوحات لكتابة بعض النصوص، وأقالم ملونة، وأوراق عمل .)والسبورة التعليمية، والحاسب اآللي (البوربوينت)، وجهاز عرض الشفافيات أثر استخدا م استراتيجية التعليم المتمايز في تنمية المهارات النحوية في مقرر اللغة العربية لدى طلبة األقسام العلمية أثر استخدا م استراتيجية التعليم المتمايز في تنمية المهارات النحوية في مقرر اللغة العربية لدى طلبة األقسام العلمية ( 111 ) الجعدي جلة العربية للعلوم ونشر األبحاث ــ مجلة المناهج وطرق التدريس ــ المجلد األول ــ العدد ال سادس عشر ــ ديسمبر 2022م المجلة العربية للعلوم ونشر األبحاث ــ مجلة المناهج وطرق التدريس ــ المجلد األول ــ العدد ال سادس عشر ــ ديسمبر 2022 م أما في تقويم البرنامج التعليمي فقد استُخدم التقويم ال قبلي من خالل طرح األسئلة في بداية املحاضرات للكشف عن خبرات الطلبة، وتهيئتهم وإثارة دافعتيهم للتعلم ، و التقويم التكويني الذي يتم خالل تدريس املهارات النحوية ، وذلك عن طريق طرح األسئلة للكشف عن مدى تحقق األهداف في كل محاضرة ، باإلضافة إلى تفعيل دور الطلبة وضمان مشاركتهم ودمجهم في املوقف التعليمي واستثارة انتباههم باستمرار ، و التقويم الختامي يتم في نهاية كل محاضرة ؛ للتأكد من تحقيق األهداف التعليمية التي وضع ت لكل مهارة، وكذلك تم استخدام تقويم ختامي للبرنامج بشكل متكامل يقيس مدى تحقق أهداف البرنامج في تنمية املهارات النحوية. كما تم تحديد الزمن املناسب لتنفيذ البرنامج التعليمي القائم على استراتيجية التعليم املتمايز حيث تم تنفيذ البرنامج يوم اإلثنين من كل أسبوع من الساعة العاشرة صباحً ا حتى الساعة الثانية عشرة ظهرًا، والجدول اآلتي يوضح دروس البرنامج التعليمي القائم على.استراتيجية التعليم املتمايز ( جدول رقم6) يوضح دروس البرنامج التعليمي القائم على استراتيجية التعليم املتمايز أما في تقويم البرنامج التعليمي فقد استُخدم التقويم ال قبلي من خالل طرح األسئلة في بداية املحاضرات للكشف عن خبرات الطلبة، وتهيئتهم وإثارة دافعتيهم للتعلم ، و التقويم التكويني الذي يتم خالل تدريس املهارات النحوية ، وذلك عن طريق طرح األسئلة للكشف عن مدى تحقق األهداف في كل محاضرة ، باإلضافة إلى تفعيل دور الطلبة وضمان مشاركتهم ودمجهم في املوقف التعليمي واستثارة انتباههم باستمرار ، و التقويم الختامي يتم في نهاية كل محاضرة ؛ للتأكد من تحقيق األهداف التعليمية التي وضع ت لكل مهارة، وكذلك تم استخدام تقويم ختامي للبرنامج بشكل متكامل يقيس مدى تحقق أهداف البرنامج في تنمية املهارات النحوية. 4- عرض النتائج ومناقشتها. ( جدول رقم6) يوضح دروس البرنامج التعليمي القائم على استراتيجية التعليم املتمايز م املحاضرة الدروس مدة املحاضرة التاريخ األولى الفاعل– .نائب الفاعل ساعتان 7/3/ 2022م الثانية املفعول به– املفعول املطلق ساعتان 14 /3/ 2022 .م الثالث ة املفعول ألجله– املفعول فيه ساعتان 21 /3/ 2022 .م الرابعة املفعول معه– االستثناء ساعتان 28 /3/ 2022 .م الخامسة الحال– التمييز ساعتان 9/5/ 2022 .م السادسة الفعل املضارع ساعتان 16 /5/ 2022 .م السابعة العدد ساعتان 23 /5/ 2022 .م م املحاضرة الدروس مدة املحاضرة التاريخ األولى الفاعل– .نائب الفاعل ساعتان 7/3/ 2022م الثانية املفعول به– املفعول املطلق ساعتان 14 /3/ 2022 .م الثالث ة املفعول ألجله– املفعول فيه ساعتان 21 /3/ 2022 .م الرابعة املفعول معه– االستثناء ساعتان 28 /3/ 2022 .م الخامسة الحال– التمييز ساعتان 9/5/ 2022 .م السادسة الفعل املضارع ساعتان 16 /5/ 2022 .م السابعة العدد ساعتان 23 /5/ 2022 .م  نتائج اإلجابة عن السؤال الثالث :"ما أثر تدريس البرنامج التعليمي القائم على استراتيجية التعليم املتمايز في تنمية املهارات النحوية لدى طلبة األقسام العلمية بكلية التربية– صنعاء."؟ تم التوصل لإلجابة عن هذا السؤال من خالل التجربة امليدانية للدراسة، وتطبيق أدواتها على عينة الدراسة، وتطبيق اختبا ر املهارات النحوية تطبيقً ا قبليًا وبعديًا، ثم معالجة البيانات إحصائيًا باستخدام البرنامج ( اإلحصائيspss )؛ وذلك لحساب الفرق بين متوسطات درجات املجموعة التجريبية في التطبيقين القبلي والبعدي الختبار املهارات النحوية، وفيما يلي تفصيل ذلك من خالل تفسير النتائج ومناقشتها في ضوء فرضية الدراسة اآلتية : o تنص الفرضية أنه :"توجد فروق دالة إحصائيًا عند مستوى الداللة ( 0.05 )بين متوسطي درجات املجموعة التجريبية في التطبيقين (القبلي والبعدي )الختبار املهارات النحوية على مستوى االختبار ككل وعلى مستوى كل مهارة مستقلة لصالح ال تطبيق ،البعدي يُعزى إلى تدريس املهارات النحوية باستراتيجية التعليم املتمايز." ّوللت ُّحق ق من صحة هذه الفرضية، حُ سِ بَ متوسطا املجموعة التجريبية )(عينة الدراسة وانحرافها املعياري في التطبيقين القبلي والبعدي الختبار امل هارات النحوية ، ثم حُ سِ بَتْ قيمة إحصاء اخت( بارt-test ) لعينتين مترابطتين الختبار الفرق اإلحصائي بين املتوسطين على مستوى االختبار ككل وعلى مستوى كل مهارة على حدة ، والجدول اآلتي .يبين هذه املؤشرات اإلحصائية ونتيجة االختبار أثر استخدا م استراتيجية التعليم المتمايز في تنمية المهارات النحوية في مقرر اللغة العربية لدى طلبة األقسام العلمية ( 112 ) الجعدي المجلة العربية للعلوم ونشر األبحاث ــ مجلة المناهج وطرق التدريس ــ المجلد األول ــ العدد ال سادس عشر ــ ديسمبر 2022 م ( جدول7) نتائج( اختبارt-test ) لعينتين مترابطتين لختبار دللة الفرق بين متو سطي مجموعة الدراسة في التطبيقين القبلي والبعدي.لختبار املهارات النحوية على مستوى املهارات مجتمعة املجموعة التطبيق العدد املتوسط النحراف درجة الحرية T الدللة ال تفسير حجم األثر التجريبية القبلي 41 11.0244 5.02736 40 20.453 0.000 دالة إحصائيًا 0.9127 ا لبعدي 41 27.3902 4.32942 ( يتضح من الجدول7) أن مجموعة الدراسة (التجريبية) حصلت في اختبار املهارات النحوية القبلي على ( املتوسط الحسابي11.0244 ( ) بانحراف معياري قدره5.02736 )، بينما حصلت في اختبار املهارات النحوية البعدي ( على املتوسط الحسابي27.3902) ب( انحراف معياري قدره4.32942 )، وملا كان املتوسط الحسابي لدرجات االختبار )البعدي أكبر من املتوسط الحسابي لدرجات االختبار القبلي لدى طلبة األقسام العلمية (الرياضيات، وعلوم الحياة بكلية التربية صنعاء، فإن ذلك يدل على أن املهارات النحوية لديهم أصبحت أعلى بعد تد ريسهم تلك املهارات ( باستراتيجية التعليم املتمايز، مما يدل على ذلك أن حجم األثر بلغ0.9127 ) وهو تأثير كبير جدً ا، إذ أنه عند ضرب ( املقدار في100 ( ) نجد أن ما يقارب91.27 ) من التباين في أداء طلبة األقسام العلمية في االختبارين، وتفسر قيمة% حجم األثر بمدى مصداقي( ة الداللة اإلحصائية لقيمةt ( )، حيث تشير قيمة اختبارt-test ( ) البالغ20.453 ) إلى وجود ( فرق إحصائي بين املتوسطين لصالح التطبيق البعدي؛ إذ أن مستوى الداللة0.000 ) صفري أقل من مستوى (الداللة املحدد في الدراسة بـ0.05 ). 4- عرض النتائج ومناقشتها. وهذا يدل على أن البرنامج التعليمي املصمم وفق استراتيجية التعليم املتمايز له تأثير كبير على تنمية املهارات النحوية لدى املجموعة التجريبية (مجموعة الدراسة) من طلبة األقسام العلمية (الرياضيات- وعلوم الحياة) بكلية التربية- .صنعاء ( جدول7) نتائج( اختبارt-test ) لعينتين مترابطتين لختبار دللة الفرق بين متو سطي مجموعة الدراسة في التطبيقين القبلي والبعدي.لختبار املهارات النحوية على مستوى املهارات مجتمعة املجموعة التطبيق العدد املتوسط النحراف درجة الحرية T الدللة ال تفسير حجم األثر التجريبية القبلي 41 11.0244 5.02736 40 20.453 0.000 دالة إحصائيًا 0.9127 ا لبعدي 41 27.3902 4.32942 ( يتضح من الجدول7) أن مجموعة الدراسة (التجريبية) حصلت في اختبار املهارات النحوية القبلي على ( املتوسط الحسابي11.0244 ( ) بانحراف معياري قدره5.02736 )، بينما حصلت في اختبار املهارات النحوية البعدي ( على املتوسط الحسابي27.3902) ب( انحراف معياري قدره4.32942 )، وملا كان املتوسط الحسابي لدرجات االختبار )البعدي أكبر من املتوسط الحسابي لدرجات االختبار القبلي لدى طلبة األقسام العلمية (الرياضيات، وعلوم الحياة بكلية التربية صنعاء، فإن ذلك يدل على أن املهارات النحوية لديهم أصبحت أعلى بعد تد ريسهم تلك املهارات ( باستراتيجية التعليم املتمايز، مما يدل على ذلك أن حجم األثر بلغ0.9127 ) وهو تأثير كبير جدً ا، إذ أنه عند ضرب ( املقدار في100 ( ) نجد أن ما يقارب91.27 ) من التباين في أداء طلبة األقسام العلمية في االختبارين، وتفسر قيمة% حجم األثر بمدى مصداقي( ة الداللة اإلحصائية لقيمةt ( )، حيث تشير قيمة اختبارt-test ( ) البالغ20.453 ) إلى وجود ( فرق إحصائي بين املتوسطين لصالح التطبيق البعدي؛ إذ أن مستوى الداللة0.000 ) صفري أقل من مستوى (الداللة املحدد في الدراسة بـ0.05 ). 4- عرض النتائج ومناقشتها. وهذا يدل على أن البرنامج التعليمي املصمم وفق استراتيجية التعليم املتمايز له تأثير كبير على تنمية املهارات النحوية لدى املجموعة التجريبية (مجموعة الدراسة) من طلبة األقسام العلمية (الرياضيات- وعلوم الحياة) بكلية التربية- .صنعاء لو لتعرف على حجم األثر الذي أحدثه املتغير املستقل (استراتيجية التعليم امل تمايز ) في املتغير التابع (تنمية املهارات النحوية ) لدى املجموعة التجريبية من طلبة األقسام العلمية بكلية التربية صنعاء في التطبيقين القبلي والبعدي، حُ سِ بَ (معامل إيتا)، والجدول اآلتي يبين قيمة حجم األثر بتطبيق مربع (إيتا) من خالل املعادلة اآلتية : 𝒕𝟐 𝒅𝒇+ 𝒕𝟐=األثر حجم ( حيثt () هي قيمة إحصائي االختبار التائي، و𝒅𝒇 .) درجة الحرية ( جدول8 ) حجم أثر البرنامج التعليمي القائم على استراتيجية التعليم املتمايز في تنمية املهارات النحوية لدى .املجموعة التجريبية من طلبة األقسام العلمية بكلية التربية صنعاء امل تغير املستقل املتغير التابع حجم األثر استراتيجية التعليم املتمايز تنمية املهارات النحوية 0.9127 ( يتضح من الجدول8 ) أن حجم انتقال أثر تدريس البرنامج التعليمي باستراتيجية التعليم املتمايز في تنمية امل هارات النحوية لدى املجموعة التجريبية قسمي (الرياضيات وع لوم الحياة) من طلبة األقسام العلمية بكلية التربية- صنعاء ( بلغ0.9127)، ويعَدُّ مرتفعًا مقارنة باملعيار الذي حدده املتخصصون بالقياس والتقويم مقداره ( 0.06 ) ، وهذا يدل على فاعلية استراتيجية التعليم املتمايز في تنمية املهارات النحوية لدى طلبة األقسام العلمية بك لية التربية .صنعاء المجلة العربية للعلوم ونشر األبحاث ــ مجلة المناهج وطرق التدريس ــ المجلد األول ــ العدد ال سادس عشر ــ ديسمبر 2022 م و يبين الجدول اآلتي رقم (9) املؤشرات اإلحصائية ونتيجة االختبار التائي الختبار الفرق اإلحصائي بين متوسطي املجموع ة التجريبية )(مجموعة الدراسة في اختبار امل هارات النحوية القبلي و البعدي على مستوى كل مهار ة على حدة (مستقلة) ، وقُرِنَتْ كل مهارة برتبتها بين بقية املهارات حسب متوسطات االختبار البعدي. 4- عرض النتائج ومناقشتها. ( جدول9 ) نتائج( اختبارt-test ) لعينتين مترابطتين لختبار دللة الفرق اإلحصائي بين متوسطي املجموعة التجريبية في التطبيقين القبلي والبعدي لختباراملهارات النحوية على مستوى كل مهارة على حدة ( جدول9 ) نتائج( اختبارt-test ) لعينتين مترابطتين لختبار دللة الفرق اإلحصائي بين متوسطي املجموعة التجريبية في التطبيقين القبلي والبعدي لختبار املهارات النحوية على مستوى كل مهارة على حدة م الـــمــــه ــــارة التطبيق املتوسط الـرتبـة درجــة الحريـة T الدللة التفسير 1 يستخرج الفاعل من أمثلة معطاة قبلي 0.3171 2 40 7.822 0.000 دالة إحصائيًا بعدي 0.9512 2 .يعرب الفاعل في أمثلة معطاة قبلي 0.3171 6 40 9.282 0.000 دالة إحصائيًا بعدي 0.8537 3 .يوضح حكم إعراب الفاعل قبلي 0.3659 1 40 7.625 0.000 دالة إحصائيًا بعدي 0.9756 4 يوضح نوع الفاعل في أمثلة .معطاة قبلي 0.2439 6م 40 6.934 0.002 دالة إحصائيًا بعدي 0.8537 5 يستخرج نائب الفاعل من أمثلة .معطاة قبلي 0.3659 5 40 5.563 0.340 غي ر دالة بعدي 0.878 6 يعرب نائب الفاعل في أمثلة .معطاة قبلي 0.2439 3 40 7.979 0.000 دالة إحصائيًا بعدي 0.9024 7 يستخرج املفعول به من أمثلة .معطاة قبلي 0.3171 1م 40 8.496 0.000 دالة إحصائيًا بعدي 0.9756 8 يعرب املفعول به في أمثلة مع.طاة قبلي 0.2927 6م 40 6.158 0.05 دالة إحصائيًا بعدي 0.8537 9 يوضح نوع املفعول به في أمثلة .معطاة قبلي 0.2439 8 40 6.870 0.000 دالة إحصائيًا بعدي 0.8049 10 يستخرج املفعول املطلق من .أمثلة معطاة قبلي 0.2439 3م 40 8.600 0.000 دالة إحصائيًا بعدي 0.9268 11 يوضح نوع املفعول املطلق في .أمثلة معطاة قبلي 0.439 1م 40 6.530 0.003 دالة إحصائيًا بعدي 0.9756 12 يستخرج النائب عن املفعول .املطلق من أمثلة معطاة قبلي 0.3902 3م 40 6.137 0.002 دالة إحصائيًا بعدي 0.9268 13 يستخرج امل فعول ألجله من أمثلة .معطاة قبلي 0.3171 6م 40 5.808 0.009 دالة إحصائيًا بعدي 0.8537 14 يعرب املفعول ألجله في أمثلة .معطاة قبلي 0.2927 5م 40 6.606 0.000 دالة إحصائيًا بعدي 0.878 15 يستخرج ظرفي الزمان واملكان من .أمثلة معطاة قبلي 0.561 8م 40 2.429 0.000 دالة إحصائيًا بعدي 0.8049 16 يميز بين الظرف املحدد واملبهم في .أمثلة معطاة قبلي 0.3902 6م 40 4.865 0.000 دالة إحصائيًا بعدي 0.8537 ( 114 ) الجعدي المجلة العربية للعلوم ونشر األبحاث ــ مجلة المناهج وطرق التدريس ــ المجلد األول ــ العدد ال سادس عشر ــ ديسمبر 2022 م م الـــمــــه ــــارة التطبيق املتوسط الـرتبـة درجــة الحريـة T الدللة التفسير 17 يميز بين الظرف املتصرف وغير .املتصرف من خالل أمثلة معطاة قبلي 0.3171 8م 40 5.048 0.01 دالة إحصائيًا بعدي 0.8049 18 يستخرج املفعول معه من أمثلة .معطاة قبلي 0.4878 2م 40 5.385 0.000 دالة إحصائيًا بعدي 0.9512 19 يعرب املفعول معه إعرابًا .صحيحً ا قبلي 0.439 5م 40 4.671 0.000 دالة إحصائيًا بعدي 0.878 20 يستخرج أركان الستثن اء من .أمثلة معطاة قبلي 0.2683 11 40 4.372 0.628 غير دالة إحصائيًا بعدي 0.7073 21 يقارن بين أنواع الستثناء من .حيث األداة واإلعراب قبلي 0.3415 2م 40 7.404 0.000 دالة إحصائيًا بعدي 0.9512 22 .يعرب الحال في أمثلة معطاة قبلي 0.4146 2م 40 6.312 0.000 دالة إحصائيًا بعدي 0.9512 23 يوضح نوع الحال من خالل أمثلة .معطاة قبلي 0.4146 6م 40 4.579 0.000 دالة إحصائيًا بعدي 0.8537 24 يبين صاحب الحال في أمثلة .معطاة قبلي 0.3171 9 40 4.706 0.000 دالة إحصائيًا بعدي 0.7805 25 يوضح نوع التمييز في أمثلة .معطاة قبلي 0.3659 12 40 2.994 0.368 غير دالة إحصائيًا بعدي 0.6829 26 .يعرب التمييز إعرابًا صحيحً ا قبلي 0.3415 6م 40 5.477 0.000 دالة إحصائيًا بعدي 0.8537 27 .يبين أدوات نصب الفعل املضارع قبلي 0.4146 7 40 4.230 0.000 دالة إحصائيًا بعدي 0.8293 28 يوضح سبب نصب الفعل املضارع بأن مضمرة وجوبًا أو .جوازًا قبلي 0.2683 7م 40 6.104 0.000 دالة إحصائيًا بعدي 0.8293 29 ًيميز بين األدوات التي تجزم فعال مضارعًا واحدً ا والتي تجزم .فعلين قبلي 0.3171 10 40 4.385 0.000 دالة إحصائيًا بعدي 0.7561 30 يعرب الفعل املضارع املجزوم .إعرابًا صحيحً ا قبلي 0.3415 4 40 - 6.343 0.000 دالة إحصائيًا بعدي 0.9024 31 يبين سبب اقتران جواب الشرط .بالفاء من خالل أمثلة معطاة قبلي 0.3659 10م 40 3.825 0.002 دالة إحصائيًا بعد ي 0.7561 32 .ًأن يكتب األعداد كتابةً صحيحة قبلي 0.1707 13 40 3.704 0.000 دالة إحصائيًا بعدي 0.5366 مجموع اختبار املهارات النحوية قبلي 11.0244 40 20.453 0.000 دالة إحصائيًا بعدي 27.3902 يتبين من الجدول السابق اآلتي : ( 115 ) المجلة العربية للعلوم ونشر األبحاث ــ مجلة المناهج وطرق التدريس ــ المجلد األول ــ العدد ال سادس عشر ــ ديسمبر 2022 م جلة العربية للعلوم ونشر األبحاث ــ مجلة المناهج وطرق التدريس ــ المجلد األول ــ العدد ال سادس عشر ــ ديسمبر 2022م جاءت املتوسطات ا لظاهرية ملجموعة الدراسة (املجموعة التجريبية) في اختبار املهارات النحوية البعدي أكبر ( من املتوسطات الظاهرية في االختبار القبلي في جميع املهارات النحوية، وبداللة إحصائية أقل من0.05 ) ما عدا بعض املهارات، وهي كالتالي : - ( حصلت املهارات ذات األرقام3- 7- 11 ) في اختبار املهارات النحوية البعدي على متوسط حسابي متحد بلغ ( 0.9756 " )، وجئن في الرتبة األولى، ونصوصهن على التوالي يوضح حكم إعراب الفاعل""، و يستخرج املفعول به من أمثلة معطاة""، و يوضح نوع املفعول املطلق في أمثلة معطاة "، بينما حصلن في االختبار القبلي على املتوسطا( ت الحسابية التالية على الترتيب0.3659 ()، و0.3171 ()، و0.439 )، وبهذا فمتوسطهن في االختبار ( البعدي أكبر من متوسطهن في االختبار القبلي، وكان مستوى الداللة0.000 )، وهو مستوى داللة صفري مقارنة ( بمستوى الداللة املحدد في الدراسة بــ0.05 )، وهذا يشير إلى وجود فرق إحصائي دال بين متوسطي التطبيقين القبلي والبعدي لصالح التطبيق البعدي في املهارات النحوية؛ مما يدل على فاعلية استراتيجية التعليم املتمايز في تنمية املهارات النحوية لدى طلبة األقسام العلمية (الرياضيات، وعلوم الحياة) بكلية التربية– .صنعاء - جاءت املهارات ذات األ( رقام1 – 18 – 21 – 22 ) في الرتبة الثانية، وحصلن في االختبار البعدي على متوسط ( حسابي متحد بلغ0.9512 ( )، بينما كانت متوسطاتهن في االختبار القبلي على الترتيب0.3171 () و0.4878 ) (و0.3415 () و0.4146 " )، ونصوصهن على التوالي يستخرج الفاعل من أمثلة معطاة""، و يستخرج املفعول معه من أمثلة معطاة""، و يقارن بين أنواع الستثناء من حيث األداة واإلعراب""، و يعرب الحال في أمثلة معطاة ،" فمتوسطهن ( الحسابي في االختبار البعدي أكبر من االختبار القبلي، وكان مستوى الداللة0.000 )، وهو مستوى داللة صفري مقارنة بمستوى الداللة املحدد في ا( لدراسة بــ0.05 )، وهذا يدل على فاعلية البرنامج القائم على )استراتيجية التعليم املتمايز في تنمية املهارات النحوية لدى طلبة األقسام العلمية (الرياضيات، وعلوم الحياة بكلية التربية– .صنعاء - ( جاءت املهارات ذات األرقام6 – 10 - 12 ) في الرتبة الثالثة، وحصلن في اال ختبار البعدي على متوسط حسابي ( متحد بلغ0.9268 ( )، بينما كانت متوسطاتهن في االختبار القبلي على الترتيب0.2439 () و0.2439 () و0.3902 ،) "ونصوصهن على التوالي،"يعرب نائب الفاعل في أمثلة معطاة"، و"يستخرج املفعول املطلق من أمثلة معطاة "و يستخرج النائب عن املفعول امل،"طلق من أمثلة معطاة فمتوسطهن الحسابي في االختبار البعدي أكبر من ( االختبار القبلي، وكان مستوى الداللة0 0.00 )، وهو مستوى داللة صفري مقارنة بمستوى الداللة املحدد في ( الدراسة بــ0.05)، وهذا يدل على فاعلية البرنامج القائم على استراتيجية التعليم املتمايز في تن مية املهارات النحوية السابقة لدى طلبة األقسام العلمية (الرياضيات، وعلوم الحياة) بكلية التربية– .صنعاء - ( جاءت املهارتان ذواتا األرقام14 - 19 ) في الرتبة الخامسة، وحصلتا في االختبار البعدي على متوسط حسابي متحد ( بلغ0.878)، بينما كان متوسطاهما في االختبار القبل( ي على الترتيب0.2927 () و0.439 )، ونصاهما على التوالي "يعرب املفعول ألجله في أمثلة معطاة""، و يعرب املفعول معه إعرابًا صحيحً ا "، فمتوسطهما الحسابيان في ( االختبار البعدي أكبر من متوسط االختبار القبلي، وكان مستوى الداللة0 0.00 )، وهو مستوى داللة صفري مقارنة بمست( وى الداللة املحدد في الدراسة بــ0.05 )، وهذا يدل على فاعلية البرنامج القائم على استراتيجية التعليم املتمايز في تنمية املهارات النحوية لدى طلبة األقسام العلمية (الرياضيات، وعلوم الحياة) بكلية التربية– .صنعاء - ( حصلت املهارات ذات األرقام4- 8- 13 - 16 - 23 - 26 ) في االختبار البعدي على متوسط حسابي متحد بلغ ( 0.8537 ( )، بينما كانت متوسطاتهن في االختبار القبلي على الترتيب0.2439 () و0.2927 ()، و0.3171 ،) أثر استخدا م استراتيجية التعليم المتمايز في تنمية المهارات النحوية في مقرر اللغة العربية لدى طلبة األقسام العلمية ( 116 ) الجعدي المجلة العربية للعلوم ونشر األبحاث ــ مجلة المناهج وطرق التدريس ــ المجلد األول ــ العدد ال سادس عشر ــ ديسمبر 2022 م (و0.3902 ()، و0.4146 ()، و0.3415 ،)، وبهذا فإن متوسطهن الحسابي في االختبار البعدي أكبر من االختبار القبلي مما يدل على وج( ود فرق إحصائي بين التطبيقين لصالح التطبيق البعدي؛ حيث كان مستوى الداللة0 0.00 ،) ( وهو مستوى داللة صفري مقارنة بمستوى الداللة املحدد في الدراسة بــ0.05 )، وهذا يدل على فاعلية البرنامج القائم على التعليم املتمايز في تنمية املهارات النحوية لدى طلبة األقسام ال)علمية (الرياضيات، وعلوم الحياة ، كما أنهن ج ئ.ن في الرتبة السادسة (و0.3902 ()، و0.4146 ()، و0.3415 ،)، وبهذا فإن متوسطهن الحسابي في االختبار البعدي أكبر من االختبار القبلي مما يدل على وج( ود فرق إحصائي بين التطبيقين لصالح التطبيق البعدي؛ حيث كان مستوى الداللة0 0.00 ،) ( وهو مستوى داللة صفري مقارنة بمستوى الداللة املحدد في الدراسة بــ0.05 )، وهذا يدل على فاعلية البرنامج القائم على التعليم املتمايز في تنمية املهارات النحوية لدى طلبة األقسام ال)علمية (الرياضيات، وعلوم الحياة ، كما أنهن ج ئ.ن في الرتبة السادسة - ( جاءت املهارتان ذواتا األرقام27 - 28 ) في الرتبة السابعة، وحصلتا في االختبار البعدي على متوسط حسابي متحد ( بلغ0.8293 ( )، بينما كان متوسطاهما في االختبار القبلي على الترتيب0.4146 () و0.2683)، ونص اهما على التوالي "يبين أدوات نصب الفعل املضارع""، و يوضح سبب نصب الفعل املضارع بأن مضمرة وجوبًا أو جوازًا ،" فمتوسطهما الحسابيان في االختبار البعدي أكبر من االختبار القبلي، وهذا يدل على وجود فرق إحصائي بين التطبيقين لصالح التطبيق البعدي؛ حيث كان مستوى الدالل( ة0 0.00 )، وهو مستوى داللة صفري مقارنة ( بمستوى الداللة املحدد في الدراسة بــ0.05 )، وهذا يدل على فاعلية البرنامج القائم على استراتيجية التعليم املتمايز في تنمية املهارات النحوية لدى طلبة األقسام العلمية (الرياضيات، وعلوم الحياة) بكلية التربية– .صنعاء - جاءت ( املهارتان ذواتا األرقام15 - 17 ) في الرتبة الثامنة، وحصلتا في االختبار البعدي على متوسط حسابي متحد ( بلغ0.8049 ( )، بينما كان متوسطاهما في االختبار القبلي على الترتيب0.561 () و0.3171 )، ونصاهما على التوالي "يستخرج ظرفي الزمان واملكان من أمثلة معطاة""، و يميز ب ين الظرف املتصرف وغير املتصرف من خالل أمثلة معطاة "، فمتوسطهما الحسابيان في االختبار البعدي أكبر من االختبار القبلي، وهذا يدل على وجود فرق إحصائي ( بين التطبيقين لصالح التطبيق البعدي؛ حيث كان مستوى الداللة0 0.00 )، وهو مستوى داللة صفري مقارنة بمستوى الداللة ا( ملحدد في الدراسة بــ0.05 )، وهذا يدل على فاعلية البرنامج القائم على استراتيجية التعليم املتمايز في تنمية املهارات النحوية لدى طلبة األقسام العلمية (الرياضيات، وعلوم الحياة) بكلية التربية– .صنعاء - ( حصلت املهارات ذات األرقام5- 20 - 25) على مستوى غير دال إحص ائيًا ونصوصهن " يستخرج نائب الفاعل من أمثلة معطاة" ،"يستخرج أركان الستثناء من أمثلة معطاة"، و"يوضح نوع التمييز في أمثلة معطاة "، مما يشير إلى ضعف عينة الدراسة في هذه املهارات، ولكن الضعف لدى طلبة األقسام العلمية في هذه املهارات يرجع إلى مستواهم العلمي الساب ق وعدم إتقانهم لهذه املهارات في املراحل السابقة ملرحلة الجامعة ونفورهم من مادة النحو، ولكن هؤالء املهارات جاءتن في الرتب قبل األخيرة، مما يدل على فاعلية استراتيجية التعليم املتمايز في تنمية تلك املهارات، كما أن هناك فرقًا كبيرًا بين املتوسط الحسابي في االخ تبار البعدي عن االختبار القبلي، وهذا الفرق بين املتوسطين وحصولهن على رتب قبل األخيرة يدل على فاعلية البرنامج القائم على استراتيجية التعليم املتمايز في إكساب طلبة األقسام العلمية (الرياضيات، وعلوم الحياة) بكلية التربية– صنعاء هذه املهارات .النحوية - جاءت امل( هارتان ذواتا األرقام29 - 31 ) في الرتبة العاشرة، وحصلتا في االختبار البعدي على متوسط حسابي متحد ( بلغ0.7561 ( )، بينما كان متوسطاهما في االختبار القبلي على الترتيب0.3171 () و0.3659 )، ونصاهما على التوالي "يميز بين األدوات التي تجزم فعالً مضارعًا واحدً ا والتي ت جزم فعلين""، و يبين سبب اقتران جواب الشرط بالفاء من خالل أمثلة معطاة "، فمتوسطهما الحسابيان في االختبار البعدي أكبر من االختبار القبلي، وهذا يدل ( على وجود فرق إحصائي بين التطبيقين لصالح التطبيق البعدي؛ حيث كان مستوى الداللة0 0.00 )، وهو مستوى صفري مقارنة بمست( وى الداللة املحدد في الدراسة بــ0.05 )، وهذا يدل على فاعلية البرنامج القائم على ( 117 ) المجلة العربية للعلوم ونشر األبحاث ــ مجلة المناهج وطرق التدريس ــ المجلد األول ــ العدد ال سادس عشر ــ ديسمبر 2022 م جلة العربية للعلوم ونشر األبحاث ــ مجلة المناهج وطرق التدريس ــ المجلد األول ــ العدد ال سادس عشر ــ ديسمبر 2022م )استراتيجية التعليم املتمايز في تنمية املهارات النحوية لدى طلبة األقسام العلمية (الرياضيات، وعلوم الحياة بكلية التربية– .صنعاء - ( جاءت املهارة ذات الرقم32 ) في الرتبة األخيرة و نصها" ،"أن يكتب األعداد كتابة صحيحة حصلت في اختبار ( املهارات النحوية البعدي على متوسط حسابي بلغ0.5366 ( )، بينما كان متوسطها في االختبار القبلي0.1707 ) ، ( وكانت قيمةt-test ( ) تساوي3.704 ( ) بمستوى داللة0.000 )، وهو مستوى داللة صفري أقل من مستوى الداللة املحدد( في الدراسة بـ0.05 )، وهذا يشير إلى وجود فرق إحصائي دال بين متوسطي درجات التطبيقين (القبلي- البعدي) الختبار مهارات املهارات النحوية لصالح التطبيق البعدي، ويدل على فاعلية استراتيجية التعليم املتمايز في تدريس املهارات النحوية لدى طلبة األقسام العلمية بكلية ا.لتربية صنعاء وال يعني أنها جاءت في الرتبة األخيرة أنها ضعيفة وإنما جاءت في الرتبة األخيرة لوجود صعوبة فيها لدى مجموعة الدراسة في املراحل الدراسية .السابقة ومن خالل التحليالت اإلحصائية السابقة يتم قبول فرضية ا لدراسة، ونصها :"توجد فروق دالة إحصائيًا عند مست وى الداللة ( 0.05 )بين متوسطي درجات املجموعة التجريبية في التطبيقين (القبلي والبعدي )الختبار املهارات النحوية على مستوى االختبار ككل وعلى مستوى كل مهارة مستقلة لصالح التطبيق ،البعدي يُعزى إلى تدريس املهارات النحوية باستراتيجية التعليم املتمايز "، وتُرفض الفر.ضية الصفرية التي تنفي وجود هذا الفرق اإلحصائي وتُعزى هذه النتيجة من وجهة نظر الباحث إلى أن استراتيجية التعليم املتمايز اعتمدت مجموعة توليفية من االستراتيجيات التي تناسب وتالئم قدرات وخصائص الطلبة، وتعدد األنشطة و الوسائل، وإثارة الدافعية واملناقشة الفاعلة بين الباحث والطلبة مما أدى إلى رفع معدالت الطلبة في أداء املهارات النحوية كما أدى إلى زيادة الثقة والود .بين الطلبة والباحث واتفقت هذه النتيجة مع أغلب الدراسات السابقة التي أشارت إلى تفوّق استراتيجية التعليم املتمايز على الطريقة التقليدية في متغير التحصيل( وإن اختلفت املادة الدراسية، كدراسات أبو شباب : 2019م ؛ الشقران : 2019م ؛ الشقران : 2019 ) ، كما اتفقت مع دراسات املحور الثاني التي أشارت إلى تفوق االختبار البعدي في إكساب املهارات النحوية كدراس تي (عفاف يوسف : 2019 م، و رائد حميد : 2019 ). )األندلس ي، أحمد (ب. ت :العقد الفريد، تحقيق : مفيد قميحة، الجزء الثاني، بيروت، دار .الكتب العلمية - )األندلس ي، أحمد (ب. ت :العقد الفريد، تحقيق : مفيد قميحة، الجزء الثاني، بيروت، دار .الكتب العلمية - ( توملينسون، كارول آن2001 :) الصف املتمايز االستجابة الحتياجات جميع طلبة الصف، ترجمة مدارس .الظهران األهلية، طبعة أولى، الظهران، دار الكتاب - ( توملينسون، كارول آن2001 :) الصف املتمايز االستجابة الحتياجات جميع طلبة الصف، ترجمة مدارس .الظهران األهلية، طبعة أولى، الظهران، دار الكتاب - ( الجوجو، ألفت2011 :) فعالية تدريس النحو في ضوء نظرية الذكاءات املتعددة في تنمية بعض املفاهيم النحوية ،لدى طالبات الصف السابع األساس ضعيفات التحصيل، مجلة جامعة األزهر بغزة، سلسلة العلوم اإلنسانية املجلد13 ، ع1 (ص1371 - 1422 .) - ( الجوجو، ألفت2011 :) فعالية تدريس النحو في ضوء نظرية الذكاءات املتعددة في تنمية بعض املفاهيم النحوية ،لدى طالبات الصف السابع األساس ضعيفات التحصيل، مجلة جامعة األزهر بغزة، سلسلة العلوم اإلنسانية املجلد13 ، ع1 (ص1371 - 1422 .) م - الحبيش ي، عبد ( الواحد2008 :) مدى إ تقان طلبة قسم اللغة العربية في كلية التربية بجامعة صنعاء للمهارات )النحوية، (رسالة ماجستير غير منشورة.، كلية التربية، جامعة صنعاء، اليمن - الحبيش ي، عبد ( الواحد2008 :) مدى إ تقان طلبة قسم اللغة العربية في كلية التربية بجامعة صنعاء للمهارات )النحوية، (رسالة ماجستير غير منشورة.، كلية التربية، جامعة صنعاء، اليمن - ( حسن، عمار2016 :)ثأ ،ر التعليم املتمايز في تحصيل طلبة قسم التربية الفنية في مادة تاري الفن، مجلة ديالي ( 71 )، ص409 - 438 . - ( حسن، عمار2016 :)ثأ ،ر التعليم املتمايز في تحصيل طلبة قسم التربية الفنية في مادة تاري الفن، مجلة ديالي ( 71 )، ص409 - 438 . - ( الحليس ي، معيض حسن2012 :)أثر استخدام استراتيجية التعليم املتمايز على التحصيل الدراس ي في مقرر اللغ ة اإلنجليزية لتالميذ الصف السادس االبتدائي، (ماجستير غير منشورة)، جامعة أم القرى، مكة املكرمة، كلية .التربية - ( الحليس ي، معيض حسن2012 :)أثر استخدام استراتيجية التعليم املتمايز على التحصيل الدراس ي في مقرر اللغ ة اإلنجليزية لتالميذ الصف السادس االبتدائي، (ماجستير غير منشورة)، جامعة أم القرى، مكة املكرمة، كلية .التربية - ( حميد، رائد حسين2019 :) أثر أنموذجي كمب وبارمان في اكتساب املفاهيم النحوية في مادة قواعد اللغة العربية لدى طالبات الصف الرابع العلمي، مجلة كلية الت ربية األساسية للعلوم التربوية واإل( نسانية، العدد45 )، ص ص382 - 409 . توصيات الدراسة .ومقترحاتها في ضوء نتائج الدراسة يوص ي الباحث ويقترح اآلتي : 1- إقامة دروات وورش عمل نصف فصلية لطلبة األقسام العلمية ملراجعة املهارات اللغوية عامة ؛ لكي تكون كتابتهم واضحة وتحدثهم سليما. 2- .تضمين استراتيجية التعليم املتمايز ضمن طرائق التدريس الحديثة لتدريسها في كليات التربية ُ 2- .تضمين استراتيجية التعليم املتمايز ضمن طرائق التدريس الحديثة لتدريسها في كليات التربية ُ 2- .تضمين استراتيجية التعليم املتمايز ضمن طرائق التدريس الحديثة لتدريسها في كليات التربية 3- إثراء املكتبات الجامعية والعامة واملدرسية بكتب تتعلق باستراتيجية التعليم املتمايز؛ ل لتعرُّف عليها بشكل .كبير 3- إثراء املكتبات الجامعية والعامة واملدرسية بكتب تتعلق باستراتيجية التعليم املتمايز؛ ل لتعرُّف عليها بشكل .كبير 4- تدريس مقرر اللغة العربية ل جميع األقسام العلمية في جميع املستويات وليس االقتصار على املستوى األول ؛ وذلك ملا لديهم من ضعف في املهارات اللغوية عامة و املهارات النحوية خاصة. 4- تدريس مقرر اللغة العربية ل جميع األقسام العلمية في جميع املستويات وليس االقتصار على املستوى األول ؛ وذلك ملا لديهم من ضعف في املهارات اللغوية عامة و املهارات النحوية خاصة. ممم 5- ول سد الفجوة البحثية في موضوع الدراسة يقترح الباحث إجراء دراسات مستقبلية في املوضوعات اآلتية 1. .إجراء دراسات تستقص ي أثر استراتيجيات التعليم املتمايز في مواد دراسية أخرى غير اللغة العربية ُأ ع ي حم يآ 1. .إجراء دراسات تستقص ي أثر استراتيجيات التعليم املتمايز في مواد دراسية أخرى غير اللغة العربية 2. ُإجراء دراسات مشابهة للدراسة الحالية ت دخل متغير.ات أخرى 2. ُإجراء دراسات مشابهة للدراسة الحالية ت دخل متغير.ات أخرى ( 118 ) جلة العربية للعلوم ونشر األبحاث ــ مجلة المناهج وطرق التدريس ــ المجلد األول ــ العدد ال سادس عشر ــ ديسمبر 2022م 3. .إجراء دراسات حول استراتيجية التعليم املتمايز في املراحل الدراسية األولى 3. .إجراء دراسات حول استراتيجية التعليم املتمايز في املراحل الدراسية األولى 4. إجراء دراسة مقارنة بين أثر استراتيجية "التعليم املتمايز" واستراتيجيات أخرى في تدريس املوضوعات .القرائية أو النحوية أو الكتابية املصادر واملراجع املصادر واملراجع - ( ابن جني أبو الفتح عثمان1913 :) .الخصائص، املجلد األول، القاهرة، دار الكتب املصرية - ( أبو مغلي، سميح2009 :) ّاألساليب الحديثة لتدريس اللغة العربية، عم.ان، دار البداية - ( أبو شباب، إيمان ساجع2019 :) أثر برنامج تدريبي مستند إلى التعلم املتمايز في التحصيل في مادة الرياضيات واالتجاهات نحوها ل دى طلبة الصف الثالث ذوي صعوبات التعلم في املدارس الحكومية في األردن، مجلة جامعة فلسطين لألبحاث والدراسات، مج9، عدد3، ص ص90 - 118 . - ( أبو شباب، إيمان ساجع2019 :) أثر برنامج تدريبي مستند إلى التعلم املتمايز في التحصيل في مادة الرياضيات واالتجاهات نحوها ل دى طلبة الصف الثالث ذوي صعوبات التعلم في املدارس الحكومية في األردن، مجلة جامعة فلسطين لألبحاث والدراسات، مج9، عدد3، ص ص90 - 118 . - أحمد، نضال ؛ ( والجهيمي أمل2016 :) فاعلية استراتيجية التعليم املتمايز وفق نموذج الفورمات في تنمية االستيعاب املفاهيمي في مادة األ حياء للصف الثاني الثانوي بمدينة الرياض، ورقة مقدمة إلى مؤتمر التميز في ( تعليم وتعلم العلوم والرياضيات األول " توجه العلوم والتقنية والهندسة والرياضياتSTEM )، جامعة امللك .سعود - أحمد، نضال ؛ ( والجهيمي أمل2016 :) فاعلية استراتيجية التعليم املتمايز وفق نموذج الفورمات في تنمية االستيعاب املفاهيمي في مادة األ حياء للصف الثاني الثانوي بمدينة الرياض، ورقة مقدمة إلى مؤتمر التميز في ( تعليم وتعلم العلوم والرياضيات األول " توجه العلوم والتقنية والهندسة والرياضياتSTEM )، جامعة امللك .سعود - )األندلس ي، أحمد (ب. ت :العقد الفريد، تحقيق : مفيد قميحة، الجزء الثاني، بيروت، دار الكتب الع )األندلس ي، أحمد (ب. ت :العقد الفريد، تحقيق : مفيد قميحة، الجزء الثاني، بيروت، دار .الكتب العلمية - ( حميد، رائد حسين2019 :) أثر أنموذجي كمب وبارمان في اكتساب املفاهيم النحوية في مادة قواعد اللغة العربية لدى طالبات الصف الرابع العلمي، مجلة كلية الت ربية األساسية للعلوم التربوية واإل( نسانية، العدد45 )، ص ص382 - 409 . - الدليمي، طه علي ؛ ( والوائلي، سعاد عبد الكريم2005 :) ،)اتجاهات حديثة في تدريس اللغة العربية، أربد (األردن .عالم الكتب الحديث - الدليمي، طه علي ؛ ( والوائلي، سعاد عبد الكريم2005 :) ،)اتجاهات حديثة في تدريس اللغة العربية، أربد (األردن .عالم الكتب الحديث - ( الدليمي، كامل محمود2004 :) ّأساليب تدريس قواعد اللغة العربية، عم ا.ن، دار املناهج - ( الرازي محمد بن أبي بكر بن عبد القادر2005 :)مختار الصحاح، ط2 .، دار عمار - ( الدليمي، كامل محمود2004 :) ّأساليب تدريس قواعد اللغة العربية، عم ا.ن، دار املناهج (ل ك أ ل)ط ل - ( الدليمي، كامل محمود2004 :) ّأساليب تدريس قواعد اللغة العربية، عم ا.ن، دار املناهج - ( الرازي محمد بن أبي بكر بن عبد القادر2005 :)مختار الصحاح، ط2 .، دار عمار ( الدليمي، كامل محمود2004 :) أساليب تدريس قواعد اللغة العربية، عم ا.ن، دار املناهج - ( الرازي محمد بن أبي بكر بن عبد القادر2005 :)مختار الصحاح، ط2 .، دار عمار أثر استخدا م استراتيجية التعليم المتمايز في تنمية المهارات النحوية في مقرر اللغة العربية لدى طلبة األقسام العلمية ( 119 ) الجعدي المجلة العربية للعلوم ونشر األبحاث ــ مجلة المناهج وطرق التدريس ــ المجلد األول ــ العدد ال سادس عشر ــ ديسمبر 2022 م جلة العربية للعلوم ونشر األبحاث ــ مجلة المناهج وطرق التدريس ــ المجلد األول ــ العدد ال سادس عشر ــ ديسمبر 2022م - ( الراعي، أمجد2014 :) فعالية استراتيجية التعليم املتمايز في تدريس الرياضيات على اكتساب املفاهيم الرياضية وامليل نحو الرياضيات لدى طالب الصف السابع األساس ي، (ماجستير غير منشورة)، الجامعة اإلسالمية، غزة. - ( زقوت، محمد شحادة1999 :)املرشد في تدريس اللغة العربية، ط2، الجامعة اإل.سالمية، غزة فلسطين - ( الراعي، أمجد2014 :)فعالية استراتيجية التعليم املتمايز في تدريس الرياضيات على اكتساب املفاهيم الرياضية وامليل نحو الرياضيات لدى طالب الصف السابع األساس ي، (ماجستير غير منشورة)، الجامعة اإلسالمية، غزة. - ( زقوت، محمد شحادة1999 :)املرشد في تدريس اللغة العربية، ط2، الجامعة اإل.سالمية، غزة فلسطين - ( الراعي، أمجد2014 :) فعالية استراتيجية التعليم املتمايز في تدريس الرياضيات على اكتساب املفاهيم الرياضية وامليل نحو الرياضيات لدى طالب الصف السابع األساس ي، (ماجستير غير منشورة)، الجامعة اإلسالمية، غزة. )األندلس ي، أحمد (ب. ت :العقد الفريد، تحقيق : مفيد قميحة، الجزء الثاني، بيروت، دار .الكتب العلمية - ( الضبعات، زكريا2007 :)طرا ّئق تدريس اللغة العربية، عم.ان، دار الفكر - ( الضبعات، زكريا2007 :)طرا ّئق تدريس اللغة العربية، عم.ان، دار الفكر ( الضبعات، زكريا2007 :)طرا ّئق تدريس اللغة العربية، عم.ان، دار الفكر - ( الطويل، السيد1991 :)من قضايا اللسان العربي"د راسة في اإلعراب والبنية"، جزء2 ، القاهرة، دار الهدى .للطباعة - ( الطويل، السيد1991 :)من قضايا اللسان العربي"د راسة في اإلعراب والبنية"، جزء2 ، القاهرة، دار الهدى .للطباعة - عاشور، راتب ؛ ( والحوامدة، محمد2007 :) ّأساليب تدريس اللغة العربية بين النظرية والتطبيق، عم ان، دار .املسيرة - عاشور، راتب ؛ ( والحوامدة، محمد2007 :) ّأساليب تدريس اللغة العربية بين النظرية والتطبيق، عم ان، دار .املسيرة - عا ،فش ي ا( بتسام بنت عباس1997 :) املهارات النحوية لدى طالبات قسم اللغة العربية بكلية التربية للبنات .بالرياض وعالقتها بالتحصيل في مقررات التخصص، (ماجستير غير منشورة)، كلية التربية للبنات بالرياض - ( عامر، فخر الدين2000 :)طرق التدريس الخاصة باللغة العربية والتربي ة اإلسالمية، ط2 ، القاهرة، دار عالم - عا ،فش ي ا( بتسام بنت عباس1997 :) املهارات النحوية لدى طالبات قسم اللغة العربية بكلية التربية للبنات .بالرياض وعالقتها بالتحصيل في مقررات التخصص، (ماجستير غير منشورة)، كلية التربية للبنات بالرياض .بالرياض وعالقتها بالتحصيل في مقررات التخصص، (ماجستير غير منشورة)، كلية التربية للبنات بالرياض - ( عامر، فخر الدين2000 :)طرق التدريس الخاصة باللغة العربية والتربي ة اإلسالمية، ط2 ، القاهرة، دار عالم .الكتب - ( عامر، فخر الدين2000 :)طرق التدريس الخاصة باللغة العربية والتربي ة اإلسالمية، ط2 ، القاهرة، دار عالم .الكتب - ( عبدالباري، ماهر شعبان2012 :) فاعلية برنامج لتنمية مهارات التفكير العليا في النحو العربي لدى طالب شعبة .اللغة العربية في كلية التربية ببنها، املجلة التربوية، الكويت - ( عبدالباري، ماهر شعبان2012 :) فاعلية برنامج لتنمية مهارات التفكير العليا في النحو العربي لدى طالب شعبة .اللغة العربية في كلية التربية ببنها، املجلة التربوية، الكويت - عبيدات، ذوقان ؛ ( وأبو السميد، سهيلة2007 :)استراتي جيات التدريس في القرن الحادي والعشرين دليل املعلم واملشرف التربوي، ط1 ّ، عم.ان، دار الفكر - عبيدات، ذوقان ؛ ( وأبو السميد، سهيلة2007 :)استراتي جيات التدريس في القرن الحادي والعشرين دليل املعلم واملشرف التربوي، ط1 ّ، عم.ان، دار الفكر - العربي، صالح عبد املجيد ؛ والعقيلي، عبد ( العزيز1986 :) أهداف واستخدامات معامل اللغات وأثرها في تنمية . )األندلس ي، أحمد (ب. ت :العقد الفريد، تحقيق : مفيد قميحة، الجزء الثاني، بيروت، دار .الكتب العلمية - ( زقوت، محمد شحادة1999 :)املرشد في تدريس اللغة العربية، ط2، الجامعة اإل.سالمية، غزة فلسطين - ( السيد، محمد أحمد1969 :) ،)دراسة مقارنة بين طرائق تدريس قواعد اللغة العربية، (ماجستير غير منشورة .جامعة أم القرى ز و رب ريس ي رم لإ زا - ( السيد، محمد أحمد1969 :) ،)دراسة مقارنة بين طرائق تدريس قواعد اللغة العربية، (ماجستير غير منشورة .جامعة أم القرى - ( السيد، محمد أحمد1969 :) ،)دراسة مقارنة بين طرائق تدريس قواعد اللغة العربية، (ماجستير غير منشورة .جامعة أم القرى السيد، محمود (ب. ت) : .في قضايا اللغة التربوية، الكويت، وكالة املطبوعات - السيد، محمود (ب. ت) : .في قضايا اللغة التربوية، الكويت، وكالة املطبوعات - شحات ة( ، حسن1993 :)تعليم اللغة العربية بين النظرية والتطبيق، ط2 ، القاهرة، الدار املصرية اللبنانية .للطباعة - شحات ة( ، حسن1993 :)تعليم اللغة العربية بين النظرية والتطبيق، ط2 ، القاهرة، الدار املصرية اللبنانية .للطباعة - شحاتة، حسن ؛ والنجار، زينت ؛ ( وعمار، حامد2003 :) معجم املصطلحات التربية والنفسية، القاهرة، الدار ا ملصرية اللبنانية، ط1. - شحاتة، حسن ؛ والنجار، زينت ؛ ( وعمار، حامد2003 :) معجم املصطلحات التربية والنفسية، القاهرة، الدار ا ملصرية اللبنانية، ط1. - ( الشقران، خالد يوسف2019 :) أثر التدريس املتمايز في اكتساب املفاهيم العلمية ومهارات عمليات العلم ،واالتجاه نحو العلوم لدى طالب الصف السابع األساس ي، (دكتوراه غير منشورة)، كلية التربية، جامعة اليرموك .إربد، األردن - ( الشقران، خالد يوسف2019 :) أثر التدريس املتمايز في اكتساب املفاهيم العلمية ومهارات عمليات العلم ،واالتجاه نحو العلوم لدى طالب الصف السابع األساس ي، (دكتوراه غير منشورة)، كلية التربية، جامعة اليرموك .إربد، األردن - الشهراني، جواهر الحق مح( مد2019 :) أثر تدريس العلوم باستخدام استراتيجية التعليم املتمايز القائم على األنشطة في تنمية االستيعاب املفاهيمي وتنمية عادات العقل لدى طالبات الصف الثاني املتوسط، مجلة البحث ( العلمي في التربية، العدد20 ( )، الجزء1)، ص ص479 - 510 . - الشهراني، جواهر الحق مح( مد2019 :) أثر تدريس العلوم باستخدام استراتيجية التعليم املتمايز القائم على األنشطة في تنمية االستيعاب املفاهيمي وتنمية عادات العقل لدى طالبات الصف الثاني املتوسط، مجلة البحث ( العلمي في التربية، العدد20 ( )، الجزء1)، ص ص479 - 510 . )األندلس ي، أحمد (ب. ت :العقد الفريد، تحقيق : مفيد قميحة، الجزء الثاني، بيروت، دار .الكتب العلمية املهارات اللغوية، الرياض، دار املري - ( عطية محسن2007 :)تدريس اللغة ّالعربية في ضوء الكفايات األدائية، عم.ان، دار املناهج - العاليلي، عبد هللا، 1974م :"الصحاح في اللغة (املصطلحات العلمية)"، ط1، .بيروت، لبنان، دار الحضارة العربية - ( عطية محسن2007 :)تدريس اللغة ّالعربية في ضوء الكفايات األدائية، عم.ان، دار املناهج - العاليلي، عبد هللا، 1974م :"الصحاح في اللغة (املصطلحات العلمية)"، ط1، .بيروت، لبنان، دار الحضارة العربية أثر استخدا م استراتيجية التعليم المتمايز في تنمية المهارات النحوية في مقرر اللغة العربية لدى طلبة األقسام العلمية( 120 ) الجعدي المجلة العربية للعلوم ونشر األبحاث ــ مجلة المناهج وطرق التدريس ــ المجلد األول ــ العدد ال سادس عشر ــ ديسمبر 2022 م جلة العربية للعلوم ونشر األبحاث ــ مجلة المناهج وطرق التدريس ــ المجلد األول ــ العدد ال سادس عشر ــ ديسمبر 2022م - علي عبده، سماح عبد ( الوهاب2009 :)تقويم برنامج إعداد معلم اللغة العربية في كلية التربية بجامعة صن عاء .في ضوء معايير الجودة الشاملة، (رسالة ماجستير غير منشورة)، كلية التربية، جامعة صنعاء، اليمن - ( الفيروز آبادي، مجد الدين محمد19 .غير منشورة)، كلية التربية، الجامعة اإلسالمية، غزة، فلسطين - الناقة، صالح ؛ ( وشي العيد، إبراهيم2009 :)صعوبات تعلم مادة قواعد الل غة العربية في املرحلة االبتدائية من وجهة نظر معلمي املادة ومعلماتها، مجلة دراسات تربوية، ع5، ص149 - 185 . - علي عبده، سماح عبد ( الوهاب2009 :)تقويم برنامج إعداد معلم اللغة العربية في كلية التربية بجامعة صن عاء .في ضوء معايير الجودة الشاملة، (رسالة ماجستير غير منشورة)، كلية التربية، جامعة صنعاء، اليمن - ( الفيروز آبادي، مجد الدين محمد19 .غير منشورة)، كلية التربية، الجامعة اإلسالمية، غزة، فلسطين ماا - علي عبده، سماح عبد ( الوهاب2009 :)تقويم برنامج إعداد معلم اللغة العربية في كلية التربية بجامعة صن عاء .في ضوء معايير الجودة الشاملة، (رسالة ماجستير غير منشورة)، كلية التربية، جامعة صنعاء، اليمن - ( الفيروز آبادي، مجد الدين محمد19 .غير منشورة)، كلية التربية، الجامعة اإلسالمية، غزة، فلسطين - الناقة، صالح ؛ ( وشي العيد، إبراهيم2009 :)صعوبات تعلم مادة قواعد الل غة العربية في املرحلة االبتدائية من وجهة نظر معلمي املادة ومعلماتها، مجلة دراسات تربوية، ع5، ص149 - 185 . - النبهان، مسلم ؛ ( والكنعاني، عبد الواحد2016 :)فاعلية استراتيجيتي الدعائم التعليمية والتعليم93 :) القاموس املحيط، بيروت، دار الفكر، ط41 . - النبهان، مسلم ؛ ( والكنعاني، عبد الواحد2016 :)فاعلية استراتيجيتي الدعائم التعليمية والتعليم93 :) القاموس املحيط، بيروت، دار الفكر، ط41 . )األندلس ي، أحمد (ب. ت :العقد الفريد، تحقيق : مفيد قميحة، الجزء الثاني، بيروت، دار .الكتب العلمية - اللقاني، أحمد ؛ وال،جمل ( علي2003 :) ،معجم املصطلحات التربوية املعرفة في املناهج وطرق التدريس، القاهرة .عالم الكتب - اللقاني، أحمد ؛ وال،جمل ( علي2003 :) ،معجم املصطلحات التربوية املعرفة في املناهج وطرق التدريس، القاهرة .عالم الكتب - ( كامل، عبد الرحمن2005 :)مهارات التفكر الالزمة لتدريس النحو : مدى توافرها لدى الطالب املعلم، وكيفية إكسابه إياها وأثرها على تحصيل ط ال ب الصف األول الثانوي العام، ا ملؤتمر العلمي السادس لكلية التربية بالفيوم التنمية املهنية املستدامة للمعلم العربي، مصر الفيوم، كلية التربية جامعة القاهرة، فرع الفيوم، مج2 ، ص : 136 . - ( كوجك، كوثر وآخرون2008 :) تنويع التدريس في الفصل دليل املعلم لتحسين طرق التعليم والتعلم في مدارس الوطن الع.ربي، بيروت، مكتب اليونسكو اإلقليمي للتربية في الدول العربية - ( كوجك، كوثر وآخرون2008 :) تنويع التدريس في الفصل دليل املعلم لتحسين طرق التعليم والتعلم في مدارس الوطن الع.ربي، بيروت، مكتب اليونسكو اإلقليمي للتربية في الدول العربية - ( مجاور، محمد صالح الدين2000 :) ،)تدريس اللغة العربية في املرحلة الثانوية (أسسه، وتطبيقاته التربوية .القاهرة، دار الفكر - ( مجاور، محمد صالح الدين2000 :) ،)تدريس اللغة العربية في املرحلة الثانوية (أسسه، وتطبيقاته التربوية .القاهرة، دار الفكر - ( املصري، عبير عمر حمدان2016 :)أثر توظيف األناشيد التعليمية في عالج صعوبات ا لقواعد النحوية لدى طالبات الصف الرابع األساس في محافظة خانيونس، (رسالة ماجستير.) - ( املصري، عبير عمر حمدان2016 :)أثر توظيف األناشيد التعليمية في عالج صعوبات ا لقواعد النحوية لدى طالبات الصف الرابع األساس في محافظة خانيونس، (رسالة ماجستير.) - املتمايز في تحصيل طالب الصف الثاني متوسط في مادة الفيزياء، (رسالة ماجستير غير منشورة)، جامعة .القادسية - املتمايز في تحصيل طالب الصف الثاني متوسط في مادة الفيزياء، (رسالة ماجستير غير منشورة)، جامعة .القادسية - ( يوسف، عفاف2019 :)أثر استراتيجية خرائط املفاهيم في تحسين مهارات القواع د النحويّة لدى طالب الصف السابع األساس ي في منطقة إربد، مجلة جامعة النجاح لألبحاث (العلوم اإلنسانية)، املجلد33 ، العدد األول، ص ص104 - 124 . - ( يوسف، عفاف2019 :)أثر استراتيجية خرائط املفاهيم في تحسين مهارات القواع د النحويّة لدى طالب الصف السابع األساس ي في منطقة إربد، مجلة جامعة النجاح لألبحاث (العلوم اإلنسانية)، املجلد33 ، العدد األول، ص ص104 - 124 . - Heacox, D. (2002) Differentiating Instruction in the Regular Classroom How to reach and teach ALL learners, grades 3-12 by. Free Spirit Publishing.p11 - Heacox, D. (2002) Differentiating Instruction in the Regular Classroom How to reach and teach ALL learners, grades 3-12 by. )األندلس ي، أحمد (ب. ت :العقد الفريد، تحقيق : مفيد قميحة، الجزء الثاني، بيروت، دار .الكتب العلمية Free Spirit Publishing.p11 - Hall, tracey, et al . (2009). Implication for UDL implementation. UDL America.p460. أثر استخدا م استراتيجية التعليم المتمايز في تنمية المهارات النحوية في مقرر اللغة العربية لدى طلبة األقسام العلمية الجعدي ( 121 )
https://openalex.org/W3170813762	https://research-repository.st-andrews.ac.uk/bitstream/10023/25513/1/Lucas_2022_Fluids_Five_wave_resonances_CC.pdf	English	null	Five-Wave Resonances in Deep Water Gravity Waves: Integrability, Numerical Simulations and Experiments	Fluids	2,021	cc-by	22,941	erlin 2,†, Dian-Yong Liu 3,‡, Shane Walsh 4, Rossen Ivanov 5 and Miguel D. Bustamante 4,* 1 School of Computing and Mathematics, Keele University, Staffordshire ST5 5BG, UK; d.lucas1@keele.ac.uk 1 School of Computing and Mathematics, Keele University, Staffordshire ST5 5BG, UK; d.lucas1@keele.ac.uk 2 Department of Ocean Engineering, Texas A&M University, 727 Ross Street, College Station, TX 77843, USA; perlin@tamu.edu 2 Department of Ocean Engineering, Texas A&M University, 727 Ross Street, College Station, TX 77843, USA; perlin@tamu.edu 3 Naval Architecture and Ocean Engineering College, Dalian Maritime University, Dalian 116026, China; dianyongl@dlmu.edu.cn 3 Naval Architecture and Ocean Engineering College, Dalian Maritime University, Dalian 116026, China; dianyongl@dlmu.edu.cn 4 School of Mathematics and Statistics, University College Dublin, Belﬁeld, D04 V1W8 Dublin, Ireland; shanewalsh11235@gmail.com 5 School of Mathematical Sciences, Technological University Dublin—City Campus, Grangegorman Lower, D07 ADY7 Dublin, Ireland; rossen.ivanov@tudublin.ie * Correspondence: miguel.bustamante@ucd.ie * Correspondence: miguel.bustamante@ucd.ie p g † Former Afﬁliation: Department of Naval Architecture and Marine Engineering, University of Michigan, Ann Arbor, MI 48109, USA. ‡ Former Afﬁliation: State Key Laboratory of Coastal and Offshore Engineering, Dalian University of Technology, Dalian 116023, China; experiments performed at the University of Michigan. Abstract: In this work we consider the problem of ﬁnding the simplest arrangement of resonant deep- water gravity waves in one-dimensional propagation, from three perspectives: Theoretical, numerical and experimental. Theoretically this requires using a normal-form Hamiltonian that focuses on 5-wave resonances. The simplest arrangement is based on a triad of wavevectors K1 + K2 = K3 (satisfying speciﬁc ratios) along with their negatives, corresponding to a scenario of encountering wavepackets, amenable to experiments and numerical simulations. The normal-form equations for these encountering waves in resonance are shown to be non-integrable, but they admit an integrable reduction in a symmetric conﬁguration. Numerical simulations of the governing equations in natural variables using pseudospectral methods require the inclusion of up to 6-wave interactions, which imposes a strong dealiasing cut-off in order to properly resolve the evolving waves. We study the resonance numerically by looking at a target mode in the base triad and showing that the energy transfer to this mode is more efﬁcient when the system is close to satisfying the resonant conditions. We ﬁrst look at encountering plane waves with base frequencies in the range 1.32–2.35 Hz and steepnesses below 0.1, and show that the time evolution of the target mode’s energy is dramatically changed at the resonance. fluids fluids erlin 2,†, Dian-Yong Liu 3,‡, Shane Walsh 4, Rossen Ivanov 5 and Miguel D. Bustamante 4,* We then look at a scenario that is closer to experiments: Encountering wavepackets in a 400-m long numerical tank, where the interaction time is reduced with respect to the plane-wave case but the resonance is still observed; by mimicking a probe measurement of surface elevation we obtain efﬁciencies of up to 10% in frequency space after including near-resonant contributions. Finally, we perform preliminary experiments of encountering wavepackets in a 35-m long tank, which seem to show that the resonance exists physically. The measured efﬁciencies via probe measurements of surface elevation are relatively small, indicating that a ﬁner search is needed along with longer wave ﬂumes with much larger amplitudes and lower frequency waves. A further analysis of phases generated from probe data via the analytic signal approach (using the Hilbert transform) shows a strong triad phase synchronisation at the resonance, thus providing independent experimental evidence of the resonance. Citation: Lucas, D.; Perlin, M.; Liu, D.-Y.; Walsh, S.; Ivanov, R.; Bustamante, M.D. Five-Wave Resonances in Deep Water Gravity Waves: Integrability, Numerical Simulations and Experiments. Fluids 2021, 6, 205. https://doi.org/ 10.3390/ﬂuids6060205 Academic Editor: Alexander I. Dyachenko Received: 6 April 2021 Accepted: 27 May 2021 Published: 1 June 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Citation: Lucas, D.; Perlin, M.; Liu, D.-Y.; Walsh, S.; Ivanov, R.; Bustamante, M.D. Five-Wave Resonances in Deep Water Gravity Waves: Integrability, Numerical Simulations and Experiments. Fluids 2021, 6, 205. https://doi.org/ 10.3390/ﬂuids6060205 Academic Editor: Alexander I. Dyachenko Received: 6 April 2021 Accepted: 27 May 2021 Published: 1 June 2021 Publisher’s Note: MDPI stays neutral Citation: Lucas, D.; Perlin, M.; Liu, D.-Y.; Walsh, S.; Ivanov, R.; Bustamante, M.D. Five-Wave Resonances in Deep Water Gravity Waves: Integrability, Numerical Simulations and Experiments. Fluids 2021, 6, 205. https://doi.org/ 10.3390/ﬂuids6060205 Academic Editor: Alexander I. Dyachenko Received: 6 April 2021 Accepted: 27 May 2021 Published: 1 June 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. erlin 2,†, Dian-Yong Liu 3,‡, Shane Walsh 4, Rossen Ivanov 5 and Miguel D. Bustamante 4,* This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ Citation: Lucas, D.; Perlin, M.; Liu, D.-Y.; Walsh, S.; Ivanov, R.; Bustamante, M.D. Five-Wave Resonances in Deep Water Gravity Waves: Integrability, Numerical Simulations and Experiments. Fluids 2021, 6, 205. https://doi.org/ 10.3390/ﬂuids6060205 1. Introduction Historically, nonlinear resonant interactions in surface water waves have focused mainly on the so-called exact resonances, deﬁned by the equations k1 ± . . . ± kN = 0, ω1 ± . . . ± ωN = 0, where N denotes the number of interacting waves, kj denote the wave-vectors, and ωj denote the frequencies where ωj = ω(kj) is provided by a dispersion relation. Early theoretical developments for these interactions gave rise to resonance interaction theory, originated by Phillips (1960) and developed by several authors (see the reviews by [1,2], and references therein). The key working hypothesis of this theory is the smallness of the wave steepness, namely the product between wave amplitude and magnitude of the wave- vector. A formal perturbation theory based on multiple-scale methods is thus developed which allows one to obtain the exact resonances as necessary conditions to avoid secular behaviour. Solutions to these exact resonance equations can be found in many cases. Case N = 3, called triad resonances, applies to capillary-gravity waves (and also to a variety of wave systems such as Charney-Hasegawa-Mima, quasi-geostrophic equations, internal waves, inertial waves, etc.); case N = 4, known as quartet resonances, applies to gravity waves (including deep water). The case N = 5 is relevant in the important context of gravity waves in one-dimensional propagation, which is the topic to be discussed in this paper. In this context, it was shown in [3] that the interaction coefﬁcients for the 4-wave resonances vanish identically. Later on, in [4] it was demonstrated that 5-wave resonances can exist and their interaction coefﬁcients are nonzero. Then, in [5] (see also [6]) the explicit calculation of all 5-wave resonances along with their interaction coefﬁcients was performed. Once solutions to the exact resonance equations are found, the dynamics of the wave amplitudes (at dominant orders) is dictated by coupled nonlinear PDE systems. In some simple scenarios the equations are integrable and can be solved analytically via the inverse scattering transform, leading to recurrent behaviour, showing periodic exchanges of energy between modes. In some scenarios, however, particularly in cases where multiple resonant interactions are coupled, the ODE and PDE systems obtained are known to display chaotic behaviour [7–10]. For practical reasons, the multiple resonant interaction dynamics is discussed us- ing stochastic approaches, quite successfully. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Keywords: water gravity waves; 5-wave resonances; pseudospectral numerical simulations; water wave tank experiments https://www.mdpi.com/journal/ﬂuids Fluids 2021, 6, 205. https://doi.org/10.3390/ﬂuids6060205 Fluids 2021, 6, 205 2 of 35 1. Introduction Such a minimal arrangement is obtained in terms of a triad of positive wavevectors: K1 + K2 = K3, Kj > 0, where the wavevectors satisfy quite speciﬁc ratios. Of course, triads are not resonant in water gravity waves because the dispersion relation is subadditive. The quintet resonances obtained represent 3 →2 processes (transforming three waves into two waves), and the interacting wavenumbers must contain positive and negative elements from the set {±K1, ±K2, ±K3}. Notably, the fact that minimal 5-wave resonances can be based on non-resonant triads was demonstrated by one of us in another one-dimensional resonant system (also with subadditive dispersion relation) of historical importance: The Fermi-Pasta-Ulam-Tsingou system [34,35]. In Section 2 we review the Hamiltonian theory in natural variables up to and including 6-wave interactions, which will be useful for numerical simulations. We then review the approach in normal-form variables, and brieﬂy describe all possible resonant quintets obtained in the simpliﬁed scenario of encountering wavevectors based on non-resonant triads. We then consider the dynamical system for such a resonant conﬁguration and demonstrate that it is not integrable in general, although it has several integrals of motion and in particular the amplitudes are bounded; by reducing to a symmetric scenario of encountering wavevectors with the same amplitudes one obtains an integrable system based on a homoclinic orbit. In Section 3 we provide detailed results of our water-wave pseudospectral numerical simulations, in two main scenarios: Encountering plane waves and encountering wave packets. For each scenario only the Fourier modes with wavevectors ±K2 and ±K3 have initial energy in them. The energy transfer to the ‘target’ Fourier modes with wavevectors ±K1 is therefore measured after a time period of nonlinear interaction, by deﬁning an ‘energy efﬁciency’ as the proportion of the total energy that goes to these target wavevectors. Several numerical experiments are conducted, off resonant and at resonance, in order to quantify the peak of efﬁciency at the resonant case. As an alternative way to measure efﬁciency, closer to the experimental way, we consider a ‘numerical probe measurement’ corresponding to what the surface elevation at a given point along the numerical tank would look like. The resulting time series is analysed in terms of frequency and time using a wavelet transform. The resulting scaleograms are shown and an alternative efﬁciency is deﬁned, this time in terms of the wavelet frequency spectrum. This provides an independent quantiﬁcation of the resonance. 1. Introduction Pioneered by Hasselmann [11–13] and Zakharov [14,15], the now established theory of wave turbulence describes the energy exchanges across scales due to resonant quartet interactions in the case of deep water surface gravity waves. A key assumption of this theory, in addition to the smallness of steepness and the limit of large box size, is an asymptotic closure of the hierarchy of cumu- lants [16], which leads to a set of evolution equations for the so-called spectrum variables, namely the individual quadratic energies of the spatial Fourier transforms of the original ﬁeld variables. This approach assumes that the phases of the Fourier transforms do not play an important role in the dynamics of energy transfers. Interesting modern departures from these theories for water waves and other wave systems consider quasi-resonant sce- narios [17,18] and ﬁnite-amplitude regimes in discrete wave turbulence for other systems (such as the barotropic vorticity equation and more recently in wave-mean ﬂow models of solar cycle modulations), where the phases interact with the spectrum variables producing interesting effects, such as precession resonance [19–21]. g p Up to and including the papers by [22–24], and the review by [1], only two experiments studied resonant interactions between wavetrains with comparable initial amplitudes and only one experiment studied the relative roles of nonlinearity and randomness for a broad spectrum of waves. Since then, experiments on triad resonances in gravity-capillary waves were performed by [25,26]. In the case of gravity waves, experiments on quartet reso- nances based on earlier work on modulational instability [27] were conducted by [28,29]. Resonant interactions in the presence of an underwater current were investigated by [30]. Experiments on persistent wave patterns and steady-state resonant waves were performed by [31,32]. Finally, experiments on degenerate quartets of oblique waves not inﬂuenced Fluids 2021, 6, 205 3 of 35 by modulational instability were conducted by [33]. All these experiments have conﬁrmed the resonant interaction theory in the case of small steepness (ka < 0.1 roughly). The lat- ter experiments also conﬁrmed the expected nonlinear corrections due to marginally off-resonance terms. In this paper we consider the simplest experimental/numerical/theoretical setup to search for a resonance in deep water gravity waves in one-dimensional propagation. Namely, we look for a 5-wave resonance using the minimum possible number of wavevec- tors (along with their negatives, representing an experiment of encountering wavepackets). 1. Introduction A widely used form of these conditions in the study of nonlinear phenomena on water waves is derived by expanding the vertical velocity at the free surface to ﬁfth order [15,36–40], leading to the system ∂tζ = 5 ∑ n=1 Qn(ζ, Φ), ∂tΦ = 5 ∑ n=1 Rn(ζ, Φ) , (1) (1) where Qn and Rn are given by where Qn and Rn are given by where Qn and Rn are given by Q1 = −LΦ , (2) Q2 = −∂x[ζ ∂xΦ] −L[ζ LΦ] , (3) Q3 = −L(ζ L[ζ LΦ] + 1 2ζ2∂2 xΦ) −∂2 x( 1 2ζ2LΦ) , (4) Q4 = −L{ζL(ζ L[ζ LΦ] + 1 2ζ2∂2 xΦ) + 1 2ζ2∂2 x[ζLΦ] −1 6ζ3∂2 xLΦ} −∂2 x{ 1 2ζ2L[ζLΦ] + 1 3ζ3∂2 xΦ} , (5) Q1 = −LΦ , (2) Q2 = −∂x[ζ ∂xΦ] −L[ζ LΦ] , (3) Q3 = −L(ζ L[ζ LΦ] + 1 2ζ2∂2 xΦ) −∂2 x( 1 2ζ2LΦ) , (4) Q4 = −L{ζL(ζ L[ζ LΦ] + 1 2ζ2∂2 xΦ) + 1 2ζ2∂2 x[ζLΦ] −1 6ζ3∂2 xLΦ} −∂2 x{ 1 2ζ2L[ζLΦ] + 1 3ζ3∂2 xΦ} , (5) , (2) ∂xΦ] −L[ζ LΦ] , (3) 2 2 2 2 Q4 = −L{ζL(ζ L[ζ LΦ] + 1 2ζ2∂2 xΦ) + 1 2ζ2∂2 x[ζLΦ] −1 6ζ3∂2 xLΦ} (5) Q5 = −L ζL{ζL(ζ L[ζ LΦ] + 1 2ζ2∂2 xΦ) + 1 2ζ2∂2 x[ζLΦ] −1 6ζ3∂2 xLΦ} −L 1 2ζ2∂2 x(ζ L[ζ LΦ] + 1 2ζ2∂2 xΦ) −1 6ζ3∂2 xL[ζLΦ] −1 24ζ4∂4 xΦ −∂2 x 1 2ζ2L(ζ L[ζ LΦ] + 1 2ζ2∂2 xΦ) + 1 3ζ3∂2 x[ζLΦ] −1 8ζ4∂2 xLΦ , (6) R1 = −g ζ , (7) R2 = −1 2{∂xΦ}2 + 1 2{LΦ}2 , (8) R3 ={LΦ}[L[ζ LΦ] + ζ ∂2 xΦ] , (9) Q5 = −L ζL{ζL(ζ L[ζ LΦ] + 1 2ζ2∂2 xΦ) + 1 2ζ2∂2 x[ζLΦ] −1 6ζ3∂2 xLΦ} −L 1 2ζ2∂2 x(ζ L[ζ LΦ] + 1 2ζ2∂2 xΦ) −1 6ζ3∂2 xL[ζLΦ] −1 24ζ4∂4 xΦ −∂2 x 1 2ζ2L(ζ L[ζ LΦ] + 1 2ζ2∂2 xΦ) + 1 3ζ3∂2 x[ζLΦ] −1 8ζ4∂2 xLΦ , (6) R1 = −g ζ , (7) Q5 = −L ζL{ζL(ζ L[ζ LΦ] + 1 2ζ2∂2 xΦ) + 1 2ζ2∂2 x[ζLΦ] −1 6ζ3∂2 xLΦ} −L 1 2ζ2∂2 x(ζ L[ζ LΦ] + 1 2ζ2∂2 xΦ) −1 6ζ3∂2 xL[ζLΦ] −1 24ζ4∂4 xΦ −∂2 x 1 2ζ2L(ζ L[ζ LΦ] + 1 2ζ2∂2 xΦ) + 1 3ζ3∂2 x[ζLΦ] −1 8ζ4∂2 xLΦ , (6) R1 = −g ζ , (7) R2 = −1 2{∂xΦ}2 + 1 2{LΦ}2 , R2 = −1 2{∂xΦ}2 + 1 2{LΦ}2 , (8) R3 ={LΦ}[L[ζ LΦ] + ζ ∂2 xΦ] , (9) { }[ [ζ ] ζ x ] R4 ={LΦ}L(ζ L[ζ LΦ] + 1 2ζ2∂2 xΦ) + ∂2 x{ 1 4ζ2{LΦ}2} + 1 2[L[ζ LΦ] + ζ ∂2 xΦ]2 + 1 2ζ{∂2 xζ}{LΦ}2 −1 2ζ2{∂xLΦ}2 , (10) R5 ={LΦ}L{ζL(ζ L[ζ LΦ] + 1 2ζ2∂2 xΦ) + 1 2ζ2∂2 x[ζLΦ] −1 6ζ3∂2 xLΦ} + ∂2 x 1 2ζ2{LΦ}L[ζLΦ] + 1 3ζ3{LΦ}∂2 xΦ −ζ2{∂xLΦ}[∂xL[ζLΦ] + 2 3ζ∂3 xΦ] + 1 6ζ3{∂2 xLΦ}{∂2 xΦ} + [L[ζLΦ] + ζ∂2 xΦ](L(ζ L[ζ LΦ] + 1 2ζ2∂2 xΦ) + 1 2ζ{∂2 xζ}{LΦ}2) , (11) (10) (11) where g is the acceleration of gravity and the Fourier transform of the linear operator L is deﬁned by: L ˆΦk(t) = −k tanh(kh) ˆΦk(t), ˆΦk(t) = 1 L Z L/2 −L/2 Φ(x, t)e−ikx dx, and h (> 0) is the water depth. 1. Introduction We also show that the resonance can be diagnosed by looking at the coherence of the quintet phases over their time evolution. g y g q p In Section 4 we provide a detailed description of the linear tank experimental setup and calibration of the two wavemakers and the two measuring probes that measure surface elevation at two symmetrically situated points. We perform 9 experiments: In each experi- ment the wavemakers are oscillated with two frequencies each: f2 and f3, corresponding to wavevectors ±K2 and ±K3, respectively. We analyse the probes’ time series (surface elevation) and produce frequency-time scaleograms as in the previous section, obtaining preliminary evidence that the resonance can be captured in this type of experiment. Finally, in Section 5 we summarise the results and discuss the challenges posed, as well as possible improvements to experiments and extensions to other scenarios. In addition, we present independent evidence of the resonance by showing that phase synchronisation is strong at the resonance, in all scenarios studied: Numerical plane waves, numerical wave packets and experimental waves. Fluids 2021, 6, 205 4 of 35 2. Deep-Water Surface Waves in One-Dimensional Propagation 2. Deep-Water Surface Waves in One-Dimensional Propagation p 2.1. Fifth-Order Hamiltonian Theory in Natural Variables We consider the evolution of the free surface of a water column (in one dimension) via the boundary conditions for the surface elevation ζ(x, t) and the velocity potential Φ(x, t). 1. Introduction Notice that in the above deﬁnitions for Qn and Rn we introduced a useful convention on the use of parenthesis: Terms of order 1, 2, 3, 4, 5 in the ﬁeld amplitudes are enclosed, respectively, by curly {}, square [], round () brackets, and so on, repeating the cycle. From here we restrict our analysis to the deep-water limit, h →∞, leaving the ﬁnite-depth case for a subsequent work. In the deep-water limit the L-operator’s action reduces to L ˆΦk(t) = −\|k\| ˆΦk(t). Computationally we will assume periodic boundary conditions in the longitudinal direction with computational box size L, so that Φ(x + L, t) = Φ(x, t), ζ(x + L, t) = ζ(x, t), for all x, t ∈R. Fluids 2021, 6, 205 5 of 35 The Hamiltonian up to and including 6-wave interactions is given by The Hamiltonian up to and including 6-wave interactions is given by H = 1 2 Z L/2 −L/2 gζ2 + Φ 5 ∑ n=1 Qn ! dx . (12) (12) Explicit expressions for this, order by order, can be found in [40]. The evolution Equations (1) are canonical Hamiltonian equations: ∂tζ = δH δΦ , ∂tΦ = −δH δζ . We note in passing that there is a relation between the above formulation and the Hamiltonian formulation using Dirichlet-Neumann operators by [41]. g p y Deﬁning the Fourier components of surface elevation and velocity potential as ζ(x, t) = ∑ k ∈( 2 π L )Z eikx ˆζk(t) , Φ(x, t) = ∑ k ∈( 2 π L )Z eikx ˆΦk(t) , (13) (13) we consider now the canonical transformation from natural variables ( ˆζk, ˆΦk) to the so- called normal variables ak. For this the quadratic part of the Hamiltonian must be diagonal in the normal variables and the Poisson bracket be preserved. The resulting transforma- tion is we consider now the canonical transformation from natural variables ( ˆζk, ˆΦk) to the so- called normal variables ak. For this the quadratic part of the Hamiltonian must be diagonal in the normal variables and the Poisson bracket be preserved. The resulting transforma- tion is ˆζk = \|k\| 4 g 1/4 (ak + a∗ −k) , (14) ˆΦk = −i g 4 \|k\| 1/4 (ak −a∗ −k) . 1. Introduction (15) (14) (15) Notice that while ˆζk and ˆΦk satisfy reality conditions ( ˆζ−k = ˆζ∗ k etc.) the normal variables ak do not. In fact, we may interpret ak and a−k as independent waves propagating in direction k and −k, respectively. Thus, the number of degrees of freedom is preserved under this linear transformation. The variables ak have dimensions L3/2T−1/2. 2.2. The Simplest Arrangement of Five-Wave Resonances 2.2. The Simplest Arrangement of Five-Wave Resonances 2.2. The Simplest Arrangement of Five-Wave Resonances Expressions for the Hamiltonian in normal variables ak are usually cumbersome. The usual approach is to further transform the system to the so-called normal form via near-identity canonical transformations relating ak to normal-form variables bk: ak = bk + ∑ k1 A(1) k,k1,k−k1bk1bk−k1 + ∑ k1 A(2) k,k1,k+k1b∗ k1bk−k1 + ∑ k1 A(3) k,k1,−k−k1b∗ k1b∗ −k−k1 + ∑ k1,k2 B(1) k,k1,k2,k−k1−k2bk1bk2bk−k1−k2 + ∑ k1,k2 B(2) k,k1,k2,k+k1−k2b∗ k1bk2bk+k1−k2 + ∑ k1,k2 B(3) k,k1,k2,k+k1+k2b∗ k1b∗ k2bk+k1+k2 + ∑ k1,k2 B(4) k,k1,k2,−k−k1−k2b∗ k1b∗ k2b∗ −k−k1−k2 + ∑ k1,k2,k3 C(1) k,k1,k2,k3,k−k1−k2−k3bk1bk2bk3bk−k1−k2−k3 + ∑ k1,k2,k3 C(2) k,k1,k2,k3,k+k1−k2−k3b∗ k1bk2bk3bk+k1−k2−k3 + ∑ k1,k2,k3 C(3) k,k1,k2,k3,k+k1+k2−k3b∗ k1b∗ k2bk3bk+k1+k2−k3 + ∑ k1,k2,k3 C(4) k,k1,k2,k3,k+k1+k2+k3b∗ k1b∗ k2b∗ k3bk+k1+k2+k3 + ∑ k1,k2,k3 C(5) k,k1,k2,k3,−k−k1−k2−k3b∗ k1b∗ k2b∗ k3b∗ −k−k1−k2−k3 , where the constant coefﬁcients A(j) k,k1,k2, B(j) k,k1,k2,k3, C(j) k,k1,k2,k3,k4 are provided explicitly in [6]. In these new variables, only resonant interactions are present by deﬁnition. For deep- water surface waves in one-dimensional propagation, it is known that the lowest-order where the constant coefﬁcients A(j) k,k1,k2, B(j) k,k1,k2,k3, C(j) k,k1,k2,k3,k4 are provided explicitly in [6]. In these new variables, only resonant interactions are present by deﬁnition. For deep- water surface waves in one-dimensional propagation, it is known that the lowest-order Fluids 2021, 6, 205 6 of 35 resonances appearing in the normal-form Hamiltonian are ﬁve-wave resonances [4,42]. More speciﬁcally: Three-wave resonances are absent; four-wave resonances are present but the corresponding interaction coefﬁcients vanish at the resonant manifold, with only Birkhoff four-wave resonances surviving; and ﬁve-wave resonances are present and most of them have nonzero interaction coefﬁcients, with the essential requirement that the interacting wavenumbers have opposite signs [5]. The calculation of the normal form up to and including resonant ﬁve-wave interactions was performed by [4–6], with two important results: 1. Explicit formulae for the ﬁve-wave resonant manifold, leading to ﬁve different types of resonance, numbered from (i) to (v). 2. Closed-form expressions for the ﬁve-wave interaction coefﬁcients evaluated at the resonant manifold. In particular, it was shown that these interaction coefﬁcients are equal to zero in resonance types (iii) and (iv), and nonzero in the resonance types (i), (ii) and (v). 2.2. The Simplest Arrangement of Five-Wave Resonances In recent results by one of us regarding the Fermi-Pasta-Ulam-Tsingou (FPUT) sys- tem with periodic boundary conditions [34,35], it has been demonstrated that ﬁve-wave resonances are effectively constructed out of three-wave “frequency-only” resonances, namely where the momentum condition for the three interacting waves is not satisﬁed. It is to be noted that in the FPUT system the dispersion relation is strictly subadditive, just like in deep water waves, so there are no exact three-wave resonances in either system. Therefore it comes as a surprise that a ﬁve-wave resonance can be based on a three-wave “mismatched” resonance, and especially with the momentum conditions involving only three different wavenumbers along with their negatives. This motivated the question of this paper: Can a ﬁve-wave resonance be found in terms of three positive wavenumbers satisfying the momentum condition, K + K′ = K′′, along with their negatives? This question has an experimental appeal too, as its answer would provide a minimal scenario involving two opposing wave trains, each consisting of just three central wavenumbers. For this question we studied exhaustively the resonant cases and found only two different resonant solutions, both coming from case (v) in [5]: • First resonant quintet: • First resonant quintet: K2 + K2 + (−K2) = K3 + (−K1), K1 : K2 : K3 = 16 : 9 : 25 , (K1 + K2 = K3) (16) (16) along with its negative version (−K2) + (−K2) + K2 = (−K3) + K1. In terms of lin- ear frequencies via the dispersion relation f (k) = p g\|k\|/2π, this resonance reads f1 : f2 : f3 = 4 : 3 : 5, where fj := f (Kj) , j = 1, 2, 3. along with its negative version (−K2) + (−K2) + K2 = (−K3) + K1. In terms of lin- ear frequencies via the dispersion relation f (k) = p g\|k\|/2π, this resonance reads f1 : f2 : f3 = 4 : 3 : 5, where fj := f (Kj) , j = 1, 2, 3. • Second resonant quintet: (17) K2 + K2 + (−K3) = K3 + (−K4), K2 : K3 : K4 = 1 : 3 : 4 , (K2 + K3 = K4) (17) along with its negative version (−K2) + (−K2) + K3 = (−K3) + K4. 2.2. The Simplest Arrangement of Five-Wave Resonances In terms of linear frequencies via the dispersion relation f (k) = p g\|k\|/2π, this resonance reads f2 : f3 : f4 = 1 : √ 3 : 2, where fj := f (Kj) , j = 2, 3, 4. There is a third resonant quintet but it is of type (iv) in the notation of [5], where the interaction coefﬁcient is equal to zero. This quintet could appear in numerical simulations using the third-order equations such as [36], so we list it anyway: • Third resonant quintet (vanishing interaction coefﬁcient): K2 + K2 + K2 = K4 + (−K2), K2 : K3 : K4 = 1 : 3 : 4 , (K2 + K3 = K4) Fluids 2021, 6, 205 7 of 35 along with its negative version (−K2) + (−K2) + (−K2) = (−K4) + K2. Notice that one of the wavenumbers (K3), along with its negative, do not appear explicitly in this resonant quintet. The proof that the ﬁve-wave resonances listed above are the only possible ones based on three wavenumbers and their negatives is elementary but long: See Appendix A for details. It is based on a direct implementation of the inequalities in the classiﬁcation given in [5], except that we allow some of the inequalities to saturate by virtue of the degeneracy of the solutions (as clearly some frequencies must coincide since we use three basic frequencies only). 2.3. Non-Integrability of the Five-Wave Resonance in the Case of Encountering Waves In this subsection we consider the normal form Hamiltonian up to ﬁve-wave reso- nances. If we consider just one of the quintets from the ﬁrst and second resonant quintets of the previous subsection, and assume all other modes in the system are initially zero, it is then possible to show that the normal form Hamiltonian up to ﬁve-wave interactions is integrable, as it will consist of Birkhoff-only four-wave terms plus one ﬁve-wave term only. The system has four degrees of freedom, and four constants of motion in involution: The Hamiltonian and three quadratic invariants. In contrast, as soon as we allow for the corresponding “negative” quintet (and assume all other modes in the system are initially zero), we obtain two interacting quintets whose Hamiltonian is not integrable. The system has six degrees of freedom, but only four constants of motion: The Hamiltonian and three quadratic invariants. 2.2. The Simplest Arrangement of Five-Wave Resonances (18) (18) Fluids 2021, 6, 205 8 of 35 The equations of motion will not be shown explicitly; they are obtained simply by following the canonical formalism: dbn dt = −i ∂H ∂b∗n , n = ±1, ±2, ±3. Is this dynamical system integrable? Notice that the quadratic and quartic terms in the Hamiltonian depend on the moduli squared \|bn\|2 of the normal form variables only, so these terms commute with any of the moduli squared (i.e., these terms do not change the energy of the evolving modes). In contrast, the quintic term depends on the quintet phases φ+ = 2φ2 + φ−2 −φ3 −φ−1 and φ−= 2φ−2 + φ2 −φ−3 −φ1 as well as the moduli, and is thus responsible for the energy exchanges across modes. We can therefore state that the system has a reduced number of degrees of freedom: 6 moduli + 2 quintic phases = 8 degrees of freedom, the 4 remaining phases being obtainable by quadrature. This result and the structure of the normal form imply that there are 4 Manley-Rowe-type quadratic invariants: I+ = \|b2\|2 + 2\|b3\|2 + \|b−3\|2, I−= \|b−2\|2 + 2\|b−3\|2 + \|b3\|2 , (19) J+ = \|b2\|2 + 2\|b−1\|2 + \|b1\|2, J−= \|b−2\|2 + 2\|b1\|2 + \|b−1\|2 . (20) (19) (20) Thus, along with the Hamiltonian, there are 5 independent invariants, leading to an effective 3-dimensional ﬁrst-order autonomous dynamical system. If we could ﬁnd one more independent invariant then we could conclude that the system is integrable. It turns out that one can simplify the search as follows. A rigorous result, motivated by the question of integrability (and clearly unrelated to the actual physical situation of low steepnesses), considers the limit of large amplitudes \|bn\| ≫1, where we can neglect the quadratic and quartic terms in the Hamiltonian (18), leading to a homogeneous system. Then, if the original system has an extra independent invariant then this new homogeneous system has an extra independent invariant (other than the 5 invariants already found). So we focus on the homogeneous system for this question of integrability. We can apply two ideas to study the homogeneous system. 2.2. The Simplest Arrangement of Five-Wave Resonances To see this, consider for example the ﬁrst resonant quintet in the previous subsection along with its negative version. That is, we have the quintets K2 + K2 + (−K2) = K3 + (−K1) and (−K2) + (−K2) + K2 = (−K3) + K1, with K1/K2 = 16/9 and K3/K2 = 25/9. ( ) ( ) It is useful to deﬁne the set of relevant wavevectors as C = {K1, K2, K3, −K1, −K2, −K3}, It is useful to deﬁne the set of relevant wavevectors as C = {K1, K2, K3, −K1, −K2, −K3}, so that the normal form amplitudes satisfy bk := 0 ⇐⇒k /∈C. Then, the Hamiltonian in the normal form variables can be obtained by gathering the analyses of the interaction coefﬁcients done in [4,5]. We get H = ∑ k1∈C ωk1b∗ k1bk1 + 1 4 ∑ k1,k2∈C Tk1k2b∗ k1bk1b∗ k2bk2 + 1 12 ∑ k1,k2,k3,k4k5∈C Tk1k2k3 k4k5 b∗ k1b∗ k2b∗ k3bk4bk5 + c.c. δk1+k2+k3 k4+k5 , where ωk = p g\|k\| is the dispersion relation, Tk1k2 = 1 4π2 k1k2 min(\|k1\|, \|k2\|), and the coefﬁcient Tk1k2k3 k4k5 (symmetric on the upper indices and the lower indices separately) is known explicitly on the resonant manifold. For our resonant quintet and its negative version we get (case (v)(a) in [5]) TK2K2−K2 K3−K1 = T−K2−K2K2 −K3K1 = −K13/4 2 K3/4 3 K1/4 1 g1/4π3/2 . Introducing the notation bn = bKn, b−n = b−Kn, for n = 1, 2, 3, we get the following explicit expression for the Hamiltonian in normal form: H = g1/2 K1/2 2 (\|b2\|2 + \|b−2\|2) + K1/2 1 (\|b1\|2 + \|b−1\|2) + K1/2 3 (\|b3\|2 + \|b−3\|2) + 1 16π2 K3 2(\|b2\|2 −\|b−2\|2)2 + K3 1(\|b1\|2 −\|b−1\|2)2 + K3 3(\|b3\|2 −\|b−3\|2)2 + K2 2 8π2 (\|b2\|2 −\|b−2\|2) K1(\|b1\|2 −\|b−1\|2) + K3(\|b3\|2 −\|b−3\|2) + K2 1K3 8π2 (\|b1\|2 −\|b−1\|2)(\|b3\|2 −\|b−3\|2) − K13/4 2 K3/4 3 K1/4 1 2 g1/4π3/2 (b∗ 2)2b∗ −2b3b−1 + (b∗ −2)2b∗ 2b−3b1 + (b2)2b−2b∗ 3b∗ −1 + (b−2)2b2b∗ −3b∗ 1 . (18) − K13/4 2 K3/4 3 K1/4 1 2 g1/4π3/2 (b∗ 2)2b∗ −2b3b−1 + (b∗ −2)2b∗ 2b−3b1 + (b2)2b−2b∗ 3b∗ −1 + (b−2)2b2b∗ −3b∗ 1 . 2.2. The Simplest Arrangement of Five-Wave Resonances First, we can use an idea that was ﬁrst applied in [43], whereby the Kovalevskaya exponents of a homogeneous dynamical system are computed, and then a theorem by Yoshida [44] is used in order to obtain the degree (in terms of the amplitudes) that a potentially new invariant of such a system would have [45]. We refer the reader to [43] for details of the method, as it is not relevant in our case because we could not establish in this way whether our system is integrable or not. On applying this method we found a Kovalevskaya exponent of 7/9, which would correspond to an invariant of degree 7/3 in the amplitudes. Unfortunately this method neither guarantees nor precludes the existence of an invariant: The result only states that if a new invariant existed, then it should be of degree 7/3 in the amplitudes. The second idea is to take advantage of the fact that the homogeneous system is effectively 3-dimensional, so Poincaré cuts can be produced. If these clearly show the existence of chaotic regions then we can safely conclude that the system is chaotic. We can use the quadratic invariants to reduce the analysis to the plane spanned by the vari- ables \|b1\|2 and \|b3\|2. The remaining degree of freedom can be chosen to be the phase φ+ = arg(b2 2b−2b∗ 3b∗ −1). Using a highly accurate numerical scheme that conserves the Hamiltonian up to O(10−11) we integrate the system for several choices of initial condi- tions and generate a Poincaré cut on the (\|b1\|2, \|b3\|2) plane at the instances when φ+ ∈2πZ. The resulting cuts are plotted in Figure 1. The evidence for chaos is clear: We can visually identify sets of islands (representing quasi-periodic motion) which are surrounded by the usual chaotic sea. We conclude that the homogeneous system is chaotic, and therefore the original system is not integrable. 9 of 35 Fluids 2021, 6, 205 Figure 1. Poincaré cuts for the homogeneous version of the system consisting of two interacting quintets made out of three central modes and their negative versions. Figure 1. Poincaré cuts for the homogeneous version of the system consisting of two interacting quintets made out of three central modes and their negative versions. 2.4. 2.2. The Simplest Arrangement of Five-Wave Resonances While it is not possible to determine from this argument alone how much energy can actually be transferred, a rigorous analysis of the reduced case where initially \|b2\| = \|b−2\|, \|b3\| = Fluids 2021, 6, 205 10 of 35 10 of 35 \|b−3\|, \|b1\| = \|b−1\| = 0 shows that it is possible to transfer all the energy from \|b2\|(= \|b−2\|) to \|b1\|(= \|b−1\|) by virtue of the conservation of J+(= J−), leading to a homoclinic orbit with limt→±∞\|b1\| = \|b2\|t=0/ √ 3 while limt→±∞\|b2\| = 0. \|b−3\|, \|b1\| = \|b−1\| = 0 shows that it is possible to transfer all the energy from \|b2\|(= \|b−2\|) to \|b1\|(= \|b−1\|) by virtue of the conservation of J+(= J−), leading to a homoclinic orbit with limt→±∞\|b1\| = \|b2\|t=0/ √ 3 while limt→±∞\|b2\| = 0. 2.2. The Simplest Arrangement of Five-Wave Resonances Analysis of the Scenario of Encountering Waves in the Small-Steepness Case In preparation for the numerical and experimental studies of the next sections, we consider a situation where the amplitudes are initially small so that wave steepnesses \|k\|5/4\|bk\|/(4g)1/4 are small (typically of order 0.1 or less). In this situation, the truncated Hamiltonian (18) approximates well the system, as terms in it appear in a hierarchical way, with quadratic terms about 100 times larger than quartic terms, and with quartic terms about 10 times larger than quintic terms, and so on if we had continued to higher orders. Despite the smallness of the quintic terms in the Hamiltonian, the energy-exchange dynamics is still controlled solely by that term so it cannot be neglected. In fact, at resonance, which is the case when this Hamiltonian describes the system, it is possible to show that the quadratic part of the Hamiltonian is conserved separately, as it is a linear combination of the four Manley-Rowe invariants (19) and (20). As a result, the sum of the quartic part and the quintic part of the Hamiltonian is conserved separately, and these two parts differ by a factor of order 10 only. As these terms are not sign-deﬁnite it is possible to have signiﬁcant transfers. We are interested in a scenario where modes b±2 and b±3 are non-zero initially, with b±1 = 0 initially. We expect that the resonant case will display active energy transfers towards the “target” modes b±1, in sharp contrast with the non-resonant cases, where there is no quintic term in the Hamiltonian and therefore no energy transfer amongst modes at this level of nonlinearity. Energy exchanges can be studied using the Manley-Rowe invariants along with Fjørtoft-like arguments, as follows. The Manley-Rowe invariants I± from Equation (19) do not provide a lot of insight except that any exchanges between the already energetic modes b±2 and b±3 must be balanced. On the other hand, the Manley-Rowe invariants J± from Equation (20) provide a clear scenario of active transfer, whereby energy in modes b±2 can be efﬁciently transferred to modes b±1. 3. Numerical Simulations of the Fifth-Order Governing Equations in Natural Variables To demonstrate excitation of f1 or a±K1 at the point of resonance we conduct a series of numerical experiments which ﬁx a±K2 and vary a±K3 as indicated in the subsequent plots, keeping the initial amplitudes To demonstrate excitation of f1 or a±K1 at the point of resonance we conduct a series of numerical experiments which ﬁx a±K2 and vary a±K3 as indicated in the subsequent plots, keeping the initial amplitudes ˆζK2 = 0.06β K2 , ˆζK3 = 0.12β K3 , ˆζK1 = 0, ˆΦK2 = ˆΦK3 = ˆΦK1 = 0 (21) a∗ −K2 = 4g K2 1/4 0.03β K2 , aK3 = a∗ −K3 = 4g K3 1/4 0.06β K3 , aK1 = a∗ −K1 = 0 (22) (21) (22) where β is an overall rescaling of the initial amplitudes and all other ak = 0 initially. Note that Equation (21) indicates that the phases of the waves are set initially to zero. Experiments, not shown, demonstrate that varying the phases has little qualitative effect on the results to follow, only adding some corrections, and maintaining the aK = a∗ −K symmetry leaves the results quantitatively identical. 3. Numerical Simulations of the Fifth-Order Governing Equations in Natural Variables Here we present results of the numerical simulations of the full PDE (1) up to and including six-wave interactions in the natural variables, in the scenario of waves with central wavenumbers K1, K2, K3 encountering their negative versions, namely the waves with central wavenumbers −K1, −K2, −K3. We will focus on demonstrating numerically the quintet resonance given by Equation (16). The evolution Equation (1) are solved using a standard pseudospectral method, dealiasing such that nmax = N/6 for resolution N and fourth order Runge-Kutta timestep- ping. In all the results that follow N = 214 and the timestep was chosen as ∆t = 0.0005. The code was validated against an existing third order code using the equations of [37], experimental data from [38,39] and monitoring conservation of the Hamiltonian. p g We consider initial conditions which are as close as possible to the experiments described in Section 4. Numerically the permitted wavenumbers K in the domain are of the form K = n2π/L, n ∈Z. At ﬁrst it might seem sensible to choose L close to that of the real experiments (35 m; see Section 4), but in reality the pertinent and comparable behaviours should be captured using the same ‘physical’ wavenumbers, or frequencies, within a similar interaction time interval in any sufﬁciently large numerical domain. It is of more importance to account for the discreteness of this wavenumber spectrum and the fact that, at signiﬁcant amplitudes, nonlinear corrections will adjust the precise location of the resonance in k. For this reason it is advantageous to use a large L which has the consequence of allowing smaller δk, and therefore a relatively higher ﬁdelity grid in k space, but naturally necessitating higher resolutions to account for a certain spatial scale, and hence temporal frequency. To this end we choose L = 400 m and consider the scaled resonance condition n1 = 16p, n2 = 9p, with p = 50 which results in resonant wavenumbers K1 = 8002π L , K2 = 4502π L , K3 = 12502π L , corresponding to linear frequencies via the dispersion relation f (k) = p g\|k\|/2π: f1 ≈1.766Hz, f2 ≈1.32Hz, f3 ≈2.21Hz. 3.1. Encountering Plane Waves In order to diagnose growth of energy in the target mode we deﬁne a measure of efﬁciency using the normal variables, themselves computed from the natural ones by inverting (14) and (15): Fluids 2021, 6, 205 11 of 35 Ek(t) = ωkaka∗ k H , (23) (23) where H is the full Hamiltonian, deﬁned in Equation (12). We show maxt∈[0,500] EK1(t) against K3 in Figure 2 for β = 1 and β = 0.75. A distinct, but narrow peak is observed near the predicted resonant K3 (marked with vertical line). The efﬁciencies are around 1.5% at the peak for β = 1, with the time series of EK(t) shown in Figure 3 demonstrating sustained growth in mode aK1 over approximately 250 s in the resonant case, and no growth off resonance. Of note is the width of the resonant peak in K3; coarser sampling of the initial wavenumber (or frequency) space will likely miss the optimal value for the resonance in the simulations. Due to nonlinear corrections the peak does not appear precisely at K3 = 1250 2π L but slightly displaced at K3 = 1248 2π L ; namely, a difference in wavelength of δλ ≈0.5 mm or in frequency δ f ≈0.0018 Hz. Figure 3 also shows the time series for the efﬁciency (or amplitude) of aK3. This shows a decrease of energy at late time which is generic across all of the calculations and only moderated slightly by the resonance. To determine which other nonlinear interactions are important in this system we examine the wavenumber spectrum as a function of time, plotted in Figure 4. Aside from K1, K2, K3 we can observe 2K2 having energy from early time, and side-bands of K3 namely K3 ± δK with δK = 2K2 −K1 growing at late times due to modulational instability. This is the reason for the decrease observed in aK3. Importantly this is a background effect, present in all cases, and does not hinder the ﬁve-wave resonance under consideration. To mimic the diagnostics available in a physical experiment we also seek evidence of resonant transfer in a time series of a numerical ‘probe’ measurement of the surface elevation, namely ζ(xp, t), where xp = L/2 + 2 m is the ﬁxed location in the domain for the probe. 3.1. Encountering Plane Waves The choice of xp is arbitrary in the plane wave case, however this choice is made to be in keeping with the experiments in Section 4. The resonance should be observed by an increase in the temporal linear frequency f1 in this measure. In order to diagnose such growth we create a frequency-time ﬁgure using a wavelet transform in the form of a ‘scaleogram’. With a duration of t = 200 s, sampling 10,000 points in time and performing a wavelet transform using Gabor wavelets with 48 oscillations, with a scale resolution of 11 frequency scales along with 64 voices per scale, we obtain Figure 5. Three cases are shown (at the same K3 as the time series of Figure 3) which demonstrates, for f3 ≈2.206 Hz, the growth in time of the signal with frequency f1, marked with a horizontal blue dashed line. A similar efﬁciency can be deﬁned using this measure deﬁned as the sum of the squares of the scaleogram signals in Figure 5, on a strip of width 0.05f1 about f1 and between t ∈[10,190], divided by the sum of squares over the whole range of frequencies, shown in Figure 6. From these data emerges a qualitatively similar result as for the spatial Fourier efﬁciency shown in Figure 2, with a distinct peak around K3 = 1248 2π L . 12 of 35 Fluids 2021, 6, 205 (a) (b) Figure 2. The maximum efﬁciency max EK1as a function of starting K3 (or f3) for the numerical experiments considered. This demonstrates how narrow the peak transfer is at resonance in the discrete numerical case. (a) We show β = 1 (blue circles), β = 0.75 (orange squares), and the encountering wave packets (‘A’ red triangle, deﬁned in Equation (24), and ‘B’ green diamonds, deﬁned in Equations (25)–(28)). Note the upper axis shows the equivalent frequency f3 = p gK3/2π and the (b) plot shows the encountering wave packet case in isolation to better visualise the change in behaviour at resonance. (b) (a) (b) (a) Figure 2. The maximum efﬁciency max EK1as a function of starting K3 (or f3) for the numerical experiments considered. This demonstrates how narrow the peak transfer is at resonance in the discrete numerical case. 3.1. Encountering Plane Waves (a) We show β = 1 (blue circles), β = 0.75 (orange squares), and the encountering wave packets (‘A’ red triangle, deﬁned in Equation (24), and ‘B’ green diamonds, deﬁned in Equations (25)–(28)). Note the upper axis shows the equivalent frequency f3 = p gK3/2π and the (b) plot shows the encountering wave packet case in isolation to better visualise the change in behaviour at resonance. (a) (b) Figure 3. Time series for the efﬁciency Ek (23) (relative amplitude) for the K1 target mode (a) and K3 (b) under plane-wave propagation. A clear increase of energy transfer is observed in K1 at the resonant frequency (f3/Hz ≈2.206, K3 = 1248 2π L ) compared to off resonance (f3/Hz = 2.25, K3 = 1298 2π L and f3/Hz = 2.15, K3 = 1185 2π L ). (a) (b) (b) (a) Figure 3. Time series for the efﬁciency Ek (23) (relative amplitude) for the K1 target mode (a) and K3 (b) under plane-wave propagation. A clear increase of energy transfer is observed in K1 at the resonant frequency (f3/Hz ≈2.206, K3 = 1248 2π L ) compared to off resonance (f3/Hz = 2.25, K3 = 1298 2π L and f3/Hz = 2.15, K3 = 1185 2π L ). 13 of 35 Fluids 2021, 6, 205 Figure 4. Time dependent spatial Fourier spectra, \|aK(t)\|, with the pertinent modes annotated, as in Figure 3 an increase of energy transfer is observed in K1 at the resonant frequency (f3/Hz ≈2.206, K3 = 1248 2π L ) compared to off resonance (f3/Hz = 2.25, K3 = 1298 2π L and f3/Hz = 2.15, K3 = 1185 2π L ). The decrease in K3 can be attributed to side-band instability, present in all cases, but not impeding the ﬁve-wave resonance. Figure 4. Time dependent spatial Fourier spectra, \|aK(t)\|, with the pertinent modes annotated, as in Figure 3 an increase of energy transfer is observed in K1 at the resonant frequency (f3/Hz ≈2.206, K3 = 1248 2π L ) compared to off resonance (f3/Hz = 2.25, K3 = 1298 2π L and f3/Hz = 2.15, K3 = 1185 2π L ). The decrease in K3 can be attributed to side-band instability, present in all cases, but not impeding the ﬁve-wave resonance. (b) f3 = 2.206 Hz (RESONANT) (a) f3 = 2.150 Hz (b) f3 = 2.206 Hz (RESONANT) (c) f3 = 2.250 Hz Figure 5. 3.1. Encountering Plane Waves Scaleograms for the plane-wave numerical probe data with t = 200 s, produced by sampling 10,000 time points and performing a wavelet transform using Gabor wavelets with 48 oscillations, with a scale resolution of 11 frequency scales along with 64 voices per scale. Vertical lines represent the initial and ﬁnal times t = 10 s, 190 s for the calculation of the efﬁciency to the target mode f1 that avoids spurious boundary effects. Horizontal lines represent the theoretical frequencies f1, f3 and f4 stemming from the dispersion relation f (k) = p g\|k\|/2π for the wavevectors K1 = K3 −K2, K3 and K4 = K3 + K2. The width about the frequencies f1 and f4 is of the order ∆f = 0.05f. (c) f3 = 2.250 Hz (a) f3 = 2.150 Hz (b) f3 = 2.206 Hz (RESONANT) (a) f3 = 2.150 Hz (c) f3 = 2.250 Hz Figure 5. Scaleograms for the plane-wave numerical probe data with t = 200 s, produced by sampling 10,000 time points and performing a wavelet transform using Gabor wavelets with 48 oscillations, with a scale resolution of 11 frequency scales along with 64 voices per scale. Vertical lines represent the initial and ﬁnal times t = 10 s, 190 s for the calculation of the efﬁciency to the target mode f1 that avoids spurious boundary effects. Horizontal lines represent the theoretical frequencies f1, f3 and f4 stemming from the dispersion relation f (k) = p g\|k\|/2π for the wavevectors K1 = K3 −K2, K3 and K4 = K3 + K2. The width about the frequencies f1 and f4 is of the order ∆f = 0.05f. 14 of 35 Fluids 2021, 6, 205 17 18 19 20 21 22 23 0.000 0.001 0.002 0.003 0.004 0.005 0.006 2.05 2.11 2.17 2.23 2.28 2.34 2.39 K3[m-1] Efficiency f3[Hz] t∈[10s,190s] Eff f1 Figure 6. Efﬁciency as a function of the experiment, parameterised by the corresponding wavevector K3, for the plane-wave case for a long time series [0, 200 s]. The efﬁciency is deﬁned as the sum of the squares of the scaleogram signals in Figure 5, on a strip of width 0.05f1 about f1 and between the vertical lines in the ﬁgure, divided by the sum of squares over the whole range of frequencies, including f2 which is not shown. An efﬁciency of 0.006 corresponds to 0.6% efﬁciency. 3.1. Encountering Plane Waves The dashed grey vertical line corresponds to the theoretical resonant case K1 : K2 : K3 = 16 : 9 : 25. 17 18 19 20 21 22 23 0.000 0.001 0.002 0.003 0.004 0.005 0.006 2.05 2.11 2.17 2.23 2.28 2.34 2.39 K3[m-1] Efficiency f3[Hz] t∈[10s,190s] Eff f1 Figure 6. Efﬁciency as a function of the experiment, parameterised by the corresponding wavevector K3, for the plane-wave case for a long time series [0, 200 s]. The efﬁciency is deﬁned as the sum of the squares of the scaleogram signals in Figure 5, on a strip of width 0.05f1 about f1 and between the vertical lines in the ﬁgure, divided by the sum of squares over the whole range of frequencies, including f2 which is not shown. An efﬁciency of 0.006 corresponds to 0.6% efﬁciency. The dashed grey vertical line corresponds to the theoretical resonant case K1 : K2 : K3 = 16 : 9 : 25. To ﬁnalise the plane-wave case we provide a piece of analysis that connects with the theoretical derivation of the Hamiltonian system in the resonant case. Recall that for the symmetric initial conditions we took, the resonant case is expected to be integrable. A useful integrability test is to plot variables that would normally behave independently, except when the system is near integrable. To this end we provide joint probability density functions over the simulation time t ∈[0, 200 s] of the quintet phase φ+(t) = 2φ2(t) + φ−2(t) −φ3(t) −φ−1(t) versus its time derivative dφ+ dt (t) in Figure 7. The resonant case, f1 = 2.206 Hz, shows a clear reduction of the system’s dimension, evidencing the onset of a coherent, near-integrable regime which we attribute to the resonance. (a) f3 = 2.150 Hz (b) f3 = 2.206 Hz (RESONANT) (c) f3 = 2.250 Hz Figure 7. Joint probability density functions of the quintet phase φ+(t) versus its time derivative dφ+ dt (t), over the simulation time t ∈[0, 200 s], for the cases f3/Hz = 2.150, 2.206 and 2.250 in the plane-wave case, corresponding to Figures 3 and 5. (a) f3 = 2.150 Hz (c) f3 = 2.250 Hz (b) f3 = 2.206 Hz (RESONANT) (a) f3 = 2.150 Hz (c) f3 = 2.250 Hz Figure 7. 3.2. Encountering Wave Packets In order to determine how reproducible the above result using plane waves may be in a physical experiment, we adapt the initial condition of the previous section into two types of spatially localised, propagating initial data. We wish to examine the joint effects of the dispersion of waves from a source (wavemaker), the limited interaction time and the limited spatial interaction region, due to the ﬁniteness of the domain. The ﬁrst such initial condition we consider is a localised wave packet at the centre of the domain containing the same wave content as in the planar case of the previous section, i.e., ζ(x, 0) = ˆζK2eiK2x + ˆζK3eiK3x e−36 x−x0 σ 30 , Φ(x, 0) = 0 , (24) (24) with x0 = L/2 and σ = L/4 giving a packet occupying half of the domain, composed of both left propagating waves (i.e., propagating towards −x direction) and right propagating waves (i.e., propagating towards +x direction). This situation is close to the plane-wave case: Setting progressively larger σ would interpolate towards plane waves. It is also analogous to starting measurements in an experiment once the waves have encountered and overlapped, but neglects any interactions which occur as they start to encounter. As the waves propagate away from the centre of the domain the region nonlinearly interacting shrinks until eventually the required waves are no longer coincident. This will be referred to as the ‘A’ conﬁguration. The ‘B’ conﬁguration has similar half-domain localised wave packets, but now two of them, which contain either left or right propagating waves in such a way as to have encountering waves at the centre, i.e., ζr(x, 0) = K2 4 g 1/4 aK2eiK2x + K3 4 g 1/4 aK3eiK3x ! e−36( x−L/4 σ ) 30 , (25) Φr(x, 0) = −i g 4 K2 1/4 aK2eiK2x + g 4 K3 1/4 aK3eiK3x ! e−36( x−L/4 σ ) 30 , (26) ζl(x, 0) = K2 4 g 1/4 a∗ −K2e−iK2x + K3 4 g 1/4 a∗ −K3e−iK3x ! e−36( x−3L/4 σ ) 30 , (27) Φl(x, 0) = i g 4 K2 1/4 a∗ −K2e−iK2x + g 4 K3 1/4 a∗ −K3e−iK3x ! 3.1. Encountering Plane Waves Joint probability density functions of the quintet phase φ+(t) versus its time derivative dφ+ dt (t), over the simulation time t ∈[0, 200 s], for the cases f3/Hz = 2.150, 2.206 and 2.250 in the plane-wave case, corresponding to Figures 3 and 5. Fluids 2021, 6, 205 15 of 35 15 of 35 A careful investigation of the source of this coherence shows that it really arises due to the increase in amplitude felt by aK1 at resonance. Off resonance the phase rotates with a greater deal of variability, since the complex amplitude is closer to the origin, than in the resonant case where aK1 has larger absolute value and therefore rotates at a more consistent rate, given by the linear frequency ωK. Notice also that in the symmetrical “encountering waves” setup studied in our numerical simulations, the symmetry φ−j = −φj is valid at all times. This leads to the identity φ+ = φ1 + φ2 −φ3, indicating that the 5-wave resonance is directly related to a phase locking between the three basic waves. 3.2. Encountering Wave Packets e−36( x−3L/4 σ ) 30 , (28) (25) (26) (27) (28) where the subscripts r and l are the right and left propagating packets, respectively, and the centre of each packet is x0 = L/4 from the centre. The full initial condition is ζ = ζr + ζl and φ = φr + φl, see upper panel of Figure 8. This conﬁguration will therefore capture more dispersion and temporal behaviour as the waves propagate across one another, but will have have a smaller overall region at any given time where the necessary waves are coincident. Between these two conﬁgurations we have convenient middle ground between the plane wave case and the full physical experiment, to help ascertain where differences are observed and perhaps show how to design better experiments in the future. Fluids 2021, 6, 205 16 of 35 16 of 35 Figure 8. Snapshots of the surface elevation ζ(x, t) at various times the ‘A’ conﬁguration (left) and the ‘B’ conﬁguration (right) of initial coincident wave packets given in Equations (24)–(28). The numerical ‘probes’ are located at x = 198 and x = 202 to be 2 m either side of the domain centre, in-keeping with the experiments of Section 4 . We perform the same parameterised sweep of f3 as in Section 3.1 to intersect the resonance condition and seek increased transfer efﬁciency. max EK1 is shown in Figure 2 along side the plane wave case and time series are shown in Figure 9. The efﬁciency is now much lower but a broader peak persists case A showing a stronger signal than B Now Figure 8. Snapshots of the surface elevation ζ(x, t) at various times the ‘A’ conﬁguration (left) and the ‘B’ conﬁguration (right) of initial coincident wave packets given in Equations (24)–(28). The numerical ‘probes’ are located at x = 198 and x = 202 to be 2 m either side of the domain centre, in-keeping with the experiments of Section 4 . Figure 8. Snapshots of the surface elevation ζ(x, t) at various times the ‘A’ conﬁguration (left) and the ‘B’ conﬁguration (right) of initial coincident wave packets given in Equations (24)–(28). The numerical ‘probes’ are located at x = 198 and x = 202 to be 2 m either side of the domain centre, in-keeping with the experiments of Section 4 . 3.2. Encountering Wave Packets We perform the same parameterised sweep of f3 as in Section 3.1 to intersect the resonance condition and seek increased transfer efﬁciency. max EK1 is shown in Figure 2 along side the plane wave case and time series are shown in Figure 9. The efﬁciency is now much lower but a broader peak persists, case A showing a stronger signal than B. Now the time series demonstrates, in both cases but less pronounced in case A, that growth into K1 is more transient and less sustained than in the plane case, the peak in \|aK1\| occurring within the ﬁrst 10–20 s. The reduction in overall efﬁciency and reduction in sustained growth is, of course, to be expected since the interaction is now localised in space and is not globally instantaneous Fluids 2021, 6, 205 17 of 35 across the domain as in the plane wave case. In addition, although the initial local surface elevations are comparable the overall energy content in the modes is reduced by the localising kernel used to create the individual packets. Finally it should be noted that the initial spectrum is broadened by the localisation, this means that ﬁrst there is a wider range of K3 experiencing resonant (or quasi-resonant) transfer, but also it is possible for more than just the single target K1 to experience resonant growth. We may take this into account by allowing more modes to contribute to the efﬁciency deﬁnition about the central K1. However a better approach, having established that some resonant interactions do persist is to return to the wavelet, scaleogram, wave probe analysis as described above and see if we can observe transfers using a local measure of the surface height. (a) ‘A’ conﬁguration (b) ‘B’ conﬁguration Figure 9. Time series for the efﬁciency Ek (23) (relative amplitude) for the K1 target mode for encountering wave-packets, case A on the left and B on the right. In case A the peak of energy transfer observed in K1 is at the frequency (f3/Hz ≈2.204, K3 = 1246 2π L ) and is here compared to off resonance (f3/Hz = 2.22, K3 = 1264 2π L and f3/Hz = 2.185, K3 = 1224 2π L ). 3.2. Encountering Wave Packets In case B the peak of energy transfer observed in K1 is at the frequency (f3/Hz ≈2.22, K3 = 1264 2π L ) and is here compared to off resonance (f3/Hz = 2.25, K3 = 1298 2π L and f3/Hz = 2.172, K3 = 1210 2π L ). (b) ‘B’ conﬁguration (a) ‘A’ conﬁguration (a) ‘A’ conﬁguration (b) ‘B’ conﬁguration (b) ‘B’ conﬁguration (a) Figure 9. Time series for the efﬁciency Ek (23) (relative amplitude) for the K1 target mode for encountering wave-packets, case A on the left and B on the right. In case A the peak of energy transfer observed in K1 is at the frequency (f3/Hz ≈2.204, K3 = 1246 2π L ) and is here compared to off resonance (f3/Hz = 2.22, K3 = 1264 2π L and f3/Hz = 2.185, K3 = 1224 2π L ). In case B the peak of energy transfer observed in K1 is at the frequency (f3/Hz ≈2.22, K3 = 1264 2π L ) and is here compared to off resonance (f3/Hz = 2.25, K3 = 1298 2π L and f3/Hz = 2.172, K3 = 1210 2π L ). Given the moderately stronger signal in the efﬁciency EK using the A initial condition, we analyse the probe measurements from this case. We now use two probe locations xp = L/2 ± 2m ﬁrst to correspond to the physical experiments in the proceeding section, but also noticing that a symmetry breaking occurs, with a more deﬁnite peak in the wavelet based efﬁciency in the left hand probe at L/2 −2m as shown in the scaleograms in Figure 10 and the ‘sum of squares’ efﬁciency in Figure 11. Figure 10 shows the same 3 cases as in Figure 9a and the initial frequency f3 is in evidence. Naturally, due to the broadening of the spectrum and the shorter time window in which the waves will be encountering the probe, the scaleogram shows a far more scattered set of frequencies. As described the left hand probe shows a discernible brightness about the target f1 line for f3 = 2.204 Hz where the peak in EK1 is also observed in Figure 2, however this is not easily seen in the right hand probe. This opens up some interesting questions about symmetry breaking and measurement for a physical experiment. 3.2. Encountering Wave Packets The symmetry breaking presumably arises due to numerical error in preparing the localised initial condition and/or due to instability of the localised initial condition, including processes due to nonlinear interactions. There are other frequencies in these plots at which a coherent signal is observed; these are due to other nonlinear interactions, e.g., the side-band instability at K3 ± δK described earlier, which we will not elaborate upon here. y p From these results we may conclude that quite long interaction times and regions will be required to make deﬁnitive observations of a ﬁve-wave resonance of this type. 18 of 35 Fluids 2021, 6, 205 (a) Left Probe, f3 = 2.185 Hz (b) Right Probe, f3 = 2.185 Hz (c) Left Probe, f3 = 2.204 Hz (d) Right Probe, f3 = 2.204 Hz (e) Left Probe, f3 = 2.220 Hz (f) Right Probe, f3 = 2.220 Hz ure 10. Scaleograms for measurements of probe 1 (left panels) and probe 2 (right panels), for three numerical wave ket experiments close to the resonance. Frequencies are displayed in the vertical axis and time in the horizontal axis zontal lines represent the theoretical frequencies f1, f3 and f4 stemming from the dispersion relation f (k) = p g\|k\|/2π he wavevectors K1 = K3 −K2, K3 and K4 = K3 + K2. The rows from top to bottom correspond to experiments with Hz = 2.185, 2.204, 2.220 chosen to correspond to the time series in Figure 9. Vertical lines represent the initial and ﬁnal s t = 10 s, 190 s for the calculation of the efﬁciency to the target mode f1 that avoids spurious boundary effects. (a) Left Probe, f3 = 2.185 Hz (b) Right Probe, f3 = 2.185 Hz (a) Left Probe, f3 = 2.185 Hz (b) Right Probe, f3 = 2.185 Hz (d) Right Probe, f3 = 2.204 Hz (c) Left Probe, f3 = 2.204 Hz (c) Left Probe, f3 = 2.204 Hz (d) Right Probe, f3 = 2.204 Hz (f) Right Probe, f3 = 2.220 Hz (e) Left Probe, f3 = 2.220 Hz (e) Left Probe, f3 = 2.220 Hz (f) Right Probe, f3 = 2.220 Hz Figure 10. Scaleograms for measurements of probe 1 (left panels) and probe 2 (right panels), for three numerical wave packet experiments close to the resonance. Frequencies are displayed in the vertical axis and time in the horizontal axis. 3.2. Encountering Wave Packets Horizontal lines represent the theoretical frequencies f1, f3 and f4 stemming from the dispersion relation f (k) = p g\|k\|/2π for the wavevectors K1 = K3 −K2, K3 and K4 = K3 + K2. The rows from top to bottom correspond to experiments with f3/Hz = 2.185, 2.204, 2.220 chosen to correspond to the time series in Figure 9. Vertical lines represent the initial and ﬁnal times t = 10 s, 190 s for the calculation of the efﬁciency to the target mode f1 that avoids spurious boundary effects. 19 of 35 Fluids 2021, 6, 205 18.0 18.5 19.0 19.5 20.0 0.00 0.02 0.04 0.06 0.08 0.10 2.11 2.14 2.17 2.2 2.23 K3[m-1] Efficiency f3[Hz] t∈[10s,190s] Eff f1 Probe 1 t∈[10s,190s] Eff f1 Probe 2 Figure 11. Efﬁciency as a function of the numerical experiment, for each probe, parameterised by the corresponding wavevector K3, for the encountering wavepackets case for a long time series [0, 200 s]. The efﬁciency is deﬁned as the sum of the squares of the scaleogram signals in Figure 10, on a strip of width 0.05f1 about f1 and between the vertical lines in the ﬁgure, divided by the sum of squares over the whole range of frequencies, including f2 which is not shown. An efﬁciency of 0.10 corresponds to 10% efﬁciency. The dashed grey vertical line corresponds to the theoretical resonant case K1 : K2 : K3 = 16 : 9 : 25. 18.0 18.5 19.0 19.5 20.0 0.00 0.02 0.04 0.06 0.08 0.10 2.11 2.14 2.17 2.2 2.23 K3[m-1] Efficiency f3[Hz] t∈[10s,190s] Eff f1 Probe 1 t∈[10s,190s] Eff f1 Probe 2 t∈[10s,190s] Eff f1 Probe 1 t∈[10s,190s] Eff f1 Probe 2 Figure 11. Efﬁciency as a function of the numerical experiment, for each probe, parameterised by the corresponding wavevector K3, for the encountering wavepackets case for a long time series [0, 200 s]. The efﬁciency is deﬁned as the sum of the squares of the scaleogram signals in Figure 10, on a strip of width 0.05f1 about f1 and between the vertical lines in the ﬁgure, divided by the sum of squares over the whole range of frequencies, including f2 which is not shown. An efﬁciency of 0.10 corresponds to 10% efﬁciency. The dashed grey vertical line corresponds to the theoretical resonant case K1 : K2 : K3 = 16 : 9 : 25. 4. Experiments We performed preliminary experiments in a water wave tank to search for this res- onance. The experimental setup, shown in Figure 12, consists of a linear wave tank (length = 35 m) with identical right-angle wedges (wavemakers) on each end that are os- cillated vertically. The water mass at rest has a depth of 0.55 m. Two probes are located symmetrically at a distance of 15.5 m from the corresponding nearest end. The distance between the probes is 4 m. Figure 12. Diagram of the experimental setup, including probe #1 and probe #2. Note N denotes north end; S indicates south end. Unit: Metres. Figure 12. Diagram of the experimental setup, including probe #1 and probe #2. Note N denotes north end; S indicates south end. Unit: Metres. See, in Appendix B, probe calibration curves and consistency tests. Encountering Wavepacket Experiments In our experiments the water depth is ﬁnite and therefore one should use the gravity- wave dispersion relation 2π f (k) = p gk tanh(kh), where g = 9.8 m s−1 is the accel- eration of gravity and h = 0.55 m is the water depth. However, for the frequencies studied (f ≥1.33 Hz) the relative error with respect to the inﬁnite-depth approximation 2π f (k) = p g\|k\| is less than 10−5 so we can use this latter approximation. Equivalently, for Fluids 2021, 6, 205 20 of 35 20 of 35 the frequencies tested, deep water can be assumed. We perform 9 experiments. On each experiment, the two wavemakers operate with the same oscillation pattern: the frequencies tested, deep water can be assumed. We perform 9 experiments. On each experiment, the two wavemakers operate with the same oscillation pattern: η(t) = A2 cos(2π f2t) + A3 cos(2π f3t) , η(t) = A2 cos(2π f2t) + A3 cos(2π f3t) , η(t) = A2 cos(2π f2t) + A3 cos(2π f3t) , consisting of the sum of two monochromatic oscillations: The ﬁrst one with a base ﬁxed frequency f2 = 1.33 Hz corresponding to wavevectors ±K2 = ±7.131 m−1 and with a ﬁxed amplitude of approximately A2 ≈0.06/K2 (so steepness is A2K2 = 0.06), while the second one has a changeable frequency f3, depending on the experiment, and a changeable amplitude A3 ≈0.12/K3 again so that the steepness is ﬁxed at 0.12. Table 1 summarises the range of values of wavemaker frequencies f3 used in the 9 experiments, with the corresponding amplitudes A3 used. Table 1. For each experiment, frequency f3 along with the wavemaker oscillation amplitude A3, chosen so that A3K3 ≈0.12, where K3 = 4π2g−1 f 2 3 is the wavenumber. In the last column, the theoretical target frequency f1 = p gK1/2π that would be observed if wavenumber K1 = K3 −K2 was produced. We highlight in boldface the resonant case f1 : f2 : f3 = 4 : 3 : 5, corresponding to Equation (16). Encountering Wavepacket Experiments Experiment f3 (Hz) A3 (cm) Target f1 (Hz) Exp 1 2.05 0.71 1.56 Exp 2 2.08 0.69 1.60 Exp 3 2.10 0.68 1.62 Exp 4 2.15 0.64 1.69 Exp 5 2.185 0.62 1.73 Exp 6 2.22 0.60 1.78 Exp 7 2.25 0.59 1.81 Exp 8 2.30 0.56 1.88 Exp 9 2.35 0.54 1.94 In each experiment, therefore, as a result of the wavemakers’ activity, two encountering wave trains with central wavevectors K2, K3, −K2, −K3 will approach the probes, and will eventually encounter each other, triggering nonlinear interactions. If the theory is correct, when K3/K2 = 25/9 the ﬁve-wave resonance should produce waves with wavevectors ±K1 = ±16K2/9, as explained in Equation (16). The frequencies are proportional to the square roots of the wavenumbers, and as explained in the text right after Equation (16), at resonance we would have f1 : f2 : f3 = 4 : 3 : 5. Now, eventually these daughter waves would reach the probes, which would record a signal with frequency f1,reso = 4f2/3. Theoretically, this ﬁve-wave resonance should occur near Experiment 6, because in that experiment we have K3 = 4π2g−1 f 2 3 = 19.85 m−1 and as K2 = 7.131 m−1, we would get K3/K2 ≈2.78 which is very close to 25/9. Similarly, the corresponding frequency of the oscillations produced by the nonlinear resonant interactions, f1 = p gK1/2π = p g(K3 −K2)/2π ≈1.78 Hz, would be very close to f1,reso = 4f2/3 ≈1.77 Hz. p g( ) y f , f Probe measurements are started when the wavepackets reach the probes. For each experiment we perform measurements on each probe, at intervals of ∆t = 0.02 s and during a time of 81.9 s, corresponding to a total of N = 4096 data points per probe per experiment. Figure 13 shows the time series for experiment 7, which is the one that shows a stronger signal for the target frequency f1, as we will see when we analyse the scalograms. From the time series we can see that the two probes’ measured oscillations are quite close and in phase: The dominant frequency in the plot is f2 = 1.33 Hz corresponding to the wavevectors ±K2. Modulations about this main frequency correspond to a combination of frequencies f3 and f1 along with a spectrum of less important frequencies, including harmonics. Encountering Wavepacket Experiments The fact that the two probes’ signals are not exactly the same is reminiscent of the symmetry breaking observed in the numerical case, and may be due to slight Fluids 2021, 6, 205 21 of 35 21 of 35 differences in the experimental conditions and the subsequent nonlinear ampliﬁcation of these differences. differences in the experimental conditions and the subsequent nonlinear ampliﬁcation of these differences. As the fastest waves are the ones with wavevectors ±K2 (at the ﬁxed frequency f2 = 1.33 Hz in the experiment), one can assume these waves will reach the probes ﬁrst. We recall that, in the experiments, the initial measurement time t = 0 corresponds precisely to the time when the waves reach the probes for the ﬁrst time. As the group velocity of the waves with wavevectors ±K3 is less than the group velocity of the waves with wavevectors ±K2, we expect to see no effect from the nonlinear interactions (and in particular no generation of ±K1 wavevectors) in the early stages. To be more precise, a simple analysis of the ﬁve-wave resonance Equation (16) shows that nonlinearly-produced wavevectors ±K1 could be measured by the probes from the moment the wavevectors ±K3 reach the probes. This time can be calculated explicitly for each experiment, giving, for experiments 1 to 9, t0 = {14.44, 15.04, 15.44, 16.44, 17.14, 17.84, 18.42, 19.42, 20.42} s. The leftmost vertical marker line in Figure 13 corresponds to this “ﬁrst measurement” time. It is also evident from Figure 13 that wave amplitudes become larger near t ≈60 s. This is due to the reﬂected wavepackets with wavevectors ±K2, which after reﬂection from the walls of the tank arrive back at the probes. After this time we expect a stronger signal for the nonlinearly-produced wavevectors ±K1 in the probes. The time this happens is t1 = 59.38 s, independent from the experiment. The rightmost vertical marker line in Figure 13 corresponds to this “second measurement” time. [ ] - - η() [] Figure 13. Plots of the probe measurements for experiment number 7. Encountering Wavepacket Experiments The vertical lines correspond to the ﬁrst time t0 the wavepackets with wavevectors ±K3 arrive at the probes (leftmost line), and the time t1 the wavepackets with wavevectors ±K2 arrive at the probes after reﬂection from the walls of the tank (rightmost line). η() [] [ ] Figure 13. Plots of the probe measurements for experiment number 7. The vertical lines correspond to the ﬁrst time t0 the wavepackets with wavevectors ±K3 arrive at the probes (leftmost line), and the time t1 the wavepackets with wavevectors ±K2 arrive at the probes after reﬂection from the walls of the tank (rightmost line). In order to understand the frequency content of these signals, we apply a wavelet transform on them. Wavelet scaleograms show the time evolution of the energy content Fluids 2021, 6, 205 22 of 35 22 of 35 over a range of frequencies. By trial and error we found that a good compromise is struck between frequency resolution and time resolution in the wavelet transform by choosing a Gabor wavelet with 96 oscillations and with the usual scale resolution of log2(4096/2) = 11 and a total of 256 voices per scale. Before analysing the real data, Figure 14 shows density- plot scaleograms corresponding to artiﬁcial signals containing only four frequencies: (29) σartif(t) = 10 sin(2π f2t) + 3 sin(2π f3t) + 0.3 sin(2π f1t) + 0.3 sin(2π f4t) , (29) where f3 is chosen from the list in Table 1 and f1, f4 are two possible target frequencies deﬁned in terms of the dispersion relation by 2π f1 = p gK1 and 2π f4 = p gK4, where K1 = K3 −K2 and K4 = K3 + K2 are target wavevectors that could be generated by nonlinear interactions in the experiments (notice that 2π f3 = p gK3 deﬁnes the wavevector K3). Apart from f2 = 1.33 Hz which is ﬁxed, these frequencies depend on the experiment (but are completely determined by it) according to Table 1. The scaleograms show spurious boundary effects at early and late times for the frequency f3. For the frequencies f1 and f4 the boundary effects are minimal. The plots show that these two frequencies are resolved within a band of width ∆f = 0.09f. Thus, a real signal from the experiments at a given frequency will be expected to have a comparable width. Encountering Wavepacket Experiments This will be useful when estimating the relative energy in a frequency. In Figures 15–17 we show the scaleograms obtained from probe 1 data (left panels) and probe 2 data (right panels). For each panel, vertical lines analogous to those in Figure 13 indicate the theoretical ﬁrst time t0 when nonlinearly produced target frequency f1 due to the ﬁve-wave resonance can be measured by the probes (leftmost line), the theoretical time t1 when the wavepackets arrive back at the probes after reﬂecting from the tank walls (middle line), and the time beyond which the scaleograms show spurious boundary effects, as evidenced in Figure 14 (rightmost line). The ﬁgures also show horizontal lines corresponding to the theoretical frequencies f1, f3, f4 derived from the wavevectors ±K1, ±K3, ±K4, and a theoretical harmonic f1 + f2. It is apparent that experiment 6 shows some activity in the f1 line, as well as the line f1 + f2. However, the next experiment (number 7) shows a more convincing signal at f1, apparently resolved, particularly at the late stages after t = t1 = 59.38 s. g In order to provide a more quantitative measure of the signals shown by the scale- ograms, we calculate the relative energy of the wavelet transforms that are plotted in the scaleograms, for the frequency f1. We do this by deﬁning an early-time energy by the sum of squares between t = t0 and t = t1 = 59.38 s, and on a strip of width ∆f = 0.09f1 about f1. We then divide this energy by the total energy over the time interval [t0, 59.38 s] and over the whole frequency range (including f2 which is not shown). The result is an “early-time efﬁciency” which we plot in Figure 18, dashed lines, for each probe. Similarly, we deﬁne a late-time energy by the sum of squares between t = t1 = 59.38 s and t = 77.92 s, and on a strip of width ∆f = 0.09f1 about f1. We then divide this energy by the total energy over the time interval [59.38 s, 77.92 s] and over the whole frequency range (including f2, not shown). The result is a “late-time efﬁciency” which we plot in Figure 18, solid lines, for each probe. Encountering Wavepacket Experiments It is evident from these plots that there is an efﬁciency peak that stands out, and although it is modest (0.5% efﬁciency) it nevertheless shows that considering the short times involved there is scope for a measurement of the theoretical ﬁve-wave resonance, perhaps in a larger tank and with larger amplitudes so that the effect can be clearly obtained. 23 of 35 Fluids 2021, 6, 205 (a) Mock Exp 1: f3 = 2.05 Hz (b) Mock Exp 2: f3 = 2.08 Hz (c) Mock Exp 3: f3 = 2.10 Hz (d) Mock Exp 4: f3 = 2.15 Hz (e) Mock Exp 5: f3 = 2.185 Hz (f) Mock Exp 6: f3 = 2.22 Hz (g) Mock Exp 7: f3 = 2.25 Hz (h) Mock Exp 8: f3 = 2.30 Hz (i) Mock Exp 9: f3 = 2.35 Hz Figure 14. Scaleograms for the artiﬁcial signal, Equation (29), mimicking three of the main frequencies found in the experiments: f1, f3, f4. The width about the frequencies f1 and f4 is of the order ∆f = 0.09f and is due to the way the scaleogram is produced. The following comments apply to all scaleogram ﬁgures in this section. The colormaps indicate the amplitude of the scaleograms. The scaleograms are produced by sampling the artiﬁcial signal using 4096 time points and performing a continuous wavelet transform using Gabor wavelets with 96 oscillations, with a scale resolution of 11 frequency scales along with 256 voices per scale. Encountering Wavepacket Experiments (a) Mock Exp 1: f3 = 2.05 Hz (b) Mock Exp 2: f3 = 2.08 Hz (c) Mock Exp 3: f3 = 2.10 Hz (c) Mock Exp 3: f3 = 2.10 Hz (b) Mock Exp 2: f3 = 2.08 Hz (a) Mock Exp 1: f3 = 2.05 Hz (b) Mock Exp 2: f3 = 2.08 Hz (e) Mock Exp 5: f3 = 2.185 Hz (c) Mock Exp 3: f3 = 2.10 Hz (f) Mock Exp 6: f3 = 2.22 Hz ( ) p f3 (d) Mock Exp 4: f3 = 2.15 Hz (d) Mock Exp 4: f3 = 2.15 Hz (f) Mock Exp 6: f3 = 2.22 Hz (e) Mock Exp 5: f3 = 2.185 Hz (h) Mock Exp 8: f3 = 2.30 Hz (i) Mock Exp 9: f3 = 2.35 Hz (g) Mock Exp 7: f3 = 2.25 Hz (h) Mock Exp 8: f3 = 2.30 Hz (i) Mock Exp 9: f3 = 2.35 Hz (g) Mock Exp 7: f3 = 2.25 Hz Figure 14. Scaleograms for the artiﬁcial signal, Equation (29), mimicking three of the main frequencies found in the experiments: f1, f3, f4. The width about the frequencies f1 and f4 is of the order ∆f = 0.09f and is due to the way the scaleogram is produced. The following comments apply to all scaleogram ﬁgures in this section. The colormaps indicate the amplitude of the scaleograms. The scaleograms are produced by sampling the artiﬁcial signal using 4096 time points and performing a continuous wavelet transform using Gabor wavelets with 96 oscillations, with a scale resolution of 11 frequency scales along with 256 voices per scale. 24 of 35 Fluids 2021, 6, 205 (a) Probe 1, Exp 1: f3 = 2.05 Hz (b) Probe 2, Exp 1: f3 = 2.05 Hz (c) Probe 1, Exp 2: f3 = 2.08 Hz (d) Probe 2, Exp 2: f3 = 2.08 Hz (e) Probe 1, Exp 3: f3 = 2.1 Hz (f) Probe 2, Exp 3: f3 = 2.1 Hz ure 15. Scaleograms for measurements of probe 1 (left panels) and probe 2 (right panels), for experiments 1, 2 and quencies are displayed in the vertical axis and time in the horizontal axis. Three of the main frequencies found in th eriments are marked in each panel: f1, f3, f4, along with f1 + f2 for reference. Encountering Wavepacket Experiments The rows from top to bottom correspon xperiments 1, 2 and 3: Starting from the top, f3/Hz = 2.05, 2.08, 2.1. The three vertical lines correspond, from left t t, to: (i) The theoretical time (which depends on the experiment) when the ﬁrst nonlinearly-produced wavepacke h central wavenumbers ±K1 arrive at the probes; (ii) the theoretical time (which is ﬁxed) when the ﬁrst wavepacke h central wavenumbers ±K2 arrive at the probes after reﬂecting from the ends of the tank; (iii) the ﬁxed time when th ﬁcial signal’s scalogram shows a signiﬁcant departure from the expected frequencies. See further comments on th rmaps and details of scaleogram calculations in the caption of Figure 14. (b) Probe 2, Exp 1: f3 = 2.05 Hz (a) Probe 1, Exp 1: f3 = 2.05 Hz (a) Probe 1, Exp 1: f3 = 2.05 Hz (b) Probe 2, Exp 1: f3 = 2.05 Hz (d) Probe 2, Exp 2: f3 = 2.08 Hz (c) Probe 1, Exp 2: f3 = 2.08 Hz (c) Probe 1, Exp 2: f3 = 2.08 Hz (d) Probe 2, Exp 2: f3 = 2.08 Hz (f) Probe 2, Exp 3: f3 = 2.1 Hz (e) Probe 1, Exp 3: f3 = 2.1 Hz (e) Probe 1, Exp 3: f3 = 2.1 Hz (f) Probe 2, Exp 3: f3 = 2.1 Hz Figure 15. Scaleograms for measurements of probe 1 (left panels) and probe 2 (right panels), for experiments 1, 2 and 3. Frequencies are displayed in the vertical axis and time in the horizontal axis. Three of the main frequencies found in the experiments are marked in each panel: f1, f3, f4, along with f1 + f2 for reference. The rows from top to bottom correspond to experiments 1, 2 and 3: Starting from the top, f3/Hz = 2.05, 2.08, 2.1. The three vertical lines correspond, from left to right, to: (i) The theoretical time (which depends on the experiment) when the ﬁrst nonlinearly-produced wavepackets with central wavenumbers ±K1 arrive at the probes; (ii) the theoretical time (which is ﬁxed) when the ﬁrst wavepackets with central wavenumbers ±K2 arrive at the probes after reﬂecting from the ends of the tank; (iii) the ﬁxed time when the artiﬁcial signal’s scalogram shows a signiﬁcant departure from the expected frequencies. See further comments on the colormaps and details of scaleogram calculations in the caption of Figure 14. Figure 15. Encountering Wavepacket Experiments Scaleograms for measurements of probe 1 (left panels) and probe 2 (right panels), for experiments 1, 2 and 3. Frequencies are displayed in the vertical axis and time in the horizontal axis. Three of the main frequencies found in the experiments are marked in each panel: f1, f3, f4, along with f1 + f2 for reference. The rows from top to bottom correspond to experiments 1, 2 and 3: Starting from the top, f3/Hz = 2.05, 2.08, 2.1. The three vertical lines correspond, from left to right, to: (i) The theoretical time (which depends on the experiment) when the ﬁrst nonlinearly-produced wavepackets with central wavenumbers ±K1 arrive at the probes; (ii) the theoretical time (which is ﬁxed) when the ﬁrst wavepackets with central wavenumbers ±K2 arrive at the probes after reﬂecting from the ends of the tank; (iii) the ﬁxed time when the artiﬁcial signal’s scalogram shows a signiﬁcant departure from the expected frequencies. See further comments on the colormaps and details of scaleogram calculations in the caption of Figure 14. Fluids 2021, 6, 205 25 of 35 luids 2021, 6, 205 25 of 35 (a) Probe 1, Exp 4: f3 = 2.15 Hz (b) Probe 2, Exp 4: f3 = 2.15 Hz (c) Probe 1, Exp 5: f3 = 2.185 Hz (d) Probe 2, Exp 5: f3 = 2.185 Hz (e) Probe 1, Exp 6: f3 = 2.22 Hz (f) Probe 2, Exp 6: f3 = 2.22 Hz Figure 16. Same description as in the caption of Figure 15, but for experiments 4, 5 and 6, corresponding to f3/Hz = 2.15, 2.185, 2.22, respectively. (b) (b) Probe 2, Exp 4: f3 = 2.15 Hz (b) (a) Probe 1, Exp 4: f3 = 2.15 Hz (b) (a) Probe 1, Exp 4: f3 = 2.15 Hz (c) P b 1 E 5 f 2 185 H (c) Probe 1, Exp 5: f3 = 2.185 Hz (d) Probe 2, Exp 5: f3 = 2.185 Hz (c) Probe 1, Exp 5: f3 = 2.185 Hz (d) Probe 2, Exp 5: f3 = 2.185 Hz (e) Probe 1, Exp 6: f3 = 2.22 Hz (f) Probe 2, Exp 6: f3 = 2.22 Hz Figure 16. Same description as in the caption of Figure 15, but for experiments 4, 5 and 6, corresponding to f3/Hz = 2.15, 2.185, 2.22, respectively. Encountering Wavepacket Experiments (e) Probe 1, Exp 6: f3 = 2.22 Hz (f) Probe 2, Exp 6: f3 = 2.22 Hz (e) Probe 1, Exp 6: f3 = 2.22 Hz (e) Probe 1, Exp 6: f3 = 2.22 Hz Figure 16. Same description as in the caption of Figure 15, but for experiments 4, 5 and 6, corresponding to f3/Hz = 2.15, 2.185, 2.22, respectively. Fluids 2021, 6, 205 26 of 35 luids 2021, 6, 205 26 of 35 (a) Probe 1, Exp 7: f3 = 2.25 Hz (b) Probe 2, Exp 7: f3 = 2.25 Hz (c) Probe 1, Exp 8: f3 = 2.3 Hz (d) Probe 2, Exp 8: f3 = 2.3 Hz (e) Probe 1, Exp 9: f3 = 2.35 Hz (f) Probe 2, Exp 9: f3 = 2.35 Hz Figure 17. Same description as in the caption of Figure 15, but for experiments 7, 8 and 9, corresponding to f3/Hz = 2.25, 2.3, 2.35, respectively. (b) (a) Probe 1, Exp 7: f3 = 2.25 Hz (b) Probe 2, Exp 7: f3 = 2.25 Hz (b) (a) Probe 1, Exp 7: f3 = 2.25 Hz (d) Probe 2, Exp 8: f3 = 2.3 Hz (c) Probe 1, Exp 8: f3 = 2.3 Hz (c) Probe 1, Exp 8: f3 = 2.3 Hz (e) Probe 1, Exp 9: f3 = 2.35 Hz (f) Probe 2, Exp 9: f3 = 2.35 Hz (f) Probe 2, Exp 9: f3 = 2.35 Hz (e) Probe 1, Exp 9: f3 = 2.35 Hz Figure 17. Same description as in the caption of Figure 15, but for experiments 7, 8 and 9, corresponding to f3/Hz = 2.25, 2.3, 2.35, respectively. 27 of 35 Fluids 2021, 6, 205 17 18 19 20 21 22 0.000 0.001 0.002 0.003 0.004 0.005 2.05 2.11 2.17 2.23 2.28 2.34 K3[m-1] Efficiency f3[Hz] t∈[59.36s, 77.92s] Eff f1 Probe 1 t∈[59.36s, 77.92s] Eff f1 Probe 2 t∈[t0,59.36s] Eff f1 Probe 1 t∈[t0,59.36s] Eff f1 Probe 2 Figure 18. Efﬁciency as a function of the experiment, for the two probes and for early (dashed lines) and late (solid lines) measurements. An efﬁciency of 0.005 corresponds to 0.5% efﬁciency. The initial time for the early measurements is the theoretical time (which depends on the experiment) when the ﬁrst nonlinearly-produced wavepackets with central wavenumbers ±K1 arrive at the probes: For the 9 experiments, t0 = {14.44, 15.04, 15.44, 16.44, 17.14, 17.84, 18.42, 19.42, 20.42} s. Encountering Wavepacket Experiments The ﬁnal time for the early measurements is the theoretical time (which is ﬁxed) when the ﬁrst wavepackets with central wavenumbers ±K2 arrive at the probes after reﬂecting from the ends of the tank. The dashed grey vertical line corresponds to the theoretical resonant case K1 : K2 : K3 = 16 : 9 : 25. 17 18 19 20 21 22 0.000 0.001 0.002 0.003 0.004 0.005 2.05 2.11 2.17 2.23 2.28 2.34 K3[m-1] Efficiency f3[Hz] t∈[59.36s, 77.92s] Eff f1 Probe 1 t∈[59.36s, 77.92s] Eff f1 Probe 2 t∈[t0,59.36s] Eff f1 Probe 1 t∈[t0,59.36s] Eff f1 Probe 2 Figure 18. Efﬁciency as a function of the experiment, for the two probes and for early (dashed lines) and late (solid lines) measurements. An efﬁciency of 0.005 corresponds to 0.5% efﬁciency. The initial time for the early measurements is the theoretical time (which depends on the experiment) when the ﬁrst nonlinearly-produced wavepackets with central wavenumbers ±K1 arrive at the probes: For the 9 experiments, t0 = {14.44, 15.04, 15.44, 16.44, 17.14, 17.84, 18.42, 19.42, 20.42} s. The ﬁnal time for the early measurements is the theoretical time (which is ﬁxed) when the ﬁrst wavepackets with central wavenumbers ±K2 arrive at the probes after reﬂecting from the ends of the tank. The dashed grey vertical line corresponds to the theoretical resonant case K1 : K2 : K3 = 16 : 9 : 25. 5. Conclusions and Discussion • On the experimental front the main difﬁculty is to ﬁne tune the amplitudes and frequencies in order to capture the resonance, but we got this from hindsight. Our preliminary experiments seem to show that the resonance exists physically, although as can be seen in Figure 18, the efﬁciency is relatively small. We learned from our more recent numerical experiments that the resonance peak has a narrow width. For this reason we propose to conduct experiments in much longer wave ﬂumes with much larger amplitudes of lower frequency waves. In addition, the study of the second resonant quintet (17) obtained theoretically, and/or the exploration of optimal initial conditions, may make it easier to ﬁnd a resonance in experiments as well as numerical simulations. In future work we plan to extend this type of minimal search to gravity-capillary waves and to gravity waves in two-dimensional propagation. We would like to end on a positive note, by reporting on the following phase-locking analysis between the three scenarios studied: Numerical encountering plane waves, nu- merical encountering wave packets, and experimental encountering waves. We will es- tablish that the triad phases get locked near our theoretical resonance, thus providing an independent piece of evidence that the resonance is indeed taking place. We explain brieﬂy how this is done. Using the probe data for each scenario and each “experiment” within a given scenario, we perform pass-band ﬁlters around each of the three important frequencies f1, f2, f3, with width 0.09f. Each one of these three ﬁltered signals is then extended via the analytic signal approach using a Hilbert transform (see, for example, [46]) in order to get the “conjugate” signal and thus calculate the “analytic phases” denoted by the time series ϕ1(t), ϕ2(t), ϕ3(t). These phases contain the fast time dependence 2π fjt, respectively. After subtracting this fast time dependence, obtain the new phases eϕj(t) = ϕ1(t) −2π fjt, j = 1, 2, 3. We then calculate the triad phase ϕ(t) := eϕ1(t) + eϕ2(t) −eϕ3(t) . (30) (30) The phase-locking value for this signal is deﬁned by (see, e.g., [47]): The phase-locking value for this signal is deﬁned by (see, e.g., [47]): R := \|⟨exp(iϕ(t))⟩t\| ∈ [0, 1], (31) R := \|⟨exp(iϕ(t))⟩t\| ∈ [0, 1], (31) where ⟨·⟩t denotes time average over the whole time series, and \| · \| denotes modulus. 5. Conclusions and Discussion A phase-locking value close to 1 denotes a situation where the corresponding phase is locked at a given value for most of the time. The resulting phase-locking values, for the different scenarios and the different experiments, are plotted in Figure 19, using similar notation as in the efﬁciency Figures 6, 11 and 18. It is evident that in each scenario the phase-locking value is maximum precisely at the resonant case (denoted in each panel with a vertical dashed line), thus conﬁrming that the 5-wave resonance not only provides larger target amplitudes but also strong phase synchronisations amongst the interacting modes. Notice that the effect of symmetry breaking is again patent in the numerical wave packets case, cf. Figure 19b, as well as in the experimental case, cf. Figure 19c. In each case, one of the probes shows a clear global maximum of phase-locking R at the resonance, while the other probe has a local maximum at the resonance and a global maximum at another place. This behaviour is consistent with the efﬁciency plots in Figures 11 and 18: The probes that show a higher efﬁciency peak in these ﬁgures, correspond precisely to the probes that show a clear and strong maximum of the phase-locking value R in Figure 19b,c. where ⟨·⟩t denotes time average over the whole time series, and \| · \| denotes modulus. A phase-locking value close to 1 denotes a situation where the corresponding phase is locked at a given value for most of the time. The resulting phase-locking values, for the different scenarios and the different experiments, are plotted in Figure 19, using similar notation as in the efﬁciency Figures 6, 11 and 18. It is evident that in each scenario the phase-locking value is maximum precisely at the resonant case (denoted in each panel with a vertical dashed line), thus conﬁrming that the 5-wave resonance not only provides larger target amplitudes but also strong phase synchronisations amongst the interacting modes. Notice that the effect of symmetry breaking is again patent in the numerical wave packets case, cf. Figure 19b, as well as in the experimental case, cf. Figure 19c. In each case, one of the probes shows a clear global maximum of phase-locking R at the resonance, while the other probe has a local maximum at the resonance and a global maximum at another place. 5. Conclusions and Discussion In this work we proposed to study the minimal resonant conﬁguration of water gravity waves in one-dimensional propagation. The resulting 5-wave resonances are based on a triad of wavevectors along with their negatives, leading to a scenario of encountering wavepackets, which has an experimental appeal. The problem poses interesting challenges in all fronts: • On the theoretical front, the 5-wave resonant manifold and the interaction coefﬁcients on this resonant manifold had been obtained in a series of papers over two decades ago [4–6], triggered by the discovery that the interaction coefﬁcients vanish identically on the 4-wave resonant manifold for one-dimensional propagation of water gravity waves [3]. We used these results to ﬁnd the simplest 5-wave resonance that can be made out of a triad of wavevectors and their negatives, and calculated its normal- form Hamiltonian. We proved that the system is not integrable, but it lacks just one constant of motion to become integrable, so symmetric scenarios can produce integrable systems. • On the front of numerical simulations of the governing partial differential equations, the equations had been obtained over ﬁve decades ago [15], as a power series in terms of the steepness effectively. The numerical implementation we needed to use in order to accurately resolve 5-wave interactions is the one that uses up to 6-wave interactions in a pseudo-spectral setting [40]. Such an implementation requires a higher-than- usual dealiasing and thus can get quite expensive in terms of the required spectral resolution. We managed to validate our implementation against some benchmarks, and were able to establish the existence of the resonance and quantify its effects in terms of the energy transferred to target modes. We considered encountering plane waves and also encountering wave packets, in a simulated tank that is 300 m long. The plane-wave case provided the most efﬁcient energy transfer in terms of Hamiltonian energy, while the wavepackets case provided a higher efﬁciency in terms of the probe measurements of surface elevation at a point along the tank. Fluids 2021, 6, 205 28 of 35 28 of 35 • On the experimental front the main difﬁculty is to ﬁne tune the amplitudes and frequencies in order to capture the resonance, but we got this from hindsight. Our preliminary experiments seem to show that the resonance exists physically, although as can be seen in Figure 18, the efﬁciency is relatively small. 5. Conclusions and Discussion Figures 6, 11 and 18, respectively. In all panels, the dashed grey vertical line corresponds to the theoretical resonant case K : K : K = 16 : 9 : 25 18.0 18.5 19.0 19.5 20.0 0.00 0.05 0.10 0.15 0.20 2.11 2.14 2.17 2.2 2.23 K3[m-1] R (phase-locking) f3[Hz] (b) Numerical wave packets 2 17 18 19 20 21 22 0.00 0.05 0.10 0.15 0.20 0.25 2.05 2.11 2.17 2.23 2.28 2.34 K3[m-1] R (phase-locking) f3[Hz] Probe 1 Probe 2 (c) Experiments (b) Numerical wave packets Figure 19. Phase-locking values of analytic-signal-approach triad phases, Equations (30) and (31), obtained from probe data, for different experiments within the three scenarios studied in this paper: (a) Encountering numerical plane waves; (b) encountering numerical wave packets; (c) encountering experimental waves. In all three scenarios, a clear local maximum of the phase-locking value is attained at the predicted resonance, corresponding to the maximum in efﬁciency for each case, cf. Figures 6, 11 and 18, respectively. In all panels, the dashed grey vertical line corresponds to the theoretical resonant case K1 : K2 : K3 = 16 : 9 : 25. Author Contributions: Conceptualization, D.L., M.P. and M.D.B.; theoretical investigation and mod- elling, D.L., S.W., R.I. and M.D.B.; numerical simulations and analysis, D.L. and M.D.B.; experimental investigation, M.P., D.-Y.L.; writing—original draft preparation, review and editing, D.L., M.P., R.I. and M.D.B. All authors have read and agreed to the published version of the manuscript. Author Contributions: Conceptualization, D.L., M.P. and M.D.B.; theoretical investigation and mod- elling, D.L., S.W., R.I. and M.D.B.; numerical simulations and analysis, D.L. and M.D.B.; experimental investigation, M.P., D.-Y.L.; writing—original draft preparation, review and editing, D.L., M.P., R.I. and M.D.B. All authors have read and agreed to the published version of the manuscript. Funding: R.I. is partially supported by the Bulgarian National Science Fund, grant KΠ -06H42/2 from 27.11.2020. Data Availability Statement: The data presented in this study are available on request from the corresponding author (miguel.bustamante@ucd.ie). Acknowledgments: We thank Wooyoung Choi for providing us with key references about the ﬁfth-order code. Acknowledgments: We thank Wooyoung Choi for providing us with key references about the ﬁfth-order code. Acknowledgments: We thank Wooyoung Choi for providing us with key references about the ﬁfth-order code. Conﬂicts of Interest: The authors declare no conﬂict of interest. 5. Conclusions and Discussion This behaviour is consistent with the efﬁciency plots in Figures 11 and 18: The probes that show a higher efﬁciency peak in these ﬁgures, correspond precisely to the probes that show a clear and strong maximum of the phase-locking value R in Figure 19b,c. 29 of 35 Fluids 2021, 6, 205 17 18 19 20 21 22 23 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 2.05 2.11 2.17 2.23 2.28 2.34 2.39 K3[m-1] R (phase-locking) f3[Hz] (a) Numerical plane waves 18.0 18.5 19.0 19.5 20.0 0.00 0.05 0.10 0.15 0.20 2.11 2.14 2.17 2.2 2.23 K3[m-1] R (phase-locking) f3[Hz] Probe 1 Probe 2 (b) Numerical wave packets 17 18 19 20 21 22 0.00 0.05 0.10 0.15 0.20 0.25 2.05 2.11 2.17 2.23 2.28 2.34 K3[m-1] R (phase-locking) f3[Hz] Probe 1 Probe 2 (c) Experiments Figure 19. Phase-locking values of analytic-signal-approach triad phases, Equations (30) and (31), obtained from probe data, for different experiments within the three scenarios studied in this paper: (a) Encountering numerical plane waves; (b) encountering numerical wave packets; (c) encountering experimental waves. In all three scenarios, a clear local maximum of the phase-locking value is attained at the predicted resonance, corresponding to the maximum in efﬁciency for each case, cf. Figures 6, 11 and 18, respectively. In all panels, the dashed grey vertical line corresponds to the theoretical resonant case K1 : K2 : K3 = 16 : 9 : 25. 17 18 19 20 21 22 23 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 2.05 2.11 2.17 2.23 2.28 2.34 2.39 K3[m-1] R (phase-locking) f3[Hz] ( ) (a) Numerical plane waves (a) Numerical plane waves 18.0 18.5 19.0 19.5 20.0 0.00 0.05 0.10 0.15 0.20 2.11 2.14 2.17 2.2 2.23 K3[m-1] R (phase-locking) f3[Hz] Probe 1 Probe 2 (b) Numerical wave packets 17 18 19 20 21 22 0.00 0.05 0.10 0.15 0.20 0.25 2.05 2.11 2.17 2.23 2.28 2.34 K3[m-1] R (phase-locking) f3[Hz] Probe 1 Probe 2 (c) Experiments Figure 19. Phase-locking values of analytic-signal-approach triad phases, Equations (30) and (31), obtained from probe data, for different experiments within the three scenarios studied in this paper: (a) Encountering numerical plane waves; (b) encountering numerical wave packets; (c) encountering experimental waves. In all three scenarios, a clear local maximum of the phase-locking value is attained at the predicted resonance, corresponding to the maximum in efﬁciency for each case, cf. 5. Conclusions and Discussion The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. Fluids 2021, 6, 205 30 of 35 30 of 35 Appendix A. Construction of 5-Wave Resonances Using Only Three Different Positive Wavevectors J1, J2, J3 (with 0 < J2 < J1 and J3 = J1 + J2) Along with Their Negatives Appendix A. Construction of 5-Wave Resonances Using Only Three Different Positive Wavevectors J1, J2, J3 (with 0 < J2 < J1 and J3 = J1 + J2) Along with Their Negatives Appendix A. Construction of 5-Wave Resonances Using Only Three Different Positive Wavevectors J1, J2, J3 (with 0 < J2 < J1 and J3 = J1 + J2) Along with Their Negatives Consider three different positive wavevectors J1, J2, J3, with 0 < J2 < J1 and J3 = J1 + J2, along with their negatives −J1, −J2 −J3. These are six wavevectors in total. Using these six wavevectors only, we want to solve the equations determining 5-wave resonances for deep water waves with dispersion relation ω(k) = p g\|k\|. As obtained in [5], and using the notation in that reference, the problem is reduced to solving the momentum equation and the frequency condition, respectively: k1 + k2 + k3 = p + q, q \|k1\| + q \|k2\| + q \|k3\| = q \|p\| + q \|q\| , for the wavevectors k1, k2, k3, p, q ∈Z. The solution given in the reference establishes ﬁve cases: (i) All wavevectors are positive. (i) All wavevectors are positive. p (ii) p, q > 0, and one of the k1, k2, k3 is negative. (ii) p, q > 0, and one of the k1, k2, k3 is negative. (iii) p, q > 0, and two of the k1, k2, k3 are negative. p, q > 0, and two of the k1, k2, k3 are negative. (iv) p, q have different signs, and k1, k2, k3 > 0. (v) p, q have different signs, and one of the k1, k2, k3 is negative. Recall that we need to use the six wavevectors mentioned at the beginning, namely we have k1, k2, k3, p, q ∈{±J1, ±J2, ±J3}. 5. Conclusions and Discussion Moreover, a wavevector cannot appear in both sides of the momentum equation, as that would lead to a triad resonance, which is known not to exist. Therefore one must impose in what follows the condition {k1, k2, k3} ∩{p, q} = ∅. (A1) (A1) Notice however that, on a given side of the momentum equation, it is possible to repeat wavevectors. Namely, it is possible to have #{k1, k2, k3} < 3 and/or #{p, q} < 2. We now consider one by one the ﬁve possible cases including all relevant subcases. It turns out that only subcases (v.1.c) and (v.2.b) below have solutions: the resonances (16) and (17) presented in this paper. (i) As all wavevectors are positive, we need to restrict k1, k2, k3, p, q ∈{J1, J2, J3}. As we must impose (A1) we conclude {k1, k2, k3} ̸= {J1, J2, J3}. Therefore we have two subcases: (i.1) k1 = k2 = k3 and (i.2) k1 = k2 ̸= k3. In subcase (i.1) the option p p = q ̸= k1 leads to the momentum equation 3k1 = 2p but the resonance condition is 3√k1 = 2√p which contradicts the momentum equation. The only other option is p ̸= q ̸= k1 ̸= p. We have the following three instances: p = q ̸= k1 leads to the momentum equation 3k1 = 2p but the resonance condition is 3√k1 = 2√p which contradicts the momentum equation. The only other option is p ̸= q ̸= k1 ̸= p. We have the following three instances: p ̸ q ̸ ̸ p g (i.1.a) k1 = k2 = k3 = J2, p = J1, q = J3. Here, the momentum equation is 3J2 = J1 + J3 which implies J2 = J1 and thus J3 = 2J2, so the resonance condition is 3√J2 = √J2 + √2J2, which has no solution. √ √ √ (i.1.b) k1 = k2 = k3 = J1 p = J2, q = J3. Here, the momentum equation is 3J1 = J2 + J3 which implies J1 = J2 and thus J3 = 2J2, so the resonance condition is 3√J2 = √J2 + √2J2, which has no solution. √ √ √ (i.1.b) k1 = k2 = k3 = J1 p = J2, q = J3. 5. Conclusions and Discussion Here, the momentum equation is 3J1 = J2 + J3 which implies J1 = J2 and thus J3 = 2J2, so the resonance condition is 3√J2 = √J2 + √2J2, which has no solution. 3√J2 = √J2 + √2J2, which has no solution. (i.1.c) k1 = k2 = k3 = J3 p = J1, q = J2. Here, the momentum equation is 3J3 = J1 + J2 which implies J3 = 0, with no solution. 3√J2 = √J2 + √2J2, which has no solution. (i.1.c) k1 = k2 = k3 = J3 p = J1, q = J2. Here, the momentum equation is 3J3 = J1 + J2 which implies J3 = 0, with no solution. √J2 √J2 + √J2, (i.1.c) k1 = k2 = k3 = J3 p = J1, q = J2. Here, the momentum equation is 3J3 = J1 + J2 which implies J3 = 0, with no solution. In conclusion subcase (i.1) has no solution. We now study subcase (i.2) k1 = k2 ̸= k3. In this subcase we must have p = q ̸= kj for all j = 1, 2, 3. We have the following six instances: (i.2.a) k1 = k2 = J2, k3 = J1, p = q = J3. Here, the momentum equation is 2J2 + J1 = 2J3 which implies J1 = 2J1, with no solution. (i.2.a) k1 = k2 = J2, k3 = J1, p = q = J3. Here, the momentum equation is 2J2 + J1 = 2J3 which implies J1 = 2J1, with no solution. (i.2.b) k1 = k2 = J1, k3 = J2, p = q = J3. Here, the momentum equation is 2J1 + J2 = 2J3 which implies J2 = 2J2, with no solution. (i.2.c) k1 = k2 = J2, k3 = J3, p = q = J1. Here, the momentum equation is 2J2 + J3 = 2J1 which implies J1 = 3J2 and thus J3 = 4J2 so the resonance condition is (i.2.a) k1 = k2 = J2, k3 = J1, p = q = J3. Here, the momentum equation is 2J2 + J1 = 2J3 which implies J1 = 2J1, with no solution. (i.2.b) k1 = k2 = J1, k3 = J2, p = q = J3. Here, the momentum equation is (i.2.b) k1 = k2 = J1, k3 = J2, p = q = J3. 5. Conclusions and Discussion The only other option is p ̸= q ̸= k1 ̸= p. We have the following three instances: (ii.1.a) k1 = k2 = J2, p = J1, q = J3. Here, the momentum condition is 2J2 = \|k3\| + J1 + J3 which implies J2 = \|k3\| + 2J1, with no solution (because J2 ≤J1). (ii.1.b) k1 = k2 = J1, p = J2, q = J3. Here, the momentum condition is 2J1 = \|k3\| + J2 + J3 which implies \|k3\| = J1 −2J2, with only solution \|k3\| = J2, leading to J1 = 3J2 and thus J3 = 4J2. Thus, the resonance condition reads 2√3J2 + √J2 = √J2 + √4J2, with no solution. (ii.1.c) k1 = k2 = J3, p = J2, q = J1. Here, the momentum condition is 2J3 = \|k3\| + J2 + J1 which implies \|k3\| = J3. The resonance condition reads 2√J3 + √J3 = √J1 + √J2, or 3√J1 + J2 = √J1 + √J2. Squaring this gives 9(J1 + J2) = J1 + J2 + 2√J1J2, thus J1 + J2 = √J1J2/4. Squaring again gives J2 1 + J2 2 + 2J1J2 = J1J2/16, with no real solution. In conclusion subcase (ii.1) has no solution. We now study subcase (ii.2) k1 ̸= k2. In this subcase we must have p = q ̸= kj for all j = 1, 2, 3. We have the following three instances: (ii.2.a) k1 = J2, k2 = J1, p = q = J3. Here, the momentum equation is J2 + J1 = \|k3\| + 2J3, which implies 0 = \|k3\| + J3, with no solution. p (ii.2.b) k1 = J2, k2 = J3, p = q = J1. Here, the momentum equation is J2 + J3 = \|k3\| + 2J1, which implies \|k3\| = 2J2 −J1, so J1 < 2J2. As \|k3\| ∈{J1, J2, J3}, we can check that \|k3\| = J3 is not possible as it implies J2 = 2J1 < 4J2, a contradiction. We can check that the two remaining choices \|k3\| = J1 or \|k3\| = J2 imply J1 = J2. So we conclude \|k3\| = J1 = J2 and thus J3 = 2J2. Thus, the resonance condition reads √J2 + √2J2 + √J2 = 2√J2, with no solution. (ii.2.c) k1 = J1, k2 = J3, p = q = J2. 5. Conclusions and Discussion Here, the momentum condition is 2J1 = \|k3\| + J2 + J3 which implies \|k3\| = J1 −2J2, with only solution \|k3\| = J2, leading to J1 = 3J2 and thus J3 = 4J2. Thus, the resonance condition reads 2√3J2 + √J2 = √J2 + √4J2, with no solution. (ii) In this case we restrict k1, k2, p, q ∈{J1, J2, J3} and k3 ∈{−J1, −J2, −J3}, with {k1, k2} ∩ {p, q} = ∅following from (A1). The momentum condition reads k1 + k2 = \|k3\| + p + q. As in case (i) we have two subcases: (ii.1) k1 = k2 and (ii.2) k1 ̸= k2. In subcase (ii.1) the option p = q ̸= k1 leads to the momentum condition 2k1 = \|k3\| + 2p, leading to \|k3\| = 2(k1 −p), so k1 > p. However, the resonance condition reads 2√k1 + p \|k3\| = 2√p, leading to p \|k3\| = 2(√p −√k1), so p > k1, a contradiction. The only other option is p ̸= q ̸= k1 ̸= p. We have the following three instances: (ii.1.a) k1 = k2 = J2, p = J1, q = J3. Here, the momentum condition is 2J2 = \|k3\| + J1 + J3 which implies J2 = \|k3\| + 2J1, with no solution (because J2 ≤J1). (ii.1.b) k1 = k2 = J1, p = J2, q = J3. Here, the momentum condition is 2J1 = \|k3\| + J2 + J3 which implies \|k3\| = J1 −2J2, with only solution \|k3\| = J2, leading to J1 = 3J2 and thus J3 = 4J2. Thus, the resonance condition reads 2√3J2 + √J2 = √J2 + √4J2, with no solution. (ii) In this case we restrict k1, k2, p, q ∈{J1, J2, J3} and k3 ∈{−J1, −J2, −J3}, with {k1, k2} ∩ {p, q} = ∅following from (A1). The momentum condition reads k1 + k2 = \|k3\| + p + q. As in case (i) we have two subcases: (ii.1) k1 = k2 and (ii.2) k1 ̸= k2. In subcase (ii.1) the option p = q ̸= k1 leads to the momentum condition 2k1 = \|k3\| + 2p, leading to \|k3\| = 2(k1 −p), so k1 > p. However, the resonance condition reads 2√k1 + p \|k3\| = 2√p, leading to p \|k3\| = 2(√p −√k1), so p > k1, a contradiction. 5. Conclusions and Discussion Here, the momentum equation is 2J3 + J1 = 2J2 which implies 3J1 = 0 with no solution (i.2.e) k1 = k2 = J3, k3 = J2, p = q = J1. Here, the momentum equation is 2J3 + J2 = 2J1 which implies 3J2 = 0, with no solution. (i 2 f) k k J k J p q J Here the momentum equation is (i.2.e) k1 = k2 = J3, k3 = J2, p = q = J1. Here, the momentum equation is 2J3 + J2 = 2J1 which implies 3J2 = 0, with no solution. p (i.2.f) k1 = k2 = J3, k3 = J1, p = q = J2. Here, the momentum equation is 2J3 + J1 = 2J2 which implies 3J1 = 0, with no solution. (i.2.f) k1 = k2 = J3, k3 = J1, p = q = J2. Here, the momentum equation is 2J3 + J1 = 2J2 which implies 3J1 = 0, with no solution. p In conclusion subcase (i.2) has no solution. Therefore, case (i) has no solution. p In conclusion subcase (i.2) has no solution. Therefore, case (i) has no solution. (ii) (ii) In this case we restrict k1, k2, p, q ∈{J1, J2, J3} and k3 ∈{−J1, −J2, −J3}, with {k1, k2} ∩ {p, q} = ∅following from (A1). The momentum condition reads k1 + k2 = \|k3\| + p + q. As in case (i) we have two subcases: (ii.1) k1 = k2 and (ii.2) k1 ̸= k2. In subcase (ii.1) the option p = q ̸= k1 leads to the momentum condition 2k1 = \|k3\| + 2p, leading to \|k3\| = 2(k1 −p), so k1 > p. However, the resonance condition reads 2√k1 + p \|k3\| = 2√p, leading to p \|k3\| = 2(√p −√k1), so p > k1, a contradiction. The only other option is p ̸= q ̸= k1 ̸= p. We have the following three instances: (ii.1.a) k1 = k2 = J2, p = J1, q = J3. Here, the momentum condition is 2J2 = \|k3\| + J1 + J3 which implies J2 = \|k3\| + 2J1, with no solution (because J2 ≤J1). (ii.1.b) k1 = k2 = J1, p = J2, q = J3. 5. Conclusions and Discussion Here, the momentum equation is 2J1 + J2 = 2J3 which implies J2 = 2J2, with no solution. 2J1 + J2 = 2J3 which implies J2 = 2J2, with no solution. (i.2.c) k1 = k2 = J2, k3 = J3, p = q = J1. Here, the momentum equation is 2J2 + J3 = 2J1 which implies J1 = 3J2 and thus J3 = 4J2, so the resonance condition is 2√J2 + √4J2 = 2√3J2, which has no solution. (i.2.c) k1 = k2 = J2, k3 = J3, p = q = J1. Here, the momentum equation is 2J2 + J3 = 2J1 which implies J1 = 3J2 and thus J3 = 4J2, so the resonance condition is 2√J2 + √4J2 = 2√3J2, which has no solution. Fluids 2021, 6, 205 31 of 35 31 of 35 (i.2.d) k1 = k2 = J1, k3 = J3, p = q = J2. Here, the momentum equation is 2J1 + J3 = 2J2 which implies J2 = 3J1 which has no solution (because J2 ≤J1). (i.2.e) k1 = k2 = J3, k3 = J2, p = q = J1. Here, the momentum equation is 2J3 + J2 = 2J1 which implies 3J2 = 0, with no solution. (i.2.f) k1 = k2 = J3, k3 = J1, p = q = J2. Here, the momentum equation is 2J3 + J1 = 2J2 which implies 3J1 = 0, with no solution. (i.2.d) k1 = k2 = J1, k3 = J3, p = q = J2. Here, the momentum equation is 2J1 + J3 = 2J2 which implies J2 = 3J1 which has no solution (because J2 ≤J1). (i.2.d) k1 = k2 = J1, k3 = J3, p = q = J2. Here, the momentum equation is 2J1 + J3 = 2J2 which implies J2 = 3J1 which has no solution (because J2 ≤J1). (i.2.d) k1 = k2 = J1, k3 = J3, p = q = J2. Here, the momentum equation is 2J1 + J3 = 2J2 which implies J2 = 3J1 which has no solution (because J2 ≤J1). (i.2.e) k1 = k2 = J3, k3 = J2, p = q = J1. Here, the momentum equation is 2J3 + J2 = 2J1 which implies 3J2 = 0, with no solution. (i.2.f) k1 = k2 = J3, k3 = J1, p = q = J2. 5. Conclusions and Discussion Here, the momentum equation is J1 + J3 = \|k3\| + 2J2, which implies \|k3\| = 2J1 −J2. As \|k3\| ∈{J1, J2, J3}, we can check that \|k3\| = J3 implies J1 = 2J2 and \|k3\| = J3 = 3J2, so the resonance condition reads √2J2 + √3J2 + √3J2 = 2√J2, with no solution. The two remaining choices \|k3\| = J2 or \|k3\| = J1 imply \|k3\| = J1 = J2 and J3 = 2J2. Thus, the resonance condition reads √J2 + √2J2 + √J2 = 2√J2, with no solution. In conclusion subcase (ii.2) has no solution. Therefore, case (ii) has no solutio In conclusion subcase (ii.2) has no solution. Therefore, case (ii) has no s (iii) This case has zero interaction coefﬁcients so it will not be considered. (iii) This case has zero interaction coefﬁcients so it will not be considered. ) This case has zero interaction coefﬁcients so it will not be considered. (iv) This case has zero interaction coefﬁcients so it will not be considered. (v) In this case we restrict k1, k2, p ∈{J1, J2, J3} and k3, q ∈{−J1, −J2, −J3}, with {k1, k2} ∩ {p} = ∅and k3 ̸= q following from (A1). Instead of considering explicitly all 27 possible instances we will use the results from [5] regarding inequalities amongst frequencies. These translate directly to inequalities amongst wavevectors, which in our notation can be summarised as: k1 ≤k2 < p, \|k3\| < \|q\|, p ̸= \|q\|. (A2) k1 ≤k2 < p, \|k3\| < \|q\|, p ̸= \|q\|. (A2) Fluids 2021, 6, 205 32 of 35 As we assume without loss of generality J2 ≤J1 < J3, it follows that p ̸= J2 and \|q\| ̸= J2 because J2 is the smallest wavenumber. Thus, there are two subcases: (v.1) In subcase (v.1) p = J3, q = −J1, inequalities (A2) imply k1, k2 < J3 and k3 = −J2, with the now strict inequality J2 < J1. There are three options: q y p (v.1.a) k1 = k2 = J1. Here the momentum condition reads 2J1 + (−J2) = J3 + (−J1), which simpliﬁes to J1 = J2, a contradiction. p (v.1.b) k1 = J2, k2 = J1. Here the momentum condition reads J2 + J1 + (−J2) = J3 + (−J1), which simpliﬁes to J1 = J2, a contradiction. (v.1.c) k1 = k2 = J2. 5. Conclusions and Discussion Here the momentum condition reads 2J2 + (−J2) = J3 + (−J1), which is identically satisﬁed (because J3 = J2 + J1). We turn to the resonance condition to ﬁnd 2√J2 + √J2 = √J1 + J2 + √J1, or √J1 + J2 = 3√J2 −√J1. Squaring this gives J1 + J2 = 9J2 + J1 −6√J1J2, or 3√J1J2 = 4J2. Squaring again gives 9J1 = 16J2. Thus J1 = 16J2/9 and J3 = 25J2/9. In summary this leads to a 5-wave resonance parameterised by K ∈Z+ as follows: 9K + 9K + (−9K) = 25K + (−16K), √ 9K + √ 9K + √ 9K = √ 25K + √ 16K . In subcase (v.2) p = J1, q = −J3, inequalities (A2) imply k1 = k2 = J2 with the now strict inequality J2 < J1, while \|k3\| < J3. There are thus two options: q y \| \| p (v.2.a) k3 = −J2. Here the momentum condition reads 2J2 + (−J2) = J1 + (−J3), which simpliﬁes to J2 = −J2, a contradiction. (v.2.a) k3 = −J2. Here the momentum condition reads 2J2 + (−J2) = J1 + (−J3), which simpliﬁes to J2 = −J2, a contradiction. (v.2.b) k3 = −J1. Here the momentum condition reads 2J2 + (−J1) = J1 + (−J3), which simpliﬁes to J1 = 3J2, thus J3 = 4J2. We turn to the resonance condition to ﬁnd 2√J2 + √3J2 = √3J2 + √4J2, which is satisﬁed. In summary this leads to a 5-wave resonance parameterised by K ∈Z+ as follows: K + K + (−3K) = 3K + (−4K), √ K + √ K + √ 3K = √ 3K + √ 4K . Appendix B. Probe Calibration and Wavemaker Tests Appendix B. Probe Calibration and Wavemaker Tests Figure A1 shows a picture of the probes arrangement in the experimental tank. Figure A1. Picture of the experimental tank with a close up on the installed probes. The wire of each capacitance probe is directly facing each wedge. Figure A1. Picture of the experimental tank with a close up on the installed probes. The wire of each capacitance probe is directly facing each wedge. Figure A2 shows the results of the calibration of the probes. A linear regression gives R2 = 0.999 for the relation between surface elevation and voltage, over a wide range of surface elevations (between −9 and 9 centimetres). Fluids 2021, 6, 205 33 of 35 33 of 35 Figure A2. Calibration of the probes. (a) Probe 1. (b) Probe 2. Figure A3 shows the results of several cases of monochromatic wavemaker oscillations over a range of frequencies to be used in the interacting experiment. On each panel, solid black lines show the wave surfaces measured by probe 1 when the north wedge oscillates monochromatically, while red dashed lines correspond to probe 2 measurements when the south wedge is oscillated. Apart from slight differences between the two probes in panels (c) and (d) (f = 2.22 Hz and f = 2.3 Hz, respectively), the waves are stable for the range of frequencies studied. It was found (ﬁgure not shown) that for frequencies above f = 2.35 Hz it was difﬁcult to get regular wave trains, so we do not consider wavemaker frequencies higher than f = 2.35 Hz. Figure A2. Calibration of the probes. (a) Probe 1. (b) Probe 2. Figure A2. Calibration of the probes. (a) Probe 1. (b) Probe 2. Figure A3 shows the results of several cases of monochromatic wavemaker oscillations over a range of frequencies to be used in the interacting experiment. On each panel, solid black lines show the wave surfaces measured by probe 1 when the north wedge oscillates monochromatically, while red dashed lines correspond to probe 2 measurements when the south wedge is oscillated. Apart from slight differences between the two probes in panels (c) and (d) (f = 2.22 Hz and f = 2.3 Hz, respectively), the waves are stable for the range of frequencies studied. Appendix B. Probe Calibration and Wavemaker Tests It was found (ﬁgure not shown) that for frequencies above f = 2.35 Hz it was difﬁcult to get regular wave trains, so we do not consider wavemaker frequencies higher than f = 2.35 Hz. Figure A3. Tests of monochromatic wave generation by each wavemaker with the corresponding measurements by the closest probes. In each panel, the solid black line (red dashed line) represents the measurement by probe 1 (probe 2) of a wave generated by a monochromatic oscillation of the north wedge (south wedge). (a) Monochromatic frequency f = 1.33 Hz. 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https://openalex.org/W2055371599	https://eprints.gla.ac.uk/219725/1/219725.pdf	English	null	The cost-effectiveness of exercise referral schemes	BMC public health	2,011	cc-by	7,843	* Correspondence: nana.anokye@brunel.ac.uk 1Health Economics Research Group (HERG), Brunel University, Uxbridge, Middlesex UB8 3PH, UK Full list of author information is available at the end of the article © 2011 Anokye et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Anokye et al. BMC Public Health 2011, 11:954 http://www.biomedcentral.com/1471-2458/11/954 Open Access Abstract Background: Exercise referral schemes (ERS) aim to identify inactive adults in the primary care setting. The primary care professional refers the patient to a third party service, with this service taking responsibility for prescribing and monitoring an exercise programme tailored to the needs of the patient. This paper examines the cost-effectiveness of ERS in promoting physical activity compared with usual care in primary care setting. Methods: A decision analytic model was developed to estimate the cost-effectiveness of ERS from a UK NHS perspective. The costs and outcomes of ERS were modelled over the patient’s lifetime. Data were derived from a systematic review of the literature on the clinical and cost-effectiveness of ERS, and on parameter inputs in the modelling framework. Outcomes were expressed as incremental cost per quality-adjusted life-year (QALY). Deterministic and probabilistic sensitivity analyses investigated the impact of varying ERS cost and effectiveness assumptions. Sub-group analyses explored the cost-effectiveness of ERS in sedentary people with an underlying condition. Results: Compared with usual care, the mean incremental lifetime cost per patient for ERS was £169 and the mean incremental QALY was 0.008, generating a base-case incremental cost-effectiveness ratio (ICER) for ERS at £20,876 per QALY in sedentary individuals without a diagnosed medical condition. There was a 51% probability that ERS was cost-effective at £20,000 per QALY and 88% probability that ERS was cost-effective at £30,000 per QALY. In sub-group analyses, cost per QALY for ERS in sedentary obese individuals was £14,618, and in sedentary hypertensives and sedentary individuals with depression the estimated cost per QALY was £12,834 and £8,414 respectively. Incremental lifetime costs and benefits associated with ERS were small, reflecting the preventative public health context of the intervention, with this resulting in estimates of cost-effectiveness that are sensitive to variations in the relative risk of becoming physically active and cost of ERS. Conclusions: ERS is associated with modest increase in lifetime costs and benefits. The cost-effectiveness of ERS is highly sensitive to small changes in the effectiveness and cost of ERS and is subject to some significant uncertainty mainly due to limitations in the clinical effectiveness evidence base. The cost-effectiveness of exercise referral schemes ana K Anokye1, Paul Trueman1, Colin Green2, Toby G Pavey2, Melvyn Hillsdon3 and Rod S Tayl Population The model considers a cohort of individuals, aged between 40-60 years, who present in a sedentary state. The age of the population was selected to reflect the evidence on the effectiveness of ERS [10]. This paper aims to examine the cost-effectiveness of ERS in promoting physical activity compared to usual care in a primary care setting. Our analysis uses pre- vious research as a point of departure, and builds on this through use of evidence synthesis and through further analysis of the cost-effectiveness of ERS in indi- viduals with pre-existing conditions, which is intended to reflect the use of ERS in practice in the UK. Modelling approach A decision analytic model was developed to examine the cost effectiveness of ERS. The model considers a cohort of individuals who are exposed to ERS compared to a control group with no ERS. The modelling framework estimates the likelihood of becoming physically active and examines the effects of physical activity/inactivity on the development of conditions which are known to be associated with level of physical activity. Specifically, the model considers the impact of ERS on the develop- ment of coronary heart disease, stroke and type II dia- betes. Whilst many other conditions are thought to be associated with physical activity, these three conditions were selected on the basis that there is robust quantifi- able evidence on the relationship between physical activ- ity and their incidence [15]. Figure 1 illustrates the model structure, which is a based on a previously devel- oped policy-relevant cost-effectiveness model [8]. This structure was reviewed against best practice principles for economic modelling and considered suitable [10]. To date, there is limited evidence on the cost-effec- tiveness of ERS. A systematic review identified four pre- vious economic evaluations [10]. These comprised three trial-based economic evaluations of ERS [11-13] and one model-based evaluation [8] of the cost-effectiveness of brief interventions in primary care to promote physical activity, including ERS. Whilst the evidence base sug- gests that exercise referral is a cost-effective intervention in sedentary but otherwise healthy populations there are a number of shortcomings associated with the evidence. First, as the authors of each of the studies acknowledge, there is significant uncertainty around estimates of cost- effectiveness, mainly due to limitations in the effective- ness evidence. Second, the evidence tends to focus on sedentary but otherwise healthy individuals, while a number of individuals are currently referred to an ERS with a diagnosed condition, such as coronary heart dis- ease or depression [14]. Background between 1 and 1.8 billion pounds is associated with the treatment of physical inactivity related diseases [6]. In spite of the negative impacts of physical inactivity, only 39% of men and 29% of women in England reported meeting the recommended level to be considered ‘physi- cally active’, as defined by guidance from the Chief Medical Officer, whilst based on accelerometer data, only 6% of men and 4% of women met the recom- mended level [7]. Insufficient physical activity is an important public health issue in England as it is associated with an increased risk of developing over 20 health conditions including coronary heart disease (CHD), cancer, dia- betes, and stroke [1-4] and is rated among the top ten leading causes of death in high-income countries [5]. In England, physical inactivity is estimated to cost the economy around 8.3 billion pounds annually, of which Over the past decade, exercise referral schemes (ERS) have become one of the most common interventions used to promote physical activity in primary care [8,9]. In an ERS, people who are sedentary and/or have risk Correspondence: nana.anokye@brunel.ac.uk 1Health Economics Research Group (HERG), Brunel University, Uxbridge, Middlesex UB8 3PH, UK Full list of author information is available at the end of the article Page 2 of 11 Page 2 of 11 Page 2 of 11 Anokye et al. BMC Public Health 2011, 11:954 http://www.biomedcentral.com/1471-2458/11/954 factor(s) for conditions known to benefit from physical activity (e.g. high blood pressure) are referred by a pri- mary care professional to a third party service (often a sports centre or leisure facility), which then prescribes and monitors an exercise programme tailored to the individual needs of the patients [9]. Effectiveness of ERS/comparator Effectiveness of ERS/comparator Evidence of the effectiveness of ERS, compared with usual care, measured in terms of the probability of mov- ing from a sedentary state to an active state, was derived from the meta-analysis conducted as part of a recent review of the effectiveness literature for ERS [10]. This was based on ‘intention-to-treat’ analyses, which adjusted for attrition, and showed ERS to be associated with a higher probability (relative risk (RR): 1.11; 95% CI: 0.99, 1.25) of being active compared with usual care. The active state is defined in line with the effectiveness literature and physical activity for health guidance [16] i. e. doing at least 90-150 min of at least moderate inten- sity physical activity per week. Thus, a sedentary lifestyle corresponds not only to non-participation in physical activity but also to participation below the requisite amount. The active state is assumed to last long enough to enable health benefits to be obtained, although this remains undefined given the inadequate evidence on the dose response relationship between the number of years being physically active and the incidence of long-term outcomes. Previous analyses of behaviour change have referred to this scenario as ‘fully engaged’ [17] to describe an individual who makes lasting changes to their lifestyle following an intervention. Treatment costs with CHD, stroke and type II diabetes Total lifetime treatment costs were estimated using pub- lished cost estimates (identified through a systematic review) for the annual cost associated with CHD, dia- betes, and stroke [8,21,22], and assumptions on age of onset and life-expectancy combined with estimates of the annual cost of treating an individual with the condi- tion [15]. It was assumed that the treatment cost of stroke, unlike the other health states was an event cost because the direct costs associated with treatment of stroke tend to occur once, rather than a recurring cost. This is acknowledged as a simplification in the model, as in reality there are likely to be acute and ongoing costs associated with stroke. Intervention and comparator ERS was assumed to take the form of a structured pro- gramme of exercise based in a leisure centre incorporat- ing monitoring of individual performance, as is mainly the case in current practice [10]. Individuals are assumed to have been referred to the scheme by a pri- mary care professional. The comparator was usual care in a primary care setting. It should be acknowledged that individuals who are not exposed to ERS may choose to become physically active. Perspective and time horizon The model adopts an NHS and personal social services perspective (third-party payer perspective), as used by the National Institute for Health and Clinical Excellence Figure 1 Diagram of model structure. Figure 1 Diagram of model structure. Figure 1 Diagram of model structure. Page 3 of 11 Anokye et al. BMC Public Health 2011, 11:954 http://www.biomedcentral.com/1471-2458/11/954 individuals is generated from the prevalence of these conditions in that population, using the HSE 2006 data to inform these probabilities (Table 1). The probability of developing the health states among active individuals are derived using RR estimates identified from literature review [8,19,20] and subsequent adjustment (dividing the probabilities for the sedentary population by the RR from the literature) of the probability for each condition in the ERS cohort. The physical activity levels and study population used to measure the RR estimates match those of the cohort under consideration in this study. (NICE) in it’s reference case for cost-effectiveness analy- sis in health technology appraisal [2]. A lifetime time horizon is adopted to capture future costs and to acknowledge the benefits of physical activity. Future costs and benefits are discounted at a rate of 3.5% per annum [2]. ERS/intervention costs h f h The cost of the ERS intervention was derived from pre- viously published research identified as part of a recently published systematic review [10] which identified a detailed micro-level costing exercise for a leisure centre based ERS [12]. Isaacs et al. [12] reported resource use in a health service/local authority that consists of provi- sion of facilities, exercise trainers and administrative support. Cost estimates are up-rated to 2010 prices, using the consumer price index, for the current analyses. The validity of the resource use and cost estimates employed for ERS were assessed by an expert advisory group (including clinicians, exercise scientists and health economists) and judged to be representative of ERS schemes in current practice. No attempt was made to estimate a net cost of the intervention which subtracts any cost savings that might result from ERS from the cost of the intervention. When this was explored in Isaacs et al. [12] and Gusi et al. [11], there was no clear evidence of a change in health care utilisation (e.g. med- ications, hospital or primary care) as result of the intervention. Risks of developing health states associated with inactivity Evidence of the effect of physical activity on the devel- opment of the outcomes considered in the model (CHD, stroke and type II diabetes) is derived from a sys- tematic review [8] and from the Health Survey for Eng- land (HSE) 2006 (survey year focused on cardiovascular disease and risk factors). HSE is the main data source on morbidities in England [18]. The probability of devel- oping CHD, stroke or type II diabetes among sedentary Assessment of uncertainty Uncertainty in parameter estimates was explored through the use of deterministic and probability sensi- tivity analyses. The deterministic sensitivity analysis included one-way, scenario and extreme values analysis. In addition, uncertainties around parameters considered to be key drivers of the cost-effectiveness of ERS were addressed simultaneously using probabilistic sensitivity analyses (PSA). The parameters that had different unit values in the 2 arms of the model (i.e. probability to be active and probability to get the disease conditions) were specified as incremental differences between the 2 arms and not absolute values. The intuition is that the Primary outcome measure (QALY) Table 1 Estimates of the inputs to the model Costs are in 2010 prices. distributions of these parameters may be correlated and hence representing them as absolute values may overes- timate the uncertainty. The distributions and their respective calculation of alpha and beta calculations were based on [25]. In cases where there were no data on standard errors the standard approach of using 10% of mean estimates as standard error was followed [26] The data adopted in the probabilistic analysis are reported in Table 2. associated with each of the conditions in the model are derived using health state values for each condition [8,23] and data on life-expectancy after onset of the condition [24]. Life-expectancy is derived by applying data on aver- age age of onset for each condition (Table 1). Primary outcome measure (QALY) The primary outcome of the economic evaluation is expressed in terms of the incremental cost per quality- adjusted life-year (QALY). Estimates of the QALYs Anokye et al. BMC Public Health 2011, 11:954 http://www.biomedcentral.com/1471-2458/11/954 Page 4 of 11 Table 1 Estimates of the inputs to the model Input Value Data source Probability of experiencing an outcome associated with physical activity Probability of experiencing CHD when active 0.014 HSE [21]; Shaper et al. [19] Probability of experiencing CHD when sedentary 0.027 HSE [21]; Shaper et al. [19] Probability of experiencing stroke when active 0.011 HSE [21]; Herman et al. [20] Probability of experiencing stroke when sedentary 0.015 HSE [21]; Herman et al. [20] Probability of experiencing type II diabetes when active 0.022 HSE [21]; NICE [8] Probability of experiencing type II diabetes when sedentary 0.044 HSE [21]; NICE [8] Inputs used in calculating QALYs/treatment costs Utility/health state value of being in CHD state 0.55 Kind et al. [22]; NICE [8] Utility/health state value of being in stroke state 0.52 Kind et al. [22]; NICE [8] Utility/health state value of being in type II diabetes state 0.7 Kind et al. [22]; NICE [8] Utility/health state value of being in a non-disease health state 0.83 Kind et al. [22]; NICE [8] Average age of cohort (in years) 50 HSE [18] Average age of mortality (in years) 84 ONS [23] Assumed average age of onset of a disease health state (in years) 55 NICE [8] Life years remaining after onset of CHD 18.41 NICE [8]; ONS [23] Life years remaining after onset of stroke 5.12 NICE [8]; ONS [23] Life years remaining after onset of type II diabetes 28.13 NICE [8]; ONS [23] Lifetime treatment costs/QALYs associated with health states (per person) Lifetime treatment costs associated with CHD state £17,728 NICE [8] Lifetime treatment costs associated with stroke state £1,965 DH [22] Lifetime treatment costs associated with type II diabetes state £50,309 Currie et al. [21] Lifetime treatment costs associated with non-disease health state - - QALYs associated with CHD state 9.94 Kind et al. [23]; NICE [8] QALYs associated with stroke state 5.15 Kind et al. [23]; NICE [8] QALYs associated with type II diabetes state 14.18 Kind et al. [23]; NICE [8] QALYs associated with non-disease health state 17.18 Kind et al. [23]; NICE [8] Costs are in 2010 prices. Subgroup analyses in individuals with pre-existing conditions Sub-group analyses included an assessment of the cost effectiveness of ERS in sedentary 40-60 year olds indivi- duals with a diagnosed condition known to benefit from physical activity. Obesity, hypertension and depression were identified as the three most common conditions reported with participation in ERS [14] and were included in the analysis. Anokye et al. BMC Public Health 2011, 11:954 http://www.biomedcentral.com/1471-2458/11/954 Page 5 of 11 Table 2 Probabilistic sensitivity analysis inputs Parameters Deterministic Standard error Distribution Alpha Beta Incremental probability to be active 0.048 0.0048 beta 95.152 1887.181 Incremental probability to experience CHD 0.013 0.0013 beta 98.687 7492.621 Incremental probability to experience stroke 0.004 0.0004 beta 99.596 24799.4 Incremental probability to experience diabetes 0.022 0.0022 beta 97.778 4346.677 Treatment discounted cost of CHD 17728.031 1772.803 gamma 100 177.280 Treatment discounted cost of stroke 1965.165 196.517 gamma 100 19.652 Treatment discounted cost of diabetes 50309.426 5030.943 gamma 100 503.094 Discounted QALY for CHD health state 9.942 0.994 gamma 100 0.099 Discounted QALY for stroke health state 5.148 0.515 gamma 100 0.051 Discounted QALY for type II diabetes health state 14.182 1.418 gamma 100 0.142 Cost of intervention 222 37.9 gamma 34.311 6.470 Source: Briggs et al (25) Table 2 Probabilistic sensitivity analysis inputs Estimating the cost-effectiveness of ERS Table 4 shows the estimated incremental cost-effective- ness ratio (ICER) of the base-case analysis, using a cohort of 1,000 individuals and a lifetime horizon. Total costs and outcomes are divided by the cohort size (1,000) to gener- ate per person estimates of costs and benefits. The ICER was calculated with respect to the standard comparator ‘usual care’. Compared with usual care, ERS is more expensive as it incurs additional mean lifetime costs of £170 per person, but is more effective leading to a lifetime mean QALY gain of 0.008 per person. The mean cost per QALY of ERS compared with usual care is £20,876. Model validation Two main procedures involving internal validation and peer review were employed to check the validity of the model [27]. The former consisted of simulating a series of changes in the input values that are likely to vary the results of the model with checks to see that the impacts on the results are expected. For example, setting all QALY parameters to zero, and checking if the output of the QALYs in each arm is zero. In addition to this, the model was replicated and compared using TreeAge and excel software, and subject to a process of internal peer review, including consistency checks, across the research team. The validation process included peer review by a modeller, unrelated to the research team, who under- stood the complexities of the model and who was able to scrutinise the spreadsheet of the model and the for- mulae behind it. Table 1 reports the derivation of the outcomes asso- ciated with physical activity. This includes the probabil- ity of experiencing an outcome (CHD, stroke or type II diabetes), utility values, and life years associated with each outcome. The estimates of lifetime treatment costs and QALYs for an individual in each health state are summarised in Table 1. Among the conditions included in the model, type II diabetes incurred the largest treatment cost and stroke the least, although it should be noted that stroke was considered as a one off clinical event whilst other chronic outcomes were associated with ongoing treat- ment costs. Estimates of effectiveness/costs of ERS Table 3 summarises the estimates of the effectiveness of ERS on physical activity levels and overall intervention costs associated with ERS. Table 3 Estimates of effectiveness and intervention costs of ERS Inputs Value Data source Effectiveness Probability of becoming active after exposure to ERS 0.345 Pavey et al. [10] Probability of becoming active after exposure to usual care 0.297 Pavey et al. [10] Intervention costs Cost of the intervention per participant to the providers £222a Pavey et al. [10] aIn 2010 prices (estimates used in model) Table 3 Estimates of effectiveness and intervention costs of ERS Inputs Value Data source Effectiveness Probability of becoming active after exposure to ERS 0.345 Pavey et al. [10] Probability of becoming active after exposure to usual care 0.297 Pavey et al. [10] Intervention costs Cost of the intervention per participant to the providers £222a Pavey et al. [10] aIn 2010 prices (estimates used in model) Table 3 Estimates of effectiveness and intervention costs of ERS Inputs Value Data source Effectiveness Probability of becoming active after exposure to ERS 0.345 Pavey et al. [10] Probability of becoming active after exposure to usual care 0.297 Pavey et al. [10] Intervention costs Cost of the intervention per participant to the providers £222a Pavey et al. [10] aIn 2010 prices (estimates used in model) Deterministic sensitivity analysis Table 5 shows the impact of the variation in parameter estimates (one-way analysis) on the cost-effectiveness of ERS. Assuming a less intensive ERS or more effective ERS resulted in an ICER below £30,000 per QALY and lower than the base-case. On the other hand, including intervention costs to participants led to an ICER above £30,000 per QALY, whilst a less effective ERS resulted Anokye et al. BMC Public Health 2011, 11:954 http://www.biomedcentral.com/1471-2458/11/954 Page 6 of 11 Table 4 Base-case cost-effectiveness results comparing ERS with usual care ERS Usual care Difference Incremental cost per QALY (ICER) Lifetime total healthcare costs per persona £2,492 £2,322 £170 £20,876 Total QALYs per person 16.743 16.735 0.008 aIn 2010 prices Table 4 Base-case cost-effectiveness results comparing ERS with usual care QALY, there is a 0.508 probability that ERS is cost- effective. This increases to 0.879 when a threshold of £30,000 per QALY is considered. in ERS being dominated by usual care (negative ICER)- that is ERS is more expensive and leads to loss of health gains. The findings of the scenario analysis are pre- sented in Table 5. In the worst case scenario, ERS was dominated by the comparator. In the best case scenario, the ICER fell to under £700 per QALY. Subgroup analysis in individuals with pre-existing conditions Table 6 shows the probabilities of experiencing the health states in the disease-specific cohorts. For each of the conditions considered, the estimated ICER is lower than the base case, reflecting the increased likelihood of developing one of the morbidities considered in the model if the individual has a pre-existing condition (Table 7). Compared with usual care, ERS in these cohorts remains more costly (albeit less so than in a general population cohort). In terms of effectiveness, ERS (compared with usual care) is more effective lead- ing to improved QALY gains which are higher than in the base case (ranging from 0.011 to 0.017). The cost per QALY of ERS compared with usual care is between £8,414 and £14,618 and thus can be considered cost- effective at the £20,000 per QALY threshold. Probabilistic sensitivity analysis A scatter plot of the probabilistic data, showing simu- lated estimates of cost difference against QALY differ- ence between ERS and usual care, is provided in Figure 2. The scatter plot shows that all the simulations gener- ated an improved effectiveness of ERS but also at higher cost (i.e. all points were in the north-east quadrant of the cost-effectiveness plane). This reflects the relatively modest uncertainty around the cost of the intervention and assumptions about the distribution of uncertainty around the estimates of effect size. A judgment on the cost-effectiveness of ERS, from a decision-maker context, will depend on the maximum amount decision makers are willing to spend to obtain an additional unit of effectiveness (in this case, a QALY). This judgement can be informed through the presentation of a cost-effectiveness acceptability curve, as presented in Figure 3. At a threshold of £20,000 per Discussion Our analysis estimates the cost-effectiveness of ERS using a cost utility analysis framework. Our base case Table 5 Cost-effectiveness results (after deterministic sensitivity analyses) comparing ERS with usual care Parameters/scenarios How data was adjusted for in the model Incremental cost per person Incremental effect per person (QALY) ICER Base case analysis - £170 0.008 £20,876 Parameters Intervention costs to participants Costs of intervention was varied from £222 to £342 (including costs to providers and participants) £290 0.008 £35,652 Less intensive ERS Costs of intervention was varied from £222 to £110 £58 0.008 £7,085 Effectiveness of ERS (based on lower limit of 95% CI) Probability of becoming active after exposure to ERS was varied from 0.336 to 0.294 £226 -0.001 Dominated Effectiveness of ERS (based upper limit of 95% CI) Probability of becoming active after exposure to ERS was varied from 0.336 to 0.371 £122 0.015 £7,947 Scenarios Worst cases of cost and effectiveness Worst case cost (£342) and worst case effectiveness (0.294) £346 -0.001 Dominated* Best cases of cost and effectiveness Best case cost (£110) and best case effectiveness (0.371) £10 0.015 £679 Worst case cost and best case effectiveness Best case cost (£110) and worst case effectiveness (0.294) £242 0.015 £15,734 Best case cost and worst case effectiveness Worst case cost (£342) and best case effectiveness (0.371) £114 -0.001 Dominated* *ERS more costly and less effective than control sults (after deterministic sensitivity analyses) comparing ERS with usual care Anokye et al. BMC Public Health 2011, 11:954 http://www.biomedcentral.com/1471-2458/11/954 Page 7 of 11 Page 7 of 11 Figure 2 Cost-effectiveness plane. Figure 3 Cost-effectiveness acceptability curve showing the probability of cost-effectiveness for ERS at varying levels of threshold. Figure 2 Cost-effectiveness plane. Figure 2 Cost-effectiveness plane. Figure 2 Cost-effectiveness plane. Figure 2 Cost-effectiveness plane. Figure 3 Cost-effectiveness acceptability curve showing the probability of cost-effectiveness for ERS at varying levels of threshold. Figure 3 Cost-effectiveness acceptability curve showing the probability of cost-effectiveness for ERS at varying lev Page 8 of 11 Anokye et al. BMC Public Health 2011, 11:954 http://www.biomedcentral.com/1471-2458/11/954 Table 6 Inputs used in the subgroup analysis model Cohort Inputs Value Data source Obese Probability of experiencing CHD when active 0.0259 HSE [28]; Hu et al. [18] Probability of experiencing CHD when sedentary 0.0376 HSE [28]; Hu et al. [18] Probability of experiencing stroke when active 0.0259 HSE [28]; Hu et al. Discussion [18] Probability of experiencing stroke when sedentary 0.0376 HSE [28]; Hu et al. [18] Probability of experiencing type II diabetes when active 0.0756 HSE [28]; Hu et al. [18] Probability of experiencing type II diabetes when sedentary 0.0986 HSE [28]; Hu et al. [18] Hypertensive Probability of experiencing CHD when active 0.060 HSE [28]; Hu et al. [29] Probability of experiencing CHD when sedentary 0.074 HSE [28]; Hu et al. [29] Probability of experiencing stroke when active 0.060 HSE [28]; Hu et al. [29] Probability of experiencing stroke when sedentary 0.074 HSE [28]; Hu et al. [29] Depressive Probability of experiencing CHD when active 0.0336 HSE [28]; Surtees et al. [30] Probability of experiencing CHD when sedentary 0.0801 HSE [28]; Surtees et al. [30] Table 6 Inputs used in the subgroup analysis model Cohort Inputs Table 6 Inputs used in the subgroup analysis model level lifetime QALY gains are relatively modest (less than 0.01 in our base case analysis). This estimate is predicated on the evidence of effectiveness derived from a meta-analysis [10], which has provided the most robust estimate to date of the effectiveness of ERS com- pared to usual care. However, it should be acknowl- edged that the cost-effectiveness analysis is attempting to capture lifetime benefits based on evidence of rela- tively modest effect sizes derived from short-term stu- dies. Any such analysis inevitably involves some assumptions about the degree to which behaviour change is lasting and fails to consider other health beha- viours which may impact on long-term outcomes. The result is that the cost-effectiveness analysis estimates that ERS has a modest lifetime cost and a marginal life- time QALY gain. Even small changes in the source data used to populate the model, particularly evidence of effect size and cost, may lead to significant changes in the resulting ICER. This can best be illustrated through consideration of the net benefit calculation. If we value each QALY gained at £30,000 and accept that our analy- sis is generating a lifetime QALY gain of approximately 0.008 in most cases, then the value of the benefits gen- erated in monetary terms is approximately £240 which exceeds the cost of the intervention. However, even a modest change in the lifetime QALY gain, to 0.007 would result in the costs exceeding the benefits making the cost-effectiveness of ERS questionable. Table 7 Cost-effectiveness results (disease specific cohorts) comparing ERS with usual care Cohort Incremental cost per person(£) Incremental effect per person(QALY) ICER (£) Obese £168 0.011 £14,618 Hypertensive £168 0.013 £12,834 Depressive £147 0.017 £8,414 Discussion In the analyses considering the ‘non-health’ outcomes, impacts of PA were captured as: (a) reduced absenteeism at work and disbenefits such as injuries and disability, and (b) process utility directly attributable to increased exer- cise. The former set of outcomes were obtained through synthesis of the literature to identify estimates of the magnitude of their associations with physical activity [10] and accounted via a descriptive cost consequences analysis. The process utility was included as a one-off ‘feel good’ benefit (QALY gain) associated with being physically active, and was estimated via regression ana- lyses using HSE 2008 data related to EQ-5D and self- reported physical activity, with uncertainty in this esti- mate tested via sensitivity analyses. Conversely, there are a number of assumptions which could be considered to be favourable to ERS, notably, the assumption relating to the lasting effect of physical activity. Sensitivity analyses provide some reassurance that the net effect of these assumptions is modest and that the incremental cost-effectiveness of ERS remains below £30,000 per QALY under most scenarios. Furthermore, our findings are largely consistent with previous analyses of ERS which have suggested that ERS results in modest increases in QALYs (via adverse health events avoided) at a relatively low cost. Previous studies have tended to conclude that ERS is a cost-effective use of resources, although they too have highlighted uncertainty in evi- dence based and the analytical framework used. Isaacs et al. [12] presented results in the form of an incremen- tal cost per unit change in SF-36 score, with the authors concluding that in comparison with controls, ERS led to an incremental cost of £19,500 per unit change in SF-36 score at 6 month follow-up. Given the outcome measure adopted in the study it is not possible to make helpful comparison with our own findings, although it should be noted that this study also found only a modest change in health status. In contrast, the study by Gusi et al. [11] showed that ERS resulted in an incremental QALY gain of 0.132 over a 6 month period as measured by change in the EQ-5D, at an incremental cost of €41 per participant, generating an ICER of €311 per QALY. Discussion assumptions result in a favourable cost-effectiveness ratio of £20,876 per QALY gained from ERS compared to usual care. The typical cost effectiveness threshold for UK ranges from £20,000 to £30,000 per QALY. However, ICERs were highly sensitive to plausible varia- tions in the relative risk for change in physical activity and cost of ERS. The cost-effectiveness of ERS appears to improve when targeted at individuals with a pre- existing condition known to benefit from increased phy- sical activity (i.e. £14,618/QALY in sedentary obese indi- viduals, £12,834/QALY in sedentary hypertensives and £8,414/QALY for sedentary individuals with depression). This suggests that it might be possible to target ERS to individuals with pre-existing conditions in whom the payoffs/impact may be higher. However, there remain some major uncertainties over whether the evidence used to populate the model, derived from the meta-ana- lysis, is applicable to these groups. There may be good reason to believe that uptake, adherence and effective- ness might differ according to the characteristics of the recipients. We have attempted to adjust the model to take into account differences in the rate of long-term ill- nesses, but no data were identified as part of the effec- tiveness review to allow for adjustment of the effect of ERS in different populations. There is a pressing need for better primary evidence to inform these uncertainties. Whilst our cost-effectiveness estimates suggest that ERS is a cost effective use of National Health Service (NHS) resources, it should be noted that the individual There are a number of limitations in the analysis that need to be acknowledged. In some respects the analysis Table 7 Cost-effectiveness results (disease specific cohorts) comparing ERS with usual care Cohort Incremental cost per person(£) Incremental effect per person(QALY) ICER (£) Obese £168 0.011 £14,618 Hypertensive £168 0.013 £12,834 Depressive £147 0.017 £8,414 Anokye et al. BMC Public Health 2011, 11:954 http://www.biomedcentral.com/1471-2458/11/954 Page 9 of 11 Page 9 of 11 The individuals in this study were obese and/or depressed and the findings may provide further evidence to suggest that physical activity can have process bene- fits, i.e. health status gains (independent of other pre- ventative effects) far greater than those suggested by our own analysis. However, no attempt was made to ascer- tain whether the benefits might be sustained beyond the study period. Discussion The findings presented by NICE [8] showed ERS compared with controls led to an incre- mental cost per person of £25.10 and a lifetime QALY gain of 0.31 per person equating to an incremental cost per QALY of £81. We are inclined to relate our findings more directly to the NICE [8] analysis because of simila- rities in the methods used in both studies. For example, the model used in our study was based on the model used by NICE [8]. The analysis conducted for NICE showed a greater QALY gain than our own findings. This might be partially explained by the inclusion of colon cancer as an additional outcome in the NICE model. In addition to this, the NICE model adopted higher estimates of the effectiveness of ERS than our analysis (RR of becoming active of 1.60 vs 1.11 herein) and there are differences in the handling of uptake and adherence between the two analyses. Coupled with a lower estimated cost of ERS this result in the NICE ana- lysis generating improved ICERs compared to our own findings. In testing our own model we sought to repro- duce the findings of the NICE model by incorporating the improved effectiveness of ERS. Despite slight differ- ences in the modelling approach it produced relatively consistent findings. Whilst we have based our approach to modelling the cost-effectiveness of ERS on the model structure used by NICE, we believe that the meta-analy- sis of effectiveness used in the current economic analysis has resulted in more robust input data and ultimately more accurate estimates of the cost-effectiveness of ERS [10]. can be considered to be conservative as it includes only a small number of conditions which are associated with physical activity. The inclusion of other conditions, such as musculoskeletal disease and mental health, are expected to further improve the cost-effectiveness of ERS. These conditions are excluded from the current analysis due to limitations in the available data on the relationship between their incidence and physical activ- ity. Additional developments of this model to adopt a wider perspective via the incorporation of ‘non-health’ outcomes associated with ERS slightly further improved the cost-effectiveness of ERS to a base case ICER of £17,032/QALY (see Pavey et al. [10] for detail). Conclusions p 5. World Health Organisation: World Health Report Geneva: WHO; 2002. This study examines the cost-effectiveness of ERS in promoting physical activity compared to usual care in a primary care setting. Using a cost utility analysis frame- work, the study uses previous research as a point of departure, and builds on this through use of evidence synthesis and through further analysis of the cost-effec- tiveness of ERS in individuals with pre-existing condi- tions, which is intended to reflect the use of ERS in practice in the UK. ERS is associated with modest increase in lifetime costs and benefits. Compared to usual care, the base-case ICER for ERS was £20,876/ QALY in sedentary individuals with at least one lifestyle risk factor and £14,618/QALY in sedentary obese indivi- duals, £12,834/QALY in sedentary hypertensives and £8,414/QALY for sedentary individuals with depression. However, cost-effectiveness of ERS is highly sensitive to small changes in the effectiveness and cost of ERS and is subject to some significant uncertainty mainly due to limitations in the clinical effectiveness evidence base. Therefore, further research on the clinical effectiveness of ERS is strongly recommended. 6. Department of Health: 2009 Annual Rep London: Department of Health; 2010. 6. Department of Health: 2009 Annual Report of the Chief Medical Officer London: Department of Health; 2010. London: Department of Health; 2010. 7. Craig R, Mindell J, Hirani V: Health Survey for England 2008. Volume 1: Physical Activity and Fitness National Health Service Information Centre; 2009. 8. National Institute for Health and Clinical Excellence (NICE): Modelling the Cost Effectiveness of Physical Activity Interventions London: NICE; 2006. 9. National Institute for Health and Clinical Excellence (NICE): Rapid Review of the Economic Evidence of Physical Activity Interventions London: NICE; 2006. 10. Pavey TG, Anokye N, Taylor AH, Trueman P, Moxham T, Fox KR, Hillsdon H, Green C, Campbell JL, Foster C, Mutrie N, Searle J, Taylor RS: The effectiveness and cost-effectiveness of exercise referral schemes: a systematic review and economic evaluation. Health Technology Assessment 2011. 10. Pavey TG, Anokye N, Taylor AH, Trueman P, Moxham T, Fox KR, Hillsdon H, Green C, Campbell JL, Foster C, Mutrie N, Searle J, Taylor RS: The effectiveness and cost-effectiveness of exercise referral schemes: a systematic review and economic evaluation. Health Technology Assessment 2011. 11. Authors’ contributions address cost effectiveness. However, there will always be a trade-off between developing a simple model, which can be populated and acknowledges its limitations ver- sus a more complex model which may be a better repre- sentation of reality but can only be partially populated and may result in greater uncertainty. The fundamental issue which needs to be addressed is improvements in the source data on the effectiveness of ERS. NKA and PT undertook the economic modelling with support from CG, drafted the first manuscript and coordinated its revision. TP and RT directed the project, analysed the data for the clinical effectiveness, helped identify data sources and assisted in drafting and revising the manuscript. MH provided an advisory role and assisted in drafting and revising the manuscript. All authors read and approved the final manuscript. Discussion However, all of the studies reported above suggest that ERS is associated with only small mean differences in lifetime costs and benefits, giving rise to the resulting ICER being very sensitive to small changes in the rela- tive risk of becoming physically active, together with small changes in other data inputs. This highlights the main limitation of this research, namely the limited evi- dence to show that ERS has a significant and lasting effect on participation in physical activity. Related to this, the model assumed that the active state last long enough to enable health benefits to be obtained and this could not be addressed in the sensitivity analysis due lack of data and the type of model used. Decision analy- tic models may not be well suited to interventions which involve complex behaviour change components. Individual level simulation models which can detect changes in individual behaviours over time may better Anokye et al. BMC Public Health 2011, 11:954 http://www.biomedcentral.com/1471-2458/11/954 Anokye et al. BMC Public Health 2011, 11:954 http://www.biomedcentral.com/1471-2458/11/954 Page 10 of 11 Page 10 of 11 Page 10 of 11 Anokye et al. BMC Public Health 2011, 11:954 http://www.biomedcentral.com/1471-2458/11/954 References 1. Department for Culture, Media and Sport (DCMS): Before, During and After: Making the Most of the London 2012 Games London: DCMS; 2008. 2. National Institute for Health and Clinical Excellence (NICE): Workplace Health Promotion: How to Encourage Employees to be Physically Active London: NICE; 2008. 3. Department of Health: Be Active, be Healthy: a Plan for Getting the Nation Moving London: Department of Health; 2009. 4. Department of Health: Choosing Activity: A Physical Activity Action Plan London: Department of Health; 2005. 4. Department of Health: Choosing Activity: A Physical Activity Action Plan London: Department of Health; 2005. 5. World Health Organisation: World Health Report Geneva: WHO; 2002. 6. Department of Health: 2009 Annual Report of the Chief Medical Officer London: Department of Health; 2010. Competing interests h h d l h The authors declare that they have no competing interests. Further research is urgently required to examine the effectiveness of ERS with a particular focus on 1) how to motivate individuals to participate in ERS; 2) identify sub-groups of the sedentary population who are most able to benefit from ERS; 3) identify factors that are likely to lead to sustained increased in physical activity and changes in lifestyle. In the absence of robust evi- dence on these, the economic case for ERS remains encouraging but ultimately equivocal. Received: 13 June 2011 Accepted: 26 December 2011 Received: 13 June 2011 Accepted: 26 December 2011 Published: 26 December 2011 Received: 13 June 2011 Accepted: 26 December 2011 Published: 26 December 2011 Published: 26 December 2011 Conclusions Gusi N, Reyes MC, Gonzalez-Guerrero JL, Herrera E, Garcia JM: Cost-utility of a walking programme for moderately depressed, obese, or overweight elderly women in primary care: a randomised controlled trial. BMC Public Health 2008, 8:231. 12. Isaacs AJ, Critchley JA, Tai SS, Buckingham K, Westley D, Harridge SDR, Smith C, Gottlieb JM: Exercise evaluation randomised trial(EXERT): a randomised trial comparing GP referral for leisure centre-based exercise, community-based walking and advice only. Health Technology Assessment 2007, 11(10). 13. Stevens W, Hillsdon M, Thorogood M, McArdle D: Cost-effectiveness of a primary care based physical activity intervention in 45-74 year old men and women: a randomised controlled trial. British Journal of Sports Medicine 1998, 32(3):236-241. 14. British Heart Foundation National Centre (BHFNC): A Toolkit for the Design, Implementation and Evaluation of Exercise Referral Schemes. Guidance for Referring Health Professionals, Exercise Referral Professionals and Exercise Referral Scheme Commissioners London: BHFNC; 2009. Acknowledgements HTA Funding/Publication: This project was funded by the NIHR Health Technology Assessment programme (project number 08/72/01) and will be published in full in the Health Technology Assessment journal, around September. See the HTA programme website for further project information. Department of Health Disclaimer: The views and opinions expressed therein are those of the authors and do not necessarily reflect those of the Department of Health. 15. Beale S, Bending M, Trueman P: An Economic Analysis of Environmental Interventions that Promote Physical Activity University of York: York Health Economics Consortium; 2007. 16. O’Donovan G, Blazevich AJ, Boreham C, Cooper AR, Crank H, Ekelund U, Fox KR, Gately P, Giles-Corti B, Gill JMR, Hamer M, McDermott I, Murphy M, Mutrie N, Reilly JJ, Saxton JM, Stamatakis E: The ABC of physical activity for health: a consensus statement from the British Association of Sport and Exercise Sciences. Journal of Sports Science 2010, 28(6):573-591. 17. Wanless D: Securing good health for the whole population.Edited by: Treasury HM 2004, Crown. No: 0947819983; Norwich: HMSO. 1Health Economics Research Group (HERG), Brunel University, Uxbridge, Middlesex UB8 3PH, UK. 2Peninsula College of Medicine and Dentistry, University of Exeter, Veysey Building, Salmon Pool Lane, Exeter EX2 4SG, UK. 3School of Sport and Health Sciences, University of Exeter, St Luke’s, Exeter EX1 2 LU, UK. Author details 1H l h E Chilcott J, Tappenden P, Rawdin A, Johnson M, Kaltenthaler E, Papaioannou D, Shippam A: Avoiding and identifying errors in health technology assessment models: qualitative study and methodological review. Health Technology Assessment 2010, 14(25). 27. Chilcott J, Tappenden P, Rawdin A, Johnson M, Kaltenthaler E, Papaioannou D, Shippam A: Avoiding and identifying errors in health technology assessment models: qualitative study and methodological review. Health Technology Assessment 2010, 14(25). 28. Joint Health Surveys Unit of Social and Community Planning Research and University College London: Health Survey for England 2006 2008, SN: 5809. 29. Hu G, Jousilahti P, Antikainen R, Tuomilehto J: Occupational, commuting, and leisure-time physical activity in relation to cardiovascular mortality among Finnish subjects with hypertension. American Journal of Hypertension 2007, 20(12):1242-1250. 30. 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https://openalex.org/W2895979052	http://sajie.journals.ac.za/pub/article/download/1899/861	English	null	DIGITALISATION OF SHOP-FLOOR OPERATIONS IN THE SOUTH AFRICAN TOOL, DIE, AND MOULD-MAKING INDUSTRY	South African journal of industrial engineering	2,018	cc-by	8,595	South African Journal of Industrial Engineering August 2018 Vol 29(2), pp 153-170 South African Journal of Industrial Engineering August 2018 Vol 29(2), pp 153-170 DIGITALISATION OF SHOP-FLOOR OPERATIONS IN THE SOUTH AFRICAN TOOL, DIE, AND MOULD- MAKING INDUSTRY M.T. Dewa1#, A.F. van der Merwe1 & S. Matope1 M.T. Dewa1#, A.F. van der Merwe1 & S. Matope1 M.T. Dewa1#, A.F. van der Merwe1 & S. Matope1 ARTICLE INFO Article details Submitted by authors 12 Jan 2018 Accepted for publication 15 Jul 2018 Available online 31 Aug 2018 Contact details Corresponding author 17628598@sun.ac.za Author affiliations 1 Department of Industrial Engineering, Stellenbosch University, South Africa # Author was enrolled for a PhD in the Department of Industrial Engineering, Stellenbosch University, South Africa DOI http://dx.doi.org/10.7166/29-2-1899 ARTICLE INFO ABSTRACT ARTICLE INFO Digitalisation has been advocated as a possible strategy to improve the competitiveness of tool, die, and mould-making (TDM) companies in the 21st century. The recent rise of digital technologies, such as Internet of Things devices, now makes digitalisation an achievable reality. This paper focuses on the digitalisation of shop-floor operations in the South African TDM industry through the development of a novel mobile data collection (MDC) tool known as a shop-floor management system (SMS). The developed SMS was deployed to, and validated in, a selected tooling company for various products. The developed system improved the shop-floor’s real-time data collection. OPSOMMING Digitalisering is voorgestel as ’n moontlike strategie wat die mededingendheid van gereedskap- en gietvorming-maatskappye in die 21ste eeu kan verbeter. Die onlangse opkoms van digitale tegnologie, soos Internet of Things-toestelle, maak nou digitalisering ’n haalbare realiteit. Hierdie artikel fokus op die digitalisering van werkswinkelvloerbedrywighede in die Suid- Afrikaanse TDM-bedryf deur die ontwikkeling van ’n nuwe mobiele data versameling instrument vir werkswinkelvloerbestuur. Die ontwikkelde stelsel is ontplooi tot, en gevalideer in, ’n geselekteerde gereedskapmaatskappy vir verskillende produkte. Die ontwikkelde stelsel het die werkswinkel-vloer se reële-tyd data- insameling verbeter. 1 INTRODUCTION Tool, die, and mould (TDM) manufacturing has long been considered a key industrial sector, as it is the sole supplier of basic production equipment. Therefore, the TDM industry in South Africa is a critical support industry to the broader manufacturing industry, bridging the gap between product development and series production. This makes the sector a high value-adding constituent in the supply of manufactured products by being at the heart of component manufacturing and by forming the backbone of the manufacturing sector [1]. TDM firms have been traditionally known to consist of small, medium or micro-enterprises (SMMEs) that are usually family-owned businesses. The European tooling industry represents an annual turnover of approximately 13,000 million Euros, and consists of over 7,000 companies, 95 per cent of which are SMMEs [2]. Likewise, over 90 per cent of TDM firms in South Africa are small businesses in the range of SMMEs [3]. However, globalisation and the rapid growth in information communication technologies (ICT) have changed views about the way production is being done in the TDM sector. Gaining a competitive edge is no longer solely determined by product quality and lower costs: customers of the tooling industry grow more demanding and rapidly change their needs. There is a general consensus among researchers and industrialists that due date conformance and delivery reliability have also become major key success factors for successful firms in the TDM industry ([4]; [5]; [6]). 153 Tool-making firms that align themselves with these current customer demands stand out on the market, and attract more clients than those that lag behind. A global survey conducted by Schuh, Pitsch, Kuhlmann and Stark [7] on the different tool-making firms (located in the 20 most important countries in the world of tooling) classified the different firms, based on their market share and competence levels, and using a ‘world of tooling’ radar, into four categories:  Allstars: a cluster of TDM companies with a high tooling competence and a big market share.  Established: a cluster of tool-making firms with a high tooling competence and a medium market size.  Rookies: a cluster of tool-making firms with a medium tooling competence and a small market size.  Rising stars: a cluster of tool-making firms with both a low tooling competence and a small market size [7].  Allstars: a cluster of TDM companies with a high tooling competence and a big market share. 1 INTRODUCTION  Established: a cluster of tool-making firms with a high tooling competence and a medium market size.  Rookies: a cluster of tool-making firms with a medium tooling competence and a small market size.  Rising stars: a cluster of tool-making firms with both a low tooling competence and a small market size [7]. g stars: a cluster of tool-making firms with both a low tooling competence and a small ket size [7]. The South African TDM sector was classified within the ‘Rising star’ category of the radar in the study, as depicted in Figure 1. This is mainly because recent results of the on-going benchmarking initiative in the South African TDM industry have shown that most firms struggle in the global market owing to intense external competition and internal shortcomings [8]. One of the identified challenges was that of poor organisational practices adopted by most observed TDM firms in South Africa. The organisational shortfalls of the South African TDM sector are a sign of poor business or operational practice on the shop-floor. According to Von Leipzig and Dimitrov [9], the results from the on-going benchmarking surveys clearly show that the initial and later stages of the tool production value stream are being neglected by South African TDM firms. In their analysis, Von Leipzig and Dimitrov [9] commented that the South African TDM sector is characterised by difficulties in data collection and manipulation. It is evident that the South African TDM sector has an intermediate potential to develop and improve its status and to become a globally recognised competitor — that is, attaining ‘Allstar’ status. The question remains: what can a South African TDM firm do to attain ‘Allstar’ status? This paper aims to answer this question by focusing specifically on how the South African TDM sector can overcome its data collection and manipulation shortcomings. Figure 1: World of tooling radar [7] Figure 1: World of tooling radar [7] 154 A study by Schuh, Pitsch, Salmen and Rittstieg [10] on the concept of ‘fast forward tooling’ identified nine critical success factors that tool-making firms can implement to improve their global competitive position (see Figure 2). One of the factors suggested in the work is that of ‘digitalisation’, which has the potential significantly to improve the competitiveness of a tooling enterprise, thereby becoming the basis for other success factors. 1 INTRODUCTION The basis for digitalisation is the recording of data; and there is a lot of data one can find in a manufacturing environment. Hence, the concept enables the recording of information from manufacturing processes and from serial production. After the data is collected, it is processed into smart data, or information that is presented in a meaningful way for managers and decision-makers to make decisions. This permits the establishment of unique know-how or sustainable knowledge [10]. As a result, a company will possess expert knowledge that can be used for future decision-making, and may be commercialised. Thus the concept of digitalisation has the potential of addressing data collection and manipulation shortcomings. Figure 2: ‘Fast forward tooling’ success factors [10] With the recent growth in ICT technology such as Internet of Things (IoT) devices, it is now possible to apply the concept of digitalisation, thus improving the flexibility, agility, efficiency, and productivity of operations in a tooling environment [11]. According to Business Insider estimates observed by Samuelson, Lanman and Pocek [12], by the year 2020 around 20 billion devices will be connected to the internet, with 40 per cent of these being IoT devices, as shown in Figure 3. Figure 2: ‘Fast forward tooling’ success factors [10] Figure 2: ‘Fast forward tooling’ success factors [10] Figure 2: ‘Fast forward tooling’ success factors [10] With the recent growth in ICT technology such as Internet of Things (IoT) devices, it is now possible to apply the concept of digitalisation, thus improving the flexibility, agility, efficiency, and productivity of operations in a tooling environment [11]. According to Business Insider estimates observed by Samuelson, Lanman and Pocek [12], by the year 2020 around 20 billion devices will be connected to the internet, with 40 per cent of these being IoT devices, as shown in Figure 3. With the recent growth in ICT technology such as Internet of Things (IoT) devices, it is now possible to apply the concept of digitalisation, thus improving the flexibility, agility, efficiency, and productivity of operations in a tooling environment [11]. According to Business Insider estimates observed by Samuelson, Lanman and Pocek [12], by the year 2020 around 20 billion devices will be connected to the internet, with 40 per cent of these being IoT devices, as shown in Figure 3. 1 INTRODUCTION Figure 3: Growth in IoT devices [12] Figure 3: Growth in IoT devices [12] Figure 3: Growth in IoT devices [12] 155 According to Schuh, Kuhlmann, Pitsch and Komorek [13], the IoT devices will greatly impact the way toolmakers will perform production in the 21st century. However, the application of IoT or current ICT technology is yet to be fully explored in terms of its relevance in the digitalisation of shop-floor operations in the South African TDM industry. As a result, the potentials of digitalisation are yet to be comprehensively explored by South African tooling companies. This paper attempts to fill this gap by developing a system for the digital transformation of operations on the tool room shop-floor. A systematic method of process analysis, selection of appropriate technologies, design and development for the digital transformation is employed while accessing its applicability in the South African TDM context. 2.1 Digitalisation In this section, the concept of digitalisation is further explored as a potential initiative to improve the competitiveness of TDM operations in the area of data collection. The benefits and perceived shortcomings of digitalisation are outlined. Mobile and cloud computing technologies are presented as supporting digitalisation and state-of-the-art research on digitalisation within the global tooling industry is shown. Then the relevance of this concept in the current tooling industry is justified. 2.1.1 Concept of digitalisation Digitalisation can be viewed as the implementation of current modern ICT to improve production processes’ efficiency and productivity. The concept has been promoted as having the potential significantly to improve the competitiveness of any manufacturing enterprise [14]. According to Stolterman and Fors [15], ‘digitalisation’, or ‘digital transformation’, refers to the changes associated with the application of digital technology to all aspects of human society [16]. The concept differs from ‘digitisation’, which refers to the conversion of analogue or manual processes to digital ones. Brennen and Kreiss [17] have defined digitalisation as “the adoption or increase in use of digital or computer technology by an organisation, industry or country”. Unfortunately, the rate of adoption of digital technologies in the manufacturing sector has been slow, with most firms using a ‘wait and see’ approach [18]. However, the few companies that have implemented digital solutions have been able to tell stories of success. Thus tool-making firms ought to view digital solutions as an opportunity rather than as a threat [11]. Digitalisation is viewed as an on-going process rather than as a one-stop destination, as firms ought continually to evaluate what is worth digitalising. This is mainly because digital technologies evolve fast in a globalised world. To be successful, one has to keep up with current technological trends, and pilot small solutions incrementally to inspire workers while improving competitiveness. The concept of digitalisation has been found to apply to different scenarios and applications in the business world. The available literature records three different views of digitalisation: those at the process level, product level, and the supply chain level. In the process-level view of digitalisation, new digital tools are adopted to streamline business activities and processes by reducing manual steps [16], while the product-level view focuses on the ability to turn existing products or services into digital variants, thus offering a competitive advantage over other tangible products [19]. The supply-chain view of digitalisation focuses on the sole use of digital technologies to connect suppliers and customers, radically transforming business procedures through connectivity. Since this study focuses on the digitalisation of shop-floor operations in a tooling environment, only the process- level view applies in this study. The main goal in process-level digitalisation is to ensure the real- time availability of information for decision-makers within an organisation [20]. 2.1.2 The digitalisation effort pays off in a lot of ways. Firstly, digitalisation improves process transparency, which is a key step to greater efficiency and profitability [21]. Anything that is recorded can easily be measured, tracked, and monitored. Secondly, since digitalisation ensures the processing of data into real-time information, often referred to as ‘smart data’, informed decision- making by production managers is supported. Eventually the productivity of the enterprise is considerably improved, since decisions can be made quickly, based on the readily available information [14]. The improved productivity will result in a substantial reduction in time spent by employees searching for information, as workers need to spend most of their time on activities that produce value. As a result, all employees become more efficient when working within an optimised process. Thirdly, with informed decisions made at all levels of the value chain, the costs associated 156 with incorrect information and erroneous decisions are largely eliminated [22]. Moreover, business integration is significantly improved by digitalisation, helping firms to reduce greatly the time spent on interruptions while simultaneously enhancing their product or service quality [23]. This is crucial in the TDM industry, where there are no complete or standard solutions on the market for tool- making firms. Thus each company is supposed to develop its own solutions, as they all have different applications and business context scenarios. Enterprise resource planning (ERP) solutions on the market usually do not fit well in a tooling environment, as they are offered with more modules than required. As a result, buying one is very expensive, and extra costs may be incurred in efforts to adapt the system to the business rules of the company using it. According to Schuh, Kuhlmann, Pitsch, and Komorek [13], digitalisation can aid in collaborative work within the tooling industry by ensuring seamless communication within value creation networks. Eventually the product’s time-to- market is improved while the focus on the customer is enhanced [24]. According to Schuh et al. [10], digitalisation in the context of tooling has to be adapted to industry- specific characteristics, which is why the industry-specific characteristics of a group need to be determined before selecting and implementing the appropriate digital technologies. 2.1.3 Although digitalisation has the potential to improve operations significantly, a few shortfalls need to be addressed. The internet is one of the central technologies in digitalisation. As a result, data security of sensitive information remains a challenge that is yet to be adequately resolved [26]. Furthermore, workers with low computer or digital literacy require sufficient training before they can implement or use any digital solutions [27]. These factors can slow down the acceptance or possible adoption of digital solutions. Digital transformation of business operations has often been perceived by business owners as a costly venture [28]. As a result, a majority of SMMEs do not consider trying to implement any new technology into their businesses. However, the cost of some digital devices has significantly reduced on the market, and do-it-yourself (DIY) open-source devices have opened new opportunities for innovative designs at little or no cost ([29]; [30]). Furthermore, digitalisation is often thought by some to be the same as ‘automation’. According to Henriette, Feki and Boughzala [19], process automation is only one part of the entire digitalisation framework. This perception only focuses on the process-level view of digitalisation yet the concept also includes the product level and the supply chain level, as discussed earlier in section 2.1.1. As a result, another misconception workers usually have is that digitalisation may lead to the loss of jobs if manual processes are replaced by semi-automated methodologies. On the contrary: digitalisation should be viewed as a way of making processes better and more effective rather than replacing human labour [16]. A thorough understanding of the currently available digital technologies can assist in eliminating the above-mentioned misconceptions about digitalisation. The next section will give a brief overview of some of the common classes of currently available digital technologies in the form of mobile devices and cloud computing platforms. 2.1.2 In a study on establishing a framework for digitalisation in production firms, Krishnan, Pujari and Sarkar [25] outlined four dimensions that digitalisation can address in manufacturing: (1) The operational excellence of a production firm can be enhanced as productivity and efficiency across processes and functions grow. (2) A firm’s agility or adaptation, its response to internal and external changes (disturbances), is improved. The dimensions of (3) innovation and (4) customer-centricity are also enhanced in a firm by digitalisation. 3 Limitations of, and misconceptions about, digitalisation 2.1.5 State-of-the-art The implementation of current ICT in the TDM industry has been slow. Some researchers have successfully implemented solutions in TDM firms using current ICT. Efforts to create such information systems for the TDM sector are on-going, with examples from Choi, Shin, Choi and Lee [6], Hu, Zhou and Li [40], Silva, Roque and Almeida [41], and Li, Wang, San, and Seng [42], among others. An understanding of the TDM industry domain is therefore paramount before digitalisation can be fully implemented in the field. The next section focuses on the research methodology adopted in developing a digital solution that suits the South African TDM sector. .4 Digitalisation through mobile devices and cloud computing platforms Mobile devices such as smartphones and tablets have become ubiquitous in everyday life [31]. These gadgets have become readily available at low prices, causing a growth in usage in our time. Modern smartphones have become programmable. They also come with a growing number of embedded sensors, such as a digital compass, accelerometer, gyroscope, GPS, microphone, and camera [32]. As a result, the range of data these devices can handle is enormous, including text messages, GPS, barcodes, QR-codes, pictures or images, audio, and video [18]. Furthermore, mobile devices can browse the internet, making them easily part of a global network of other objects. Mobile data can be accessed anywhere at any time in near-real time [33]. Mobile devices are well-suited for applications in which data collection is done repeatedly and conducted in a distributed way; and a large percentage of the data types collected are quantitative in nature [34]. According to Mell and Grance [35], cloud computing is a model for enabling ubiquitous, convenient, on-demand network access to a shared pool of configurable computing resources (e.g., networks, 157 157 servers, storage, applications, and services) that can be rapidly provisioned and released with minimal management effort or service provider interaction. This model of technology is a broad field of study with numerous resources that allow the sharing of data over distributed systems. Google has the most commonly used cloud computing resources in the form of Google Drive [36]. Microsoft Azure is another commonly used platform for Windows users [37]. Cloud computing platforms have a wide usage in many domains, including business [38], healthcare, and manufacturing [39]. servers, storage, applications, and services) that can be rapidly provisioned and released with minimal management effort or service provider interaction. This model of technology is a broad field of study with numerous resources that allow the sharing of data over distributed systems. Google has the most commonly used cloud computing resources in the form of Google Drive [36]. Microsoft Azure is another commonly used platform for Windows users [37]. Cloud computing platforms have a wide usage in many domains, including business [38], healthcare, and manufacturing [39]. 3 RESEARCH METHODOLOGY This section outlines the research design and methods used in the study. According to Krishnan et al. [25], a systematic approach, which involves analysis, design, development, and testing, is required to achieve the digital transformation of a business’s operations. This design strategy entails analysis of the current state of an industry, identification of processes requiring improvement, and careful selection of appropriate technologies to bring about the digital transformation. Thus knowledge engineering and systems engineering methods were used in the study. Knowledge engineering was used to establish the industry-specific characteristics of the South African TDM sector by establishing the processes requiring digitalisation. The systems engineering methodology was employed for the development of the digital solution. This methodology involved selection of the appropriate digital technologies, and the design, development, testing, and validation of the final solution. The systems engineering methodology was chosen because, according to Hoefler and Marm [43], this approach allows for a systematic route from problem definition to solution realisation, which results in a prototype being developed and tested. The agile approach of software development was used for the design and creation of the digital solution. According to Spataru [44], the agile approach simplifies the development process. This is because development is conducted incrementally for each module, unlike in the waterfall approach, which follows a hierarchy of long steps [44]. Firstly, a case study for a selected TDM company was used to test and validate the solution. At this stage, real-world scenarios and data were used to test the robustness of the system. Secondly, the available development platforms (software and hardware) were compared, using set criteria for the purpose of selecting the best platform. A questionnaire was designed to verify the selected technology, and decision matrices were developed, following Satterlee, E., McCullough, Dawson and Cheung [34], for the selection of the software and hardware. The remaining stages, which involved designing, developing, and testing the system, were implemented in one selected company, as different tooling firms produce a wide range of products and services. As a result, the industry-specific characteristics vary from one company to another [10]. So case-study-specific data related to the selected firm was used. After developing and testing a complete digital solution, the same company chosen during the design phase was used for the validation process, which followed three distinct phases:  System preparation;  System deployment; and  Product selection and testing. 4.1 Selected processes The experts who were consulted were questioned about the tooling functions that greatly impacted the due date reliability or speed of a project. The experts all alluded to the following critical functions:  Cost estimation  Project management — planning, scheduling, and control  Production  Project management — planning, scheduling, and control  Production  Project management — planning, scheduling, and control  Production It was concluded, therefore, that improving the data collection in the domains of cost estimation, process planning, and production functions would significantly improve business operations in a tooling environment. The cost estimation function was identified as crucial because research has shown that, in the mould-making industry, usually less than 10 per cent of all offers turn into orders ([45]; [46]; [47]; [48]). As a result, accurate and timely cost estimation before tool, die, and mould production is a key attribute for sustaining global competitiveness. Due to the skill shortages that the South African TDM industry has experienced, the majority of tool-making firms take a long time to quote for a job. Furthermore, results from benchmarking exercises at these firms have shown that the majority of the generated quotes lacked accuracy, due to the methods employed [45]. For effective and timely cost estimation, the parameters to be considered must be known, together with the decision heuristics to be followed. Furthermore, the project management of orders in a tooling environment is another complex task that requires great skill and expertise. This is mainly because operational disturbances such as rush orders or breakdowns can complicate the life of project managers. Carefully prepared process plans and schedules can easily become invalid, thus needing to be continually revised [49]. For a project to finish within the budgeted time, efficient project management techniques should be employed. All value-adding activities in a tool room need careful procedural conformance and great skill. As a result, administrative work on the shop floor can be a nightmare if it is not well-designed and well-managed. The time taken for each operation is a critical parameter, as it will help to give feedback on the progress of orders. Furthermore, knowledge of manufacturing task times serves as a valuable input during the cost estimation of the same job in the future. After establishing these system functions, the key parameters for each function were derived. 3 RESEARCH METHODOLOGY 158 RESULTS AND DISCUSSION 4 This section outlines the major research findings of the study. The method of knowledge engineering was employed, with field experts from five different tool-making firms being were interviewed to obtain results on:  Tool-room processes that could be significantly improved through digitalisation;  Parameters for the identified processes; and  Derivation of user needs and specifications. The five experts who were visited were mainly from injection mould design and press metal tool production specialist firms within the Western Cape Province tool, die and mould-making (TDM) and plastics manufacturing industries. The mean number of employees for the observed population of companies was 10.4. The tooling expert from company D had the greatest amount of experience (40 years’ experience in the tooling industry), as shown in Table 1. The average experience was found to be 28.6 years. Table 1: Experts’ experience in years Expert from company A B C D E Experience of the expert (years) 19 28 23 40 33 Table 1: Experts’ experience in years Figure 4: System use case model The major system functions derived are highlighted in grey. Though the cost estimation, process planning, and production cases are the major functions, the human resources function plays a crucial role in defining the required operations and resources. The database design and development was the first stage in setting up the system. The entity identification for each class was derived, and an entity relationship diagram (ERD) was developed, as shown in Figure 5. The ‘Google Sheets’ platform was used in developing the backend database for each of the tables. The mobile application for the system was developed using the AppSheet environment. 4.1 Selected processes Since labour-related costs are the greatest contributor to the overall tool cost, monitoring and control of 159 production time is crucial. One of the consulted experts indicated that over 60 per cent of the tool’s costs are labour-related. production time is crucial. One of the consulted experts indicated that over 60 per cent of the tool’s costs are labour-related. 4.2 Parameter and user derivation The system functions and parameters for the identified processes were also derived. Cost estimation, project planning, and production functions were deemed to be crucial in contributing to shop-floor management and to meeting delivery due dates. As a result, the main system users will be cost estimators, project managers, and artisans in a tool-making environment. The use case model generated in Figure 4, and the information in Table 2, summarise the key findings in the derivation process. Figure 4: System use case model 4.3 Overview of shop-floor management system The overall system architecture and functionality is illustrated in Figure 6. With the use of web- based cloud computing platforms, the captured data can be collected in a distributed manner. This simplifies the methodology, as information can be accessed in real time at any place. Mobile devices speed up shop-floor data collection, reducing the costs incurred in preparing and printing paper- based documents. Furthermore, the conditional logic set in the mobile devices simplifies the end- user’s collection process, thus reducing the effort involved in recording the data manually. 160 Figure 5: Entity relationship diagram (ERD) Figure 5: Entity relationship diagram (ERD) 161 Table 2: System users, functions, and parameters Users Functions Parameters Data for each parameter Cost estimator  Cost estimation  Quoting  Labour costs Number of hours for:  Data management  Mould design  Computer simulations  Advanced engineering  CNC machining  General shop labour  CMM parts  Material costs Material type and weight required for:  Mould base  Copper (electrodes)  Cavity core steel  Components  Texture  Tooling (cutters and inserts)  Sample material  Service costs Number of hours required for:  Heat treating and plating  Manifold hot-runner system Project manager  Planning  Material types and dimensions  Material types  Length, width, and height of material  Mass required  Purchasing  Supplier lists  Supplier costs  Contractors list  Contractor costs  Price of all materials  Prices of all services  Scheduling  Available resources — workers and machines  Available dates and times Production workers  Manufacturing Value-added time  Set-up time  Operation time Non-value added time  Set-up downtime  Operation downtime  Operational disturbance reason The responses of the five experts (represented in Table 3 and Table 4 respectively) validated the Table 2: System users, functions, and parameters The responses of the five experts (represented in Table 3 and Table 4 respectively) validated the need to use mobile and cloud-computing technologies during shop-floor operations in South African TDM firms. The developed system was integrated with the tooling companies’ existing Microsoft Excel spreadsheets. The developed system was integrated with the tooling companies’ existing Microsoft Excel spreadsheets. Table 3: Existing capacity analysis Question Summary of responses from experts Implications Availability of mobile devices (smart phones, tablets, etc.) No. However, most workers have their own devices. 4.3 Overview of shop-floor management system If workers have their own mobile devices, there may be no need to invest in buying devices. Availability of workers skilled in the use of mobile devices and having ICT expertise Yes. Few workers in a tooling environment are skilled in ICT. Usually the tool designers are good at using computer technology (because they use computers every day) There may be a need to train workers how to use digital technologies if they are introduced. 162 ENQUIRY COST ESTIMATE QUOTATION ORDER (AGREE ON DELIVERY DATE) ARTISAN JOB START JOB END JOB LOCATION  JOB CARDS  SPECIFICATIONS/ DRAWINGS  MATERIALS AND RESOURCES  SCHEDULE QUOTING PROCESS PLANNING PROCESS DATA PC Based Job/Data Tracking Costing feedback Costing feedback Feedback Feedback Load the job onto the SMS Load the job onto the SMS Real-time feedback to customer Real-time feedback to customer Manufacturing process Job specification from customer Job specification from customer Estimated vs. Actual cost analysis Estimated vs. Actual cost analysis MICROSOFT CORPORATION Production Automatic data feedback via smart phone Automatic data feedback via smart phone Actual operation times Production schedule Actual operation times Production schedule Load the job onto the SMS Load the job onto the SMS Real-time feedback to customer Real-time feedback to customer Estimated vs. Actual cost analysis Estimated vs. Actual cost analysis Feedback Feedback Manufacturing process Manufacturing process Figure 6: Schematic diagram of SMS overview Figure 6: Schematic diagram of SMS overview Table 4: Software and hardware requirements Question Summary of responses from experts Software implications Hardware implications Integration with existing data management platforms Yes. Some firms already use Excel and Pastel for data management. The selected software platform should be compatible with many platforms, especially Microsoft Excel. The selected device must be compatible with the software platform chosen. Ability to take photos, graphics, or video Yes. During the order definition stage, there may be a need to take a photo of the sample product or part. In some cases, toolmakers may need to take a video of the process on a machine. The selected software platform should support capturing and storage of pictures and videos in the dataset. The selected devices should have built-in cameras for taking photos and videos. They should also have the capability, and a large enough screen, to display graphics and videos. Need to record GPS data No None None Need to collect data from the field Yes. 4.3 Overview of shop-floor management system The engineering drawing is also supposed to be captured during the production process. The selected software platform should support the capturing of PDF files for the engineering drawings. The selected devices should be able to connect to either a cellular or a wireless network (Wi-Fi). 163 Table 4 (cont.): Software and hardware requirements Question Summary of responses from experts Software implications Hardware implications Complex filter logic in data collection Yes The logic for the cost estimation and production data collection processes is complex. The selected software platform should support skip logic during the development phase. The selected devices must have a reasonable amount of processing power. Need for external support during development Yes. The researcher and respondents conducting or participating in the study are not software developers, so will need help during the development phase. The selected software platform should have a dedicated support service (or community) that will assist in the development phase. None (software-related question) Need for merging with external data sets Yes. There is a need to integrate the collected data with financial systems in the future. The selected software platform should be compatible with existing data sets. None (software-related question) Table 4 (cont.): Software and hardware requirements 4.4 SMS module navigation The developed SMS is a web-based mobile solution, as shown in the design specification overview in Figure 6. The mobile solution has five different sub-modules that were deployed in different parts of a tooling firm. Figure 7 shows the main switchboard for the system. Each user obtained only the module relevant to their operations. Figure 7: Shop-floor management system (SMS) main switchboard modules Scheduling Sales Resources Production Dashboard Figure 7: Shop-floor management system (SMS) main switchboard modules The resources module is used to prepare the system by recording all of the company’s resources, which include worker details, machines, and assets. The users can also define common disturbances experienced and daily operations conducted. These are also used by other modules of the system during the quoting, planning, and production phases. This part (the resources module) is specifically deployed and made available to the human resources department of the business. The navigation flowchart for the resources module is illustrated in Figure 8 below. The sales module is deployed to the sales and cost estimation teams of the tooling business. 4.3 Overview of shop-floor management system The module allows for customer details and job specifications for each order or job to be captured after a customer’s enquiry. Upon receipt of an enquiry, the cost estimators determine the price of the job through parametric entry of the operations, materials, and services rendered per order. Upon completion of the costing, automated emails are generated and sent to the client with details of the quoted prices and the terms. This process significantly reduces the time spent on other administrative duties, such as generating quotes and invoices. The navigation flowchart for the sales module is illustrated in Figure 9 below. A common trend in the tooling business derived during the analysis was that of repeat orders occurring in the future. Due to this trend, the system also allows for the duplication of an old order so that the cost estimation process is not repeated. Upon approval by the customer, the order details are made available to the scheduling module of the system. 164 Main switchboard or resources icon Resources interface Machine details update Shop-floor operations update Employee details update Human Resources Figure 8: Resources module navigation flowchart Human Resources Resources interface Figure 8: Resources module navigation flowchart 165 Main switchboard or sales icon Customer details Order entry and status update Cost estimation Customer details update Materials description Operations description Services description Cost Estimatior Sales and Purchasing Figure 9: Sales module navigation flowchart In the world of tooling, cost estimation is one of the most complex tasks; it requires expert knowledge for an accurate quote to be generated. Thus the system employs intelligent algorithms to derive certain costing parameters. The pseudo-code shown in Figure 10 illustrates the decision- making algorithm employed by the system during the estimation process: Figure 9: Sales module navigation flowchart In the world of tooling, cost estimation is one of the most complex tasks; it requires expert knowledge for an accurate quote to be generated. Thus the system employs intelligent algorithms to derive certain costing parameters. 4.3 Overview of shop-floor management system The pseudo-code shown in Figure 10 illustrates the decision- making algorithm employed by the system during the estimation process: 165 BEGIN (Cost estimation interface) SELECT required operation; FOR selected operation(s): Find the required relevant machine and operator; Automatically display the machine and operator rate; Automatically compute operation cost End loop; SELECT required material(s); FOR selected material(s): Find unit material cost; Automatically display the unit cost and use quantity defined to compute material cost; End loop; If required: SELECT service(s) required; FOR selected service(s): Find, display and compute service cost; End loop; Compute total operational cost and save; Send automated emails to client and cost estimator; END BEGIN (Cost estimation interface) SELECT required operation; FOR selected operation(s): Find the required relevant machine and operator; Automatically display the machine and operator rate; Automatically compute operation cost End loop; SELECT required material(s); FOR selected material(s): Find unit material cost; Automatically display the unit cost and use quantity defined to compute material cost; End loop; If required: SELECT service(s) required; FOR selected service(s): Find, display and compute service cost; End loop; Compute total operational cost and save; Send automated emails to client and cost estimator; END Figure 10: Pseudo-code for decision-making intelligence during estimation Figure 10: Pseudo-code for decision-making intelligence during estimation Process planners or project managers can access all approved jobs from the scheduling module. In this platform, raw material purchases from suppliers are also facilitated. After the materials are received, tasks are assigned to the shop-floor toolmakers. Once deployed, each toolmaker can view the tasks assigned to them via the production module. The navigation flowchart for the scheduling module is illustrated in Figure 11 below. Main switchboard or scheduling icon Scheduling interface Complete tasks Task assignment Work In Progress tasks Assign worker and start/enddDate Project Manager Figure 11: Scheduling module navigation flowchart The production module is specifically deployed to the workers on the shop floor for the time-based entry and accounting of tasks done. In real time, workers can enter the start and stop times for their jobs. Workers can also input data on any recurring disturbances occurring during production. Once disturbances are recorded, the managers receive triggers of the event via email. Figure 12 below illustrates the production module navigation flow chart. Figure 11: Scheduling module navigation flowchart The production module is specifically deployed to the workers on the shop floor for the time-based entry and accounting of tasks done. In real time, workers can enter the start and stop times for their jobs. Workers can also input data on any recurring disturbances occurring during production. Once disturbances are recorded, the managers receive triggers of the event via email. Figure 12 below illustrates the production module navigation flow chart. 166 Main switchboard or Production icon Production interface Completed tasks Work-In-Progress and askt status update Toolmaker details Time sheet entry Toolmaker Figure 12: Production module navigation flowchart Figure 12: Production module navigation flowchart The dashboard module of the system is deployed to the top management only. The platform facilitates the real-time monitoring of shop-floor activities on key performance order-related indicators, which include due date conformance and order percentage progress. The managers can also view, in real time, costing analytics such as actual cost incurred versus estimated cost, labour hours per employee, and machine run times. The navigation flowchart for the dashboard module is illustrated in Figure 13 below. Although this is the main part that top management uses to observe shop-floor events, they requested to have access to the other four modules as well. CONCLUSION 5 The shop-floor management system (SMS) developed here is unique in addressing the requirements of South African TDM firms, which include:  Interfacing shop-floor workers with tool-room managers;  Real-time shop-floor data collection by toolmakers;  Distributed information sharing in a tool-room environment;  Generation of order progress reports;  Information storage of order histories;  Automated computation of actual costs incurred;  Automated reporting and email functions; and  Cost analytics of estimated vs actual costs per job. The developed SMS application was only implemented within one company. However, since the tool is a distributed system using mobile devices, future work can be conducted on extending the application to the domain of TDM customers and suppliers. In that way, customers will be able to enter their requirements and request quotations, while suppliers can consistently communicate about the services and products they supply (with current updated prices). Thus future work can also be done in deploying the application to TDM customers and suppliers. Furthermore, according to Christensen [50], the introduction of new technologies to improve business operations brings in some “disruptive characteristics”. All new technologies can have negative implications, especially during the introduction phases. Since the SMS is a web-based solution, threats associated with the internet are a possibility, and the absence of a good network can affect functionality. Therefore, further work can also be conducted in investigating all the disruptive characteristics of implementing the SMS in a South African tooling environment. Such an effort will help to fine-tune the solution to improve its operational effectiveness. 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https://openalex.org/W1991718747	https://europepmc.org/articles/pmc5936563?pdf=render	English	null	Applied K Fertilizer Use Efficiency in Pineapples Grown on a Tropical Peat Soil Under Residues Removal	The scientific world journal/TheScientificWorldjournal	2,005	cc-by	3,947	Applied K Fertilizer Use Efficiency in Pineapples Grown on a Tropical Peat Soil Under Residues Removal Osumanu H. Ahmed, Husni M.H. Ahmad, Hanafi M. Musa, Anuar A. Rahim, and Syed Omar S. Rastan Department of Land Management, Faculty of Agriculture, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia nu H. Ahmed, Husni M.H. Ahmad, Hanafi M. Musa, Anuar A. Rahim, and mar S. Rastan ent of Land Management Faculty of Agriculture Universiti Putra Malaysia 43400 Osumanu H. Ahmed, Husni M.H. Ahmad, Hanafi M. Musa, Anuar A. Rahim, and Syed Omar S. Rastan Department of Land Management, Faculty of Agriculture, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia E-mail: osman60@hotmail.com Received November 13, 2004; Revised December 20, 2004; Accepted December 21, 2004; Published January 21 2005 In Malaysia, pineapples are grown on peat soils, but most K fertilizer recommendations do not take into account K loss through leaching. The objective of this study was to determine applied K use efficiency under a conventionally recommended fertilization regime in pineapple cultivation with residues removal. Results showed that K recovery from applied K fertilizer in pineapple cultivation on tropical peat soil was low, estimated at 28%. At a depth of 0–10 cm, there was a sharp decrease of soil total K, exchangeable K, and soil solution K days after planting (DAP) for plots with K fertilizer. This decline continued until the end of the study. Soil total, exchangeable, and solution K at the end of the study were generally lower than prior values before the study. There was no significant accumulation of K at depths of 10–25 and 25–45 cm. However, K concentrations throughout the study period were generally lower or equal to their initial status in the soil indicating leaching of the applied K and partly explained the low K recovery. Potassium losses through leaching in pineapple cultivation on tropical peat soils need to be considered in fertilizer recommendations for efficient recovery of applied K. YWORDS: potassium fertilizers, pineapples, tropical peat soils, residues, leaching DOMAINS: agronomy, soil systems, plant sciences, environmental technology, ecosystems management, waste management policy, crop science Research Article TheScientificWorldJOURNAL (2005) 5, 42–49 ISSN 1537-744X; DOI 10.1100/tsw.2005.9 Research Article TheScientificWorldJOURNAL (2005) 5, 42–49 ISSN 1537-744X; DOI 10.1100/tsw.2005.9 Corresponding author. ©2005 with author. INTRODUCTION Large-scale production of pineapples on tropical peat soils is characterized by in situ burning of crop residues before subsequent replanting. Following the 1997/1998 economic loss in the agricultural sector due to fires and haze in South-East Asia[1,2,3], in situ burning of crops residues such as pineapple residues is being discouraged. Studies on product development from pineapple residues have shown some 42 Ahmed et al.: Potassium Fertilizer Use Efficiency TheScientificWorldJOURNAL (2005) 5, 42–49 impressive and promising results[4,5,6], but the existing K fertilization program for this residue management practice came out of studies[7,8,9,10,11,12,13] that did not take K leaching into account. impressive and promising results[4,5,6], but the existing K fertilization program for this residue management practice came out of studies[7,8,9,10,11,12,13] that did not take K leaching into account. Potassium deficiency can be severe, particularly in pineapple cultivation on peat soils where K loss through leaching can be high due to low clay and the absence of mineral matter[14,15]. Potassium fixation is almost absent in peat soils[16] and despite their high cation exchange capacity, these soils do not readily adsorb exchangeable K[16]; a substantial amount of the total available K is always present in the soil solution and is hence strongly mobile and prone to leaching[16]. There is the need to quantify the total amount of K taken up by the plant (summation of K in roots, stem, leaves, peduncle, fruit, and crown), remaining in the soil after cultivation, and losses through leaching from applied K fertilizers on peat soils under pineapple cultivation. This assessment will be useful in evaluating K requirements of pineapples on peat soils, thereby contributing to the reduction of environmental pollution by excessive and unbalanced K fertilizer uses. The economic significance of efficient K use to non-K fertilizer-producing countries such as Malaysia, whose fertilizer use and import bill are higher than N[17], cannot be overemphasized. The import bill for K fertilizer (2001) has been has estimated at US$129.74 million per year (2001)[17]. This study was carried out to determine applied K use efficiency under a conventionally recommended fertilization regime in pineapple cultivation with residues removal. RESULTS AND DISCUSSION The experimental plots did not significantly differ in their initial K forms (total, exchangeable, and soil solution). The bulk density of peat soils depends on the amount of compaction, the botanical composition of the materials, their degree of decomposition, and the mineral and moisture contents at the time of sampling[16]. Bulk densities at the depths of 0–10, 10–25, and 25–45 cm were 0.16, 0.23 and 0.13 g cm–3, and were typical of Umbro Saprists peat soil[16]. But the relatively high bulk densities at the depths of 0–10 and 10–25 cm may be due to cultivation and compaction, subsidence of peat soil, of the surface layers on drainage[16]. The fact that the pineapple estate has been under use for about 36 years and has good drainage systems supports this observation. The lower bulk density at the depth of 25–45 cm may be due to partial decomposition of plant materials. Observation has shown that the bulk density in most tropical peat soils is higher at the surface layers than the subsurface layers as the former surfaces tend be more sapric (full decomposition) than the latter layers[16]. This has been associated with climate, height of water table, and oxidation[16]. Perhaps materials at the depth of 25–45 cm may have been lignified. The distribution of K in the pineapple plant is presented in Table 2. There was no significant difference in the K uptake for roots, crown, and peduncle regardless of treatment difference, but this uptake was significantly different for stem, leaves, and fruit of the fertilized plants than the unfertilized plants. There was also significant difference across treatments (Table 2). p g The total K uptake for KF and NKF were 498.84 and 342.50 kg ha–1 (average across treatments), and with a total K rate of 554 kg ha–1, K recovery was calculated (using the stated formula in materials and methods) to be only 28.22%. Not discounting the contribution of diffusion to this recovery[23,24], probably without the rooting system of pineapple, this recovery would have been lower. During growth, it is known that the adventitious roots of pineapples form a short and compact system at the stem base, with numerous strong roots and limited branching. However, under ideal conditions, the soil root system could spread up to 1–2 m laterally and 0.85 m in depth[25]. MATERIALS AND METHODS The study was conducted in a pineapple estate in Johor, Malaysia on Umbro Saprists peat soil. The area has an annual precipitation of about 2000 mm. Monthly minimum and maximum temperatures are of 23 and 31˚C, and relative humidity ranged from 70 to 90%/month. Two treatments were used. Treatment one was on plots with K fertilizer (KF) and treatment two was on plots without K fertilization (NKF). Each experimental plot size was 4 × 12 m, with 300 suckers of cv Gandul (the most commonly grown variety) planted in each plot. The experimental plots were laid out in a randomized, complete block design with four replications. p Potassium was applied as KCl (49.8% K) to the KF plots. K fertilization schedules and rates were adapted to practices in the estate. At 83 days after planting (DAP), K was applied at 89 kg ha–1 K. Another 89 kg ha–1 K was applied at 144 DAP. At 209 DAP, 188 kg ha–1 K was applied to the KF plots and the same rate was also applied at 263 DAP (Table 1). Normal estate N and P fertilization programs were followed. All other plant management procedures and schedules of the estate were also followed. TABLE 1 The Usual N and P Fertilization Program of the Pineapple Estate Number of Days of Application After Planting Fertilizer rate (kg ha–1) 83 144 209 263 (Total) N 176 176 176 176 (704) P 11 11 7 7 (36) TABLE 1 The Usual N and P Fertilization Program of the Pineapple Estate TABLE 1 The Usual N and P Fertilization Program of the Pineapple Estate The Usual N and P Fertilization Program of the Pineapple Estate Number of Days of Application After Planting Before the start of the experiment, pineapple residues were manually removed from the study area by slashing, raking, and removing old plants of the previous crop. Before removing the crop residues, peat soil samples were taken at depths of 0–10, 10–25, and 25–45 cm using peat soil auger and analyzed for total K, exchangeable K, and soil solution K using the dry ash, double acid, and squeeze 43 Ahmed et al.: Potassium Fertilizer Use Efficiency TheScientificWorldJOURNAL (2005) 5, 42–49 methods[18,19,20], and atomic absorption spectroscopy (AAS). Subsequent peat soil samplings were done 48, 83, 144, 263, 365, 417, and 446 DAP. These samples were also analyzed for K forms mentioned using the aforementioned procedures. % fertilizer nutrient recovery = (TNF) – (TNU)/R × 100 where TNF = total nutrient uptake from fertilized plots, TNU = total nutrient uptake from unfertilized plots, and R = rate of fertilizer nutrient applied. MATERIALS AND METHODS Peat core samplers of 7.5 cm diameter were used to collect peat soil samples at the stated depths and standard procedures were used to determine the bulk density of the experimental plots, before crop residues removal. methods[18,19,20], and atomic absorption spectroscopy (AAS). Subsequent peat soil samplings were done 48, 83, 144, 263, 365, 417, and 446 DAP. These samples were also analyzed for K forms mentioned using the aforementioned procedures. Peat core samplers of 7.5 cm diameter were used to collect peat soil samples at the stated depths and standard procedures were used to determine the bulk density of the experimental plots, before crop residues removal. At 466 DAP, fresh fruits were harvested from the experimental plots excluding guard rows. A day before harvesting, three plants were randomly selected from the plots, uprooted, and partitioned into roots, stem, leaves, peduncle, fruit, and crown. This partitioning was done to enable calculation of the total uptake of K by the plants. These parts were oven dried at 60˚C until constant weights were attained and dry weights determined. Dry ash method was used to extract K from these tissues and AAS used to determine K concentrations in the tissues. Based on the plant density, a simple proportion was used to quantify K uptake in the different parts of pineapple plant per hectare. The K uptake in the plant parts for the fertilized and unfertilized treatments was compared by T test using the Statistical Analysis System[21]. The K recovery was calculated according to the formula of[22]: % fertilizer nutrient recovery = (TNF) – (TNU)/R × 100 RESULTS AND DISCUSSION It therefore could be that unlike in mineral soils, the roots of pineapple in the peat soil were not very restricted or confined to the tilled area because of the relatively low bulk density. This might have allowed the roots under the fertilized condition to access larger volume of water and plants nutrients than those under the unfertilized condition. In addition, increased K diffusion gradient under the fertilized condition might have also facilitated the uptake of K in the fertilized plots[23,24]. 44 TheScientificWorldJOURNAL (2005) 5, 42–49 Ahmed et al.: Potassium Fertilizer Use Efficiency TABLE 2 Potassium Distribution in Pineapple Plant Parts Parts KF (kg ha–1) NKF (kg ha–1) Roots 0.32a 0.29a Crown 10.99a 11.72a Peduncle 22.42a 23.47a Stem 136.92a 78.53b Leaves 139.23a 87.61b Fruit 188.96a 140.88b Total 498.84 a 342.50b Note: Same alphabet within rows indicates no significant difference between treatment means using T test at p = 0.05. TABLE 2 Potassium Distribution in Pineapple Plant Parts TABLE 2 Note: Same alphabet within rows indicates no significant difference between treatment means using T test at p = 0.05. There was a sharp decrease of soil total K, exchangeable K, and soil solution K at 0–10 cm, after 263 DAP for plots with K fertilizer (Table 3). This decline continued until the end of the study such that the three different forms of soil K at the end of the study were generally lower than those before the study. There was, however, no evidence of significant accumulation of these K forms at depths of 10–25 and 25–45 cm. There seemed to be no corresponding significant accumulation of the K forms at deeper depths. Potassiaum concentrations throughout the study period were generally lower or equal to their initial status in the soil, indicating leaching. A comparison between the initial concentrations of total (Table 4), exchangeable (Table 5), soil solution (Table 6), and those at the end of the study showed that the latter concentrations were generally lower than the former. These observations suggest that the low K recovery of K could be attributed to leaching. Due to the low clay[14] and absence of mineral matter[15], K fixation that is noticeable in mineral soils is almost absent in peat soils. Although the cation exchange capacity of these soils is high, exchangeable K is not readily adsorbed. RESULTS AND DISCUSSION Other studies have shown that despite the high exchange capacity values (150–200 cmol [+] kg–1 of organic soil), low mineral content of organic soils facilitated loss of applied fertilizer K remaining in the soil[18]. Note: Same alphabet within columns indicates no significant difference between means using T test at p = 0.05. CONCLUSION Recovery of K in pineapple cultivation on tropical peat soil is low (28.22%) partly due to leaching loss. This loss through leaching needs to be considered in fertilizer recommendations for efficient recovery of soil applied K. 45 TheScientificWorldJOURNAL (2005) 5, 42–49 Ahmed et al.: Potassium Fertilizer Use Efficiency TABLE 3 Concentrations of Three Different Forms of Soil K at Different Stages of Sampling for Three Different Depths Stage of Sampling (DAP) 0* 48 144 263 365 417 466 Total K (mg kg–1) 0–10 cm KF 883a 475a 1450a 2900a 550a 375a 273a NKF 883a 467a 450b 300b 225b 325a 305a 10–25 cm KF 575a 400a 600a 525a 475a 300a 167a NKF 675a 325a 500a 225b 225b 175a 143a 25–45 cm KF 550a 275a 350a 750a 300a 300a 240a NKF 633a 350a 400a 250b 275a 250a 195a Exchangeable K (mg kg–1) 0–10 cm KF 423a 503a 880a 3330a 538a 260a 290a NKF 540a 513a 408b 320b 147b 260a 278a 10–25 cm KF 460a 468a 635a 698a 623a 360a 330a NKF 417a 535a 623a 343b 238b 215a 155a 25–45 cm KF 423a 453a 445a 450a 395a 383a 383a NKF 383a 447a 430a 383a 393a 300a 245b Solution K (mg kg–1) 0–10 cm KF 173a 180a 287a 1593a 320a 313a 98a NKF 182a 148a 67b 94b 68b 160b 93a 10–25 cm KF 89a 60a 102a 173a 160a 175a 100a NKF 95a 68a 82a 36b 53b 85a 33a 25–45 cm KF 114a 58a 54a 77a 130a 210a 70a NKF 89a 68a 41a 56a 80a 150a 48a * Before planting. Note: Same alphabet within columns indicates no significant difference between means using T test at p = 0.05. TABLE 3 Concentrations of Three Different Forms of Soil K at Different Stages of Sampling for Three Different Depths 46 TheScientificWorldJOURNAL (2005) 5, 42–49 Ahmed et al.: Potassium Fertilizer Use Efficiency TABLE 4 Comparison Between Soil Total K Before and After the Study Treatment K (mg kg–1) Before study After study 0–10 cm KF 883a 273b NKF 883a 305b 10–25 cm KF 575a 167b NKF 675a 143b 25–45 cm KF 633a 143b NKF 550 a 240b Note: Same alphabet within rows indicates no significant difference between means before and after study using T test at p = 0.05. CONCLUSION TABLE 4 Comparison Between Soil Total K Before and After the Study TABLE 4 Comparison Between Soil Exchangeable K Before and After the Study Comparison Between Soil Exchangeable K Before and After the Study Treatment K (mg kg–1) Before study After study 0–10 cm KF 423a 290a NKF 540a 278b 10–25 cm KF 460a 330a NKF 417a 155b 25–45 cm KF 423a 383a NKF 383a 245b Note: Same alphabet within rows indicates no significant difference between means before and after study using T test at p = 0.05. 47 TheScientificWorldJOURNAL (2005) 5, 42–49 Ahmed et al.: Potassium Fertilizer Use Efficiency TABLE 6 Comparison Between Soil Solution K Before and After the Study Treatment K (mg kg–1) Before study After study 0–10 cm KF 173a 98b NKF 182a 93b 10–25 cm KF 89a 100a NKF 95a 33b 25–45 cm KF 114a 70a NKF 89 a 48a Note: Same alphabet within rows indicates no significant difference between means before and after study using T test at p = 0.05. 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Prentice Hall, Englewood Cliffs, NJ. 24. Mengel, K. and Kirby, E.A. (1987) Principles of Plant Nutrition. International Potash Institute, CH-Eorbla-fen-Bern. 25. D’Eeckenbrugge, G.C. and Leal, F. (2003) Morphology, anatomy and taxonomy. In The Pineapple: Botany Production and Uses. Bartholomew, D.P., Paull, R.E., and Rohrbach, KG., Eds. CAB International, New York. pp 13–32. This article should be referenced as follows: Ahmed, O.H., Husni, M.H.A., Hanafi, M.M., Anuar, A.R., Syed Omar, S.R. (2005) Applied K fertilizer use efficiency in pineapples grown on a tropical peat soil under residues removal. TheScientificWorldJOURNAL 5, 42–49. 19. Cottenie, A. (1980) Soil testing and plant testing as a basis of fertilizer recommendation. FAO Soils Bull. 38, 70–73. 20. Bailey, D., Nelson, P.V., Fonteno, W.C., Ji-We On Le, and Jin-Sheng (1996) Breakthrough plug research, ph, fertilization and nutrition. Floraculture Int. REFERENCES (Jan), 18–19. 19. Cottenie, A. (1980) Soil testing and plant testing as a basis of fertilizer recommendation. FAO Soils Bull. 38, 70–73. 20. Bailey, D., Nelson, P.V., Fonteno, W.C., Ji-We On Le, and Jin-Sheng (1996) Breakthrough plug research, ph, fertilization and nutrition. Floraculture Int. (Jan), 18–19. 21. SAS (2001) SAS/STAT Software. SAS Institute, Cary, NC. 15 ( ) , y, 22. Pomares-Gracia, F. and Pratt, P.F. (1987). Recovery of 15N-labelled fertilizer from manured and sludge-amended soils. Soil Sci. Soc. Am. J. 42, 717–720. 22. Pomares-Gracia, F. and Pratt, P.F. (1987). Recovery of 15N-labelled fertilizer from manured and sludge-amended soils. Soil Sci. Soc. Am. J. 42, 717–720. 23. Miller, R. and Donahue, R.L. (1995) Soils in our Environment. Prentice Hall, Englewood Cliffs, NJ. soils. Soil Sci. Soc. Am. J. 42, 717–720. 23. Miller, R. and Donahue, R.L. (1995) Soils in our Environment. Prentice Hall, Englewood Cliffs, NJ Miller, R. and Donahue, R.L. (1995) Soils in our Environment. 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https://openalex.org/W4394717258	https://www.researchsquare.com/article/rs-4198044/latest.pdf	English	null	Synthesis of Y zeolite via acid-catalyzed hydrolysis of tetraethylorthosilicate	Research Square (Research Square)	2,024	cc-by	4,137	Synthesis of Y zeolite via acid-catalyzed hydrolysis of tetraethylorthosilicate Bosen Zhou Jiangsu Ocean University Yinghao Zhao Jiangsu Ocean University Zhengbo Lai Jiangsu Ocean University Hualan Zhou Bosen Zhou Jiangsu Ocean University Yinghao Zhao Jiangsu Ocean University Zhengbo Lai Jiangsu Ocean University Hualan Zhou Jiangsu Ocean University Jing Zhu Jiangsu Ocean University ShengKang Wang Jiangsu Ocean University Yibo Zhao Jiangsu Ocean University Jiangsu Ocean University Jing Zhu Jiangsu Ocean University ShengKang Wang Jiangsu Ocean University Yibo Zhao Jiangsu Ocean University 1 Introduction Zeolite, a crystalline aluminum silicate with a well-defined micro-porous structure, plays a crucial role in energy development and chemical production and application.[1] Zeolite is mostly used in heterogeneous catalysis and adsorption separation, and the discovery of zeolite has brought breakthrough innovations to these two fields.[2] Among them, Y zeolite is one of the most important zeolites, which belongs to the FAU family and has 3D 12-member ring (MR) channels and spherical super-cages. Due to the large pore opening and strong acidity of Y zeolite, the discovery and application of Y zeolite has completely changed the fluid catalytic cracking (FCC) and hydro-cracking processes in refineries.[3] IIn recent years, FAU zeolite has also shown great potential as an efficient catalyst for the conversion of biomass into fuels and related chemicals.[4] High frame SAR has been proven to be the decisive factor for strong acidity and good thermal stability of Y zeolite, which are two of the most important properties for catalytic applications.[5] In the past decades of research, people have been trying to improve the SAR of Y zeolite. Post-synthesis dealumination is a common method for the preparation of high silicon Y zeolite, which usually uses steam/acid leaching method to remove Al atoms from the skeleton.[6] However, in the post-treatment process, the decrease in crystallinity and the loss of mass as well as the generation of dealumination gradient are always unavoidable. Therefore, the direct synthesis of high-silicon Y zeolite is undoubtedly the most attractive method compared with the post-treatment method.[7] Zeolite Y synthesis is done in different ways but often takes place under hydrothermal conditions [8-10]. In this method, different templates were applied for the synthesis of FAU zeolites with excellent performance including expensive templates such as crown ethers, silylated polymer, N-methylpyridinium iodide, modified carbon, and amphiphilic organosilane [11-14]. Most of these templates are commercially unavailable or costly, and many template agents not only can't be recycled but also cause harm to the environment. Thus, it is of great significance to use effective green template-free direct synthesis of Y zeolite. Wang et al.[15] Researched the introduction of hydroxyl radicals (•OH) in the synthesis system can significantly enhance the formation of Si–O–Si bond in the nucleation stage, so as to synthesize NaY zeolite with SAR of 6.35. Keywords: License:   This work is licensed under a Creative Commons Attribution 4.0 International License. R d F ll Li License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: No competing interests reported. Additional Declarations: No competing interests reported. Page 1/13 Abstract The siliceous precursor was hydrolyzed from tetraethylsilicic acid (TEOS) under acidic conditions, then sodium aluminate and sodium hydroxide were added, and Y zeolite was obtained by hydrothermal crystallization under alkaline conditions. The key synthesis parameters, such as reactant molar ratios, crystallization temperature and time were changed to optimize the synthesis conditions. The obtained products were characterized by XRD, SEM, N2 adsorption and ICP elemental analysis. The well- crystallized Y zeolite was successfully synthesized by hydrolysis of TEOS catalyzed by sulfuric acid at low crystallization temperature 85℃ which has a SiO2/Al2O3 ratio（SAR）of 5.55. 2.1 Zeolite Synthesis The following is a typical synthesis procedure. A total of 10.0 g TEOS was mixed with 32.5 g deionized water in a beaker. Under vigorous stirring, sulphuric acid was slowly dropped into the TEOS solution to obtain a pH value of 1.0. The obtained mixture A was stirred at 20 °C for 20 h to get a complete hydrolysis of TEOS. Solution B was prepared by adding successively 10.0 g of sodium hydroxide and 10.0 g sodium aluminate into 12 g of deionized water under stirring.Then, solution B was added into solution A to make the gel mixture. Aging was conducted with stirring at room temperature for 12 h. The molar composition of the synthesis gel was 1 Na2O: 1 Al2O3: 10 SiO2: 160 H2O. Finally, the slurry was transferred into a Teflon-lined stainless steel autoclave and left to crystallize statically at 85 °C. Products from the synthesis were separated by centrifugation, washed with deionized water and air-dried at 100 °C . The obtained as-synthesized samples were calcined at 550 °C for 5 h in air stream to give the as- calcined Y. 2.1 Materials Zeolite Y was synthesized using sodium aluminate (14% Al2O3, 25% Na2O and 61% H2O, Macklin), sodium hydroxide (NaOH, 98%, Merck), sulfuric acid (99.3%, purity) and tetraethyl orthosilicate (98%, Aladdin). 1 Introduction Ren et al.[16] Co2+ was introduced into the synthesis system, and the Y zeolite with a small crystal size and a SAR of 6.15 was obtained.Meng et al.[17] proposed a kind of hydrothermal synthesis of Y zeolite using coal fly ash as raw material without organic guiding agent. Although the use Page 2/13 of industrial waste coal fly ash can reduce the harm to the environment, the catalytic performance of the synthesized zeolite Y will become worse and the high temperature calcination is required to handle the coal fly ash, which increases the cost of synthesis. of industrial waste coal fly ash can reduce the harm to the environment, the catalytic performance of the synthesized zeolite Y will become worse and the high temperature calcination is required to handle the coal fly ash, which increases the cost of synthesis. The dissolution of silica precursors is one of the important factors in zeolite synthesis, through which silicate intermediates are formed and then nucleation and crystallization. Various silica sources have been used for the synthesis of Y including water glass, fumed silica, TEOS, colloidal silica, and sodium metasilicate. It is well known that silicon sources TEOS can be hydrolyzed not only in alkaline media but also in acidic media. At present, MCM-22, ZSM-5 and Silicalite-1 can be successfully synthesized by acid- catalyzed hydrolysis of TEOS. In this work, high crystallinity Y zeolite was synthesized at 85℃ by acid-catalyzed hydrolysis of TEOS. Effects of gel compositions and crystallization conditions are investigated in detail. Compared to the usually used hydrothermal route, synthesis by acid-catalyzed hydrolysis of TEOS shows the benefits of a lower crystallization temperature and high SiO2/Al2O3 ratio in solid phase. 3.1 Effect of Crystallization Temperature and Crystallization Time The effect of hydrothermal temperature on the crystallization of zeolite Y was evaluated by synthesis from 70 to 95 °C without using any template. The mole oxide compositions of the gels were 7 Na2O: 1 Al2O3: 10 SiO2: 300 H2O. The prepared gels were aged at temperatures of 30 °C for 24 h and then crystallized for 24 h. Figure 1 shows the crystallization rate curves of the products synthesized at different temperatures.It can be seen from the crystallization curve that different temperatures have a great influence on the nucleation stage of crystals.With the increase of crystallization temperature, the induction period of nucleation is shortened.However, it can be seen from the slope of the crystallization curve that the slopes of several curves are basically the same, indicating that different crystallization temperatures have little effect on the growth rate of crystals. With the increase of crystallization temperature, the crystal growth time is shortened. The zeolite Y in the figure is a sample that crystallizes for 32 hours at a crystallization temperature of 85° C. Noticeably, at a low temperature of 70 °C, highly crystallized Y zeolite could be obtained with a longer crystallization time 80 hours. Early efforts under hydrothermal conditions revealed that Y zeolite was created mostly at 100 °C, and impurities like P zeolite were observed at a lower temperature 80°C[18]. The present result convinced that the synthesis method by acid-catalyzed hydrolysis of tetraethylorthosilicate allows the generation of pure Y zeolite at a very low crystallization temperature 70 °C, though needing a longer crystallization time. The SEM images of Y products at different crystallization stages are shown in Figure 2. It can be seen that the particle size of Y zeolite synthesized at different crystallization temperatures is also different.At the same crystallization temperature, the particle size of the product increases with the extension of crystallization time.When the temperature increases from 80℃ to 85℃ , the product size increases from 1.6 μm to 2.4 μm with crystallization time 32 hours while from 3.6 μm to 4 μm with crystallization time 48 hours. This is because the particle size of zeolite depends on the nucleation rate and growth rate. At low temperature, it is beneficial to promote the formation of crystal nuclei and control the growth rate of grains. 2.3 Zeolite Characterization X-ray diffraction (XRD) patterns of the prepared zeolites were determined by Philips PW1730 diffractometer using Cu Kα radiation (40 kV, 40 mA). In this method, the prepared sample with the highest Page 3/13 peak intensities was assumed to have 100% crystallinity, while the crystallinities of the other prepared samples were normalized by considering the ratios of their peak intensities corrected by the width at half height to those of the sample with the highest crystallinity. peak intensities was assumed to have 100% crystallinity, while the crystallinities of the other prepared samples were normalized by considering the ratios of their peak intensities corrected by the width at half height to those of the sample with the highest crystallinity. The surface area and pore volume of the prepared zeolites were determined by the nitrogen physisorption using a Micromeritics ASAP 2010 automated system. 3.1 Effect of Crystallization Temperature and Crystallization Time With the increase of temperature, the growth process of crystal nucleus is obviously faster, and the nucleation process is inhibited, and the particle size of the product is increased [19-20]. 3.2 Effect of Initial SiO2/Al2O3 ratio Amount 3.2 Effect of Initial SiO2/Al2O3 ratio Amount 3.2 Effect of Initial SiO2/Al2O3 ratio Amount 3.2 Effect of Initial SiO2/Al2O3 ratio Amount Page 4/13 Page 4/13 The effect of SiO2/Al2O3 ratio on the crystallization of Y zeolite was tested, when the mole oxide com- positions of the gels were 7 Na2O: 1 Al2O3: X SiO2: 300 H2O, in which X varied from 6.0 to 17.0. Figure 3 shows the XRD pattern and crystallinity of zeolite prepared with different SiO2/Al2O3 ratios of initial gels. As shown in Figure 3, pure Y zeolites were obtained at the SiO2/Al2O3 ratios from 6 to 15, in which the sample with SiO2/Al2O3 12 exhibited the highest crystallinity. Table 1 shows the physical and chemical properties of Y zeolite. According to the ICP analysis, SiO2/Al2O3 ratios for final Y solids obtained with initial gel SiO2/Al2O3 ratios of 6, 9, 12 and 15 were 2.47, 3.89, 5.55 and 4.93, respectively. The BET surface areas were 494, 641, 743 and 567 m2/g, respectively. The lower surface areas at SiO2/ Al2O3 ratio of 6 correspond to its lower crystallinity as shown in Fig.3.When the SiO2/Al2O3 ratio was as low as 6 and as high as 15, the XRD Patterns in Fig. 3 both showed much lowered crystallinities of Y phase. On the other hand, the much higher SiO2/Al2O3 ratio of 12 cause a comparatively high crystallinity of Y zeolite.When the SiO2/Al2O3 ratio was up to 17, the XRD showed that the synthesized zeolite Y is an amorphous product. This is because the excessive addition of Si in the feed ratio leads to the decrease of the relative content of sodium in the mother liquor, which reduces the participation of silicon and aluminum in the structure of zeolite and eventually generates an amorphous product. 1 Textural parameters and of the samples obtained at different Si/Al ratio. Samples SiO2/Al2O3 Radio SBET, m2/gc Vmicro, cm3/gd VTot, Cm3/ge NaY-1 6a(2.47)b 494 0.18 0.22 NaY-2 9(3.89) 641 0.24 0.27 NaY-3 12 (5.55) 743 0.27 0.31 NaY-4 15 (4.93) 567 0.21 0.24 Table 1 Textural parameters and of the samples obtained at different Si/Al ratio. a SiO2/Al2O3 ratio in the initial gel. b SiO2/Al2O3 ratio calculated from ICP-AES analysis. c BET surface area. d Microporous surface area evaluated by the t- plot method. e Total pore volume calculated by the single- point method at P/P0=0 95 e Total pore volume calculated by the single- point method at P/P0=0.95. point method at P/P0=0.95. 3.3 Effect of Initial Na2O Amount It is well known that the alkalinity of the initial gel is an important parameter affecting the crystallization of zeolite.Thus, the mole oxide compositions of the gels were X Na2O: 1 Al2O3: 10 SiO2: 300 H2O, in which X varied from 3.0 to 10.0 to evaluate the influence of gel’s alkalinities. Fig. 5 shows the XRD patterns of the resultant as-calcined samples. When the Na2O content was 3 ~ 8.5, pure Y products can be obtained. In this range, crystallinities increased with the increase of Na2O content due to rapid dissolution of silicon in high alkalinity media. At low alkalinity ( Na2O content was 3), a poorly crystallized Y phase was Page 5/13 observed because the low concentration of hydroxyl ions could not depolymerize the silica source to provide sufficient solubilized aluminosilicate species for nucleation[21-24]. In contrast, high alkalinity (Na2O content was 8.5) reduces crystallinity, which may be due to the presence of excessive hydroxyl ions leading to dissolution of the already formed zeolite nuclei. A higher Na2O content up to 10 caused occurrence of P zeolite crystals. Accordingly, the Na2O content range from 4.5 to 8.5 is optimal for producing the pure phase of Y zeolite. 3.4 Effect of Initial H2O Amount The water content in the synthetic system will directly affect the concentration of each substance in the gel, thus affecting the crystallization process of zeolite. Fig. 6 shows the XRD patterns for the samples synthesized with various H2O content with the mole oxide compositions of the gels were 7 Na2O: 1 Al2O3: 10 SiO2: X H2O, in which X varied from 200 to 640. At the very low H2O content of 200, the very The water content in the synthetic system will directly affect the concentration of each substance in the gel, thus affecting the crystallization process of zeolite. Fig. 6 shows the XRD patterns for the samples synthesized with various H2O content with the mole oxide compositions of the gels were 7 Na2O: 1 Al2O3: 10 SiO2: X H2O, in which X varied from 200 to 640. At the very low H2O content of 200, the very concentrated gel created only p zeolite crystals. Because the low water content in the synthetic gel was, the induction period can be shortened, the nucleation rate and crystal growth rate can be accelerated, which was conducive to rapid crystallization, but too little water content in the synthetic system cannot form a uniform gel, resulting in difficult to form Y zeolite products and easy to appear impurity crystals. Also, the much diluted gel mixture (H2O content 600) created only p zeolite crystals, which is ascribed to the very slow nucleation rate arising for the longer distance between nutrients in this diluted solution. When H2O contents were changed from 300 to 480, pure and highly crystallized Y solids were produced. However, the slightly diluted gel mixture (H2O content 480) resulted in a lower crystallinity of Y which due to the high-water content generated the low concentration of reactants in the synthetic system leading to slowing crystallization rate. Consequently, the moderate range of H2O content from 300 to 480 is suitable for synthesizing zeolite Y[25]. Conclusion The aim of this study is to synthesize Y zeolite by hydrolyzing regular acid tetraethyl orthosilicate (TEOS) as silicon source, and the effects of crystallization temperature, crystallization time and Na2O content of seeding on the crystal size and SiO2/Al2O3 ratio of NaY zeolite was investigated. It is found that higher the crystallization temperature and the longer the crystallization time, the more ideal the crystal growth than the nucleation, the larger the Y zeolite crystal produced. 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"Effects of synthesis parameters on organic template-free preparation of zeolite Y." Journal of Inorganic and Organometallic Polymers and Materials 31.6 (2021): 2501-2510. Figures Page 8/13 Page 8/13 Figure 1 Calculated crystallinity of the prepared samples crystalized at various temperatures and times. Calculated crystallinity of the prepared samples crystalized at various temperatures and times. Page 9/13 SEM images of Y zeolite with seeding prepared at different temperatures and times (a)80 ℃ 32h, (b) 80 ℃ 48h, (c) 85 ℃ 32h, (d) 85 ℃ 48h. Page 9/13 Figure 2 SEM images of Y zeolite with seeding prepared at different temperatures and times (a)80 ℃ 32h, (b) 80 ℃ 48h, (c) 85 ℃ 32h, (d) 85 ℃ 48h. Figure 2 SEM images of Y zeolite with seeding prepared at different temperatures and times (a)80 ℃ 32h, (b) 80 ℃ 48h, (c) 85 ℃ 32h, (d) 85 ℃ 48h. Page 10/13 Figure 3 The effect of SiO2/Al2O3 ratio in the initial gel on the XRD patterns of the prepared samples. Figure 3 Figure 3 The effect of SiO2/Al2O3 ratio in the initial gel on the XRD patterns of the prepared samples. Page 11/13 Figure 4 The effect of Na2O ratio in the initial gel on the XRD patterns of the prepared samples. Figure 4 The effect of Na2O ratio in the initial gel on the XRD patterns of the prepared samples. Page 12/13 Page 12/13 Figure 5 The effect of H2O/Al2O3 ratio in the initial gel on the XRD patterns of the prepared samples. Figure 5 The effect of H2O/Al2O3 ratio in the initial gel on the XRD patterns of the prepared samples. Page 13/13
https://openalex.org/W3088691777	https://revistas.uptc.edu.co/index.php/ingenieria/article/download/11762/9627	English	null	Adaptation, Comparison, and Improvement of Metaheuristic Algorithms to the Part-of-Speech Tagging Problem	Revista Facultad de Ingeniería/Revista Facultad de Ingeniería	2,020	cc-by	5,916	Adaptation, Comparison, and Improvement of Metaheuristic Algorithms to the Part-of-Speech Tagging Problem Miguel-Alexis Solano-Jiménez; José-Julio Tobar-Cifuentes; Luz-Marina Sierra-Martínez; Carlos-Alberto Cobos-Lozada Conflict of interest: The authors state there is no conflict of interest. Re ista Fac ltad de Ingeniería (Re Fac Ing ) Vol 29 (54) e11762 2020 T nja Bo acá Colombia 1 Universidad del Cauca (Popayán-Cauca, Colombia). miguelsolano@unicauca.edu.co. ORCID: 0000-0003- 1936-3488 2 Universidad del Cauca (Popayán-Cauca, Colombia). josej@unicauca.edu.co. ORCID: 0000-0002-5436-0816 3 Ph. D. Universidad del Cauca (Popayán-Cauca, Colombia). lsierra@unicauca.edu.co. ORCID: 0000-0003- 3847-3324 4 Ph. D. Universidad del Cauca (Popayán-Cauca, Colombia). ccobos@unicauca.edu.co. ORCID: 0000-0002- 6263-1911 Miguel-Alexis Solano-Jiménez; José-Julio Tobar-Cifuentes; Luz-Marina Sierra-Martínez; Carlos-Alberto Cobos- L d Miguel-Alexis Solano-Jiménez; José-Julio Tobar-Cifuentes; Luz-Marina Sierra-Martínez; Carlos-Alberto Cobos- Lozada Tagging Problem Miguel-Alexis Solano-Jiménez1 José-Julio Tobar-Cifuentes2 Luz-Marina Sierra-Martínez3 Carlos-Alberto Cobos-Lozada4 Abstract Part-of-Speech Tagging (POST) is a complex task in the preprocessing of Natural Language Processing applications. Tagging has been tackled from statistical information and rule-based approaches, making use of a range of methods. Most recently, metaheuristic algorithms have gained attention while being used in a wide variety of knowledge areas, with good results. As a result, they were deployed in this research in a POST problem to assign the best sequence of tags (roles) for the words of a sentence based on information statistics. This process was carried out in two cycles, each of them comprised four phases, allowing the adaptation to the tagging problem in metaheuristic algorithms such as Particle Swarm Optimization, Jaya, Random-Restart Hill Climbing, and a memetic algorithm based on Global-Best Harmony Search as a global optimizer, and on Hill Climbing as a local optimizer. In the consolidation of each algorithm, preliminary experiments were carried out (using cross-validation) to adjust the parameters of each algorithm and, thus, evaluate them Revista Facultad de Ingeniería (Rev. Fac. Ing.) Vol. 29 (54), e11762. 2020. Tunja-Boyacá, Colomb L-ISSN: 0121-1129, e-ISSN: 2357-5328, DOI: https://doi.org/10.19053/01211129.v29.n54.2020.117 Adaptation, Comparison, and Improvement of Metaheuristic Algorithms to the Part-of-Speech Tagging Problem on the datasets of the complete tagged corpus: IULA (Spanish), Brown (English) and Nasa Yuwe (Nasa). The results obtained by the proposed taggers were compared, and the Friedman and Wilcoxon statistical tests were applied, confirming that the proposed memetic, GBHS Tagger, obtained better results in precision. The proposed taggers make an important contribution to POST for traditional languages (English and Spanish), non-traditional languages (Nasa Yuwe), and their application areas. Keywords: computational intelligence; computational linguistics; evolutionary computing; heuristic algorithms; natural language processing; parts of speech tagging; search methods. g ( g ) ( ) j y L-ISSN: 0121-1129, e-ISSN: 2357-5328, DOI: https://doi.org/10.19053/01211129.v29.n54.2020.11762 evista Facultad de Ingeniería (Rev. Fac. Ing.) Vol. 29 (54), e11762. 2020. Tunja-Boyacá, Colombia. Resumen La identificación de partes del discurso (Part-of-Speech Tagging, POST) es una tarea compleja en las aplicaciones de procesamiento de lenguaje natural. Ha sido abordada desde enfoques basados en información estadística y reglas, haciendo uso de distintos métodos y, últimamente, se destacan los algoritmos metaheurísticos obteniendo buenos resultados. Por ello, se involucran en esta investigación para asignar la mejor secuencia de etiquetas (roles) para las palabras de una oración, basándose en información estadística. Este proceso se desarrolló en 2 ciclos, donde cada ciclo tuvo 4 fases para la adaptación al problema de etiquetado en los algoritmos metaheurísticos Particle Swarm Optimization, Jaya, Random-Restart Hill Climbing, y un algoritmo memético basado en Global-Best Harmony Search como optimizador global, y en Hill Climbing como optimizador local. Se realizaron experimentos preliminares (utilizando validación cruzada), para ajustar los parámetros de cada algoritmo y luego ejecutarlos sobre los datasets completos de los corpus etiquetados IULA (castellano), Brown (inglés) y Nasa Yuwe (Nasa). Los resultados obtenidos por los etiquetadores propuestos se compararon mediante las pruebas estadísticas no paramétricas de Friedman y Wilcoxon, ratificando que el memético propuesto, GBHS Tagger, obtiene mejores resultados Miguel-Alexis Solano-Jiménez; José-Julio Tobar-Cifuentes; Luz-Marina Sierra-Martínez; Carlos-Alberto Cobos- Lozada de precisión. Los etiquetadores propuestos se convierten en un aporte muy importante para el POST, tanto para lenguas tradicionales (Inglés y Castellano), no tradicionales (Nasa Yuwe), y sus áreas de aplicación. Palabras clave: algoritmos heurísticos; computación evolutiva; etiquetado de partes del discurso; inteligencia computacional; lingüística computacional; métodos de búsqueda; procesamiento de lenguaje natural. Revista Facultad de Ingeniería (Rev. Fac. Ing.) Vol. 29 (54), e11762. 2020. Tunja-Boyacá, Colombia. L-ISSN: 0121-1129, e-ISSN: 2357-5328, DOI: https://doi.org/10.19053/01211129.v29.n54.2020.11762 Resumo A identificação de partes do discurso (Part-of-Speech Tagging, POST) é uma tarefa complexa nas aplicações de processamento de linguagem natural. Tem sido abordada desde enfoques baseados em informação estatística e regras, fazendo uso de distintos métodos e, ultimamente, destacam-se os algoritmos metaheurísticos obtendo bons resultados. Por isso, envolvem-se nesta pesquisa para assignar a melhor sequência de etiquetas (papéis) para as palavras de uma oração, baseando-se em informação estatística. Este processo desenvolveu-se em 2 ciclos, onde cada ciclo teve 4 fases para a adaptação ao problema de etiquetado nos algoritmos metaheurísticos Particle Swarm Optimization, Jaya, Random-Restart Hill Climbing, e um algoritmo mimético baseado em Global-Best Harmony Search como otimizador global, e em Hill Climbing como otimizador local. Realizaram-se experimentos preliminares (utilizando validação cruzada), para ajustar os parâmetros de cada algoritmo e depois executá-los sobre os datasets completos dos corpus etiquetados IULA (castelhano), Brown (inglês) e Nasa Yuwe (Nasa). Os resultados obtidos pelos etiquetadores propostos compararam-se mediante as provas estatísticas não paramétricas de Friedman e Wilcoxon, ratificando que o mimético proposto, GBHS Tagger, obtém melhores resultados de precisão. Os etiquetadores propostos convertem-se em um aporte muito importante para o POST, tanto para línguas tradicionais (Inglês e Castelhano), não tradicionais (Nasa Yuwe), e suas áreas de aplicação. Revista Facultad de Ingeniería (Rev. Fac. Ing.) Vol. 29 (54), e11762. 2020. Tunja-Boyacá, Colombia. L-ISSN: 0121-1129, e-ISSN: 2357-5328, DOI: https://doi.org/10.19053/01211129.v29.n54.2020.11762 Adaptation, Comparison, and Improvement of Metaheuristic Algorithms to the Part-of-Speech Tagging Problem Palavras chave: algoritmos heurísticos; computação evolutiva; etiquetado de partes do discurso; inteligência computacional; linguística computacional; métodos de busca; processamento de linguagem natural. Revista Facultad de Ingeniería (Rev. Fac. Ing.) Vol. 29 (54), e11762. 2020. Tunja-Boyacá, Colombia. L-ISSN: 0121-1129, e-ISSN: 2357-5328, DOI: https://doi.org/10.19053/01211129.v29.n54.2020.11762 Revista Facultad de Ingeniería (Rev. Fac. Ing.) Vol. 29 (54), e11762. 2020. Tunja-Boyacá, Colomb L-ISSN: 0121-1129, e-ISSN: 2357-5328, DOI: https://doi.org/10.19053/01211129.v29.n54.2020.117 Miguel-Alexis Solano-Jiménez; José-Julio Tobar-Cifuentes; Luz-Marina Sierra-Martínez; Carlos-Alberto Cobos- Lozada g ( g ) ( ) j y L-ISSN: 0121-1129, e-ISSN: 2357-5328, DOI: https://doi.org/10.19053/01211129.v29.n54.2020.117 Revista Facultad de Ingeniería (Rev. Fac. Ing.) Vol. 29 (54), e11762. 2020. Tunja-Boyacá, Colombia. L-ISSN: 0121-1129, e-ISSN: 2357-5328, DOI: https://doi.org/10.19053/01211129.v29.n54.2020.11762 evista Facultad de Ingeniería (Rev. Fac. Ing.) Vol. 29 (54), e11762. 2020. Tunja-Boyacá, Colombia. I. INTRODUCTION Metaheuristic algorithms are being applied every day in a variety of areas of knowledge. It is not unusual, therefore, to use them in the problem of Part-of-speech Tagging (POST) or Identification. This is a complex task of great importance in Natural Language, given the challenges it faces, such as: the ambiguity of words, the size of the tag set, and the tagging of unknown words [1, 2]. Metaheuristic algorithms in the tagging problem (POST) have been used to assign the best sequence of tags (roles) for the words of a sentence, based on both statistical information and rules of transformation to solve this problem, obtaining outstanding results in contrast to traditional approaches. Related work includes: 1) Alhasan and Al-taani [3], who represented the tagging problem as a graph, the nodes are the possible tags of a sentence and use the optimization algorithm by Bee Colony Optimization (BCO), which finds the best solution path. 2) Sierra Martínez, Cobos and Corrales [4] proposed a memetic algorithm for tagging based on Global- Best Harmony Search (GBHS) that includes knowledge of the problem through a local optimization strategy based on the Hill Climbing algorithm. 3) Forsati & Shamsfard [5] presented two improvements to the HSTAgger tagger based on the Harmony Search metaheuristic, called HSTAgger (I) and HSTAgger (II), which increase search efficiency and improve the selection of new solutions for harmony memory. 4) Ekbal and Saha [6] addressed the tagging problem using single- objective and multiobjective optimization based on the Simulated Annealing-Based Multiobjective Optimization Algorithm proposed in [7], exploiting the search capacity of the simulated annealing algorithm. Said metaheuristic approaches have been applied to corpus tagged in English, the Brown Corpus [8], the Penn Treebank Corpus [9], and other non-traditional languages such as Arabic with the KALIMAT corpus [10], Bengali (Bangladesh) [11], Hindi (India) [12], Telugu (India) [13], and Nasa Yuwe (an indigenous language of Colombia) [14]. Generally, these proposals use the Petrov tag set [15]. Metaheuristic algorithms solve problems using a search process (exploration and exploitation) of optimal solutions for a particular problem [16]. Thus, memetic algorithms [17] use population-based search to explore solutions, and local search Adaptation, Comparison, and Improvement of Metaheuristic Algorithms to the Part-of-Speech Tagging Problem based on neighborhood for the exploitation of promising solutions [18, 19]. They also add knowledge of the problem to solve it. I. INTRODUCTION Table 1 describes the metaheuristics studied in this research for their subsequent adaptation to the tagging problem. Table 1. Metaheuristic algorithms studied to adapt to the tagging problem. Metaheuristic algorithm Description Random- Restart Hill Climbing (RRHC) [20] Simple state metaheuristic that improves Hill Climbing (HC) [17]. It seeks to prevent HC from being trapped in local optimum by performing repetitive explorations in the problem space, which are generated randomly until the stop criterion occurs or a better solution is not found. Particle Swarm Optimization (PSO) [21] Population metaheuristic motivated by the intelligent collective behavior of swarms in nature. Each potential solution is called a particle, the set of particles is known as a swarm, and the position of each particle changes depending on its own experience and the experience of the swarm [22]. Jaya [23] Population metaheuristic that seeks to find the best solution in the shortest possible time, but is also always trying to get away from failure, generating an optimal balance between exploration and exploitation. Jaya is a novel, simple and efficient algorithm for optimization problem solving with and without restrictions. GBHS Tagger [4] Memetic algorithm adapted to the tagging problem [4], based on the Global-Best Harmony Search (GBHS) metaheuristic, which has the following parameters [17]: HMS (Harmonic Memory Size), NI (number of improvisations ), HCMR (Harmonic Memory Consideration Rate), and PARMin, PARMax (Tone Adjustment Rate). GBHS Tagger includes knowledge of the tagging problem using a local optimizer, adapted from the Hill Climb (HC) metaheuristic [17], which is applied to the best harmony in harmonic memory (HM). In addition to the GBHS parameters, three more parameters are defined: ProbOpt, which controls the percentage of times the local optimization process is carried out; MaxNeighbors, which defines the number of neighbors used in the local optimization process, and the parameter Alpha, which controls whether the components of each harmony in the population are randomly generated from their possible tags or taken from the tag with higher probability. Table 1. Metaheuristic algorithms studied to adapt to the tagging problem. Revista Facultad de Ingeniería (Rev. Fac. Ing.) Vol. 29 (54), e11762. 2020. Tunja-Boyacá, Colombia. L-ISSN: 0121-1129, e-ISSN: 2357-5328, DOI: https://doi.org/10.19053/01211129.v29.n54.2020.11762 I. INTRODUCTION In the present work, the adaptation of several metaheuristic algorithms to the tagging problem was carried out, using the representation of the solution proposed in [4], in order to propose improvements to the memetic presented in the same work, at the same time that it was sought to evaluate its performance on the corpus in Castilian IULA [24], English Brown [8] and Nasa Yuwe [14]. The rest of the article is organized as follows: Section 2 presents the methodology used; Section 3 details the adaptation of the selected metaheuristics to the tagging problem; Section 4 shows the results of the experiments carried out, and, finally, Section 5 presents the discussion, conclusions and future work. II. METHODOLOGY This section describes the dataset used for the evaluation of the algorithms, the activities carried out in each phase of the cycles of the Iterative Research Pattern (IRP) methodology [25], used for carrying out this work, and how the experiments were set up. I. INTRODUCTION Figure 1 shows the representation of the solution used for this investigation, which consists of: 1) a first vector of the size of the number of words in a sentence (one position per word), which contains the tags assigned to each word, from position 0 to position n-1 (T0, T1, … , Tn−1); 2) a second vector containing the cumulative probability of each tagged word, and its relationship with its predecessor and successor, and 3) a field that stores the value of the fitness function, calculated as shown in Figure 1, adapted from [5]. In GBHS Tagger, the selected context for the word to be tagged is a trigram (predecessor, word to tag, successor). Figure 1 shows the representation of the solution used for this investigation, which consists of: 1) a first vector of the size of the number of words in a sentence (one position per word), which contains the tags assigned to each word, from position 0 to position n-1 (T0, T1, … , Tn−1); 2) a second vector containing the cumulative probability of each tagged word, and its relationship with its predecessor and successor, and 3) a field that stores the value of the fitness function, calculated as shown in Figure 1, adapted from [5]. In GBHS Tagger, the selected context for the word to be tagged is a trigram (predecessor, word to tag, successor). Miguel-Alexis Solano-Jiménez; José-Julio Tobar-Cifuentes; Luz-Marina Sierra-Martínez; Carlos-Alberto Cobos- Lozada Miguel-Alexis Solano-Jiménez; José-Julio Tobar-Cifuentes; Luz-Marina Sierra-Martínez; Carlos-Alberto Cobos- Lozada Fig. 1. Representation of the solution [4]. T0 T1 T2 T3 … Ti … Tn-1 0 1 2 3 n-1 P0 P1 P2 P3 … Pi … Pn-1 −1, 1 Probabilities for each tag Tags assigned to each word i Fig. 1. Representation of the solution [4]. In the present work, the adaptation of several metaheuristic algorithms to the tagging problem was carried out, using the representation of the solution proposed in [4], in order to propose improvements to the memetic presented in the same work, at the same time that it was sought to evaluate its performance on the corpus in Castilian IULA [24], English Brown [8] and Nasa Yuwe [14]. Revista Facultad de Ingeniería (Rev. Fac. Ing.) Vol. 29 (54), e11762. 2020. Tunja-Boyacá, Colomb L-ISSN: 0121-1129, e-ISSN: 2357-5328, DOI: https://doi.org/10.19053/01211129.v29.n54.2020.117 A. Used Method Two cycles were used for this research. The first cycle focused on the adaptation of the metaheuristic algorithms to the tagging problem and the selection of the best one; the second cycle focused on the adaptation of the selected metaheuristic , Comparison, and Improvement of Metaheuristic Algorithms to the Part-of-Speech Tagging Problem algorithms to the tagging problem and the proposal of a new version of the memetic algorithm. Table 2 describes the activities carried out in each phase. Table 2. Description of the methodology. Observation Identification Development Testing Cycle 1 Review of metaheuristics and investigation of corpus and set of tags. Selection of corpus Design and implementation of the database Execution of experiments with the complete dataset. Mapping of tags to universal tagging. Adaptation and implementation of the metaheuristic algorithms to the POST. Execution of non- parametric statistical tests. Study of selected metaheuristic algorithms. Configuration and execution of experiments on small datasets. Analysis and discussion of results Cycle 2 Research into memetic algorithms and discrete implementations. Adaptation and implementation of the PSO, Jaya, Random-Restart Hill Climbing (RRHC), and HC algorithms to the tagging problem. Implementation of the final versions of the proposed taggers. Execution of full experiments and tests. Study and understanding of the memetic GBHS Tagger. Integration of HC Tagger to the memetic algorithm. Analysis of results and issuance of conclusions. Experiment setup on small datasets and parameter tuning. Execution of the experiments and development of the new version of the memetic. Table 2. Description of the methodology. B. Dataset and Experimental Setup As part of this work, the IULA (Spanish), Brown (English) and Nasa Yuwe tagged corpus were integrated into a single database designed and developed in SQL Server. The experiments were carried out on this database and, for their execution (both preliminary and complete), a client-server model was used, in which the clients (machines) request the tasks to be carried out. Each task receives the phrase and the algorithm that it must run and evaluate. Likewise, each task is executed 30 times (repetitions of the experiment) on the local machine. Once the task is finished, the results are recorded in the cloud database. Revista Facultad de Ingeniería (Rev. Fac. Ing.) Vol. 29 (54), e11762. 2020. Tunja-Boyacá, Colombia. L-ISSN: 0121-1129, e-ISSN: 2357-5328, DOI: https://doi.org/10.19053/01211129.v29.n54.2020.11762 Revista Facultad de Ingeniería (Rev. Fac. Ing.) Vol. 29 (54), e11762. 2020. Tunja-Boyacá, Colombia. L-ISSN: 0121-1129, e-ISSN: 2357-5328, DOI: https://doi.org/10.19053/01211129.v29.n54.2020.11762 III. RESULTS In the first instance of this section, the adaptation of the algorithms to the tagging problem and a new version of the memetic GBHS Tagger (GBHS4Tagger) are presented. In the second instance, the experiments and the results obtained with the Miguel-Alexis Solano-Jiménez; José-Julio Tobar-Cifuentes; Luz-Marina Sierra-Martínez; Carlos-Alberto Cobos- Lozada Miguel-Alexis Solano-Jiménez; José-Julio Tobar-Cifuentes; Luz-Marina Sierra-Martínez; Carlos-A Lozada proposed taggers are shown. It is highlighted that all the adapted algorithms use the representation of the solution presented in [4], described in Figure 1. A. Proposed JayaTagger A discrete version of Jaya, called DJaya and proposed by [27], was used, it is free of parameters. The adaptation consisted in moving towards the best-known solution and moving away from the worst solution. Handling of the worst solution parameter was varied. The JayaTagger algorithm only handles three parameters: 𝑜𝑝𝑢𝑙𝑎 𝑖𝑜𝑛𝑆𝑖𝑧𝑒, MaxGenerations and 4. The latter controls the new solution from selecting a tag of the worst solution 𝑋𝑊, making the algorithm simple to implement and evaluate. In Figure 2, the proposed JayaTagger pseudocode is presented. Fig. 2. JayaTagger pseudocode Fig. 2. JayaTagger pseudocode Fig. 2. JayaTagger pseudocode Revista Facultad de Ingeniería (Rev. Fac. Ing.) Vol. 29 (54), e11762. 2020. Tunja-Boyacá, Colombia. L-ISSN: 0121-1129, e-ISSN: 2357-5328, DOI: https://doi.org/10.19053/01211129.v29.n54.2020.11762 B. Proposed PSOTagger The adaptation proposed is done according to the following parameters (a discrete version of PSO [26] was used): 𝑊, that selects a random tag for each dimension of a particle; 𝐶1, that selects the tag of the best particle history for that word; 𝐶2, that Adaptation, Comparison, and Improvement of Metaheuristic Algorithms to the Part-of-Speech Tagging Problem selects the tag of the best global of the swarm for each dimension of the particle, and , that maintains the components of the current particle. Additionally, PSOTagger involves the parameters 𝑜𝑝𝑢𝑙𝑎 𝑖𝑜𝑛𝑆𝑖𝑧𝑒 and 𝑀𝑎𝑥𝐺𝑒𝑛𝑒𝑟𝑎 𝑖𝑜𝑛𝑠 from its original version. The tuning of the 𝑊, 𝐶1, 𝐶2, and parameters in PSO was carried out experimentally with cross validation of 5 folders and a small dataset as a sample of the evaluation dataset. The PSOTagger pseudocode is presented in Figure 3. Fig. 3. PSOTagger pseudocode. Fig. 3. PSOTagger pseudocode. Fig. 3. PSOTagger pseudocode. Fig. 3. PSOTagger pseudocode. Revista Facultad de Ingeniería (Rev. Fac. Ing.) Vol. 29 (54), e11762. 2020. Tunja-Boyacá, Colombia. L-ISSN: 0121-1129, e-ISSN: 2357-5328, DOI: https://doi.org/10.19053/01211129.v29.n54.2020.11762 C. Proposed Random-Restart Hill Climbing (RRHC) Tagger The adaptation of the RRHCTagger algorithm to the tagging problem was carried out as follows. 1) The parameters: 𝑛_𝑟𝑒𝑠 𝑎𝑟 controls the number of restarts of solution 𝑆; 𝑟𝑜𝑏, list that stores the probabilities of the possible tags of a word; 𝐴𝑐 𝑖𝑣𝑒𝐼𝑛𝑑𝑒𝑥, a list that stores the positions of words that have more than one tag, Miguel-Alexis Solano-Jiménez; José-Julio Tobar-Cifuentes; Luz-Marina Sierra-Martínez; Carlos-Alberto Cobos- Lozada and 𝑆 𝑎 𝑢𝑠𝑇𝑟𝑖𝑔𝑟𝑎𝑚, a list that stores the words selected to make a stochastic improvement. 2) The solution is stochastically improved, after a certain number of iterations without obtaining improvements, the algorithm saves the current result and the solution is restarted again (n_restart parameter), selecting another word from all the possibilities. 3) A tabu memory was implemented, which saves the words that were selected in the solution restart. In Figure 4, the proposed RRHCTagger pseudocode is presented. Fig. 4. RRHCTagger pseudocode. Fig. 4. RRHCTagger pseudocode. Revista Facultad de Ingeniería (Rev. Fac. Ing.) Vol. 29 (54), e11762. 2020. Tunja-Boyacá, Colombia. L-ISSN: 0121-1129, e-ISSN: 2357-5328, DOI: https://doi.org/10.19053/01211129.v29.n54.2020.11762 Revista Facultad de Ingeniería (Rev. Fac. Ing.) Vol. 29 (54), e11762. 2020. Tunja-Boyacá, Colombia. L-ISSN: 0121-1129, e-ISSN: 2357-5328, DOI: https://doi.org/10.19053/01211129.v29.n54.2020.11762 D. Proposed GBHS4Tagger The GBHS4Tagger algorithm is based on the GBHSTagger memetic algorithm proposed in [4] and its improvement consists of the following steps. 1) The Hill Climbing (HC) algorithm was adapted to the tagging problem involving two Adaptation, Comparison, and Improvement of Metaheuristic Algorithms to the Part-of-Speech Tagging Problem neighborhoods. The first one selects a random word, regardless of the condition, and the second selects the word with the lowest probability. These neighborhoods are controlled with the 𝑟𝑜𝑏 parameter. 2) The proposed HCTagger was incorporated into GBHS Tagger 2 [4] as a local optimizer and, thus, the new memetic version called GBHS4Tagger. In Figure 5, the proposed HCTagger pseudocode is shown and in Figure 6, the proposed GBHS4Tagger is shown. Fig. 5. HCTagger pseudocode Fig. 5. HCTagger pseudocode Revista Facultad de Ingeniería (Rev. Fac. Ing.) Vol. 29 (54), e11762. 2020. Tunja-Boyacá, Colombia. L-ISSN: 0121-1129, e-ISSN: 2357-5328, DOI: https://doi.org/10.19053/01211129.v29.n54.2020.11762 Revista Facultad de Ingeniería (Rev. Fac. Ing.) Vol. 29 (54), e11762. 2020. Tunja-Boyacá, Colombia. L-ISSN: 0121-1129, e-ISSN: 2357-5328, DOI: https://doi.org/10.19053/01211129.v29.n54.2020.11762 E. Experiments with the proposed taggers To carry out the experiments, in the first instance, an adjustment (fine-tuning) of the tagging parameters was carried out using a small dataset (sample) of 5000 sentences, in order to select the best combinations of parameters of each algorithm. Table 3 shows the distribution of the sentences in the test and training datasets for each folder, with which the experiments were carried out on each complete corpus, as seen in Table 5. All the experiments were executed using cross-validation of 5 folders, except for the Nasa Yuwe corpus, with which Leave-One-Out was used, since the dataset has only 175 sentences. Miguel-Alexis Solano-Jiménez; José-Julio Tobar-Cifuentes; Luz-Marina Sierra-Martínez; Carlos-Alberto Cobos- Lozada Fig. 6. GBHS4Tagger pseudocode. Adapted from [4]. Fig. 6. GBHS4Tagger pseudocode. Adapted from [4]. Table 3. Test and training data set for the experiments. Data sets IULA corpus Brown corpus Test Data Folder with Training data Sentences in the Test data Words in the Test data Words in the Training data Sentences in the Test data Words in the Test data Words in the Training data 1 2, 3, 4, 5 8416 16316 65521 10595 23105 45113 2 1, 3, 4, 5 8416 16357 65480 10600 22852 45199 3 1, 2, 4, 5 8416 16466 65371 10600 23130 45009 4 1, 2, 3, 5 8416 16290 65547 10600 22929 45130 5 1, 2, 3, 4 8415 16408 65429 10603 23111 45025 Nasa Yuwe corpus The Nasa Yuwe corpus used Leave-One-Out, a method that takes one phrase as test data and the remaining 174 phrases as training data. This process is repeated for all the sentences in the corpus. It is a commonly used evaluation method when the dataset is small. Table 3. Test and training data set for the experiments. Revista Facultad de Ingeniería (Rev. Fac. Ing.) Vol. 29 (54), e11762. 2020. Tunja-Boyacá, Colombia. L-ISSN: 0121-1129, e-ISSN: 2357-5328, DOI: https://doi.org/10.19053/01211129.v29.n54.2020.11762 Revista Facultad de Ingeniería (Rev. Fac. Ing.) Vol. 29 (54), e11762. 2020. Tunja-Boyacá, Colombia. L-ISSN: 0121-1129, e-ISSN: 2357-5328, DOI: https://doi.org/10.19053/01211129.v29.n54.2020.11762 , Comparison, and Improvement of Metaheuristic Algorithms to the Part-of-Speech Tagging Problem In Table 4, following, the configuration of the algorithms for the experiments carried out with each corpus is presented. In Table 4, following, the configuration of the algorithms for the experiments carried out with each corpus is presented. Table 4. Configuration of the tagging algorithms for the experiments. Algorithm Parameters defined for the IULA corpus Parameters defined for the Brown corpus Parameters defined for the Nasa Yuwe corpus PSOTagger W = 0.3, C1 = 0.15, C2 = 0.45 and P = 0.1 W = 0.3, C1 = 0.15, C2 = 0.45 and P = 0.1 W = 0.3, C1 = 0.2, C2 = 0.4 and P = 0.1. JayaTagger P4 = 4 P4 = 3 P4 = 4. RRHCTagger n_restart = 6 n_restart = 4. n_restart = 4. GBHS4Tagger Prob = 0.7 Prob = 0.7. Prob = 0.75. Table 4. Configuration of the tagging algorithms for the experiments. Table 4. Configuration of the tagging algorithms for the experiments. In Table 5, the results of the experiments carried out on the three complete corpuses are presented. It can be seen that GBHS4Tagger surpassed the other algorithms in precision value, in the IULA and Brown corpus, with the Nasa Yuwe corpus being the second best. It should be noted that the adapted algorithms obtained very good results for this problem, but there are differences between the precision values obtained in each algorithm, which allow us to appreciate that some algorithms perform better than others, as established in the second theorem of No Free Lunch Theorems for Optimization (NFLT) [28]. Table 5. Results of the experiments. Results IULA corpus BROWN corpus Nasa Yuwe corpus Algorithm Phrases Precision Std dev Phrases Precision Std dev Phrases Precision Std dev GBHS4Tagger 42079 97.5403 5.4795 52998 94.8803 6.0289 175 61.4185 15.5199 RRHCTagger 42079 97.2678 5.5006 52998 94.5342 6.0106 175 63.7096 16.2249 GBHS Tagger2 42079 97.4306 5.7844 52998 94.8236 6.2051 175 60.0749 15.5199 JayaTagger 42079 96.8592 5.5963 52998 93.0519 6.3054 175 57.084 15.977 PSOTagger 42079 96.7738 5.7844 52998 89.6933 7.0810 175 55.936 16.052 Table 6 shows the ranking of each algorithm in the experiments carried out in each corpus once the Friedman NxN non-parametric statistical test has been applied, obtaining a p value smaller than 0.05, therefore, it makes the ranking statistically significant, complementing the evaluation of the algorithms. Miguel-Alexis Solano-Jiménez; José-Julio Tobar-Cifuentes; Luz-Marina Sierra-Martínez; Carlos-Alberto Cobos- Lozada Table 6. Friedman test results - IULA corpus. In Table 4, following, the configuration of the algorithms for the experiments carried out with each corpus is presented. Made with KEEL software [29] IULA corpus Brown corpus Nasa Yuwe Algorithm Ranking Algorithm Ranking Algorithm Ranking GBHS4Tagger 1 GBHS4Tagger 1 RRHCTagger 1 GBHS Tagger2 2 GBHS Tagger2 2 GBHS4Tagger 2 2 RRHCTagger 3 RRHCTagger 3 GBHS Tagger2 3 JayaTagger 4 JayaTagger 4 JayaTagger 4 PSOTagger 5 PSOTagger 5 PSOTagger 5 Table 6. Friedman test results - IULA corpus. Made with KEEL software [29] Additionally, the Wilcoxon test showed, with a significance level of 90%, that the results obtained for the winning algorithms, GBHS4Tagger for Spanish and English, and RRHCTagger for Nasa Yuwe, are better in contrast with the other proposed taggers. Revista Facultad de Ingeniería (Rev. Fac. Ing.) Vol. 29 (54), e11762. 2020. Tunja-Boyacá, Colombia. L-ISSN: 0121-1129, e-ISSN: 2357-5328, DOI: https://doi.org/10.19053/01211129.v29.n54.2020.11762 This work was partially financed by Universidad del Cauca. This work was partially financed by Universidad del Cauca. V. DISCUSSION AND CONCLUSIONS This work achieved the adaptation of the metaheuristic algorithms PSO, Jaya, and RRHC to the problem of part of speech tagging (POST), taking into account the characteristics of each algorithm, and performing the parameter adjustment required for each algorithm on each corpus, obtaining competitive results with respect to one of the state-of-the-art algorithms. It was also possible to propose an improvement to the state-of-the-art GBHS Tagger 2 memetic algorithm, which continued to demonstrate that the performance of the tagger improves by including knowledge of the problem, as seen in the IULA (Spanish) and Brown (English) corpus. Consequently, the presented research reinforced the idea that metaheuristic approaches are capable of performing tagging with good results, with acceptable resources and times. Metaheuristic algorithms should continue to be used for tagging on other traditional and non-traditional languages, and seek new improvements for the proposed taggers in combination with other optimization techniques that improve the results of the tagging. Revista Facultad de Ingeniería (Rev. Fac. Ing.) Vol. 29 (54), e11762. 2020. Tunja-Boyacá, Colomb L-ISSN: 0121-1129, e-ISSN: 2357-5328, DOI: https://doi.org/10.19053/01211129.v29.n54.2020.117 Adaptation, Comparison, and Improvement of Metaheuristic Algorithms to the Part-of-Speech Tagging Problem Adaptation, Comparison, and Improvement of Metaheuristic Algorithms to the Part-of-Speech Tagging Problem ACKNOWLEDGMENTS The authors express especially gratitude to Colin McLachlan for suggestions relating to the English text. The authors express especially gratitude to Colin McLachlan for suggestions relating to the English text. AUTHOR’S CONTRIBUTION Miguel-Alexis Solano-Jiménez: Formal Analysis, Data curation, Investigation, Methodology, Software, Validation, Visualization, Writing – original draft, Writing – review & editing. Jose-Julio Tobar-Cifuentes: Formal Analysis, Data curation, Investigation, Methodology, Software, Validation, Writing – original draft, Writing – review & editing. Luz-Marina Sierra-Martínez: Conceptualization, Methodology, Supervision, Project administration, Writing -original draft, Writing – review & editing. Carlos-Alberto Cobos-Lozada: Conceptualization, Methodology, Supervision, Project administration, Writing -original draft, Writing – review & editing. REFERENCES [1] T. Güngör, Handbook of Natural Language Processing (2 Edition ), 2011. [1] T. Güngör, Handbook of Natural Language Processing (2 Edition ), 2011. [1] T. 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https://openalex.org/W172263345	https://journals.iucr.org/e/issues/2012/10/00/wm2664/wm2664.pdf	Latin	null	Bis[4-amino-3,5-bis(pyridin-2-yl)-4<i>H</i>-1,2,4-triazole-κ<sup>2</sup><i>N</i><sup>1</sup>,<i>N</i><sup>5</sup>]diaquacobalt(II) bis(perchlorate)	Acta crystallographica. Section E	2,012	cc-by	4,123	Related literature For the synthesis of the adpt ligand, see: Geldard & Lions (1965). For background to the coordination chemistry of the adpt ligand, see: Meng et al. (2009). For intramolecular hydrogen bonds in the adpt ligand, see: Kitchen et al. (2008). For other Co(II) coordination compounds with the same ligand, see: Keij et al. (1984); Peng et al. (2006); Garcı´a- Couceiro et al. (2009); White et al. (2010). We thank the NSFC (21061009) and the National Students of Innovation and Entrepreneurship Training Programs (111012608) for their ﬁnancial support. Supplementary data and ﬁgures for this paper are available from the IUCr electronic archives (Reference: WM2664). metal-organic compounds Experimental Crystal data [Co(C12H10N6)2(H2O)2](ClO4)2 Mr = 770.38 Monoclinic, P21=c a = 8.5839 (17) A˚ b = 12.950 (3) A˚ c = 14.975 (5) A˚ = 114.34 (2) V = 1516.7 (7) A˚ 3 Z = 2 Mo K radiation = 0.82 mm1 T = 293 K 0.04 0.03 0.01 mm Data collection Bruker SMART APEX CCD diffractometer Absorption correction: multi-scan (SADABS; Sheldrick, 1996) Tmin = 0.971, Tmax = 0.992 10155 measured reﬂections 2681 independent reﬂections 2336 reﬂections with I > 2(I) Rint = 0.038 Reﬁnement R[F 2 > 2(F 2)] = 0.050 wR(F 2) = 0.121 S = 1.07 2681 reﬂections 223 parameters H-atom parameters constrained max = 0.87 e A˚ 3 min = 0.50 e A˚ 3 Acta Crystallographica Section E Structure Reports Online ISSN 1600-5368 Acta Crystallographica Section E Structure Reports Online ISSN 1600-5368 Mi Feng, Yu-Fei Ji, Sheng-Li Liang and Zhi-Liang Liu* College of Chemistry and Chemical Engineering, Inner Mongolia University, Hohhot, People’s Republic of China Correspondence e-mail: cezlliu@imu.edu.cn Received 23 July 2012; accepted 8 September 2012 Key indicators: single-crystal X-ray study; T = 293 K; mean (C–C) = 0.005 A˚; R factor = 0.050; wR factor = 0.121; data-to-parameter ratio = 12.0. Key indicators: single-crystal X-ray study; T = 293 K; mean (C–C) = 0.005 A˚; R factor = 0.050; wR factor = 0.121; data-to-parameter ratio = 12.0. In the title structure, [Co(C12H10N6)2(H2O)2](ClO4)2, the CoII atom lies on an inversion centre and is coordinated in a slightly distorted octahedral geometry by four N atoms from two 4-amino-3,5-bis(pyridin-2-yl)-4H-1,2,4-triazole (adpt) ligands in equatorial positions and two O atoms from two water molecules in axial positions. An intramolecular N— H N interaction stabilizes the molecular conformation. Intermolecular N—H O and O—H O interactions invol- ving the perchlorate counter-anions extend the monomeric compound into a two-dimensional network parallel to the bc plane. Table 1 Hydrogen-bond geometry (A˚ , ). Table 1 Hydrogen-bond geometry (A˚ , ). D—H A D—H H A D A D—H A N6—H6B N5 0.89 2.29 2.897 (4) 125 N6—H6B O3i 0.89 2.39 2.989 (4) 124 O1—H1A O4 0.85 2.16 2.782 (4) 130 O1—H1B O2ii 0.85 2.22 2.983 (4) 150 O1—H1B O5ii 0.85 2.58 3.284 (5) 141 Symmetry codes: (i) x; y þ 3 2; z þ 1 2; (ii) x þ 1; y þ 1 2; z þ 1 2. Data collection: SMART (Bruker, 2001); cell reﬁnement: SAINT (Bruker, 2001); data reduction: SAINT; program(s) used to solve structure: SHELXS97 (Sheldrick, 2008); program(s) used to reﬁne structure: SHELXL97 (Sheldrick, 2008); molecular graphics: DIAMOND (Brandenburg & Putz, 2006); software used to prepare material for publication: publCIF (Westrip, 2010). metal-organic compounds Supplementary data and ﬁgures for this paper are available from the IUCr electronic archives (Reference: WM2664). Peng, M. X., Hong, C. G., Tan, C. K., Chen, J. C. & Tong, M. L. (2006). J. Chem. Crystallogr. 36, 703–707. Sheldrick, G. M. (1996). SADABS. University of Go¨ttingen, Germany. Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122. Westrip, S. P. (2010). J. Appl. Cryst. 43, 920–925. White, N. G., Feltham, H. L. C., Gandolﬁ, C., Albrecht, M. & Brooker, S. (2010). Dalton Trans. 39, 3751–3758. Acta Cryst. (2012). E68, m1272–m1273 References Brandenburg, K. & Putz, H. (2006). DIAMOND. Crystal Impact GbR, Bonn, Germany. Bruker (2001). SMART and SAINT. Bruker AXS Inc., Madison, Wisconsin, USA. Garcı´a-Couceiro, U., Castillo, O., Cepeda, J., Luque, A., Pe´rez-Yanez, S. & Roma´n, P. (2009). Inorg. Chim. Acta, 362, 4212–4218. Geldard, J. F. & Lions, F. (1965). J. Org. Chem. 30, 318–319. Keij, F. S., de Graaff, R. A. G., Haasnoot, J. G. & Reedijk, J. (1984). J. Chem. Soc. Dalton Trans. pp. 2093–2097. Kitchen, J. A., Noble, A., Brandt, C. D., Moubaraki, B., Murray, K. S. & Brooker, S. (2008). Inorg. Chem. 47, 9450–9458. Meng, Z.-S., Yun, L., Zhang, W.-X., Hong, C.-G., Herchel, R., Ou, Y.-C., Leng, J.-D., Peng, M.-X., Lin, Z.-J. & Tong, M.-L. (2009). Dalton Trans. pp. 10284– 10295. m1272 Feng et al. Acta Cryst. (2012). E68, m1272–m1273 doi:10.1107/S1600536812038573 metal-organic compounds Feng et al. [Co(C12H10N6)2(H2O)2](ClO4)2 m1273 Acta Cryst. (2012). E68, m1272–m1273 supporting information Acta Cryst. (2012). E68, m1272–m1273 [https://doi.org/10.1107/S1600536812038573] Bis[4-amino-3,5-bis(pyridin-2-yl)-4H-1,2,4-triazole-κ2N1,N5]diaquacobalt(II) bis(perchlorate) Acta Cryst. (2012). E68, m1272–m1273 [https://doi.org/10.1107/S1600536812038573] Bis[4-amino-3,5-bis(pyridin-2-yl)-4H-1,2,4-triazole-κ2N1,N5]diaquacobalt(II) bis(perchlorate) a Cryst. (2012). E68, m1272–m1273 [https://doi.org/10.1107/S1600536812038573] Mi Feng, Yu-Fei Ji, Sheng-Li Liang and Zhi-Liang Liu Mi Feng, Yu-Fei Ji, Sheng-Li Liang and Zhi-Liang Liu S1. Comment Recently, 4-amine-3,5-di-2-pyridyl-1,2,4-triazol (adpt) has been used as a potential multidentate ligand to generate novel metal-organic complexes due to containing five N coordination sites and three potentially conjugated aromatic rings (Meng et al., 2009). Such complexes with adpt have interesting properties for potential applications in the fields of magnetic materials (Keij et al., 1984). Several Co(II) compounds containing adpt have been reported previously (Keij et al., 1984; Peng et al., 2006; García-Couceiro et al., 2009; White et al., 2010). Herein, the synthesis and crystal structure of the title complex [Co(C12H10N6)2(H2O)2](ClO4)2, (I), is reported. As shown in Figure 1, compound (I) consists of one Co(II) atom located on an inversion centre, two adpt ligands, two water molecules and two isolated perchlorate counter anions. The Co(II) is six-coordinated by four N atoms from two adpt ligands and two O atoms from two water molecules, giving a slightly distorted octahedral coordination environment. The equatorial plane is defined by four N atoms from two adpt ligands with a chelate formation, and the axial positions are occupied by two O atoms of water molecules. The dihedral angle between the non-coordinated pyridine ring and the coordinating pyridine ring is 11.94 (16) ° and that between the coordinating pyridine ring and the triazole ring is 6.76 (6)°. In the mononuclear unit, an intramolecular N—H···N hydrogen-bonding interaction between the NH2 group attached to the the triazole ring and the non-coordinating N atom of pyridine is observed (Kitchen et al., 2008). Intermolecular N—H···O and O—H···O hydrogen-bonding interactions exist between the amine group and the coordinating water molecules, respectively, with the O atoms of the isolated perchlorate counter anions. In the crystal, the molecular entities are linked by O—H···O hydrogen bonds generating chains along the b axis. These chains in turn aggregate into a two-dimensional network parallel to the bc plane (Fig. 2). S2. Experimental 4-Amino-3,5-bis(pyridin-2-yl)-4H-1,2,4-triazole (abpt, 5mmol) was dissolved in 20 ml mixture solution of water and methanol (1:1, v/v). Then Co(ClO4)2`4H2O (5 mmol) was added to the above solution. The resulting solution was stirred for 3 h at room temperature. Upon slow evaporation of the solvent, dark red block-shaped crystals formed from the filtrate in a few days. The used 4-amine-3,5-di-2-pyridyl-1,2,4-triazol (adpt) was synthesized according to the previously reported procedure (Geldard & Lions, 1965). S3. Refinement Acta Cryst. (2012). E68, m1272–m1273 Figure 1 g The molecular structure of the title compound, with atom labels and 30% probability displacement ellipsoids for non-H atoms. N—H···N and O—H···O interactions are shown as dashed lines. g The molecular structure of the title compound, with atom labels and 30% probability displacement ellipsoids for non-H atoms. N—H···N and O—H···O interactions are shown as dashed lines. The molecular structure of the title compound, with atom labels and 30% probability displacement ellipsoids for non-H atoms. N—H···N and O—H···O interactions are shown as dashed lines. Figure 2 The crystal packing of the title compound. O—H···O interactions are shown as dashed lines. S3. Refinement All H atoms were placed in geometrically idealized positions and constrained to ride on their parent atoms with C—H = 0.93 Å and with Uiso(H) = 1.2Ueq(C). H atoms bonded to N and O atoms were located in a difference map and refined with a fixed distance of O—H = 0.85 and N—H = 0.89 Å, and with Uiso(H) = 1.2Ueq(N,O). Acta Cryst. (2012). E68, m1272–m1273 sup-1 supporting information Figure 1 The molecular structure of the title compound, with atom labels and 30% probability displacement ellipsoids for non-H atoms. N—H···N and O—H···O interactions are shown as dashed lines. Refinement Refinement Refinement on F2 Least-squares matrix: full R[F2 > 2σ(F2)] = 0.050 wR(F2) = 0.121 S = 1.07 2681 reflections 223 parameters 0 restraints Primary atom site location: structure-invariant direct methods Secondary atom site location: difference Fourier map Hydrogen site location: inferred from neighbouring sites H-atom parameters constrained w = 1/[σ2(Fo2) + (0.0587P)2 + 2.3812P] where P = (Fo2 + 2Fc2)/3 (Δ/σ)max < 0.001 Δρmax = 0.87 e Å−3 Δρmin = −0.50 e Å−3 Secondary atom site location: difference Fourier map Hydrogen site location: inferred from neighbouring sites H-atom parameters constrained w = 1/[σ2(Fo2) + (0.0587P)2 + 2.3812P] where P = (Fo2 + 2Fc2)/3 (Δ/σ)max < 0.001 Δρmax = 0.87 e Å−3 Δρmin = −0.50 e Å−3 Secondary atom site location: difference Fourier map Hydrogen site location: inferred from neighbouring sites H-atom parameters constrained w = 1/[σ2(Fo2) + (0.0587P)2 + 2.3812P] where P = (Fo2 + 2Fc2)/3 (Δ/σ)max < 0.001 Δρmax = 0.87 e Å−3 Δρmin = −0.50 e Å−3 Figure 2 g The crystal packing of the title compound. O—H···O interactions are shown as dashed lines. g The crystal packing of the title compound. O—H···O interactions are shown as dashed lines. The crystal packing of the title compound. O—H···O interactions are shown as dashed lines. Bis[4-amino-3,5-bis(pyridin-2-yl)-4H-1,2,4-triazole- κ2N1,N5]diaquacobalt(II) bis(perchlorate) Crystal data [Co(C12H10N6)2(H2O)2](ClO4)2 Mr = 770.38 Monoclinic, P21/c Hall symbol: -P 2ybc a = 8.5839 (17) Å b = 12.950 (3) Å c = 14.975 (5) Å β = 114.34 (2)° V = 1516.7 (7) Å3 Z = 2 F(000) = 786 Dx = 1.687 Mg m−3 Acta Cryst. (2012). E68, m1272–m1273 sup-2 sup-2 Acta Cryst. (2012). E68, m1272–m1273 supporting information Mo Kα radiation, λ = 0.71073 Å Cell parameters from 3601 reflections θ = 2.2–27.9° µ = 0.82 mm−1 T = 293 K Block, dark red 0.04 × 0.03 × 0.01 mm Data collection Bruker SMART APEX CCD diffractometer Radiation source: fine-focus sealed tube Graphite monochromator ω scans Absorption correction: multi-scan (SADABS; Sheldrick, 1996) Tmin = 0.971, Tmax = 0.992 10155 measured reflections 2681 independent reflections 2336 reflections with I > 2σ(I) Rint = 0.038 θmax = 25.0°, θmin = 2.2° h = −7→10 k = −15→15 l = −17→15 Mo Kα radiation, λ = 0.71073 Å Cell parameters from 3601 reflections θ = 2.2–27.9° µ = 0.82 mm−1 T = 293 K Block, dark red 0.04 × 0.03 × 0.01 mm Data collection Bruker SMART APEX CCD diffractometer Radiation source: fine-focus sealed tube Graphite monochromator ω scans Absorption correction: multi-scan (SADABS; Sheldrick, 1996) Tmin = 0.971, Tmax = 0.992 10155 measured reflections 2681 independent reflections 2336 reflections with I > 2σ(I) Rint = 0.038 θmax = 25.0°, θmin = 2.2° h = −7→10 k = −15→15 l = −17→15 Refinement Refinement on F2 Least-squares matrix: full R[F2 > 2σ(F2)] = 0.050 wR(F2) = 0.121 S = 1.07 2681 reflections 223 parameters 0 restraints Primary atom site location: structure-invariant direct methods Secondary atom site location: difference Fourier map Hydrogen site location: inferred from neighbouring sites H-atom parameters constrained w = 1/[σ2(Fo2) + (0.0587P)2 + 2.3812P] where P = (Fo2 + 2Fc2)/3 (Δ/σ)max < 0.001 Δρmax = 0.87 e Å−3 Δρmin = −0.50 e Å−3 T = 293 K Block, dark red 0.04 × 0.03 × 0.01 mm supporting information supporting information H1B 0.5093 0.6085 0.3630 0.043* O2 0.6254 (4) 0.2589 (3) 0.1740 (3) 0.0633 (10) O3 0.4116 (4) 0.3823 (2) 0.1213 (2) 0.0555 (9) O4 0.6210 (4) 0.3837 (2) 0.2840 (2) 0.0475 (8) O5 0.4126 (4) 0.2533 (3) 0.2297 (2) 0.0687 (11) C1 0.2804 (4) 0.6823 (2) 0.4928 (2) 0.0181 (7) C2 0.1401 (4) 0.7437 (3) 0.4777 (2) 0.0229 (7) H2 0.1537 0.8123 0.4977 0.027* C3 −0.0217 (4) 0.7004 (3) 0.4318 (3) 0.0273 (8) H3 −0.1184 0.7398 0.4211 0.033* C4 −0.0381 (4) 0.5988 (3) 0.4024 (2) 0.0261 (8) H4 −0.1457 0.5692 0.3704 0.031* C5 0.1082 (4) 0.5415 (3) 0.4210 (2) 0.0228 (7) H5 0.0969 0.4724 0.4023 0.027* C6 0.4598 (4) 0.7148 (2) 0.5385 (2) 0.0171 (7) C7 0.7014 (4) 0.7965 (2) 0.5981 (2) 0.0190 (7) C8 0.8325 (4) 0.8777 (2) 0.6329 (2) 0.0205 (7) C9 0.9991 (4) 0.8547 (3) 0.6470 (3) 0.0285 (8) H9 1.0293 0.7879 0.6378 0.034* C10 1.1191 (5) 0.9332 (3) 0.6750 (3) 0.0334 (9) H10 1.2307 0.9207 0.6828 0.040* C11 1.0696 (5) 1.0310 (3) 0.6914 (3) 0.0313 (8) H11 1.1477 1.0851 0.7107 0.038* C12 0.9036 (5) 1.0464 (3) 0.6786 (3) 0.0287 (8) H12 0.8726 1.1116 0.6915 0.034* Atomic displacement parameters (Å2) U11 U22 U33 U12 U13 U23 Co1 0.0183 (3) 0.0139 (3) 0.0231 (3) −0.0002 (2) 0.0096 (3) −0.0014 (2) Cl1 0.0309 (5) 0.0211 (4) 0.0282 (5) −0.0011 (3) 0.0160 (4) −0.0031 (3) N1 0.0186 (14) 0.0168 (14) 0.0212 (13) −0.0010 (11) 0.0103 (11) 0.0001 (11) N2 0.0157 (14) 0.0183 (14) 0.0245 (14) −0.0012 (11) 0.0058 (12) −0.0021 (11) N3 0.0174 (14) 0.0143 (13) 0.0244 (14) 0.0001 (11) 0.0083 (12) −0.0013 (11) N4 0.0191 (14) 0.0131 (13) 0.0185 (13) 0.0010 (10) 0.0091 (11) −0.0003 (10) N5 0.0233 (15) 0.0187 (14) 0.0297 (16) −0.0012 (12) 0.0086 (13) −0.0012 (12) N6 0.0262 (16) 0.0185 (15) 0.0424 (18) 0.0023 (12) 0.0128 (14) −0.0011 (13) O1 0.0389 (15) 0.0206 (12) 0.0312 (13) 0.0066 (11) 0.0191 (12) 0.0034 (10) O2 0.0474 (19) 0.070 (2) 0.073 (2) 0.0008 (17) 0.0256 (17) −0.0440 (19) O3 0.062 (2) 0.0395 (18) 0.0509 (18) 0.0045 (16) 0.0092 (16) 0.0161 (14) O4 0.0537 (19) 0.0519 (18) 0.0393 (16) −0.0126 (15) 0.0217 (14) −0.0229 (14) O5 0.060 (2) 0.096 (3) 0.0425 (18) −0.046 (2) 0.0130 (16) 0.0131 (18) C1 0.0217 (17) 0.0179 (16) 0.0181 (16) −0.0007 (13) 0.0116 (14) 0.0017 (12) C2 0.0237 (18) 0.0196 (17) 0.0283 (18) 0.0016 (14) 0.0138 (15) −0.0002 (14) C3 0.0205 (18) 0.0293 (19) 0.035 (2) 0.0053 (15) 0.0145 (16) 0.0059 (15) C4 0.0165 (17) 0.032 (2) 0.0295 (18) −0.0049 (15) 0.0090 (15) −0.0010 (15) C5 0.0240 (18) 0.0206 (17) 0.0268 (17) −0.0019 (14) 0.0134 (15) −0.0012 (14) C6 0.0212 (17) 0.0156 (16) 0.0175 (15) −0.0007 (13) 0.0109 (13) 0.0007 (12) H1B 0.5093 0.6085 0.3630 0.043* O2 0.6254 (4) 0.2589 (3) 0.1740 (3) 0.0633 (10) O3 0.4116 (4) 0.3823 (2) 0.1213 (2) 0.0555 (9) O4 0.6210 (4) 0.3837 (2) 0.2840 (2) 0.0475 (8) O5 0.4126 (4) 0.2533 (3) 0.2297 (2) 0.0687 (11) C1 0.2804 (4) 0.6823 (2) 0.4928 (2) 0.0181 (7) C2 0.1401 (4) 0.7437 (3) 0.4777 (2) 0.0229 (7) H2 0.1537 0.8123 0.4977 0.027* C3 −0.0217 (4) 0.7004 (3) 0.4318 (3) 0.0273 (8) H3 −0.1184 0.7398 0.4211 0.033* C4 −0.0381 (4) 0.5988 (3) 0.4024 (2) 0.0261 (8) H4 −0.1457 0.5692 0.3704 0.031* C5 0.1082 (4) 0.5415 (3) 0.4210 (2) 0.0228 (7) H5 0.0969 0.4724 0.4023 0.027* C6 0.4598 (4) 0.7148 (2) 0.5385 (2) 0.0171 (7) C7 0.7014 (4) 0.7965 (2) 0.5981 (2) 0.0190 (7) C8 0.8325 (4) 0.8777 (2) 0.6329 (2) 0.0205 (7) C9 0.9991 (4) 0.8547 (3) 0.6470 (3) 0.0285 (8) H9 1.0293 0.7879 0.6378 0.034* C10 1.1191 (5) 0.9332 (3) 0.6750 (3) 0.0334 (9) H10 1.2307 0.9207 0.6828 0.040* C11 1.0696 (5) 1.0310 (3) 0.6914 (3) 0.0313 (8) H11 1.1477 1.0851 0.7107 0.038* C12 0.9036 (5) 1.0464 (3) 0.6786 (3) 0.0287 (8) H12 0.8726 1.1116 0.6915 0.034* Atomic displacement parameters (Å2) U11 U22 U33 U12 U13 U23 Co1 0.0183 (3) 0.0139 (3) 0.0231 (3) −0.0002 (2) 0.0096 (3) −0.0014 (2) Cl1 0.0309 (5) 0.0211 (4) 0.0282 (5) −0.0011 (3) 0.0160 (4) −0.0031 (3) N1 0.0186 (14) 0.0168 (14) 0.0212 (13) −0.0010 (11) 0.0103 (11) 0.0001 (11) N2 0.0157 (14) 0.0183 (14) 0.0245 (14) −0.0012 (11) 0.0058 (12) −0.0021 (11) N3 0.0174 (14) 0.0143 (13) 0.0244 (14) 0.0001 (11) 0.0083 (12) −0.0013 (11) N4 0.0191 (14) 0.0131 (13) 0.0185 (13) 0.0010 (10) 0.0091 (11) −0.0003 (10) N5 0.0233 (15) 0.0187 (14) 0.0297 (16) −0.0012 (12) 0.0086 (13) −0.0012 (12) N6 0.0262 (16) 0.0185 (15) 0.0424 (18) 0.0023 (12) 0.0128 (14) −0.0011 (13) O1 0.0389 (15) 0.0206 (12) 0.0312 (13) 0.0066 (11) 0.0191 (12) 0.0034 (10) O2 0.0474 (19) 0.070 (2) 0.073 (2) 0.0008 (17) 0.0256 (17) −0.0440 (19) O3 0.062 (2) 0.0395 (18) 0.0509 (18) 0.0045 (16) 0.0092 (16) 0.0161 (14) O4 0.0537 (19) 0.0519 (18) 0.0393 (16) −0.0126 (15) 0.0217 (14) −0.0229 (14) O5 0.060 (2) 0.096 (3) 0.0425 (18) −0.046 (2) 0.0130 (16) 0.0131 (18) C1 0.0217 (17) 0.0179 (16) 0.0181 (16) −0.0007 (13) 0.0116 (14) 0.0017 (12) C2 0.0237 (18) 0.0196 (17) 0.0283 (18) 0.0016 (14) 0.0138 (15) −0.0002 (14) C3 0.0205 (18) 0.0293 (19) 0.035 (2) 0.0053 (15) 0.0145 (16) 0.0059 (15) C4 0.0165 (17) 0.032 (2) 0.0295 (18) −0.0049 (15) 0.0090 (15) −0.0010 (15) C5 0.0240 (18) 0.0206 (17) 0.0268 (17) −0.0019 (14) 0.0134 (15) −0.0012 (14) C6 0.0212 (17) 0.0156 (16) 0.0175 (15) −0.0007 (13) 0.0109 (13) 0.0007 (12) sup-4 Acta Cryst. Special details Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell e.s.d.'s are taken into account individually in the estimation of e.s.d.'s in distances, angles and torsion angles; correlations between e.s.d.'s in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s. planes. Refinement. Refinement of F2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F2, conventional R-factors R are based on F, with F set to zero for negative F2. The threshold expression of F2 > σ(F2) is used only for calculating R-factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F2 are statistically about twice as large as those based on F, and R- factors based on ALL data will be even larger. Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) x y z Uiso/Ueq Co1 0.5000 0.5000 0.5000 0.0181 (2) Cl1 0.51836 (11) 0.31957 (6) 0.20373 (6) 0.0255 (2) N1 0.2655 (3) 0.5819 (2) 0.46508 (18) 0.0182 (6) N2 0.7370 (3) 0.6987 (2) 0.59168 (19) 0.0203 (6) N3 0.5833 (3) 0.64755 (19) 0.55294 (19) 0.0188 (6) N4 0.5287 (3) 0.80947 (19) 0.56599 (18) 0.0165 (6) N5 0.7829 (4) 0.9719 (2) 0.6482 (2) 0.0247 (6) N6 0.4322 (4) 0.9001 (2) 0.5607 (2) 0.0295 (7) H6A 0.3984 0.9278 0.5012 0.044 H6B 0.4971 0.9452 0.6054 0.044* O1 0.5064 (3) 0.54303 (18) 0.36619 (16) 0.0287 (6) H1A 0.5952 0.5177 0.3628 0.043* Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) x y z Uiso*/Ueq omic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) sup-3 Acta Cryst. (2012). E68, m1272–m1273 supporting information (2012). E68, m1272–m1273 supporting information C7 0.0218 (17) 0.0181 (16) 0.0193 (16) −0.0002 (13) 0.0107 (14) 0.0002 (13) C8 0.0214 (17) 0.0196 (17) 0.0198 (16) −0.0013 (13) 0.0077 (14) −0.0007 (13) C9 0.0248 (19) 0.0256 (19) 0.0339 (19) −0.0015 (15) 0.0111 (16) −0.0073 (15) C10 0.0230 (19) 0.039 (2) 0.038 (2) −0.0043 (16) 0.0120 (17) −0.0086 (17) C11 0.028 (2) 0.029 (2) 0.031 (2) −0.0115 (16) 0.0065 (16) −0.0048 (16) C12 0.029 (2) 0.0191 (18) 0.0338 (19) −0.0032 (15) 0.0089 (16) −0.0063 (15) Geometric parameters (Å, º) Co1—N3 2.079 (3) N6—H6B 0.8901 Co1—N3i 2.079 (3) O1—H1A 0.8500 Co1—O1i 2.102 (2) O1—H1B 0.8500 Co1—O1 2.102 (2) C1—C2 1.381 (4) Co1—N1 2.141 (3) C1—C6 1.466 (4) Co1—N1i 2.141 (3) C2—C3 1.389 (5) Cl1—O2 1.413 (3) C2—H2 0.9300 Cl1—O5 1.416 (3) C3—C4 1.376 (5) Cl1—O4 1.428 (3) C3—H3 0.9300 Cl1—O3 1.446 (3) C4—C5 1.385 (5) N1—C5 1.341 (4) C4—H4 0.9300 N1—C1 1.355 (4) C5—H5 0.9300 N2—C7 1.316 (4) C7—C8 1.470 (4) N2—N3 1.372 (4) C8—C9 1.389 (5) N3—C6 1.319 (4) C9—C10 1.384 (5) N4—C6 1.350 (4) C9—H9 0.9300 N4—C7 1.367 (4) C10—C11 1.390 (5) N4—N6 1.420 (4) C10—H10 0.9300 N5—C8 1.342 (4) C11—C12 1.372 (5) N5—C12 1.350 (5) C11—H11 0.9300 N6—H6A 0.8901 C12—H12 0.9300 N3—Co1—N3i 180.0 H1A—O1—H1B 109.5 N3—Co1—O1i 91.15 (10) N1—C1—C2 122.4 (3) N3i—Co1—O1i 88.85 (10) N1—C1—C6 111.5 (3) N3—Co1—O1 88.85 (10) C2—C1—C6 126.1 (3) N3i—Co1—O1 91.15 (10) C1—C2—C3 118.4 (3) O1i—Co1—O1 180.000 (1) C1—C2—H2 120.8 N3—Co1—N1 77.24 (10) C3—C2—H2 120.8 N3i—Co1—N1 102.76 (10) C4—C3—C2 119.6 (3) O1i—Co1—N1 88.45 (9) C4—C3—H3 120.2 O1—Co1—N1 91.55 (9) C2—C3—H3 120.2 N3—Co1—N1i 102.76 (10) C3—C4—C5 119.0 (3) N3i—Co1—N1i 77.24 (10) C3—C4—H4 120.5 O1i—Co1—N1i 91.55 (9) C5—C4—H4 120.5 O1—Co1—N1i 88.45 (9) N1—C5—C4 122.3 (3) N1—Co1—N1i 180.000 (1) N1—C5—H5 118.8 O2—Cl1—O5 108.9 (3) C4—C5—H5 118.8 O2—Cl1—O4 109.47 (19) N3—C6—N4 109.2 (3) sup-5 Acta Cryst. (2012). supporting information E68, m1272–m1273 supporting information supporting information pp g O5—Cl1—O4 111.4 (2) N3—C6—C1 120.4 (3) O2—Cl1—O3 108.0 (2) N4—C6—C1 130.3 (3) O5—Cl1—O3 108.8 (2) N2—C7—N4 110.1 (3) O4—Cl1—O3 110.2 (2) N2—C7—C8 123.2 (3) C5—N1—C1 118.3 (3) N4—C7—C8 126.7 (3) C5—N1—Co1 125.6 (2) N5—C8—C9 123.3 (3) C1—N1—Co1 116.1 (2) N5—C8—C7 117.4 (3) C7—N2—N3 106.5 (3) C9—C8—C7 119.3 (3) C6—N3—N2 108.6 (2) C10—C9—C8 118.7 (3) C6—N3—Co1 114.6 (2) C10—C9—H9 120.7 N2—N3—Co1 136.4 (2) C8—C9—H9 120.7 C6—N4—C7 105.7 (3) C9—C10—C11 118.7 (3) C6—N4—N6 124.2 (3) C9—C10—H10 120.7 C7—N4—N6 130.1 (3) C11—C10—H10 120.7 C8—N5—C12 117.0 (3) C12—C11—C10 118.9 (3) N4—N6—H6A 109.3 C12—C11—H11 120.6 N4—N6—H6B 109.2 C10—C11—H11 120.6 H6A—N6—H6B 109.5 N5—C12—C11 123.5 (3) Co1—O1—H1A 109.3 N5—C12—H12 118.2 Co1—O1—H1B 109.3 C11—C12—H12 118.2 N3—Co1—N1—C5 −179.1 (3) Co1—N3—C6—N4 −173.37 (18) N3i—Co1—N1—C5 0.9 (3) N2—N3—C6—C1 176.9 (3) O1i—Co1—N1—C5 −87.5 (3) Co1—N3—C6—C1 3.0 (4) O1—Co1—N1—C5 92.5 (3) C7—N4—C6—N3 0.0 (3) N3—Co1—N1—C1 −0.9 (2) N6—N4—C6—N3 −179.7 (3) N3i—Co1—N1—C1 179.1 (2) C7—N4—C6—C1 −175.8 (3) O1i—Co1—N1—C1 90.7 (2) N6—N4—C6—C1 4.4 (5) O1—Co1—N1—C1 −89.3 (2) N1—C1—C6—N3 −3.6 (4) C7—N2—N3—C6 −1.0 (3) C2—C1—C6—N3 176.9 (3) C7—N2—N3—Co1 171.1 (2) N1—C1—C6—N4 171.8 (3) O1i—Co1—N3—C6 −89.3 (2) C2—C1—C6—N4 −7.7 (5) O1—Co1—N3—C6 90.7 (2) N3—N2—C7—N4 1.0 (3) N1—Co1—N3—C6 −1.1 (2) N3—N2—C7—C8 −177.6 (3) N1i—Co1—N3—C6 178.9 (2) C6—N4—C7—N2 −0.6 (3) O1i—Co1—N3—N2 99.0 (3) N6—N4—C7—N2 179.1 (3) O1—Co1—N3—N2 −81.0 (3) C6—N4—C7—C8 177.8 (3) N1—Co1—N3—N2 −172.8 (3) N6—N4—C7—C8 −2.4 (5) N1i—Co1—N3—N2 7.2 (3) C12—N5—C8—C9 0.2 (5) C5—N1—C1—C2 0.3 (4) C12—N5—C8—C7 −178.9 (3) Co1—N1—C1—C2 −178.0 (2) N2—C7—C8—N5 −171.5 (3) C5—N1—C1—C6 −179.2 (3) N4—C7—C8—N5 10.2 (5) Co1—N1—C1—C6 2.5 (3) N2—C7—C8—C9 9.4 (5) N1—C1—C2—C3 −0.4 (5) N4—C7—C8—C9 −169.0 (3) C6—C1—C2—C3 179.1 (3) N5—C8—C9—C10 −2.3 (5) C1—C2—C3—C4 −0.4 (5) C7—C8—C9—C10 176.8 (3) C2—C3—C4—C5 1.3 (5) C8—C9—C10—C11 2.3 (5) C1—N1—C5—C4 0.6 (5) C9—C10—C11—C12 −0.4 (5) O5—Cl1—O4 111.4 (2) N3—C6—C1 O2—Cl1—O3 108.0 (2) N4—C6—C1 O5—Cl1—O3 108.8 (2) N2—C7—N4 O4—Cl1—O3 110.2 (2) N2—C7—C8 C5—N1—C1 118.3 (3) N4—C7—C8 C5—N1—Co1 125.6 (2) N5—C8—C9 C1—N1—Co1 116.1 (2) N5—C8—C7 C7—N2—N3 106.5 (3) C9—C8—C7 C6—N3—N2 108.6 (2) C10—C9—C8 C6—N3—Co1 114.6 (2) C10—C9—H9 N2—N3—Co1 136.4 (2) C8—C9—H9 C6—N4—C7 105.7 (3) C9—C10—C11 C6—N4—N6 124.2 (3) C9—C10—H10 C7—N4—N6 130.1 (3) C11—C10—H10 C8—N5—C12 117.0 (3) C12—C11—C10 N4—N6—H6A 109.3 C12—C11—H11 N4—N6—H6B 109.2 C10—C11—H11 H6A—N6—H6B 109.5 N5—C12—C11 Co1—O1—H1A 109.3 N5—C12—H12 Co1—O1—H1B 109.3 C11—C12—H12 N3—Co1—N1—C5 −179.1 (3) Co1—N3—C6—N4 N3i—Co1—N1—C5 0.9 (3) N2—N3—C6—C1 O1i—Co1—N1—C5 −87.5 (3) Co1—N3—C6—C1 O1—Co1—N1—C5 92.5 (3) C7—N4—C6—N3 N3—Co1—N1—C1 −0.9 (2) N6—N4—C6—N3 N3i—Co1—N1—C1 179.1 (2) C7—N4—C6—C1 O1i—Co1—N1—C1 90.7 (2) N6—N4—C6—C1 O1—Co1—N1—C1 −89.3 (2) N1—C1—C6—N3 C7—N2—N3—C6 −1.0 (3) C2—C1—C6—N3 C7—N2—N3—Co1 171.1 (2) N1—C1—C6—N4 O1i—Co1—N3—C6 −89.3 (2) C2—C1—C6—N4 O1—Co1—N3—C6 90.7 (2) N3—N2—C7—N4 N1—Co1—N3—C6 −1.1 (2) N3—N2—C7—C8 N1i—Co1—N3—C6 178.9 (2) C6—N4—C7—N2 O1i—Co1—N3—N2 99.0 (3) N6—N4—C7—N2 O1—Co1—N3—N2 −81.0 (3) C6—N4—C7—C8 N1—Co1—N3—N2 −172.8 (3) N6—N4—C7—C8 N1i—Co1—N3—N2 7.2 (3) C12—N5—C8—C9 C5—N1—C1—C2 0.3 (4) C12—N5—C8—C7 Co1—N1—C1—C2 −178.0 (2) N2—C7—C8—N5 C5—N1—C1—C6 −179.2 (3) N4—C7—C8—N5 Co1—N1—C1—C6 2.5 (3) N2—C7—C8—C9 N1—C1—C2—C3 −0.4 (5) N4—C7—C8—C9 C6—C1—C2—C3 179.1 (3) N5—C8—C9—C10 C1—C2—C3—C4 −0.4 (5) C7—C8—C9—C10 C2—C3—C4—C5 1.3 (5) C8—C9—C10—C1 C1—N1—C5—C4 0.6 (5) C9—C10—C11—C Acta Cryst. sup-6 Acta Cryst. (2012). E68, m1272–m1273 Acta Cryst. (2012). E68, m1272–m1273 supporting information (2012). E68, m1272–m1273 sup-6 supporting information Co1—N1—C5—C4 178.8 (2) C8—N5—C12—C11 1.9 (5) C3—C4—C5—N1 −1.4 (5) C10—C11—C12—N5 −1.8 (6) N2—N3—C6—N4 0.6 (3) Symmetry code: (i) −x+1, −y+1, −z+1. Hydrogen-bond geometry (Å, º) D—H···A D—H H···A D···A D—H···A N6—H6B···N5 0.89 2.29 2.897 (4) 125 N6—H6B···O3ii 0.89 2.39 2.989 (4) 124 O1—H1A···O4 0.85 2.16 2.782 (4) 130 O1—H1B···O2iii 0.85 2.22 2.983 (4) 150 O1—H1B···O5iii 0.85 2.58 3.284 (5) 141 Symmetry codes: (ii) x, −y+3/2, z+1/2; (iii) −x+1, y+1/2, −z+1/2. supporting information Co1—N1—C5—C4 178.8 (2) C8—N5—C12—C11 1.9 (5) C3—C4—C5—N1 −1.4 (5) C10—C11—C12—N5 −1.8 (6) N2—N3—C6—N4 0.6 (3) Symmetry code: (i) −x+1, −y+1, −z+1. Co1—N1—C5—C4 178.8 (2) C8—N5—C12—C11 1.9 (5) C3—C4—C5—N1 −1.4 (5) C10—C11—C12—N5 −1.8 (6) N2—N3—C6—N4 0.6 (3) Symmetry code: (i) −x+1, −y+1, −z+1. Hydrogen-bond geometry (Å, º) D—H···A D—H H···A D···A D—H···A N6—H6B···N5 0.89 2.29 2.897 (4) 125 N6—H6B···O3ii 0.89 2.39 2.989 (4) 124 O1—H1A···O4 0.85 2.16 2.782 (4) 130 O1—H1B···O2iii 0.85 2.22 2.983 (4) 150 O1—H1B···O5iii 0.85 2.58 3.284 (5) 141 S t d (ii) +3/2 +1/2 (iii) +1 +1/2 +1/2 Hydrogen-bond geometry (Å, º) Hydrogen-bond geometry (Å, º) sup-7 Acta Cryst. (2012). E68, m1272–m1273
https://openalex.org/W4312686790	https://sfs.elpub.ru/jour/article/download/266/265	Russian	null	Corporate social responsibility of a city-forming and system-forming enterprise: analysis and directions of development	Sibirskaâ finansovaâ škola	2,022	cc-by	5,474	Менеджмент _ Менеджмент _ 130 УДК 005.35: 65.017.12 DOI: https://doi.org/10.34020/1993-4386--2022-1-130-137 2 Согласно «Критериям и порядку включения организаций в перечень системообразующих организаций российской экономики» (утв. протоколом заседания Правительственной комиссии по повышению устойчивости развития российской экономики (далее – ПК) от 10.04.2020 г. № 7кв; действ. в ред. от 19.06.2020) в Перечень системообразующих организаций (СОО) российской экономики, включение организаций в Перечень СОО осуществляется на основании отраслевых пока зателей по предложениям соответствующих федеральных органов исполнительной власти. Среди критериев для включе ния организации в Перечень СОО выделим превышение минимальных значений отраслевых показателей, определенных ПК отдельно для каждой отрасли (например, для автомобильной промышленности определены минимальные значения по размеру выручки (20 000 млн руб.) и численности персонала в организации (1 000 чел.), а также наличие одного или нескольких из следующих оснований, характеризующих деятельность организации-претендента (ОП) на включение в Пе речень СОО: а) ОП должна быть градообразующей организацией, оказывающей существенное влияние на развитие ре гиона; б) ОП осуществляет деятельность по обеспечению транспортной доступности удаленных территорий; в) признано доминирующее положение ОП на рынке определенного товара в соответствии со статьей 5 Федерального закона от 26 июля 2006 г. № 135-ФЗ «О защите конкуренции». Решение о включении ОП в Перечень СОО принимает ПК. 1 Согласно постановлению Правительства РФ от 2 апреля 2020 г. № 409 (ред. от 07.11.2020) «О мерах по обеспече нию устойчивого развития экономики», к градообразующим организациям относятся организации, у которых среднее ко личество застрахованных лиц согласно данным расчетов по страховым взносам за последние четыре отчетных периода составляет более 5000 человек. КОРПОРАТИВНАЯ СОЦИАЛЬНАЯ ОТВЕТСТВЕННОСТЬ ГРАДООБРАЗУЮЩЕЙ И СИСТЕМООБРАЗУЮЩЕЙ ОРГАНИЗАЦИИ: АНАЛИЗ И НАПРАВЛЕНИЯ РАЗВИТИЯ Э.Р. Назарова Финансовый университет при Правительстве Российской Федерации (Финансовый университет), Уфа, Россия А.Ю. Шеина Уфимский филиал Финансового университета, Уфа, Россия Л.С. Брусенцова Уфимский филиал Финансового университета, Уфа, Россия Градообразующие и системообразующие организации играют важную роль в социально-э кономическом развитии региона, за счет их налоговых поступлений формируются местные и региональные бюджеты, они предоставляют рабочие места жителям региона, формируют социальную среду и существенно влияют на обеспечение качества жизни населения. Одним из базовых элементов такого рода организаций является система корпоративной социаль ной ответственности, эффективное функционирование которой обеспечивает не только устойчивое развитие бизнеса, но и улучшение социальной среды территории и повышение качества жизни ее жителей. В данной статье рассмотрен опыт реализации основных на правлений системы корпоративной социальной ответственности в ПАО «НЕФАЗ» (входит в группу ПАО «КАМАЗ»). Особое внимание уделено приоритетным направлениям кадровой политики ПАО «НЕФАЗ» (мотивации персонала, развитию персонала, социальной политике, охране труда) и ее влиянию на стратегическое развитие рассматриваемой организации как резидента территории опережающего социально-экономического развития, монопрофильно го муниципального образования (моногорода) «Нефтекамск». Ключевые слова: градообразующие и системообразующие организации, территория опережающего социально-экономического развития, ПАО «НЕФАЗ», корпоративная социальная ответственность. ные) бюджеты, предоставляют рабочие места насе лению региона, формируют социально-экономиче скую среду, «качественные характеристики и сте пень развитости которой оказывают большую сте пень воздействия на уровень жизни населения» [1]. Градообразующие1 и системообразующие2 ор ганизации играют важную роль в социально-эконо мическом развитии экономики региона, они форми руют значимые объемы налоговых поступлений в местные бюджеты и субфедеральные (региональ 1 Согласно постановлению Правительства РФ от 2 апреля 2020 г. № 409 (ред. от 07.11.2020) «О мерах по обеспече нию устойчивого развития экономики», к градообразующим организациям относятся организации, у которых среднее ко личество застрахованных лиц согласно данным расчетов по страховым взносам за последние четыре отчетных периода составляет более 5000 человек. 2 Согласно «Критериям и порядку включения организаций в перечень системообразующих организаций российской экономики» (утв. протоколом заседания Правительственной комиссии по повышению устойчивости развития российской экономики (далее – ПК) от 10.04.2020 г. № 7кв; действ. в ред. от 19.06.2020) в Перечень системообразующих организаций (СОО) российской экономики, включение организаций в Перечень СОО осуществляется на основании отраслевых пока зателей по предложениям соответствующих федеральных органов исполнительной власти. 3 Утв. 29 декабря 2014 г. (действ. в ред. от 11.06.2021). 4 Публичное акционерное общество «НЕФАЗ» (ПАО «НЕФАЗ») входит в группу ПАО «КАМАЗ» и является крупнейшим в России заводом по производству спецнадстроек на шасси КАМАЗ. Производственные площади ПАО «НЕФАЗ» располо жены в г. Нефтекамск (Республика Башкортостан). В числе крупнейших акционеров ПАО «НЕФАЗ»: ПАО «КАМАЗ» (доля в уставном капитале составляет 50,02 %), Республика Башкортостан (доля в уставном капитале составляет 28,50 %). ПАО «НЕФАЗ» производит специальные надстройки на шасси автомобилей, пассажирские и вахтовые автобусы, трол лейбусы, электробусы, емкостно-наливную технику, бортовые прицепы и полуприцепы, запасные части. Выпускаемая ПАО «НЕФАЗ» продукция отвечает самым высоким международным стандартам. 5 «НЕФАЗ» – социально эффективное предприятие. URL: http://kama-avtodetal.ru/about/news/news 10418.html. КОРПОРАТИВНАЯ СОЦИАЛЬНАЯ ОТВЕТСТВЕННОСТЬ ГРАДООБРАЗУЮЩЕЙ И СИСТЕМООБРАЗУЮЩЕЙ ОРГАНИЗАЦИИ: АНАЛИЗ И НАПРАВЛЕНИЯ РАЗВИТИЯ Среди критериев для включе ния организации в Перечень СОО выделим превышение минимальных значений отраслевых показателей, определенных ПК отдельно для каждой отрасли (например, для автомобильной промышленности определены минимальные значения по размеру выручки (20 000 млн руб.) и численности персонала в организации (1 000 чел.), а также наличие одного или нескольких из следующих оснований, характеризующих деятельность организации-претендента (ОП) на включение в Пе речень СОО: а) ОП должна быть градообразующей организацией, оказывающей существенное влияние на развитие ре гиона; б) ОП осуществляет деятельность по обеспечению транспортной доступности удаленных территорий; в) признано доминирующее положение ОП на рынке определенного товара в соответствии со статьей 5 Федерального закона от 26 июля 2006 г. № 135-ФЗ «О защите конкуренции». Решение о включении ОП в Перечень СОО принимает ПК. Сибирская финансовая школа январь-март 1’2022 Менеджмент 131 Деятельность такого рода организаций (компаний) обусловливает повышение социальной ответствен ности бизнеса, развитие коммунальной инфра структуры, социально-культурной сферы муници пального образования (моногорода), обеспечение приоритетности соблюдения интересов его жите лей. 1. КСО реализуется при осуществлении органи зацией социальных инвестиций, а не посредством только благотворительности. 2. КСО предполагает использование триединого подхода к реализации направлений КСО, базирую щегося на применении трех подходов – экономиче ском, социальном и экологическом. 3. Реализация КСО осуществляется в интересах как самой организации, так и ее заинтересованных сторон. В соответствии с Федеральным законом № 473- ФЗ «О территориях опережающего социально-эко номического развития в Российской Федерации»3, постановлением Правительства РФ от 12 февраля 2019 г. № 129 «О создании территории опережаю щего социально-экономического развития «Нефте камск», в муниципальном образовании городском округе городе Нефтекамске Республики Башкорто стан создана территория опережающего социаль но-экономического развития (ТОСЭР) «Нефте камск». Кроме того, согласно постановлению Пра вительства РФ от 29 июля 2014 г. № 709 «О крите риях отнесения муниципальных образований Рос сийской Федерации к монопрофильным (моногоро дам) и категориях монопрофильных муниципаль ных образований Российской Федерации (моного родов) в зависимости от рисков ухудшения их соци ально-экономического положения» и утвержденно му в установленном порядке Перечню российских моногородов, Нефтекамск признан монопрофиль ным муниципальным образованием (моногородом), а главным основанием получения такого статуса является функционирование в Нефтекамске градо образующей и системообразующей организации – ПАО «НЕФАЗ»4, на объектах которого трудится зна чительная часть жителей Нефтекамска, которые, не понаслышке, знают о роли корпоративной соци альной ответственности (КСО) в бизнесе и соци ально-экономическом развитии города. Отметим, что в 2019 г. градообразующая организация г. Не фтекамска ПАО «НЕФАЗ» было включена в Реестр социально ответственных работодателей Респу блики Башкортостан. Всего в реестре находится бо лее ста организаций. 1. Мотивация персонала 1. Мотивация персонала КОРПОРАТИВНАЯ СОЦИАЛЬНАЯ ОТВЕТСТВЕННОСТЬ ГРАДООБРАЗУЮЩЕЙ И СИСТЕМООБРАЗУЮЩЕЙ ОРГАНИЗАЦИИ: АНАЛИЗ И НАПРАВЛЕНИЯ РАЗВИТИЯ Приоритетными направлениями кадровой поли тики ПАО «НЕФАЗ» являются мотивация персона ла, развитие персонала, социальная политика, ох рана труда. Остановимся на указанных направлени ях более подробно. 1. Мотивация персонала Система мотивации персонала направлена на привлечение и удержание квалифицированного персонала, повышение заинтересованности работ ников в результатах труда, росте производительно сти труда [5]. Данные таблицы 1 свидетельствуют о следую щем: 1) рост производительности труда в 2020 г. в стоимостном выражении на 1 человека составил 30 % (для сравнения, в 2019 г. – 52,7 %); такой рост был обеспечен за счет увеличения объемов произ водства, повышения мотивации персонала и обе спечения эффективности использования рабочего времени; в н с о 2 а 2 с 6 с ч н п ю л п ч н к в н Динамика показателей прои ПАО «НЕФАЗ» за 20 Год Численность, чел. С 2018 5 132 2019 5 367 2020 5 784 * Источник: составлено авторами по материалам собра https://nefaz.ru/shareholders/materialy-sobraniya-aktsionerov/ (да Анализ показателей подготовки работников ПАО «НЕФАЗ» Наименование 1 Обучение рабочей профессии (подготовка работников) Переподготовка работников по рабочей профессии Обучение второй профессии Развивающее обучение на курсах повышения квалификаци работников Повышение квалификации руководителей специалисто Менеджмент _ 132 Кадровая политика ПАО «НЕФАЗ» направлена на создание сплоченного трудового коллектива, ко торый наилучшим образом способствовал бы со вмещению целей и приоритетов организации и его работников. Успех организации напрямую зависит от квалификации, ответственности и производи тельности труда работников [4]. 2) среднесписочная численность ПАО «НЕФАЗ» в 2020 г. составила 5 784 чел., что на 7,8 % выше уров ня 2019 г.; среднесписочная численность в 2019 г. со ставила 5 367 чел., что на 4,6 % выше уровня 2018 г.; 3) за счет увеличения численности расходы на оплату труда увеличились: в 2020 г. они составили 2 756,4 млн руб., что на 11,2 % выше, чем в 2019 г.; а в 2019 г. расходы на оплату труда составили 2 478,4 млн руб., что на 21,2 % выше, чем в 2018 г.; средняя заработная плата в 2020 г. составила 39 677 руб., что на 3,2 % выше уровня 2019 г., в 2019 г. средняя заработная плата составила 38 439 руб., что на 15,9 % выше уровня 2018 г. 3) за счет увеличения численности расходы на оплату труда увеличились: в 2020 г. они составили 2 756,4 млн руб., что на 11,2 % выше, чем в 2019 г.; а в 2019 г. 2. Развитие персонала Система мотивации персонала направлена на привлечение и удержание квалифицированного персонала, повышение заинтересованности работ ников в результатах труда, росте производительно сти труда [5]. Большое внимание уделяется профессиональ ной переподготовке персонала: функционирует кор поративный учебный центр, где работники повыша ют свою квалификацию, проходят обучение для по лучения второй профессии. Данные таблицы 1 свидетельствуют о следую щем: Развитие персонала включает следующий ком плекс мер: наставничество; переподготовка, полу чение смежных профессий; ротация; оценка; обуче ние, повышение квалификации [6]. 1) рост производительности труда в 2020 г. в стоимостном выражении на 1 человека составил 30 % (для сравнения, в 2019 г. – 52,7 %); такой рост был обеспечен за счет увеличения объемов произ водства, повышения мотивации персонала и обе спечения эффективности использования рабочего времени; Для более детального анализа рассмотрим по казатели по обучению, повышению квалификации в разрезе образовательных мероприятий и выявим наиболее значимые изменения. Таблица 1 Таблица 1 Динамика показателей производительности труда ПАО «НЕФАЗ» за 2018–2020 годы* Год Численность, чел. Средняя заработная плата, руб. / мес. Производительность труда, тыс. руб. / чел. 2018 5 132 33 160 2 408 2019 5 367 38 439 3 677 2020 5 784 39 677 4 781 * Источник: составлено авторами по материалам собраний акционеров ПАО «НЕФАЗ» за 2018-2020 годы URL: https://nefaz.ru/shareholders/materialy-sobraniya-aktsionerov/ (дата обращения: 21.12.2021). Динамика показателей производительности труда ПАО «НЕФАЗ» за 2018–2020 годы* * Источник: составлено авторами по материалам собраний акционеров ПАО «НЕФАЗ» за 2018-2020 годы URL: https://nefaz.ru/shareholders/materialy-sobraniya-aktsionerov/ (дата обращения: 21.12.2021). * Источник: составлено авторами по материалам собраний акционеров ПАО «НЕФАЗ» за 2018-2020 годы URL: https://nefaz.ru/shareholders/materialy-sobraniya-aktsionerov/ (дата обращения: 21.12.2021). КОРПОРАТИВНАЯ СОЦИАЛЬНАЯ ОТВЕТСТВЕННОСТЬ ГРАДООБРАЗУЮЩЕЙ И СИСТЕМООБРАЗУЮЩЕЙ ОРГАНИЗАЦИИ: АНАЛИЗ И НАПРАВЛЕНИЯ РАЗВИТИЯ Внесенным в данный Реестр организациям, ориентированным на развитие си стем корпоративной социальной ответственности, оказываются различные виды государственной поддержки5. 4. КСО не является каким-то отдельным элемен том деятельности организации, а включена в ее стратегию развития и основную деятельность. 5. Результаты КСО должны быть оценены как самой организацией, так и ее заинтересованными сторонами (стейкхолдерами) и обществом в целом. В современной научной литературе, как прави ло, рассматривается четыре основных аспекта КСО: a) экономический аспект (заработная плата, пенсионные отчисления, социальные выплаты, по вышение квалификации сотрудников, взаимодей ствие с государством и др.); б) экологический аспект (использование сырья, воды, энергии, выбросы и отходы, сопровождающие производство продукции, работ, услуг); в) социальный аспект (соблюдение прав работ ников, обеспечение их безопасности и организация труда); г) информационный аспект (открытая отчет ность организации в области КСО) [2]. Из всего вышеперечисленного можно сделать вывод, что концепция КСО заключается в том, что экономическая эффективность производства не мо жет быть самоцелью бизнеса, она должна способ ствовать и гармоничному развитию общества в це лом». Корпоративная социальная ответственность яв ляется одним из приоритетных направлений дея тельности ПАО «НЕФАЗ», реализуемых как по отно шению к решению проблем территории присутствия (так называемая внешняя социальная ответствен ность), так и в отношении развития внутренней сре ды организации (внутренняя или корпоративная со циальная ответственность). Рассмотрим векторы реализации КСО. По мнению О.А. Сапрыкиной и М.В. Кургановой, «главными отличительными особенностями, харак теризующими понятие КСО, являются следующие характеристики. Большое внимание в организации уделяется развитию кадрового потенциала, одним из направ лений которого является оценка результативности работника [3]. 3 Утв. 29 декабря 2014 г. (действ. в ред. от 11.06.2021). 4 Публичное акционерное общество «НЕФАЗ» (ПАО «НЕФАЗ») входит в группу ПАО «КАМАЗ» и является крупнейшим в России заводом по производству спецнадстроек на шасси КАМАЗ. Производственные площади ПАО «НЕФАЗ» располо жены в г. Нефтекамск (Республика Башкортостан). В числе крупнейших акционеров ПАО «НЕФАЗ»: ПАО «КАМАЗ» (доля в уставном капитале составляет 50,02 %), Республика Башкортостан (доля в уставном капитале составляет 28,50 %). ПАО «НЕФАЗ» производит специальные надстройки на шасси автомобилей, пассажирские и вахтовые автобусы, трол лейбусы, электробусы, емкостно-наливную технику, бортовые прицепы и полуприцепы, запасные части. Выпускаемая ПАО «НЕФАЗ» продукция отвечает самым высоким международным стандартам. 5 Сибирская финансовая школа январь-март 1’2022 32 Менеджмент _______________ Кадровая политика ПАО «НЕФАЗ» направлена на создание сплоченного трудового коллектива, ко торый наилучшим образом способствовал бы со вмещению целей и приоритетов организации и его работников. Успех организации напрямую зависит от квалификации, ответственности и производи тельности труда работников [4]. КОРПОРАТИВНАЯ СОЦИАЛЬНАЯ ОТВЕТСТВЕННОСТЬ ГРАДООБРАЗУЮЩЕЙ И СИСТЕМООБРАЗУЮЩЕЙ ОРГАНИЗАЦИИ: АНАЛИЗ И НАПРАВЛЕНИЯ РАЗВИТИЯ расходы на оплату труда составили 2 478,4 млн руб., что на 21,2 % выше, чем в 2018 г.; средняя заработная плата в 2020 г. составила 39 677 руб., что на 3,2 % выше уровня 2019 г., в 2019 г. средняя заработная плата составила 38 439 руб., что на 15,9 % выше уровня 2018 г. Приоритетными направлениями кадровой поли тики ПАО «НЕФАЗ» являются мотивация персона ла, развитие персонала, социальная политика, ох рана труда. Остановимся на указанных направлени ях более подробно. Таблица 2 Сибирская финансовая школа январь-март 1’2022 Таблица 2 Анализ показателей подготовки и повышения квалификации работников ПАО «НЕФАЗ» за 2018–2020 гг., чел.* Наименование 2018 г. Уд. вес, % 2019 г. Уд. вес, % 2020 г. Уд. вес, % 1 2 3 4 5 6 7 Обучение рабочей профессии (подготовка работников) 24 1,0 1 0,02 70 1,06 Переподготовка работников по рабочей профессии 54 2,26 248 5,32 185 2,80 Обучение второй профессии 116 4,85 145 3,11 92 1,39 Развивающее обучение на курсах повышения квалификации работников 148 6,19 340 7,29 340 5,12 Повышение квалификации руководителей, специалистов и служащих (РСиС) 11 0,46 478 10,25 958 14,48 Анализ показателей подготовки и повышения квалификации работников ПАО «НЕФАЗ» за 2018–2020 гг., чел.* _ Менеджмент 133 1 2 3 4 5 6 7 Повышение квалификации в целях получения разряда по рабочей профессии 148 6,18 298 6,39 292 4,41 Подконтрольное внешнее обучение 27 1,13 59 1,26 1 568 23,70 Подконтрольное внутреннее обучение 427 17,85 2143 45,94 2 610 39,46 Проведение производственной практики студентов 252 10,53 247 5,28 217 3,28 Проведение экскурсий 590 24,67 528 11,32 182 2,75 Проведение лабораторно-практических работ для студентов 541 22,62 157 3,37 18 0,28 Проведение обучение мастеров по программе «Школа Мастера» 26 1,09 0 – 27 0,42 Сотрудничество с КНИТУ-КХТИ, обучение на базовой кафедре 28 1,17 21 0,45 56 0,85 Всего 2 392 100,0 4 665 100,0 6 615 100,0 * Источник: составлено авторами по материалам собраний акционеров ПАО «НЕФАЗ» за 2018-2020 годы. URL: https://nefaz.ru/shareholders/materialy-sobraniya-aktsionerov/ (дата обращения: 21.12.2021). Окончание табл. 2 * Источник: составлено авторами по материалам собраний акционеров ПАО «НЕФАЗ» за 2018-2020 годы. URL: https://nefaz.ru/shareholders/materialy-sobraniya-aktsionerov/ (дата обращения: 21.12.2021). * Источник: составлено авторами по материалам собраний акционеров ПАО «НЕФАЗ» за 2018-2020 годы. URL: https://nefaz.ru/shareholders/materialy-sobraniya-aktsionerov/ (дата обращения: 21.12.2021). В ходе анализа выявлено, что численность ра ботников, прошедших обучение, повышение квали фикации по сравнению с 2019 г. возросла на 1 950 чел. (для сравнения, в 2018 г. – на 4 223 чел.). При этом наиболее значимыми были признаны: под контрольное внутренне обучение (39,46 % от общего числа), подконтрольное внешнее обучение (23,7 %) и повышение квалификации руководителей, специа листов и служащих (14,48 %). Остальные показатели составили от 0,28 до 5,12 %. Если сопоставить пока затели 2018 г. и 2020 г. работников, которые прошли подконтрольное внешнее обучение, то можно убе диться, что численность выросла более чем на 22,57 %, или на 1 541 человек. Таблица 2 Так же рост произо шел на 14,02 % (или на 947 чел.) по повышению ква лификации руководителей, специалистов и служа щих, а также на 21,61 % (или на 2 183 чел.) по под контрольному внутреннему обучению. тельстве Российской Федерации и Республики Башкортостан, руководствуясь локальными норма тивными актами, ПАО «НЕФАЗ» ориентировано на удовлетворение запросов и интересов работников, а также повышение их благосостояния. ПАО «НЕФАЗ» включен в Реестр социально от ветственных работодателей Республики Башкорто стан. Корпоративная социальная ответственность в ПАО «НЕФАЗ» реализуется также и по таким на правлениям, как «Забота», «Молодежь», «Оздо ровление персонала», «Женщины», «Дети», «Не государственное пенсионное обеспечение», «Куль турно-массовая работа» (в табл. 3 приведены ре зультаты анализа реализации указанных направ лений КСО в 2018-2020 гг.). Данные таблицы 3 свидетельствуют, что затра ты на реализацию направлений корпоративной со циальной ответственности в ПАО «НЕФАЗ» в целом составили в 2018 г. 163 462,4 тыс. руб., в 2019 г. – 170 021,4 тыс. руб., в 2020 г. – 178 360,3 тыс. руб. Сопоставление расходов 2018 г. и 2019 г. позволило сделать выводы о том, что в 2019 г. расходы вырос ли более чем на 4,0 % (или на 6 559,0 тыс. руб.). Расходы в 2020 г. выросли на 8 338,9 тыс. руб. (или на 0,9 %) по сравнению с 2019 г. Таблица 3 Таблица 3 Анализ реализации направлений корпоративной социальной ответственности в ПАО «НЕФАЗ» за 2018–2020 гг.* Направления 2018 г., тыс. руб. 2019 г., тыс. руб. % к 2018 г. Откл. в сумме 2020 г., тыс. руб. % к 2019 г. Откл. в сумме 1. Направление «Забота», всего, в том числе: 55 494,5 50 558,5 91,1 –4 936,0 52 383,6 103,6 +1 825,1 – материальная помощь работникам, пенсионерам; 16 700,0 13 100,0 78,4 –3 600,0 13 724,6 104,8 +6 246 – помощь по семейным обстоятельствам, погребение; 3 060,6 3 135,4 102,4 +74,8 1 463,0 46,7 –1 672,4 – ко дню пожилых людей и инвалидов; 1 725,1 1 697,0 98,3 –28,1 1 787,8 105,4 +90,8 – в честь Дня Машиностроителя; 231,2 496,8 0,47 +265,6 452,0 91,0 –44,8 – с выходом на пенсию; 9 761,3 5 342,5 54,7 –4 418,8 4 936,6 92,4 –405,9 – денежная компенсация за проезд на работу 24 016,3 26 786,8 111,5 +2 770,5 30 019,6 112,1 +3 232,8 2. Направление «Молодежь» (всего): культурно- массовые, спортивно- оздоровительные мероприятия, спартакиады, велопробеги 8 947,9 8 585,4 95,9 –362,5 10 889,9 126,8 +2 304,5 3. Направление «Женщины»: компенсация матерям по уходу за ребенком в возрасте от 1,5 до 3 лет 34,8 27,9 80,2 –6,9 13,6 17,0 –66,6 4. Направление «Оздоровление персонала» (всего): 97 300,0 109 940,0 113,0 +12 640,0 114 100,0 103,8 +4 160,0 – компенсация на питание; 83 900,0 102 900,0 122,6 +19 000,0 103 300,0 100,4 +400,0 – санаторно-курортное лечение 13 400,0 7 040,0 52,5 –6 360,0 10 800,0 153,4 +3 760,0 5. Направление «Дети»: – лагеря, санатории; – новогодние подарки 1 400,0 700,0 50,0 –700,0 700,0 100,0 н/д 6. Негосударственное пенсионное обеспечение 285,2 209,6 73,6 –75,6 273,2 130,3 +63,6 Всего 163 462,4 170 021,4 104,0 +6 559,0 178 360,3 104,9 +8 338,9 * Источник: составлено авторами по материалам собраний акционеров ПАО «НЕФАЗ» за 2018-2020 годы. URL: https://nefaz.ru/shareholders/materialy-sobraniya-aktsionerov/ (дата обращения: 21.12.2021). Анализ реализации направлений корпоративной социальной ответственности в ПАО «НЕФАЗ» за 2018–2020 гг.* * Источник: составлено авторами по материалам собраний акционеров ПАО «НЕФАЗ» за 2018-2020 годы. URL: https://nefaz.ru/shareholders/materialy-sobraniya-aktsionerov/ (дата обращения: 21.12.2021). * Источник: составлено авторами по материалам собраний акционеров ПАО «НЕФАЗ» за 2018-2020 годы. URL: https://nefaz.ru/shareholders/materialy-sobraniya-aktsionerov/ (дата обращения: 21.12.2021). горячее питание в столовых получали 95 % от явочной численности работников, по отноше нию к 15 % от всего числа работников было орга низовано диетическое питание. Денежная ком пенсация на питание за 2018 г. 3. Социальная политика Социальная политика является неотъемлемой частью кадровой политики ПАО «НЕФАЗ» и на правлена на обеспечение конкурентных преиму ществ на рынке труда, создание эффективной си стемы социальной защиты работников. 4. Охрана труда р ру В ПАО «НЕФАЗ» большое внимание уделяется охране труда. Все рабочие места аттестованы и обе спечены необходимыми защитными средствами. В ПАО «НЕФАЗ» большое внимание уделяется охране труда. Все рабочие места аттестованы и обе спечены необходимыми защитными средствами. Для снижения уровня заболеваемости и повыше ния производительности труда реализовано направ ление «Оздоровление персонала», и это направление имеет наибольший удельный вес в 2020 г. – 63,97 % всех расходов. Расходы на «Оздоровление персона ла» составили 114 100,0 тыс. руб., или 103,8 % от об щей суммы расходов за 2020 г. По сравнению с пока зателями за 2018 г. расходы на «Оздоровление персо нала» увеличились на 16 800,0 тыс. руб., или 4,4 %. Согласно действующим нормативным актам, ра ботники проходят за счет завода периодический ме дицинский осмотр; осуществляется контроль за со стоянием условий и охраны труда; ведется система тическая работа по улучшению условий труда. ПАО «НЕФАЗ» – социально ответственная ор ганизация. Основываясь на трудовом законода Сибирская финансовая школа январь-март 1’2022 Менеджмент _ 134 Таблица 3 Таблица 3 руб., оказанная материальная помощь участникам Великой отечественной войны и ко Дню пожилых людей и инвалидов возросла на 90,8 тыс. руб. Выплата компенсации за проезд к месту работу и обратно общественным транспор том увеличилась в 2020 г. на 3 232,8 тыс. руб. (или на 12,1 %) по сравнению с 2019 г. Исходя из проведенного анализа рассмотрен ных направлений, можно сделать вывод, что корпо ративная социальная ответственность ПАО «НЕ ФАЗ» реализуется в целях регулирования социаль но-трудовых отношений, повышения мотивации персонала к обеспечению роста производительно сти и качества труда, сохранения и привлечения высококвалифицированных специалистов, поддер жания лояльности персонала и направлена на реа лизацию целей стратегического развития ПАО «НЕ ФАЗ». Противоположная картина сложилась по вы платам в честь Дня машиностроителя, расходы на проведение которого в 2020 г. составили 452,0 тыс. руб., что на 44,8 тыс. руб. меньше уров ня 2019 г. В ПАО «НЕФАЗ» выплачиваются выход ные пособия в связи с выходом на пенсию по старо сти – в 2020 г. на эти цели было потрачено 4 936,6 тыс. руб., что на 405,9 тыс. руб. меньше по сравнению с 2019 г. Самый низкий уровень испол нения по выплатам в 2020 г. составили расходы по семейным обстоятельствам (на погребение) – они снизились на 1 672,4 тыс. руб. (или на 46,7 %) по сравнению с 2019 г. Благодаря проведенным мероприятий, программ социального характера, ПАО «НЕФАЗ» является не однократным участником и призером регионального этапа Всероссийского конкурса «Российская органи зация высокой социальной эффективности» в номи нациях «За развитие кадрового потенциала в орга низациях производственной сферы», «За развитие социального партнерства в организациях производ ственной сферы», «За создание и развитие рабочих мест в организациях производственной сферы» и «За формирование здорового образа жизни в орга низациях производственной сферы». ПАО «НЕФАЗ» получил почетный диплом за первое место в конкур се на звание «Лучшая первичная профсоюзная орга низация Роспрофпром-Башкортостан» по итогам ра боты в 2020 году. Третье значимое направление КСО приходится на направление «Молодежь». За 2020 г. расходы составили 10 889,9 тыс. руб. (доля в общем объе ме затрат социальной политики – 6,1 %), что на 2 304,5 тыс. руб. больше, чем в 2019 г. А в 2019 г. затраты составили 8 585,4 тыс. руб., что на 362,5 тыс. руб. (или на 4,1 %) ниже уровня 2018 г. Основная задача направления «Молодежь» заклю чается в привлечении и закреплении в ПАО «НЕ ФАЗ» молодых работников, обеспечении возможно сти профессионального и карьерного роста, вовле чении в проектную деятельность и процессы обуче ния. Таблица 3 составила Особое внимание в системе корпоративной социальной ответственности в ПАО «НЕФАЗ» уде ляется добровольному медицинскому страхова нию и компенсации стоимости горячего питания. Что касается последнего, то надо отметить, что Сибирская финансовая школа январь-март 1’2022 Менеджмент 135 83 900 тыс. руб., за 2019 г. – 102 900 тыс. руб., за 2020 г. – 103 300 тыс. руб. 83 900 тыс. руб., за 2019 г. – 102 900 тыс. руб., за 2020 г. – 103 300 тыс. руб. ные мероприятия, благотворительные акции ко Дню знаний, массовые катания на коньках в Ледо вом дворце, организуются спартакиады, велопро беги. Общий объем расходов на эти мероприятия составил 8 947,9 тыс. руб. В ПАО «НЕФАЗ» организовано реабилитацион но-восстановительное лечение работников и их де тей по программе «Мать и дитя», а также работни ков, чей труд связан с вредными условиями труда. На это в 2018 г. было направлено 13 400 тыс. руб., в 2019 г. – 7 040 тыс. руб., а в 2020 г. – 10 800 тыс. руб. Сумма финансирования по направлению «Жен щины» в 2020 г. составила 13,6 тыс. руб., расходы сокращены на 66,6 тыс. руб., или на 83 % по срав нению с 2019 г. Организация детского отдыха – один из приори тетов социальной политики ПАО «НЕФАЗ». В рам ках направления «Дети» в 2018 г. организован от дых 285 детей работников в детских оздоровитель ных лагерях. На корпоративные новогодние подар ки детям работников ПАО «НЕФАЗ» выделено 1 400 тыс. руб., приобретено 3 500 подарков. В 2019 г. сумма финансирования на эти расходы сократилась и составила 700,0 тыс. руб., что на 50 % ниже уровня 2018 г. В 2020 г. по данному на правлению расходы остались на уровне 2019 г. Второе значимое направление корпоративной социальной ответственности приходится на на правление «Забота», в 2020 г. составило 29,4 % от общей суммы затрат (178 360,3 тыс. руб.), в 2018 г. – 33,9 % (больше на 4,2 %, чем в 2019 г.). По сравнению с 2019 г. затраты в 2020 г. увеличены на 6 246,0 тыс. руб., или на 3,6 %. Другим значимым направлением корпоратив ной социальной ответственности в ПАО «НЕФАЗ» является направление «Забота», в рамках которого оказывается материальная помощь как работникам завода, так и пенсионерам, оказавшимся в трудной жизненной ситуации (на оплату операций). Ее раз мер в 2020 г. по сравнению с 2019 г. увеличился на 6 246,0 тыс. Таблица 3 Проводятся культурно-массовые мероприятия, в том числе фестивали, спортивно-оздоровитель Анализ реализации внешней социальной ответ ственности ПАО «НЕФАЗ» показывает, что органи зация принимает активное участие в решении со циальных проблем территории. Городской округ город Нефтекамск Республики Башкортостан относится к категории монопро фильных муниципальных образований, в которых имеются риски ухудшения социально-экономиче ского положения (категория 2), согласно Перечню монопрофильных муниципальных образований январь-март 1’2022 январь-март 1’2022 Сибирская финансовая школа Менеджмент _ 136 Российской Федерации (моногородов), утвержден ному распоряжением Правительства Российской Федерации от 29 июля 2014 г. № 1398-р6. Среди основных проблем можно выделить проблему с демографией и благоустройством города. анализа проблем и принятии решений о финанси ровании проектов первостепенной важности для Территории опережающего социально-экономиче ского развития «Нефтекамск». Партнерство в решении социальных задач развития территории позволит решать проблемы, требующие значительных финансовых вложений (порой недоступных для отдельных организаций), творчества, инициативы и «свежего» видения, а также широких связей предпринимательской сети участников интеграционной структуры. Помимо ПАО «НЕФАЗ» в Нефтекамске функ ционируют следующие организации: ООО «Не фтекамский механический завод», ГК «ИНТЕХ», ООО «АрсХимСнаб», АО «Амзинский лесокомби нат», ООО «Нефтекамский завод нефтепромысло вого оборудования» – подразделение в составе АФК «Система», ООО «Нефтекамский трубный за вод», ООО «Нефтекамский машиностроительный завод», ООО «Завод Высоковольтного Оборудова ния «Электро-Н», ООО «Нефтекамский электро механический завод», АО «Нефтекамская кожга лантерейная фабрика», Текстильная фабрика ООО «Малыш», АО «Искож», Завод металлокров ли ООО «Призма». Объединенное целью гармоничного развития социальной среды Территории опережающего со циально-экономического развития «Нефтекамск», в рамках корпоративной социальной ответствен ности участников, партнерство станет объедине нием возможностей. Литература В соответствии с вышеупомянутым поста новлением Правительства РФ № 129 «О создании территории опережающего социально-экономиче ского развития «Нефтекамск», ТОСЭР создана в границах моногорода Нефтекамск. Данный ста тус обеспечивает функционирование особого пра вового режима предпринимательской деятельно сти, предусматривающего предоставление префе ренций организациям-резидентам ТОСЭР в части освобождения от налога на имущество, снижения ставок по налогу на прибыль и страховым взносам. 1. Другова Г.А. Проблема взаимодействия в системе моногород – градообразующее предпри ятие // Молодой ученый. 2017. № 19 (53). С. 123– 127. 1. Другова Г.А. Проблема взаимодействия в системе моногород – градообразующее предпри ятие // Молодой ученый. 2017. № 19 (53). С. 123– 127. 2. Курганова М.В., Сапрыкина О.А. Корпора тивная социальная ответственность: понятие, сущность, эволюция теорий // Сибирская финан совая школа. 2020. № 1 (137). С. 18–26. 2. Курганова М.В., Сапрыкина О.А. Корпора тивная социальная ответственность: понятие, сущность, эволюция теорий // Сибирская финан совая школа. 2020. № 1 (137). С. 18–26. 3. Морозов А.А. Корпоративная социальная от ветственность в контексте социально-экономиче ского развития общества // Российское предприни мательство. 2018. Том 19, № 10. С. 3119–3128. Это дает основания полагать, что список рези дентов в границах моногорода Нефтекамск расши рится, что даст возможность к расширению рабо чих мест и увеличению экономического потенциа ла города. 4. Веснин В.Р., Кафидов В.В. Корпоративное управление: учебник. М.: Инфра-М, 2018. 272 с. 5. Царитова К.Г. Корпоративная социальная ответственность в системе управления устойчи вым развитием организации: учеб. пособие. М.: Русайнс, 2020. 73 с. Несмотря на то, что градообразующей и систе мообразующей организацией является ПАО «НЕ ФАЗ», видится целесообразным создание обще ственной интеграционной структуры, в состав ко торой войдут вышеперечисленные организации Нефтекамска, представители муниципалитета, инициативные группы горожан для совместного 6. Аникеева О.П. Социальная ответственность бизнеса и международная конкурентоспособность: учебник и практикум для вузов. 2-е изд., перераб. и доп. М.: Юрайт, 2020. 169 с. Сведения об авторах Назарова Эльвира Руслановна – магистрант Уфимского филиала ФГОБУ ВО «Финансовый университет при Правительстве Российской Федерации» (Финансовый университет), Уфа, Россия. e-mail: alru8@mail.ru Шеина Анастасия Юрьевна – канд. экон. наук, доцент кафедры экономики, менеджмента и маркетинга Уфимского филиала Финансового университета, Уфа, Россия. e-mail: ms.sheina90@mail.ru Шеина Анастасия Юрьевна – канд. экон. наук, доцент кафедры экономики, менеджмента и маркетинга Уфимского филиала Финансового университета, Уфа, Россия. e-mail: ms.sheina90@mail.ru Брусенцова Лилия Самимовна – канд. социол. наук, доцент кафедры экономики, менеджмента и маркетинга Уфимского филиала Финансового университета, Уфа, Россия. e-mail: lbrusbox@mail.ru Брусенцова Лилия Самимовна – канд. социол. наук, доцент кафедры экономики, менеджмента и маркетинга Уфимского филиала Финансового университета, Уфа, Россия. Литература e-mail: lbrusbox@mail.ru 6 Распоряжение Правительства РФ от 29 июля 2014 года N 1398-р «Перечень монопрофильных муниципальных об разований Российской Федерации (моногородов)» URL: http://government.ru/docs/all/92337/ (дата обращения 18.12.2021). январь-март 1’2022 Менеджмент 137 CORPORATE SOCIAL RESPONSIBILITY OF A CITY-FORMING AND SYSTEM-FORMING ENTERPRISE: ANALYSIS AND DIRECTIONS OF DEVELOPMENT Socialnaja otvetstvennost biznesa and mezhdunarodnaja konkurentosposobnost [Social responsibility of business and international competitiveness], Moscow: Yurait, 2020, 169 p. 6. Anikeeva O.P. Socialnaja otvetstvennost biznesa and mezhdunarodnaja konkurentosposobnost [Social responsibility of business and international competitiveness], Moscow: Yurait, 2020, 169 p. CORPORATE SOCIAL RESPONSIBILITY OF A CITY-FORMING AND SYSTEM-FORMING ENTERPRISE: ANALYSIS AND DIRECTIONS OF DEVELOPMENT E. Nazarova Financial University under the Government of the Russian Federation (Financial University), Ufa, Russia A. Sheina Ufa Branch of the Financial University, Ufa, Russia L. Brusentsova Ufa Branch of the Financial University, Ufa, Russia City-forming and system-forming organizations play an important role in the socio-economic development of the region, local and regional budgets are formed due to their tax revenues, they provide jobs to residents of the region, form the social environment and significantly affect the quality of life of the population. One of the basic elements of such organizations is the corporate social responsibility system, the effective functioning of which ensures not only sustainable business development, but also improvement of the social environment of the territory and improvement of the quality of life of its residents. This article discusses the experience of implementing the main directions of the corporate social responsibility system in PJSC "NEFAZ" (part of the group of PJSC "KAMAZ"). Particular attention is paid to the priority areas of the personnel policy of PJSC NEFAZ (staff motivation, staff development, social policy, labor protection) and its impact on the strategic development of the organization in question as a resident of the territory of advanced socio-economic development, a single-industry municipality (single-industry town) "Neftekamsk". Key words: city-forming and system-forming organizations, the territory of advanced socio-economic development, PJSC "NEFAZ", corporate social responsibility. Key words: city-forming and system-forming organizations, the territory of advanced socio-economic development, PJSC "NEFAZ", corporate social responsibility. References 4. Vesnin V.R., Kafidov V.V. Korporativnoe upravlenie [Corporate Governance], Moscow: INFRA-M, 2018, 272 p. 1. Drugova G.A. The problem of interaction in the system of single-industry city-city-forming enterprise, Molodoi uchenyi, 2017, No.19 (53), pp. 123-127. (In Russ.) 5. Tsaritova K.G. Korporativnaja social'naja otvetstvennost' v sisteme upravlenija ustojchivym razvitiem organizacii [Corporate social responsibility in the management system of sustainable development of the organization], Moscow: Rusains, 2020, 73 p. 5. Tsaritova K.G. Korporativnaja social'naja otvetstvennost' v sisteme upravlenija ustojchivym razvitiem organizacii [Corporate social responsibility in the management system of sustainable development of the organization], Moscow: Rusains, 2020, 73 p. 2. Kurganova M.V., Saprykina O.A. Corporate social responsibility: concept, essence, theory evolution, Sibirskaya finansovaya shkola, 2020, No. 1 (137), pp. 18–26. (In Russ.) 3. Morozov A.A. Corporate social responsibility in the context of social and economic development of so ciety, Rossijskoe predprinimatel'stvo, 2018, Tom 19, No. 10, pp. 3119-3128. (In Russ.) 6. Anikeeva O.P. About the authors El'vira R. Nazarova – graduate student of the Ufa branch of Financial University under the Government of the Russian Federation (Financial University), Ufa, Russia. e-mail: alru8@mail.ru Anastasiya Yu. Sheina – Candidate of Economic Sciences, Associate Professor of the Department of Economics, Management and Marketing Ufa Branch of Financial University, Ufa, Russia. e-mail: ms.sheina90@mail.ru Anastasiya Yu. Sheina – Candidate of Economic Sciences, Associate Professor of the Department of Economics, Management and Marketing Ufa Branch of Financial University, Ufa, Russia. e-mail: ms.sheina90@mail.ru Liliya S. Brusentsova – Candidate of Sociological Sciences, Associate Professor of the Department of Economics, Management and Marketing, Ufa Branch of Financial University, Ufa, Russia. e-mail: lbrusbox@mail.ru Сибирская финансовая школа январь-март 1’2022 Сибирская финансовая школа январь-март 1’2022
https://openalex.org/W4242772392	https://www.qeios.com/read/9GN6D5/pdf	English	null	Hepatitis B Virus Surface Antibody	Definitions	2,020	cc-by	71	Qeios · Definition, February 7, 2020 Open Peer Review on Qeios Open Peer Review on Qeios Hepatitis B Virus Surface Antibody National Cancer Institute Qeios ID: 9GN6D5 · https://doi.org/10.32388/9GN6D5 Source National Cancer Institute. Hepatitis B Virus Surface Antibody. NCI Thesaurus. Code C62796. An antibody produced by B lymphocytes to the hepatitis B surface antigen as part of the immune response to hepatitis B viral infection. Qeios ID: 9GN6D5 · https://doi.org/10.32388/9GN6D5 1/1
https://openalex.org/W1966490056	https://storage.googleapis.com/jnl-su-j-tbcpm-files/journals/1/articles/176/submission/proof/176-1-2262-1-10-20221031.pdf	English	null	Ultrafine particles over Eastern Australia: an airborne survey	Tellus. Series B, Chemical and physical meteorology	2,015	cc-by	14,821	S C A M S C A M ABSTRACT Ultrafine particles (UFP) in the atmosphere may have significant impacts on the regional water and radiation budgets through secondary effects on cloud microphysics. Yet, as these particles are invisible for current remote sensing techniques, knowledge about their three-dimensional distribution, source strengths and budgets is limited. Building on a 40-yr-old Australia-wide airborne survey which provides a reference case study of aerosol sources and budgets, this study presents results from a new airborne survey over Eastern Australia, northern New South Wales and Queensland. Observations identified apparent changes in the number and distribution of major anthropogenic aerosol sources since the early 1970s, which might relate to the simultaneously observed changes in rainfall patterns over eastern Queensland. Coal-fired power stations in the inland areas between Brisbane and Rockhampton were clearly identified as the major sources for ultrafine particulate matter. Sugar mills, smelters and shipping along the coast close to the Ports of Townsville and Rockhampton were comparable minor sources. Airborne Lagrangian plume studies were applied to investigate source strength and ageing properties within power station plumes. Significant changes observed, compared to the measurements in the 1970s, included a significant increase in the number concentration of UFP related to coal-fired power station emissions in the sparsely populated Queensland hinterland coincident with the area with the most pronounced reduction in rainfall. Keywords: power station, emission, coal, aerosol, precipitation, regional climate impact tion levels for remote (background) and polluted continen- tal and maritime air masses. However, the local and re- gional variability is high due to local sources or long range transport (Bigg and Turvey, 1978; O’Dowd et al., 2009). Particles in a size range from a few nm to 100 nm have lifetimes of a few hours to about a week in the atmos- phere before they are removed by dry or wet deposition (Jaenicke, 1978; Heintzenberg, 1989). Within this time they can be transported over hundreds of kilometres. Thus linking UFP to climate processes requires coupling ob- servations with transport models and understanding of particlecloud interaction processes. Tellus B 2015. # 2015 W. Junkermann and J. M. Hacker. This is an Open Access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), allowing third parties to copy and redistribute the material in any medium or format and to remix, transform, and build upon the material for any purpose, even commercially, provided the original work is properly cited and states its license. 1 Citation: Tellus B 2015, 67, 25308, http://dx.doi.org/10.3402/tellusb.v67.25308 Corresponding author. email: Wolfgang.Junkermann@kit.edu Responsible Editor: Karle Ha¨ meri, University of Helsinki, Finland. Ultrafine particles over Eastern Australia: an airborne survey By WOLFGANG JUNKERMANN1,2 and JORG M. HACKER2, 1Karlsruhe Institute of Technology, IMK-IFU, Garmisch-Partenkirchen, Germany; 2School of the Environment, Flinders University, Adelaide, Australia (Manuscript received 26 June 2014; in final form 30 March 2015) (Manuscript received 26 June 2014; in final form 30 March 2015) on the material for any purpose, even commercially, provided the original work is properly cited and states its license. Citation: Tellus B 2015, 67, 25308, http://dx.doi.org/10.3402/tellusb.v67.25308 W. JUNKERMANN AND J. M. HACKER In this study, we evaluate current and historical detailed airborne observations of UFP and source identification with transport models over a region in Australia, to consider how they might be associated with significant temporal trends in precipitation distribution. In previous investigations over Western Australia (WA), the authors found observational evidence that locally emitted UFP are able to influence regional CCN number concentrations, cloud microphysics and rainfall patterns, but were not able to quantitatively separate aerosol-related processes from those caused by land use changes (Junkermann et al., 2009; Nair et al., 2011). In WA negative rainfall trends have been observed since about 1970 despite a concurrent increase in absolute water vapour content (dewpoint) in the atmosphere (Lucas, 2010; BOM, 2014). In this case, the increase of available total water in the atmosphere did not lead to more rainfall as may be expected by a higher turnover rate of water in a warmer atmosphere. The observed reduction in rainfall may instead be caused by a more local physical effect such as a regional- scale suppression of rainfall by enhanced CCN. To come to a firm conclusion, though, would require considerably more observations, analysis and modelling. The timing of the rainfall decline, however, coincides with the initiation of increased ultrafine particle production related to anthropo- genic activities, which might therefore be a significant factor for regional climate change. Due to the importance of rainfall variability for ecosystem functioning and agricultural food and feed production, the interaction between aerosols and clouds has been a matter of interest for many decades. Detailed investigations of aerosols (CCN) and clouds and their interaction in the natural atmosphere were already performed by Twomey and co-workers (Twomey, 1960, 1974; Twomey and Warner, 1967; Warner, 1968). Yet, many questions still remain unclear in some cases even whether certain aerosol effects lead regionally to more or to less precipitation. Individual process studies sometimes revealed contradictory results (Hobbs et al., 1970; Rosenfeld, 2000; Rosenfeld et al., 2006, 2008; Bigg, 2008; Qian et al., 2009). But, the majority of the studies report a delayed raindrop production and subsequent regional reduction of rainfall with increasing particle numbers. It is clear, though, that despite the high importance for the local to regional water cycle, even our current knowledge about source strengths of UFP, their contribution to CCN budgets, and their three-dimensional distribution is still rather limited (Kerminen et al., 2012; Andreae, 2013). W. JUNKERMANN AND J. M. HACKER 2 three, decades. Such long time series for aerosols are available for the South Pole, Samoa and for Cape Grim (since 1957), with a few shorter data sets for European and US locations, but not for continental Australia (Gras, 1995; Bigg, 2008; Asmi et al., 2013). they do not interact with radiation but might play a role for heterogenic chemical reactions affecting for example the budget of nitrogen oxides (Heikes and Thompson, 1983). Particles in size ranges larger than 40 nm can act as cloud condensation nuclei (CCN) (Twomey, 1977; Andreae, 2009; Kerminen et al., 2012). Ageing and growth to CCN sizes normally requires less than 10 hours (Laaksonen et al., 2005). As such they have the potential to modify the regional water cycle. A larger number of CCN competing for the available condensable water leads to smaller droplet sizes and increases the brightness of clouds (Twomey, 1974). Moreover, as smaller droplets need more time to coagulate to raindrops, cloud lifetime can also be affected (Albrecht, 1998). Slowing down raindrop production may significantly delay or even suppress precipitation from certain cloud types (Lee et al., 1980; Givati and Rosenfeld, 2004; Teller and Levin, 2006; Konwar et al., 2010). As a result the temporal, spatial and intensity of rainfall and its redistribution might change. In case the minimum droplet diameter for raindrop formation is not reached, an initial local reduction of rainfall can be expected within and downwind of the source areas of UFP, but as the water remains in the atmosphere either as water vapour or as sub-raindrop size cloud droplets rain will be formed elsewhere under more favourable conditions potentially leading to an increase of vigorous rain (Li et al., 2011; Rosenfeld et al., 2014a). Most of the current aerosol (CCN) cloud interaction studies are thus based on numerical simulations (Lohmann and Feichter, 2005; Teller and Levin, 2006; Spracklen et al., 2008, 2011; Merikanto et al., 2009; Khain, 2009). In these simulations, the major uncertainties in estimating the effects on CCN availability are the unknown size and number size distribution of regional scale particulate emissions, their historic development and the partitioning between primary emissions and secondary production (Lee et al., 2013). Another still challenging aspect of such simulations is how to include local point sources into the model grid cells (Stevens and Pierce, 2013). 1. Introduction There is clear evidence that the distribution of ultrafine aerosols (B100 nm) in the planetary boundary layer (PBL) is highly heterogeneous as a consequence of their compara- tively short lifetime and the high spatial, temporal and intensity variability of their sources. While fine (100 nm to 2.5 mm) and coarse (2.510 mm) particles are detectable from satellites under cloud-free conditions and therefore allow an estimation of the regional to global distribution, the distribution of ultrafine particles (UFP) (B100 nm) is more uncertain because they are not directly measurable using remote sensing techniques (Rosenfeld et al., 2014b). However, in situ techniques are robust. After Aitken (1888) developed the first particle counter, a large number of observations are now available to define typical concentra- Although too small to be visible and thus not directly interacting with shortwave radiation, UFP can have im- portant implications within the climate system. Produced either from gas-to-particle conversion in the atmosphere, or directly emitted from various sources, they rapidly grow from an initial nm-size at rates in the range of 210 nm/h (Kulmala and Kerminen, 2008; Suni et al., 2008; Andreae, 2013; Kulmala et al., 2013). Within the first hours of growth, (page number not for citation purpose) W. JUNKERMANN AND J. M. HACKER 2. Flight strategy and instrumentation From 24 to 30 August 2012, UFP and related meteorolo- gical parameters were surveyed along a flight track from Adelaide, SA via Broken Hill and Bourke in NSW to Chinchilla in Queensland (Qld) (Fig. 1). From Chinchilla to Rockhampton, several Lagrangian flight patterns were flown in the plumes of coal-fired power stations for a more detailed regional investigation measuring particle numbers, size distributions and in-situ winds in several cross-sections up to 200 km downwind from the sources. A long-distance flight followed along the Queensland coast and hinterland up to Cape Flattery about 40 km north of Cooktown in Far North Queensland (FNQ). The complete aerosol ins- trumentation on-board of the research aircraft consisting of scanning mobility particle sizer (SMPS), condensation particle counter (CPC) and OPC (see below) was then relo- cated into a vehicle, and ground-based mobile measurements were made along a similar route in the opposite direction starting in Mareeba (Qld) on 31 August and finishing in Adelaide on 12 September 2012. These measurements mainly focused on background particle numbers and size distributions along the transect but also included several days of measurements at fixed locations (Myella Farm in the Queensland outback and coastal locations at Townsville and Lennox Head, both affected by shipping emissions). The data from the coast are not presented here. A further direct observation of coal-fired power stations in Queensland emitting UFP was made by one of the authors in 2011. High number concentrations of UFP in the vicinity of Stanwell and Callide power stations (see Figs. 1 and 4) were found using a single condensation nucleus counter (TSI, model 3670) mounted on the back seat of a personal car (for details see below). With southsouth-westerly winds of 2 m s1, derived from Hybrid Single-Particle Lagrangian Integrated Trajectory Model (HYSPLIT) (Draxler and Rolph, 2013), 80009000 particles were measured directly upwind, 50 000 particles cm3 at the lookout close to emission height of the Callide smoke stack and 2 km downwind, and 35 000 particles cm3 on the ground 15 km downwind of the power station. Table 1 and Fig. 1 give an overview of the flight/road tracks and patterns and regional survey locations. The background colours of the map in Fig. 1 indicate the rainfall trends for the time interval from 1970 to 2010 with brown colours showing negative, green colours positive trends (source: Australian Bureau of Meteorology, BOM, 2014). ULTRAFINE PARTICLES OVER EASTERN AUSTRALIA ULTRAFINE PARTICLES OVER EASTERN AUSTRALIA 3 3 however, the area affected by rainfall change in Queensland is not subject to large-scale changes in land use and surface roughness. Therefore, the apparent rainfall changes are less likely explainable by such land surface changes. It is thus interesting to investigate whether anthropogenic re- gional ultrafine aerosol emissions may also have signifi- cantly increased over Queensland within the 40 yr of rainfall decline since 1970, which would then strengthen the hypothesis, initially raised by Bigg (2008), that changes in rainfall distribution are indeed linked to a change in aerosol abundance. sources of ultrafine aerosols and (2) their possible impor- tance to the overall ultrafine particle budget. The remote Queensland outback serves in our study as a natural laboratory with low background concentrations and a rather limited number of anthropogenic sources. For the current study, we compared our observations to an air- borne survey of aerosol sources over Australia in the early 1970s, documenting aerosol sources and typical concentra- tion levels all over Australia within the first years of acceleration of anthropogenic climate change (Bigg and Turvey, 1978). Sources of cloud-active particles over Queensland could be gas to particle conversion (nucleation) from local emissions over the forest systems or over the Great Barrier Reef (GBR) both related to biogenic activity, anthropo- genic emissions such as vehicle traffic, shipping or industry or rather a combination of both (Kulmala et al., 2013). As no major land-use changes have occurred in these areas since the 1970s, a change in the former two possible contributions is unlikely whereas there were substantial changes to be expected in the latter ones. For example fossil fuel burning industries as they are installed in the area have been identified previously to be strong emitters of primary UFP which may then grow into CCNs (Bigg and Turvey, 1978; Ayers et al., 1979; Junkermann et al., 2011a, 2011b). Results from satellite remote sensing analyses of cloud properties over Australia by Rosenfeld (2000), Rosenfeld et al. (2006) indicate smaller cloud droplets in clouds affected by the plumes of such anthropogenic sources in South Australia (SA), Victoria and New South Wales (NSW), which means that they are indeed sources for cloud-active particles and that these emissions are then transported on a regional scale. W. JUNKERMANN AND J. M. HACKER The Australian Bureau of Meteorology (BOM, 2014) is reporting similar trends in water vapour and rainfall patterns for both, the Western Australian (WA) wheat belt and an area about 300 km wide along the North-Eastern Australian (Queensland) coast extending from Brisbane for about 1000 km to the north towards Townsville. In both regions, temperature and dewpoint have increased since about 1970 while precipitation and the number of rainfall days have decreased. In Queensland, this trend is even more pronounced than in WA. In contrast to Western Australia, As aerosol size and number distributions, clouds and precipitation are all highly variable, the interaction of aerosols, clouds and rainfall is difficult to investigate using observational data from short term field or process studies. To derive statistical significant trends and possible correla- tions would require time series covering several, at least ULTRAFINE PARTICLES OVER EASTERN AUSTRALIA 5 Table 1. Overview of ﬂight- and vehicle-based measurements Date Flight track Flight hours/km Remarks 24.8.2012 Broken Hill Bourke 2:40 480 km Ferry SA-QLD with plume 25.8.2012 Bourke Chinchilla 3:40 580 km Ferry SA-QLD with plume 25.8.2012 Chinchilla local 1:20 Local plume study sunset 26.8.2012 Chinchilla Tangool 4:05 450 km Regional scale plume study high UV 27.8.2012 Thangool Rockhampton 4:10 570 km Regional scale plume study high UV 28.8.2012 Rockhampton Proserpine 4:20 620 km Plume/regional transect 29.8.2012 Proserpine Mareeba 3:50 500 km Regional transect, background 30.8.2012 Mareeba Cape Flattery and back 3:55 500 km Regional background Rainforest/sea Vehicle tracks 5.9.2012 Townsville Myella 9:00 700 km Regional transect, remote/coal area 6.9.2012 Myella 20:00 Local/rural, coal area 7.9.2012 Myella Biloela to Hervey-Bay 8:00 500 km Coal area 9.9.2012 12.9.2012 Lennox-Head Adelaide 28:00 1800 km Northern NSW transect (background) Table 1. Overview of ﬂight- and vehicle-based measurements data in spite of the given low time resolution of some of the instruments (see below). and compared and supported by the in-situ wind mea- surements from the aircraft. To determine the vertical extent of the PBL, as well as its vertical mixing state, occasional short profiles into the free troposphere were flown. The top of the PBL can be identified from a rapid change in total particle (10 nm) count rates, temperature and dewpoint (Bigg and Turvey, 1978; Junkermann et al., 2009, 2011a). The flight sections from Adelaide to Chinchilla across inland NSW and Queensland (Qld), as well as along the coast north of Cairns were planned to quantify background conditions and possible natural production of ultrafine particulate matter, including nucleation mode aerosols from biogenic emissions from the coastal rainforest, the eucalyptus forests of the inland areas or from the GBR. Based on the findings by Kulmala et al. (2004) that biogenic nucleation typically takes place in the late morning hours and continues for several hours, all flights with the excep- tion of one evening flight at Kogan Creek were scheduled for take-off between 10 AM and 11 AM local time and flight-times of about 4 hours. This is also the time of day when airborne measurements in the mixed boundary layer are comparable to ground-based measurements. 2. Flight strategy and instrumentation The airborne platform used was a motorglider, one of Flinders University’s two ECO-Dimonas (www.airbornere search.org.au). This aircraft is specifically suited to fly at low altitudes in the PBL with a cruising speed of about 90 kts (45 m/s) and is also able to climb within a few minutes to higher levels into the free troposphere. The ability to cruise at low airspeed yields high spatial resolution Based on these initial results and further observations of high ultrafine particle concentrations downwind of other coal-fired power stations (Junkermann et al., 2011b) we performed an airborne study along the Queensland coast and over the coal mining area of the Queensland hinterland to investigate (1) the source strength of individual major W. JUNKERMANN AND J. M. HACKER 4 BRISBANE CAPE FLATTERY MAREEBA Mt. ISA BILOELA CHINCHILLA GLADSTONE KOGAN CREEK CALLIDE STANWELL TOWNSVILLE SYDNEY TREND IN ANNUAL TOTAL RAINFALL mm/10y COLLINSVILLE MILLMERAN ROCKHAMPTON TARONG MYELLA 50.0 40.0 30.0 20.0 15.0 10.0 5.0 0.0 –5.0 –10.0 –15.0 –20.0 –30.0 –40.0 –50.0 G R E A T B A R R I E R R E E F Map of Eastern Australia showing the airborne (blue) ﬂight patterns and ground vehicle-based measurements (brown red power stations are marked with a symbol and red text, the daily ﬂight sections and airﬁelds are identiﬁed by black show the main wind direction during the survey days. Background colour contours show the decadal trend of rainfall betw 10, brown decreasing, green increasing annual rainfall (data provided by the Australian Bureau of Meteorology). Fig. 1. Map of Eastern Australia showing the airborne (blue) ﬂight patterns and ground vehicle-based measurements (brown, dotted). Coal-ﬁred power stations are marked with a symbol and red text, the daily ﬂight sections and airﬁelds are identiﬁed by black text. The arrows show the main wind direction during the survey days. Background colour contours show the decadal trend of rainfall between 1970 and 2010, brown decreasing, green increasing annual rainfall (data provided by the Australian Bureau of Meteorology). ULTRAFINE PARTICLES OVER EASTERN AUSTRALIA W. JUNKERMANN AND J. M. HACKER 6 back-country roads. Interference from other cars is limited and can easily be omitted from the data set. Possible dis- turbances from other cars or from the own exhaust would be directly visible from enhanced particle numbers in the CPC and OPC and the typical size distribution of fresh car emissions, resembling a characteristic increase in the lowest SMPS size bins below 10 nm. width of particle plumes (from total number concentration) to identify strong sources in larger distances based on an independent study of plume dispersion (Bigg et al., 1978) and were further limited by using a single Pollack particle counter only without size information. The size resolving technology now available (fast SMPS, GRIMM, Ainring, Germany) allows a far more detailed direct source identi- fication and allocation on a regional scale especially in the first few hours after emission. The aerosol and meteorological instrumentation was previously used for airborne ultrafine aerosol measurements over Western Australia in 2006/2007 (Junkermann et al., 2009) and 2011. Since 2007 it is also standard instrumen- tation on the KIT microlight aircraft (Junkermann, 2005; Junkermann, 2011a, 2011b). The aircraft was equipped with a combination of meteorological sensors and particle instrumentation, con- sisting of a BAT-probe (Hacker and Crawford, 1999) for high-resolution wind and 3D-turbulence, air temperature, humidity, and pressure measurements, a GRIMM (Ainring, Germany) Wide Range Aerosol Spectrometer (WRAS) for size distribution measurements from 4.5 nm to 20 mm and an additional totally independent fast (1 s) condensa- tion particle counter (CPC) with a 10 nm counting threshold (TSI 3010). The WRAS SMPS consists of a Vienna DMA and a GRIMM CPC3 (model 5403) measuring the ultrafine particle size distribution from 4.5 nm to 350 nm within 2 min, giving approximately 2.5 km horizontal resolution. The WRAS-Optical Particle Spectrometer (OPS, GRIMM, model 1.108) which measures from 300 nm to 20 mm has a time resolution of 6 s. The instruments were mounted on the aircraft in underwing pods, allowing installation of very short air intake lines. Forward pointing stainless steel tips with a diameter in accordance with isokinetic sampl- ing requirements at the nominal cruise speed of the aircraft were fitted at the tip of the intakes. (Junkermann et al., 2011a). W. JUNKERMANN AND J. M. HACKER Particle numbers agree within 5% measured with the CPC and the SMPS as long as concentrations are constant during the 2 min required for an SMPS scan and the majority of the particles is larger than the lower cut-off size (10 nm) of the CPC. The CPC thus serves also as an online high time resolution reference instrument for the SMPS (Wiedensohler et al., 2012). SMPS data were analysed according to ISO 15900:2009 (ISO, 2009) includ- ing multiple charge correction. All aerosol and selected meteorological data are displayed on an online display in the aircraft. ULTRAFINE PARTICLES OVER EASTERN AUSTRALIA For four days, the flight route basically followed the coastline north of Rockhampton, either on-shore and a few km further in- land over the forests or off-shore over the sea, to investigate background concentrations and size distributions over dif- ferent possible source regions. The depth of the mixed layer can be derived in-situ with an accuracy of 50 m or about 5% with a PBL height of 1000 m. Although this depth usually still increases during the earlier part of the day, it becomes near constant from about noon onwards (O’Dowd et al., 2009). The accuracy of the wind measurements is about 90.3 m/s (Hacker and Crawford, 1999; Metzger et al., 2011). Optimum meteorologi- cal conditions for flying budget studies are usually occurring around the hours of solar noon (11:0014:00) and with mean wind speeds of about 510 m/s. Under these condi- tions the uncertainty for emission rate estimates is in the order of 920 to 940%, mainly depending on the accuracy of the transport velocity measurement, the estimation of the average concentration within the plume cross-section and the PBL height (Lelieveld et al., 1987). Based on particle size distributions and HYSPLIT back- trajectories it was possible to identify individual sources of particles by inspecting the modes of the ultrafine particle size distribution (Junkermann et al., 2011a). This is different to the approach of Bigg and Turvey (1978), who used the Lagrangian flight patterns for emission rate estimates were flown downwind of several Queensland power stations supported by real-time readouts from the sensors serving for flight guidance. Air mass origin and history was derived from HYSPLIT back trajectories (Draxler and Rolph, 2013) 3. Results 3.1. Flight sector Broken Hill to Chinchilla (SA/NSW/QLD) 3.1. Flight sector Broken Hill to Chinchilla (SA/NSW/QLD) Using a vehicle for ground based surveys is possible with the very low traffic intensity on many of the Australian ULTRAFINE PARTICLES OVER EASTERN AUSTRALIA 7 7 0 2000 4000 6000 8000 5 36 266 5 36 266 21 124 dN/dln(dp) [cm–3] Diameter [nm] a) b) MAX (13200 /9) 6 11 15 32 29 Fig. 2. (a) Average size distribution of ultraﬁne particles between Broken Hill and Bourke (NSW) (averages over more than 100 size distributions). Blue bars: Average size distribution of the background particle size distributions (55 size distributions) for the road transect 50100 km south of the ﬂight path under northerly winds between Nyngan and Broken Hill for comparison. (b) Red: Average distribution for the ﬁrst 2 hours of the leg from Bourke to Chinchilla. Blue: average size for the last hour before landing. Black: last 30 min. Black dotted line: highest number concentration (13 200 cm3) size distribution for the last hour. Numbers indicate the size in nm of individual modes. Distance to Port Augusta power station for the graphs in both panels: Panel (a) 350800 km; Panel (b) red 8501100 km, blue 12001400 km. For more details, see text and Fig. 1. Fig. 2. (a) Average size distribution of ultraﬁne particles between Broken Hill and Bourke (NSW) (averages over more than 100 size distributions). Blue bars: Average size distribution of the background particle size distributions (55 size distributions) for the road transect 50100 km south of the ﬂight path under northerly winds between Nyngan and Broken Hill for comparison. (b) Red: Average distribution for the ﬁrst 2 hours of the leg from Bourke to Chinchilla. Blue: average size for the last hour before landing. Black: last 30 min. Black dotted line: highest number concentration (13 200 cm3) size distribution for the last hour. Numbers indicate the size in nm of individual modes. Distance to Port Augusta power station for the graphs in both panels: Panel (a) 350800 km; Panel (b) red 8501100 km, blue 12001400 km. For more details, see text and Fig. 1. therefore most likely related to long-range transport from the two large sources in SA with primary emissions (Rosenfeld, 2000; Junkermann et al., 2011b) and additional photochemical secondary aerosol production of UFP from remaining sulphur emissions (Lonsdale et al., 2012) even on the second day of transport and more than 800 km downwind. 3.1. Flight sector Broken Hill to Chinchilla (SA/NSW/QLD) Although initially planned as a clean continental air background reference section the ferry flight from Adelaide via Broken Hill and Bourke to Chinchilla encountered a continuous moderate tailwind with unanticipated coinci- dental tracking of pollution from sources of UFP near Port Augusta (power station) and Port Pirie (industrial complex with copper, lead and zinc smelters) in SA. These particle sources had been identified earlier by Rosenfeld (2000) as sources of cloud relevant particles and a detailed survey would have been useful, but time and weather constraints did not allow for more sophisticated flight patterns. The emissions from these sources were trans- ported by south-westerly winds over 1200 km within 24 hours more or less along the flight track as confirmed by HYSPLIT trajectories. Airborne measurements were only taken from Broken Hill onwards because occasional rain showers prevented earlier instrument operation. Par- ticle numbers reached about 6000/cm3 during the first day on the track from Broken Hill to Bourke with fairly constant size distributions and number concentrations within930% (Fig. 2a). During the flight on the second day between Bourke and Chinchilla concentrations varied from 2500/cm3 within the first 2 hours to 9000/cm3 within the last 2 hours with variable size distributions, featuring a significant number of fresh nucleation mode aerosol, far above the typical concentrations for an Australian background aerosol of 7001000 cm3 (Bigg and Turvey, 1978; Suni et al., 2008) (Fig. 2b). No local sources for UFP upwind of the aircraft position in the sparsely inhabited area could be identified, neither visually nor from HYSPLIT back trajectories or in situ wind measure- ments. Also the Australian National Pollution Inventory (NPI, 2015) does not indicate any significant sulphur emissions in the area. The measured concentrations were In the vehicle, a personal car, the aerosol instruments taken from the aircraft were installed on the back seat and powered from the car generator. Air was drawn to the instruments via a 120 cm long stainless steel tubing with 2.5 mm inner diameter extending 1012 cm above the car roof. Residence time in the inlet line was B0.15 s. This inlet is not isokinetic for fine particles; however, it works well for ultrafines as confirmed by direct comparison of the SMPS total particle count with inlet line to the CPC counts without inlet line. 3.1. Flight sector Broken Hill to Chinchilla (SA/NSW/QLD) Sulphur dioxide to sulphuric acid conversion in the atmosphere is not fast enough to convert more than an upper limit of 20% of the SO2 in a power station plume within 1 d (Cantrell and Whitby, 1978). Even during one or two consecutive days of transport sulphuric acid can thus be expected to be produced from remaining SO2, leading to additional gas to particle formation within the plume. Plume dispersion rapidly reduces the SO2 mixing ratios during the transport. After 800 km a plume width of about 200 km could be expected (Bigg, 1978). The Australian National Pollution Inventory now published the data for the 2012 SO2 and H2SO4 annual emissions for Port Augusta (750 kg/ h) and Port Pirie (7200 kg/h). The resulting diluted SO2 concentration, calculated based on winds and mixed layer depth derived from HYSPLIT analysis is in the order of 0.3 ppb (6 * 109 molecules cm3) sufficient for a rapid daylight production of the 106 sulphuric acid molecules cm3 required for particle formation (Kulmala et al., 2013). The co-emitted H2SO4 from Port Augusta is probably too reactive for long range transport. Dilution alone would result in 4 * 106 molecules cm3. However, for this flight leg we neither know the exact plume location, nor do we have any data on sulphur dioxide concentrations or other substances required for particle growth. therefore most likely related to long-range transport from the two large sources in SA with primary emissions (Rosenfeld, 2000; Junkermann et al., 2011b) and additional photochemical secondary aerosol production of UFP from remaining sulphur emissions (Lonsdale et al., 2012) even on the second day of transport and more than 800 km downwind. Sulphur dioxide to sulphuric acid conversion in the atmosphere is not fast enough to convert more than an upper limit of 20% of the SO2 in a power station plume within 1 d (Cantrell and Whitby, 1978). Even during one or two consecutive days of transport sulphuric acid can thus be expected to be produced from remaining SO2, leading to additional gas to particle formation within the plume. Plume dispersion rapidly reduces the SO2 mixing ratios during the transport. After 800 km a plume width of about 200 km could be expected (Bigg, 1978). The Australian National Pollution Inventory now published the data for the 2012 SO2 and H2SO4 annual emissions for Port Augusta (750 kg/ h) and Port Pirie (7200 kg/h). 3.3. Particle budgets and source strengths As described in Bigg and Turvey (1978), Lelieveld et al. (1987) and Junkermann et al. (2011a, 2011b), particle emission source strengths can be derived from airborne plume cross-section measurements multiplied with the aver- age wind speed measured by the aircraft during the plume transects with an accuracy of about 92550% (Lelieveld et al., 1987). This accuracy depends on the accuracy of the wind measurement and the estimate of the total plume cross- section (horizontal extend and PBL height). The height of the PBL is derived at least once during each flight from vertical profile measurements of particles, potential tem- perature and water vapour that are also used to confirm, As described in Bigg and Turvey (1978), Lelieveld et al. (1987) and Junkermann et al. (2011a, 2011b), particle emission source strengths can be derived from airborne plume cross-section measurements multiplied with the aver- age wind speed measured by the aircraft during the plume transects with an accuracy of about 92550% (Lelieveld et al., 1987). This accuracy depends on the accuracy of the wind measurement and the estimate of the total plume cross- section (horizontal extend and PBL height). The height of the PBL is derived at least once during each flight from vertical profile measurements of particles, potential tem- perature and water vapour that are also used to confirm, that the PBL is well mixed. Finally it is also compared to HYSPLIT reanalysis data that also contain the diurnal behaviour of the mixed layer depth. The total number of particles within the plume cross-section was calculated from the number concentration of particles integrated over the area of the plume (all data exceeding twice the back- ground) and background particle concentrations were sub- tracted. From previous studies in Germany, China and Australia, including the one at the Port Augusta power station (Junkermann et al., 2011a, 2011b), a particle flux within the first 2 hours of transport in the order of 2 * 1018 particles per second per 600 MW of power generation was derived using this method. Very similarly, the estimates for the Queensland power stations were 3 * 1018 s1 directly downwind of Kogan Creek (750 MW) on the evening of Results of the two plume studies at Kogan Creek are shown in Fig. 3. W. JUNKERMANN AND J. M. HACKER 8 Particle numbers in size bins of the nucleation mode (B15 nm) were either very low or not detected. Particle numbers in size bins of the nucleation mode (B15 nm) were either very low or not detected. panel. Concerning particle number concentrations, sizes and particle growth, all investigated plumes showed very simi- lar characteristics to observations elsewhere (Junkermann et al., 2011a, 2011b), with very high number concentrations of UFP mainly in the nucleation mode and predominately aged particles in the accumulation mode outside of the plume area. Upwind of individual power stations and outside of the plumes, particle numbers were typically well below 30005000 cm3. In contrast to the expectation that without further particle production coagulation during transport would lead to a loss of particles in ageing plumes such as these, increasing total numbers within the plume cross-sections were found for daytime hours. This can be explained by additional gas-to-particle conversion from co-emitted particle precursor gases like sulphur dioxide, nitrogen oxides, etc. following conversion of SO2 to SO¼ 4 via OH radical chemistry (Lonsdale et al., 2012; Pietika¨ inen et al., 2014). Such particle production in sulphur-rich plumes is dependent on the SOX/NOX mixture and on the background pollution levels (Lonsdale et al., 2012). The conditions in Queensland with low background pollution are most favourable for such in-plume nucleation mode particle generation. 3.2. Power station surveys Between Chinchilla, Qld, and north of Rockhampton, Qld, the survey flights concentrated on characterising the emissions and their temporal behaviour from the power stations at Kogan Creek (near Chinchilla), Callide (near Biloela) and Gladstone (near Rockhampton) (Figs. 3 and 4). Particle production via sulphuric acid is well known in the engineering community and is the cause of opacity at the chimney (Srivastava et al., 2004; Brachert et al., 2013, 2014). However, the resulting particle emission or formation rate downwind of the chimney is not well known as airborne measurements are required (Pueschel and Van Valin, 1978; Whitby et al., 1978; Junkermann et al., 2011a, 2011b). After arriving at Chinchilla on 25 August, an evening survey was flown with south-westerly winds downwind of the Kogan Creek Power Station taking off 30 min before and landing 30 min after sunset. The plume was identified and was crossed three times at distances between 30 and 50 km downwind of the power station with particle number concentrations up to 35 000 cm3 and geometrical mean diameters between 14 and 19 nm (Fig. 3). The next day, 26 August, under sunny conditions and a generally weak south-easterly wind, a late morning/ noon Lagrangian survey of the Kogan Creek plume was flown (take-off at 11:00 local time). Daytime surveys of the plumes from the power stations near Biloela (Callide), Rockhampton (Stanwell) and Gladstone followed on 27 August. 3.1. Flight sector Broken Hill to Chinchilla (SA/NSW/QLD) The resulting diluted SO2 concentration, calculated based on winds and mixed layer depth derived from HYSPLIT analysis is in the order of 0.3 ppb (6 * 109 molecules cm3) sufficient for a rapid daylight production of the 106 sulphuric acid molecules cm3 required for particle formation (Kulmala et al., 2013). The co-emitted H2SO4 from Port Augusta is probably too reactive for long range transport. Dilution alone would result in 4 * 106 molecules cm3. However, for this flight Figure 2a shows the average of all size distributions of Day 1 (August 24); Fig. 2b shows average size distributions for the first 2 hours, the last hour and the last 30 min of Day 2 before landing at Chinchilla (August 25). All measurements were taken within the boundary layer at approximately 600900 m AGL. Figure 2a for comparison also shows the background particle size distribution of air originating from the area below and to the north of the flight path measured 2 weeks later during the vehicle transect on the ground under clear northerly advection (average of all undisturbed ground based size distributions between Nyngan and Broken Hill). The vehicle-based background measurements in the area south of the flight transect under northerly winds (only data from daytime measurements between 10:00 local time and early afternoon with well-mixed boundary layers were used) always showed lower particle number concentration than what was observed on the aircraft and indicated aged continental particles. They confirm, that no significant sources of UFP exist along that part of the flight. Total particle numbers on this section of the ground-based survey were always in the range of about 10001800 cm3, very similar to previous observations over pristine forested areas in Western Australia (Junkermann et al., 2009). 3.3. Particle budgets and source strengths Within the first cross-section late morn- ing we measured 100.000 particles cm3, concentrations that are otherwise typical for high levels of urban pollution (Bae et al., 2010). Winds were low at about 22.5 m/s and PBL increased from 900 estimated after the first plume transect to about 1600 m in the last cross-section. HYSPLIT reanalysis and in situ estimates agree within 100 m. Figure 4 shows the results of the Callide and Gladstone plume sur- veys with maximum number concentrations in the plume cross-sections together with the mean geometrical size in nm. On that day (27 August 2012), the Stanwell power station plume could not be located (probably caused by flying the survey legs too close to the source). Stanwell power station nevertheless had been already identified as one of the major sources in the 2011 preliminary study mentioned above and was also identified again later during the vehicle-based survey. Number concentrations measured (using fast CPC and total SMPS number concentrations) are shown in the upper panels of Figs. 3 and 4, size distributions measured in the centre of the plume from the SMPS in the lower 25 August, and again within 10% of this estimate for the at that day (however constant within 20% in all three plum dN/dln(dp) [cm–3] Diameter [nm] (4.5 – 350) 0 50000 100000 150000 6 9 13 21 33 52 83 138 242 0 6 9 13 21 33 52 83 138 242 Fig. 3. Top: Airborne survey in the vicinity of the Kogan Creek Power Station near Chinchilla, Qld. Shown are data from an evenin ﬂight on 25 August 2012 (blue) around sunset with prevailing south-westerly winds (three plume cross-sections between 30 and 54 km distance) and from the morning ﬂight (take-off at 11:00 local time) on 26 August 2012 with prevailing south-easterly winds (yellow, plum cross-sections at 12, 48 and 130 km downwind of the power station). Numbers inside the plume cross-sections indicate the maximum particle concentration cm3 in the plume centre and the geometric mean diameter of the particle size distribution. Also shown is the ﬂigh track (red). Bottom: Size distributions measured within the centre of the ﬁrst (12 km) and last (130 km) plume crossing, blue line with yellow inﬁll for daytime and blue line with blue inﬁll for the sunset survey. The colours indicate the respective plume study, yellow/orange ﬁrs transect, red last transect, aged plume. 3.3. Particle budgets and source strengths ULTRAFINE PARTICLES OVER EASTERN AUSTRALIA ULTRAFINE PARTICLES OVER EASTERN AUSTRALIA 9 dN/dln(dp) [cm–3] Diameter [nm] (4.5 – 350) 0 50000 100000 150000 6 9 13 21 33 52 83 138 242 0 6 9 13 21 33 52 83 138 242 dN/dln(dp) [cm–3] Diameter 0 50000 100000 150000 6 9 13 21 33 52 83 138 242 Fig. 3. Top: Airborne survey in the vicinity of the Kogan Creek Power Station near Chinchilla, Qld. Shown are data from an evening ﬂight on 25 August 2012 (blue) around sunset with prevailing south-westerly winds (three plume cross-sections between 30 and 54 km distance) and from the morning ﬂight (take-off at 11:00 local time) on 26 August 2012 with prevailing south-easterly winds (yellow, plume cross-sections at 12, 48 and 130 km downwind of the power station). Numbers inside the plume cross-sections indicate the maximum particle concentration cm3 in the plume centre and the geometric mean diameter of the particle size distribution. Also shown is the ﬂight track (red). Bottom: Size distributions measured within the centre of the ﬁrst (12 km) and last (130 km) plume crossing, blue line with yellow inﬁll for daytime and blue line with blue inﬁll for the sunset survey. The colours indicate the respective plume study, yellow/orange ﬁrst transect, red last transect, aged plume. Fig. 3. Top: Airborne survey in the vicinity of the Kogan Creek Power Station near Chinchilla, Qld. Shown are data from an evening ﬂight on 25 August 2012 (blue) around sunset with prevailing south-westerly winds (three plume cross-sections between 30 and 54 km distance) and from the morning ﬂight (take-off at 11:00 local time) on 26 August 2012 with prevailing south-easterly winds (yellow, plume cross-sections at 12, 48 and 130 km downwind of the power station). Numbers inside the plume cross-sections indicate the maximum particle concentration cm3 in the plume centre and the geometric mean diameter of the particle size distribution. Also shown is the ﬂight track (red). Bottom: Size distributions measured within the centre of the ﬁrst (12 km) and last (130 km) plume crossing, blue line with yellow inﬁll for daytime and blue line with blue inﬁll for the sunset survey. The colours indicate the respective plume study, yellow/orange ﬁrst transect, red last transect, aged plume. 3.3. Particle budgets and source strengths 25 August, and again within 10% of this estimate for the first plume transect close to the power station (about 20 km) in the late morning of 26 August. Due to the low wind speed at that day (however constant within 20% in all three plume transects) and the varying PBL mixed layer depth between 860 m for the first transect and 1600 m for the third one, W. JUNKERMANN AND J. M. HACKER W. JUNKERMANN AND J. M. HACKER 10 the plume budget is less precise, but still estimated to be better than 940%. The estimate for the first plume transect 1630 MW) around midday of 26 August was 6 * 1018 s (plume width 23 km, wind speed 9 m/s, PBL 900 m, averag 40000 80000 120000 6 7 9 12 16 21 27 36 43 57 75 101 138 192 273 Diameter [nm] (4.5 – 350) dN/dln(dp) [cm–3] 0 nm nm Fig. 4. Airborne survey in the vicinity of the Biloela (Callide) and Gladstone power stations, for the survey ﬂight on 26 August 2012 from Thangool to Rockhampton, take-off at 11:00 local time. Top: Plume cross-sections for the Callide power station (yellow, 20, 50 and 80 km downwind of the power station) and for the combination of sources at Gladstone (green). The dashed lines show the HYSPLIT back trajectories and the modelled age of the three plumes. The star marks Myella Farm, the location for ground-based measurements shown i Fig. 5. Bottom: Size distributions measured in the centre of the plumes (using the same colour scheme as in the top diagram, yellow Callide green Gladstone). Shown are averages of the three size distributions with the highest number concentration in the plume centre. The colour indicate the respective plume study, yellow/orange: ﬁrst transects; red: last transect ageing Callide plume; green: Gladstone plume. nm nm 40000 80000 120000 6 7 9 12 16 21 27 36 43 57 75 101 138 192 273 Diameter [nm] (4.5 – 350) dN/dln(dp) [cm–3] 0 Fig. 4. Airborne survey in the vicinity of the Biloela (Callide) and Gladstone power stations, for the survey ﬂight on 26 August 2012 from Thangool to Rockhampton, take-off at 11:00 local time. Top: Plume cross-sections for the Callide power station (yellow, 20, 50 and 80 km downwind of the power station) and for the combination of sources at Gladstone (green). 3.4. Investigations of background air and of possible biogenic or shipping related sources north of Rockhampton These values of primary emissions from cross-sections close to the source and at sunset can be taken as a lower limit of the total particle emission rates as the source for these primary emissions inside the flue gas system is independent of any environmental conditions. Secondary production of new particles is than additionally continu- ing within the plumes due to the co-emitted SO2 reacting with OH radicals under favourable (sunny) conditions (Lonsdale et al., 2012) and may increase the total par- ticle number flux in ageing plumes. During daytime, this secondary aerosol production in sulphur rich plumes which is dependent on the SO2/NOx ratio and other variables, not only compensates for coagulation losses, but also leads to a further increase up to a doubling of the particle flux under sunny, clear sky conditions as can be seen in Fig. 3 showing the plume transect at about 130 km distance from Kogan Creek which equals approximately 8 hours of transport after emission. The aim of the survey for another 1000 km, from Rockhampton via Cairns and Townsville to Cape Flattery near Cooktown was to characterise background conditions and to search for other possible ultrafine particle sources, either from (biogenic) nucleation within or above the rain- forests (Suni et al., 2008), from the GBR (Bigg and Turvey, 1978), or from other sources, not identified previously. The flight path basically followed the eastern escarpment of the Great Dividing Ranges, with some minor excursions to the western side of the mountains due to low cloud and showers along the coast between Townsville and Cairns (Fig. 1). Most of the time, easterly winds prevailed leading to advection of maritime air masses. Particle number concentrations rapidly declined towards concentrations of about 2500 particles cm3 or less north of Rockhampton with winds turning more easterly (Figs. 5 and 6). Figure 5 shows the highly variable particle size distributions at Myella in the plume-affected area 100 km downwind of the two large power stations Callide and Stanwell, and comparison to the ‘background’ north of Rockhampton. Figure 6 shows a comparison of back- ground size distributions in the more remote areas. UFP in the smallest size bins were no longer dominating in all these ‘background’ size distributions. Total particle number Primary particles are emitted at sizes of a few nm (Srivastava et al., 2004; Junkermann et al., 2011a; Brachert et al., 2013, 2014). 3.4. Investigations of background air and of possible biogenic or shipping related sources north of Rockhampton Observed growth rates derived from mean geometric diameter in the plume centre, an emission size of 8 nm, and the travelling time from the source derived from HYSPLIT agree with those reported from previous ground based and airborne studies with an initial rate of 38 nm/h up to a diameter of about 2530 nm (Kulmala et al., 2004; Suni et al., 2008; Junkermann et al., 2011a). The exact size of the primary emitted particles cannot be exactly specified, Brachert et al. (2014) esti- mated a minimum of 8 nm from a power station stack model, Junkermann et al. (2011a) extrapolated from air- borne plume data 4 nm. The current study indicates a range of 68 nm. The final, initial size in the atmos- phere may depend on the initial ambient conditions in the plume, temperature and humidity, at the exit of the chimney, when hot and humid flue gas is cooled down and dried rapidly. Growth rates, derived from the size distributions, depend strongly on the mixture of primary emitted and additional secondarily produced particles. As shown in the bottom panels of Figs. 3 and 4, the main size modes within the distributions shift slowly towards larger particles (Junkermann et al., 2011a), with the low size bins being refilled during the day by new particle formation (NPF) within the plumes. Laaksonen et al., (2005) report particle growth to CCN sizes within about 10 hours. During the Lagrangian plume studies we were not able to follow the plumes for that long a time, except for the unanticipated and incomplete case study of the Port Augusta plume. In all cases, however, there was no indication that growth would cease after a few hours only. dN/dln(dp) [cm–3] Diameter [nm] 0 6 9 13 21 33 52 83 138 242 10000 20000 30000 Fig. 5. Average size distributions measured on the ground at Myella Farm (see Figs. 1 and 3 for locations), 100 km west of Biloela (Callide power station) during daylight hours. Blue: ﬁrst hour; green: average of low concentration (B6000 cm3) measurements; red and purple: averages of two half hour episodes with 12000/cm3. Black: average of all measurements during the day. HYSPLIT trajectories indicate that Stanwell and Callide power stations are the major ultraﬁne particle sources. 3.3. Particle budgets and source strengths The dashed lines show the HYSPLIT back- trajectories and the modelled age of the three plumes. The star marks Myella Farm, the location for ground-based measurements shown in Fig. 5. Bottom: Size distributions measured in the centre of the plumes (using the same colour scheme as in the top diagram, yellow Callide, green Gladstone). Shown are averages of the three size distributions with the highest number concentration in the plume centre. The colours indicate the respective plume study, yellow/orange: ﬁrst transects; red: last transect ageing Callide plume; green: Gladstone plume. 1630 MW) around midday of 26 August was 6 * 1018 s1 (plume width 23 km, wind speed 9 m/s, PBL 900 m, average number concentration 30 000 cm3, estimated error 930%) the plume budget is less precise, but still estimated to be better than 940%. The estimate for the first plume transect downwind of the larger station Callide (Callide A, B and C, ULTRAFINE PARTICLES OVER EASTERN AUSTRALIA 11 3.4. Investigations of background air and of possible biogenic or shipping related sources north of Rockhampton 3.5. Emissions from other sources It is interesting to note that at no stage during the current survey with maritime air masses over the forests of the Great Dividing Ranges, nor during all our previous airborne surveys over the forests of Western Australia in 2006, 2007 and 2011 (Junkermann et al., 2009, unpublished data from the 2011 campaign during north-westerly air mass advection from the Indian Ocean) were any signatures of biogenic NPF detected, nor were any ultrafine particle emissions found related to open cut mining activities. ‘Typical’ NFP events show nucleation mode particle number concentrations exceeding 10 000 cm3, as observed over Italy (Laaksonen et al., 2005), Finland (O’Dowd et al., 2009) and Western Australia (Junkermann et al., 2009) with the same instrumentation as used in our study. Very similar to the Bigg and Turvey (1978) survey, all major particle events in the present study with significantly enhanced ultrafine particle numbers could clearly be traced back to industrial installations burning sulphur-containing fossil fuel. Also essential for the particle budget within the first transects close to the power stations is the observation that no significant differences in the particle flux close to the power stations were found between day and night (i.e. at sunset and late morning hours). This further con- firms the hypothesis that a significant fraction of the initial steps of the nucleation process and also the particle pro- duction already takes place inside the power stations’ smoke stacks and is not photochemistry-driven (Srivastava et al., 2004; Junkermann et al., 2011a, 2011b; Lonsdale et al., 2012; Brachert et al., 2014). Primary particles from such processes, once emitted into the atmosphere, are no longer distinguishable from particles originating from secondary atmospheric nucleation. Due to the elevated emission height of the power station plumes, ground-based measurements in the direct vicinity of the power stations rarely find high particle numbers. Yet, increased particle concentrations were always detectable on the ground approximately 15 to 20 km downwind. Fig. 6. Background size distributions over the rain forest north of Cairns (red) during the ﬂight to Cape Flattery (Lizard Island) and the return ﬂight to Cairns over the open water (dark blue). Also shown are background size distributions measured over the hinterland forests north of Rockhampton (light blue) and during the vehicle transect in inland northern NSW (green) (average over 50 size distributions/about 2 ﬂight hours or 200 km, road transect 80 distributions 500 km or 8 hours). W. JUNKERMANN AND J. M. HACKER W. JUNKERMANN AND J. M. HACKER 12 12 0 400 800 1200 1600 2000 Diameter [nm] dN/dln(dp) [cm–3] 6 9 13 21 33 52 83 138 242 Fig. 6. Background size distributions over the rain forest north of Cairns (red) during the ﬂight to Cape Flattery (Lizard Island) and the return ﬂight to Cairns over the open water (dark blue). Also shown are background size distributions measured over the hinterland forests north of Rockhampton (light blue) and during the vehicle transect in inland northern NSW (green) (average over 50 size distributions/about 2 ﬂight hours or 200 km, road transect 80 distributions 500 km or 8 hours). 0 400 800 1200 1600 2000 Diameter [nm] dN/dln(dp) [cm–3] 6 9 13 21 33 52 83 138 242 However, with the low shipping intensity between the coast and the Reef in this area, a continuous and significant effect of ship emissions was too low to be quantified. Yet, several days of ground-based measurements on the shore north of Townsville and at Lennox Head, the eastern-most point of Australia south of Brisbane, as well as further measurements downwind of the port of Townsville indicate that ship emissions can occasionally and locally increase the particle numbers at the coast to approximately 10 000 cm3. 3.4. Investigations of background air and of possible biogenic or shipping related sources north of Rockhampton The ﬁlled blue curve shows the background size distribution inland between Rockhampton and Cairns (average over 50 size distributions/from about 2 ﬂight hours or 200 km) for comparison. dN/dln(dp) [cm–3] Diameter [nm] 0 6 9 13 21 33 52 83 138 242 10000 20000 30000 Diameter [nm] Fig. 5. Average size distributions measured on the ground at Myella Farm (see Figs. 1 and 3 for locations), 100 km west of Biloela (Callide power station) during daylight hours. Blue: ﬁrst hour; green: average of low concentration (B6000 cm3) measurements; red and purple: averages of two half hour episodes with 12000/cm3. Black: average of all measurements during the day. HYSPLIT trajectories indicate that Stanwell and Callide power stations are the major ultraﬁne particle sources. The ﬁlled blue curve shows the background size distribution inland between Rockhampton and Cairns (average over 50 size distributions/from about 2 ﬂight hours or 200 km) for comparison. 4. Summary and discussion This study presents airborne and ground/vehicle-based mobile measurements of the spatial distribution of UFP serving as possible precursors of CCN over inland parts of Northern NSW and along the Queensland coastal areas. This area is an ideal natural laboratory to investigate emissions from an array of coal-fired power stations located in a relatively sparsely inhabited, predominately agricultural and partially forested region. Power stations have clearly been identified as major sources for significant primary emissions of UFP. Natural and biogenic sources for UFP under discussion and addressed within this study were those from forests or from the GBR, as well as from forest fires. The current study confirmed the general apportion- ment of major anthropogenic aerosol sources over Australia found by Bigg and Turvey (1978) 40 yr ago to the burning of sulphur-containing fossil fuel in power stations and smelters. However, significant changes in the number and locations of these major sources were detected. Since 1974, when only Callide A was operational, eight new large coal- fired power stations were commissioned and existing sta- tions (Callide) were substantially enlarged. Most of the ‘new’ stations are now located in remote areas of the Queensland hinterland. The results presented here revealed that these ‘new’ coal-fired power stations commissioned between 1966 (Callide) and 2008 (Kogan Creek) are major sources for ultrafine particle number concentrations along the Queensland coast and within the first 200 km of the hinterland. It was not possible to locate or quantify any other comparable sources for UFP over 10 flying days and 3600 km distance. During the survey 40 yr ago, Callide A was operational while the Gladstone power station had just been commissioned in the year of the measurements (1976), but the exact date of when its operations started is not known. Both sources could also have easily been missed during the survey with the technology available at the time, consisting of airborne condensation nuclei (CN) number measurements, in situ winds and synoptic weather charts. HYSPLIT back trajectory re-analyses available now for the years of the Bigg and Turvey (1978) measure- ments indicate that even the 1976 GBR measurements might Conducting plume studies at different times of the day using Lagrangian flight patterns, it was possible to estimate the source strength of primary emissions from individual power stations. ULTRAFINE PARTICLES OVER EASTERN AUSTRALIA ULTRAFINE PARTICLES OVER EASTERN AUSTRALIA 13 have been affected by long range transport from these older power stations. Bigg and Turvey (1978) reported occasional source strengths of up to 1019 s1 and Warner (1968) discussed a possible effect of sugar cane fires on rainfall. However, as already pointed out in Bigg and Turvey (1978), these sources are not continuous and therefore less important for the total long-term budget than continuous 24 hours, 365 d industrial emissions. In addition, new environmental protection reg- ulations have diminished the occurrence of such anthropo- genic fires significantly over recent years. Bigg and Turvey (1978) reported occasional source strengths of up to 1019 s1 and Warner (1968) discussed a possible effect of sugar cane fires on rainfall. However, as already pointed out in Bigg and Turvey (1978), these sources are not continuous and therefore less important for the total long-term budget than continuous 24 hours, 365 d industrial emissions. In addition, new environmental protection reg- ulations have diminished the occurrence of such anthropo- genic fires significantly over recent years. During several days of measurements during our study over ‘clean’ inland and coastal regions outside of the coal mining and coal burning areas, mainly under the advec- tion of maritime air masses, no signatures of biogenic NPF were found from the GBR or such as reported from other Australian forest environments (Suni et al., 2008) despite favourable meteorological conditions for such a nucleation (Baranizadeh et al., 2014). Anthropogenic plumes however, could always be traced within the PBL, based on the size distributions, real time in-situ wind measurement and HYSPLIT trajectories over several hundreds of kilometres, typically following the local orography. 3.5. Emissions from other sources concentrations north of Townsville were generally lower over the inland forested and agricultural areas than over the open sea (Fig. 6, averages of 50 size distributions for both areas). The particle size distributions and total numbers observed during this part of the survey were very similar to particles found over the pristine Western Australian forests (Junkermann et al., 2009 and unpublished data from a campaign in March 2011). Particle numbers between 1000 and 1700 cm3 were measured over the rainforest at the escarpment and slightly, but not significantly higher numbers (up to 2500 cm3, 50 size distributions each) within the marine boundary layer below 500 m over the sea. Such particle levels are typical for Australian clean conditions (Suni et al., 2008; Junkermann et al., 2009) but are nevertheless larger than the clean background con- centrations reported 40 yr ago (Bigg and Turvey, 1978). Size modes indicated aged particles larger than 20 nm. Obvious signatures in the size distributions, indicating NPF, i.e. particles in the smallest size ranges between 4.5 and 15 nm were neither found over the sea nor over land in these air masses which were not affected by sulphur emissions from fossil fuel burning industries, even hundreds of kilometres upwind. As shown in Fig. 6, the size distribu- tions over the sea have slightly more particles in the lower size ranges. All particle concentrations along the coast were significantly lower than those over the areas downwind of the power stations. The slightly higher fraction of smaller particles over the sea might originate from ship emissions. Forest fires, common in Australia for centuries (Crowley and Garnett, 2012), can occasionally be a locally dominat- ing source of particulate matter. Measurements of particle emission factors (Janha¨ ll et al., 2010) are scarce, but W. JUNKERMANN AND J. M. HACKER Within the surveyed plumes of the current study, 2 hours after emission the majority of the particles was still smaller (Figs. 24). Most of the emitted particles are thus expected to further grow to CCN relevant sizes within a few more hours (Laaksonen et al., 2005; Kerminen et al., 2012) during transport of up to hundreds of kilometres [see also the extension of the plumes in Ayers et al. (1979), Rosenfeld (2000) and Rosenfeld et al. (2006)]. Based on the findings by Bigg and Turvey (1978), it was expected to identify at least some particle production from the biogenic activity of the GBR. However, as that study already claims that the reported particle emission was a sporadic event only and not reproducible from additional long term local measurements made on an island between the Reef and the continent, it can be hypothesised, that the reported activity may have rather been the result of long range transport from either Callide or Gladstone power stations instead of from the GBR. This hypothesis is supported by assessments based on HYSPLIT back trajectories for the time of the 1976 research flights. Considering recent model calculations (Teller and Levin, 2006; Zhao et al., 2012) and experimental evidence from other studies (Rosenfeld 2000; Rosenfeld et al., 2006), the anthropogenic ultrafine particle production is well within the order of magnitude that can after further growth to CCN sizes contribute to rainfall modifications suspected previously (Warner, 1968; Pueschel and Van Valin, 1978; Rosenfeld 2000; Rosenfeld et al., 2006; Bigg, 2008; Junkermann et al., 2009, 2011b) independently of whether 10% (Bigg and Turvey, 1978) or 30% (Andreae, 2009) of the CN, measured by the CPC’s, survive. The Australian Bureau of Meteorology reports clear negative trends in precipitation over land within the area since 1970 (BOM, 2014) following 80 yr without rainfall trends from 1890 to 1970. Among the changes reported by the Australian Bureau of Meteorology since 1970 are: a reduction of rainfall days along the Sunshine Coast in SE-Queensland by up to 8 d per decade; a change from low intensity rain events to longer, or more severe drought events; and a shift towards more vigorous single rain events. All of these patterns are consistent with expected consequences of en- hanced ultrafine particle number concentrations impact on cloud microphysics and radiation (Teller and Levin, 2006; Feingold and Siebert, 2009; Li et al., 2011). W. JUNKERMANN AND J. M. HACKER Within the surveyed plumes of the current study, 2 hours after emission the majority of the particles was still smaller (Figs. 24). Most of the emitted particles are thus expected to further grow to CCN relevant sizes within a few more hours (Laaksonen et al., 2005; Kerminen et al., 2012) during transport of up to hundreds of kilometres [see also the extension of the plumes in Ayers et al. (1979), Rosenfeld (2000) and Rosenfeld et al. (2006)]. Emissions associated with industrial activities and mining have previously been reported to show high CCN source strength. A typical example from Australia is the work of Ayers et al., (1979) who studied the industrial source located at Mt. Isa, Qld, with a CCN source strength of 2 * 1018 s1, making it the most significant source in North Queensland in 19761978 and claiming that its produc- tion rate was 0.1% of the global CCN production at that time. Interestingly, the particle budget studies presented in the current study show that even this source, which is still active today, though with reduced sulphur emissions (Manins et al., 2001), is now only one of several large sources for UFP in Queensland and is, although still one of the largest Australian SO2 sources (NPI), in terms of ultrafine particle number source strength, surpassed by the agglom- Detailed discussions about the production of CCN from UFP and their dependence on size and chemistry are beyond the scope of this paper. They can be found in Pierce and Adams (2007), Zhang et al. (2012), Kerminen et al. (2012) and Westervelt et al. (2013). The conditions in Queensland with high availability of sulphur dioxide and only marginal concentration of fine particulate matter (particles between 100 nm and 2.5 mm, PM 2.5) favour the survival and growth of both, primary emitted or secondary produced, UFP into highly hygroscopic particles (Lonsdale et al., 2012). A threshold of 40 nm is considered as a reasonable proxy for such sulphur containing CCN (King et al., 2007; Andreae, 2009). This threshold would also be in agreement with the clear signature of cloud modifica- tion observed from satellites (Rosenfeld, 2000) already after less than 2 hours from Port Augusta and Port Pirie, two sources that we investigated in October 2014 during another flight campaign (not published yet) and found to emit 3.2 * 1018 and 5.3 * 1018 particles s1 respectively. 4. Summary and discussion Furthermore, these measurements made it possible to address the question of whether the UFP in the plumes are emitted as a result of primary production within the power station itself or are produced later through secondary processes in the atmosphere from gas-to-particle conversion. This question is important for the overall budget of particle production because primary emission runs 24 hours per day while photochemical production is active only during the hours of sunshine. In contrast to previous plume studies which concentrated either on ‘dirty’ (Pueschel and Van Valin, 1978; Whitby et al., 1978) or on only ‘partially cleaned’ power stations (Brock et al., 2002; Zaveri et al., 2010), the current study investigated emissions from state-of-the-art ‘clean’ power stations, their size distributions and the temporal development within the ageing plumes. Using highly mobile (in a three-dimensional sense) airborne measurements and size distributions avail- able in real-time during the flights as the primary criteria for source apportionment, it was possible to follow plumes over hundreds of kilometres in Queensland and most probably even for more than 1000 km from SA into south-west Queensland. The particle emission rates derived from the plume studies are well within a range that was observed elsewhere with about 23 * 1018 s1 for a typical size power station with a capacity of 700 MW. The particle source strength of the individual power stations was thus in the same order of magnitude as the ultrafine particle emissions estimated for one of the major cities in Australia in 1976 by Bigg and Turvey (1978), Perth in Western Australia. As above, HYSPLIT back trajectory calculations for 1976 confirmed that at that time at least one coal-fired power station Muja (900 MW) upwind of Perth contributed significantly to these pre- vious measurements (Bigg et al., 2015). Within the plumes, increasing total particle numbers were found which is in agreement with the current understanding that within such W. JUNKERMANN AND J. M. HACKER 14 eration of power stations in the hinterland between Brisbane and Rockhampton. sulphur and nitrogen rich plumes further particle produc- tion continues during daytime following initial photoche- mical conversion of SO2 into sulphuric acid (Lonsdale et al., 2012). This additional particle formation com- pensates a loss of particles due to coagulation under the favourable conditions over Queensland. Such a behaviour was already reported for some of the industrial plumes during the 19741976 surveys of Bigg and Turvey (1978) but was not further investigated at that time due to missing information about the aerosol size distributions in these early studies. The absence of biogenic nucleation events during the current campaign does not mean that such biogenic nuclea- tion does not take place in Queensland. It is only an indication that, biogenic nucleation events are either not every day phenomena in the region, occurring at night, or are too low in concentration (Suni et al., 2008) to be detectable from the aircraft flying in the mid or upper half of the mixed boundary layer. Enhanced nucleation mode particles found in previous airborne campaigns could be traced back to ground-based salt lake emissions over ag- ricultural land, which are probably also linked to sulphur chemistry. All primary nucleation mode particle emissions were due to anthropogenic burning of fossil fuel. early studies. Detailed discussions about the production of CCN from UFP and their dependence on size and chemistry are beyond the scope of this paper. They can be found in Pierce and Adams (2007), Zhang et al. (2012), Kerminen et al. (2012) and Westervelt et al. (2013). The conditions in Queensland with high availability of sulphur dioxide and only marginal concentration of fine particulate matter (particles between 100 nm and 2.5 mm, PM 2.5) favour the survival and growth of both, primary emitted or secondary produced, UFP into highly hygroscopic particles (Lonsdale et al., 2012). A threshold of 40 nm is considered as a reasonable proxy for such sulphur containing CCN (King et al., 2007; Andreae, 2009). This threshold would also be in agreement with the clear signature of cloud modifica- tion observed from satellites (Rosenfeld, 2000) already after less than 2 hours from Port Augusta and Port Pirie, two sources that we investigated in October 2014 during another flight campaign (not published yet) and found to emit 3.2 * 1018 and 5.3 * 1018 particles s1 respectively. References Aitken, J. 1888. On the number of dust particles in the atmosphere. Trans. Roy. Soc. Edinb. 35(Pt.1), 119. Albrecht, B. A. 1998. Aerosols, cloud microphysics, and fractional cloudiness. Science. 254, 12271230. Andreae, M. O. 2009. Correlation between cloud condensation nuclei concentration and aerosol optical thickness in remote and polluted regions. Atmos. Chem. Phys. 9, 543556. polluted regions. Atmos. Chem. Phys. 9, 543556. Andreae, M. O. 2013. The aerosol nucleation puzzle. Science. 339, 911. DOI: 10.1126/science.1233798. Given the good historical data base since 1900 on meteorological variables, the long term homogeneous land- scape and marginal land use change together with the high production rate of additional UFP originating from well- defined sources, which allows to reconstruct the ‘laboratory notebook’ of this experiment, the area of this study would offer a unique opportunity for a regional-scale modelling study investigating aerosolcloudprecipitation interac- tion with aerosol and cloud microphysics resolving models (Vogel et al., 2009; Zhao et al., 2012; Matsui et al., 2014), supported by more extensive in-situ measurements of source strengths including detailed size and mass distributions and the partitioning between nucleation and accumulation mode emissions which are essential for CCN production (Pierce and Adams, 2009; Wang and Penner, 2009). Asmi, A., Coen, C., Ogren, M., Andrews, J. A., Sheridan, E. and co-authors. 2013. Aerosol decadal trends Part 2: In-situ aerosol particle number concentrations at GAW and ACTRIS stations. Atmos. Chem. Phys. 13, 895916. DOI: 10.5194/acp-13- 895-2013. Ayers, G. P., Bigg, E. K. and Turvey, D. E. 1979. Aitken particle and cloud condensation nucleus ﬂuxes in the plume from an isolated industrial source. J. Appl. Meteorol. 187, 449459. Bae, M.-S., Schwab, J. J., Hogrefe, O., Frank, B. P., Lala, G. G. and co-authors. 2010. Characteristics of size distributions at urban and rural locations in New York. Atmos. Chem. Phys. 10, 45214535. Baranizadeh, E., Arola, A., Hamed, A., Nieminen, T., Mikkonen, S. and co-authors. 2014. The effect of cloudiness on new-particle formation: investigation of radiation levels. Boreal Environ. Res. 19(Suppl B), 343354. ( ; g ) Finally we would like to add that we did not find a ‘new’ source of nucleation mode particles. Significant CCN production in such plumes was already reported by Pueschel and Van Valin (1978) and Whitby et al. (1978). Emission reduction measures, however, lead to a shift of the emitted particle size distribution towards and an intensification of the nucleation mode (Junkermann et al., 2011a, 2011b). W. JUNKERMANN AND J. M. HACKER However, from the few and short-term measurements presented in this study, a direct causality relationship cannot be established. Emissions associated with industrial activities and mining have previously been reported to show high CCN source strength. A typical example from Australia is the work of Ayers et al., (1979) who studied the industrial source located at Mt. Isa, Qld, with a CCN source strength of 2 * 1018 s1, making it the most significant source in North Queensland in 19761978 and claiming that its produc- tion rate was 0.1% of the global CCN production at that time. Interestingly, the particle budget studies presented in the current study show that even this source, which is still active today, though with reduced sulphur emissions (Manins et al., 2001), is now only one of several large sources for UFP in Queensland and is, although still one of the largest Australian SO2 sources (NPI), in terms of ultrafine particle number source strength, surpassed by the agglom- ULTRAFINE PARTICLES OVER EASTERN AUSTRALIA 5. Acknowledgement It is, however, interesting to note, that the observed negative trend in rainfall commenced at the same time when the first intense particle source in the area (Callide) began to operate and that the rainfall trend is negatively correlated to the growing total emission of UFP from the increased number of major ultrafine particle sources installed within the area. The authors gratefully acknowledge the NOAA Air Resources Laboratory (ARL) for the provision of the HYSPLIT transport and dispersion model and/or READY website (http://www.ready.noaa.gov) used in this publi- cation. Financial support for this campaign was pro- vided by the KIT International Excellence Fund, contract # 120120/17. According to a regional modelling study by Zhao et al. (2012) covering an area similar in size to eastern Queensland, the aerosol-cloud-precipitation system is par- ticularly sensitive to injection of additional aerosol particles into the boundary layer just below the cloud base. Adding aerosols acting as CCN into that model layer resulted in a significant reduction of rainfall up to 1500 km down- wind. As the power station stacks with 200 m height are emitting the flue gas with UFP into elevations above 300 m above ground, upward mixing to the layers where cloud formation and also longer-range transport takes place can be expected to be rather efficient. 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https://openalex.org/W2736243600	http://www.journalijar.com/uploads/493_IJAR-18105.pdf	English	null	MIES AND MINIMALISM: A PERFECT COALESCENCE.	International journal of advanced research	2,017	cc-by	2,624	MIES AND MINIMALISM: A PERFECT COALESCENCE. Srikanth Sakleshpur Venkatesh. University of Illinois at Urbana-Champaign. …………………………………………………………………………………………………….... Manuscript Info Abstract ……………………. ……………………………………………………………… Manuscript History Received: 19 April 2017 Final Accepted: 21 May 2017 Published: June 2017 Key words: - Minimalism, Architecture, „less is more‟, Mies van der Rohe, Farnsworth house The renowned architect, Mies van der Rohe, revolutionized the sphere of architecture by adopting the famous aphorism „less is more‟ in his buildings. The concept of Minimalism, which was seen in artworks, was adopted into architecture. He borrowed from the art theory which talks about the object qualities and aesthetics. Mies buildings called „masterpieces‟ encompass this theory which show that architecture can also be an art, just like painting or sculpture. With specific reference to one of Mies‟s very famous „work of art‟, the „Farnsworth house‟, this paper will try to illustrate how the concept „less is more‟ is beautifully incorporated into the architecture of this building, thereby showcasing the perfect coalescence that exists between Mies and minimalism. C Ri ht IJAR 2017 All i ht d ……………………………………………………………… The renowned architect, Mies van der Rohe, revolutionized the sphere of architecture by adopting the famous aphorism „less is more‟ in his buildings. The concept of Minimalism, which was seen in artworks, was adopted into architecture. He borrowed from the art theory which talks about the object qualities and aesthetics. Mies buildings called „masterpieces‟ encompass this theory which show that architecture can also be an art, just like painting or sculpture. With specific reference to one of Mies‟s very famous „work of art‟, the „Farnsworth house‟, this paper will try to illustrate how the concept „less is more‟ is beautifully incorporated into the architecture of this building, thereby showcasing the perfect coalescence that exists between Mies and minimalism. Copy Right, IJAR, 2017,. All rights reserved. …………………………………………………………………………………………………….... Introduction: - Ludwig Mies Van der Rohe, born in the year 1886 in the German town of Aachen, is considered the most influential architect of the 20th Century. His apprenticeship with Peter Behrens, a German architect and designer, was one of the turning points in his life. Under him he discovered the new way of looking at architecture, especially learning the use of materials like steel, and glass. The adage „less is more‟ was unveiled to him in Behrens office. ISSN: 2320-5407 ISSN: 2320-5407 Int. J. Adv. Res. 5(6), 1236-1239 Journal Homepage: - www.journalijar.com Article DOI: 10.21474/IJAR01/4529 DOI URL: http://dx.doi.org/10.21474/IJAR01/4529 MIES AND MINIMALISM: A PERFECT COALESCENCE. In 1930, Mies became the Director of „Bauhaus‟- a German art school, which later added architecture as a medium of art in its program, and developed the Bauhaus style of Architecture. Mies adopted this new style which favored the use of steel, glass, and reinforced concrete. In 1933, when the Nazi regime closed the art school, Mies shifted to the United States in 1938, and become of one of the most innovative leader of this modern movement in architecture called minimalism. (Clemence, 2006). Introduction: - To quote Macarthur (2002), “The „look‟ of the object, in this case the Farnsworth House, connected with the people, and came to be called a „poem‟, an artwork, just like a painting or a sculpture.” To begin with, let‟s discuss how minimalism in architecture is perceived as an art: As Macarthur (2002) states, minimalism in visual art is a known fact. However, modern art, which considers architecture too as a form of art, sees the emergence of minimalist architecture, which assumes that, like in visual art, buildings are physical objects and the „look‟ of the object is very important. Though buildings are seen as simple objects, whereas other visual arts like painting and sculpture are seen as real artwork, minimalist architecture sees the building as an aesthetically created object, which is considered artistic by the look they provide, thus making them a critical object and not just a material one. However, some architects believe that a building can aspire to be a form of artwork only if it not designed for utility. This raises the question whether all buildings should be then considered as just buildings or as artworks? Mertins (2014) says that Minimalist architecture tries to provide an answer to it by displaying the works of architect Mies van der Rohe, who believed that the method of construction itself was an art. It was in the concept of built object. Buildings, like trees, were objects, and if the material used on them, looked back at the person looking at them, then that architecture was art. Mies‟s connection to minimalism can be better understood by examining the different principles of minimalistic art: Though minimalism in architecture was also considered art, different tenets to minimalism in art are seen. It is generally seen as a materialist undertaking. The East-coast of USA sees minimalism as being more materialist, whereas the West- coast perceives it as being more idealist. Donald Judd, an American artist believes in the “research of truth with simple forms”. He also adds that, “To make good things, you have to have some sort of belief”. Idealist minimalism has a more spiritual approach. Mies van der Rohe‟s minimalism intersects between materialist and idealist (Chave, 2008). According to Mertins (2014), though Mies‟s architecture is considered as philosophical, it is also very scientific in character. A self-taught person, Mies studied both philosophy and science, and he put this education into his profession. Introduction: - This minimalist movement, which was first visible in the visual art, like sculpture and painting, arose in the 1950s. It was characterized by the use of simple and massive forms, which were aesthetically appealing. The artistic creation was not perceived within any pre-determined fore bonding, but was self-contained (Mertins, 2014). Mies van der Rohe adopted this movement in architecture at a time when it was the age of rationalization, structural rigidity, and mass production. His buildings were simple in form, had minimum use of material like steel, glass, and concrete, but were aesthetically immaculate. They were neither an imitation of any works from the past nor were they based on any social ideas or individual emotions. They were like a moment in history. They were self- referential, like any work of art (Mertins, 2014). According to Chave (2008), Minimalism in art has two tenets – materialistic and idealistic, wherein the idealist adopts a spiritual approach, which is almost „auratic‟ in nature. Mies adopted both these tenets and created a minimalist style of his own, which for almost a century, proved very popular. For him, architecture was „sacred‟, Corresponding Author: - Srikanth Sakleshpur Venkatesh. Address: - University of Illinois at Urbana-Champaign. 1236 ISSN: 2320-5407 ISSN: 2320-5407 Int. J. Adv. Res. 5(6), 1236-1239 Int. J. Adv. Res. 5(6), 1236-1239 meaning divine and meant for a secular society, but supported by science and adopted to the ever-changing aspect of the universe. His buildings were discernible physical objects that when looked at, shaped one‟s perceptions and affections towards them. Vasilski (2016) adds that the simplicity of objects that minimalist art postulates, are also seen in Mies‟s works. For him, the simplicity lay in the built form, relying only on the essential, by using minimum material for his structures, but without really compromising on the utility (Chave 2008). This paper shows how the concept of „less is more‟ adopted by the architect Mies van der Rohe could be a benchmark for minimalist architecture. How from one medium of expression, that is minimalist art, Mies transcended it and evolved the modern movement of minimalist architecture. With specific reference to one of Mies‟s very famous „work of art‟ in the United States, the Farnsworth House, this paper will try to illustrate how the concept „less is more‟ is beautifully incorporated in this building, thus showcasing the perfect coalescence that is visible between Mies and minimalism. ISSN: 2320-5407 ISSN: 2320-5407 ISSN: 2320-5407 His architecture was considered philosophical. He postulated that it was possible to integrate cosmos with humanity‟s place in it. Spatial fluidity and continuity from interior to exterior was an example of harmony between nature and the living world. Thus, minimalism in architecture provided Mies with a concept of sensation to perception through tangible physical objects, which were pure and simple. In this way, Mies van der Rohe‟s buildings coalesce perfectly with the aphorism he adopted very early is his career, „less is more‟: a tenet for minimalist design and architecture. This paragraph examines how the essential elements seen in Mies‟s buildings integrate the aphorism „less is more‟ perfectly in them. According to Vasilski (2016) all the elements associated with minimalism that we enumerated above, like simplicity in design over complexity are visible in Mies‟s works. Chave (2008) says that buildings built with essential elements, like steel and glass, create harmony with free flowing open spaces, and provide clarity, utility, and effect through the aesthetic appeal that they carry, by making use invisible qualities like natural light, sky, earth, and air, thus providing the sublime spiritual experience. Vasilski (2016) states that this experience is observed in Mies‟s masterpieces. Whether it is the Barcelona Pavilion or the Farnsworth house or the Seagram Building, which are a pure and open „skin and bone‟ architecture, wherein the steel structure is the „bone‟ and the glass about it is the „skin but also some of the foremost examples of functionalist aesthetic buildings (Mertins, 2014). The Farnsworth House, one of the best examples of minimalism among Mies van der Rohe‟s „masterpieces‟ in the United States: In the design of this house, Mies‟s motto „less is more‟ reaches cathartic dimensions. Located on a 60-acres site, it is the only private home ever designed and built by Mies for his friend Dr. Edith Farnsworth at Plano, Illinois between 1945 and 1951. Made completely of steel and glass, Farnsworth House essence lay more on its spiritual qualities than functional qualities. According to Clemence 2006, Mies said that materials used were the grammar of architecture. And unless grammar was mastered, one could not speak good prose and much less become a good poet. Though, Mies‟s architecture generally invokes the objective mind, Farnsworth House implores both the subjective and objective mind, as art is a mixture of both. And Farnsworth house was an architectural poem. ISSN: 2320-5407 According to Mies, a poem is a spiritual creation that never ceases to delight. This house was built in such a way that it seemed to fly above the ground. There seemed to be no visual barrier between the interior and the exterior (Clemence, 2006). Additionally, we shall deliberate how the Farnsworth house looks suspended among trees in an undisturbed landscape. It‟s almost seems part of nature. Mies states, “…we should attempt to bring nature, houses, and human beings together into higher unity. If you view nature through glass walls of Farnsworth House, it gains a more profound significance than if viewed from outside…it becomes a part of a larger whole” (Clemence, 2006). Meant to be a weekend retreat, this glass box pavilion, containing „almost nothing‟, but just one room, provided an unobstructed space that was flexible to use. The space contains certain freestanding components and one central wood core that provided veiled spaces meant for sleeping, cooking, sitting, dressing, and eating. The house in 2006 was earmarked as a National historic landmark (Clemence, 2006). Introduction: - His concept of sacred architecture, in a secular world, but primed by science, is very evident in his buildings like the S.R Crown Hall at IIT campus in Chicago and the Seagram Building in New York. The philosophy behind Mies‟s minimalism: If one argues Mies architecture as being philosophical, then it could mean that it also aspires to make the world a better place. It decrees that the space we live in should be inspiring. Architecture is something that should be experienced to be understood. Immanuel Kant, a German philosopher avers that minimalism in architecture and the philosophy of experience of life, go hand in hand. It means that the space one lives in is essential to understand architecture. What we perceive and what we feel in a living space is important (Vasilski, 2016). Furthermore, if the space that we live in is essential to understand architecture, then understanding different elements of the building structure is important in order to perceive and feel the space we live in. In this context, Vasilski (2016) postulates that in minimalism, light is also an important structural element. In the space we live in, one can experience this light as proximity to time. And at the same time, in the silence one feels the space. Providing purity, harmony, transparency, and simplicity of life is the ultimate goal of minimalist architecture. According to Mertins (2014), Mies van der Rohe also adheres to this notion. He was drawn to the critical philosophy of Immanuel Kant, the German philosopher. He believed that the search for the essence by shedding the superfluous was possible. 1237 Int. J. Adv. Res. 5(6), 1236-1239 Int. J. Adv. Res. 5(6), 1236-1239 Int. J. Adv. Res. 5(6), 1236-1239 Int. J. Adv. Res. 5(6), 1236-1239 Conclusion: - Mies van der Rohe, beautifully encompassed the aphorism „less is more‟, which is the tenet of minimalist design and architecture, in his buildings, during the period of rationalization and mass production, and brought about a new vision in the sphere of architecture (Mertins, 2014). The simplicity in basic geometrical forms, the use of simple materials like glass and steel, and invisible qualities like natural light, sky, earth, and air, all portray a sense of order of simple and clean spaces. This is the only art form that allows you to experience the „live‟ movement, that one can discern „inside‟ the object, which in this case is the building (Vasilski, 2016). According to Mies, architecture could never become complacent, hinting that a building should not be something that you „search‟, but something you should „find‟. This was possible only if the building was built by virtue of transparency, economy, and obviousness. And this is what one would call the aesthetic experience of minimalism, and that‟s found in the works of Mies Van der Rohe (Mertins, 1994). His buildings, often referred as „skin and bone‟ architecture, as he used glass enclosures on steel structures, became pioneers of minimalist architecture expression. His various typologies, like the skyscraper typology, visible in the 1238 ISSN: 2320-5407 Int. J. Adv. Res. 5(6), 1236-1239 Int. J. Adv. Res. 5(6), 1236-1239 skyscraper the Seagram building; the pavilion typology, visible in buildings like the Barcelona pavilion and Farnsworth house, and a combined typology of both skyscraper and pavilion typologies, visible in the Chicago Federal Center, have set a benchmark in the world of minimalist architecture (Rosenfield, 2016). Mies postulated that the primary objective of architecture is to unequivocally represent its epoch, and that it‟s the role of the architect to bring to light and articulate the significance of the time. Till today, this postulation of his has credence among the generations of architects he has inspired and continues to inspire (Mertins, 2014). And his buildings bear testimony to this conviction. References: - 1. Chave. C. A. (2008) Revaluing Minimalism: Patronage, Aura, and Place, The Art Bulletin, 90(3). p.466-486. 2. Clemence, P. (2006) Mies van der Rohe's Farnsworth House, Schiffer Pub, p.93. 3. Macarthur, J. (2002) The Look of the Object: Minimalism in Art and Architecture, Then and Now, Architectural Theory Review, 7(1) p.137-148. y ( ) p 4. Mertins, D. (Ed.) (1994) The Presence of Mies, Princeton Architectural Press, p.270. 5. Mertins, D. (2014) Mies, Phaidon Press, p.544 7. Rosenfield, K. (2016) Mies van der Rohe: „Architecture as language‟. Retrieved from www.archdaily.com/782034/ 8. Schulze, F., & Windhorst, E. (2012) Mies van der Rohe: A Critical Biography, The University of Chicago Press, p.512. 9. Vasilski, D. (2016) On minimalism in architecture - space as experience, Spatium, 1(36), p.61-66. 1239
https://openalex.org/W2899893323	https://opinvisindi.is/bitstream/20.500.11815/1800/1/ece3.4746.pdf	English	null	Linking warming effects on phenology, demography, and range expansion in a migratory bird population	Ecology and evolution	2,019	cc-by	9,742	Abstract Changing climatic conditions have been linked to changes in phe- nology (Parmesan & Yohe, 2003; Visser, Holleman, & Gienapp, 2006), distribution (Chen, Hill, Ohlemüller, Roy, & Thomas, 2011; This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2019 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. José A. Alves1,2 \| Tómas G. Gunnarsson2 \| William J. Sutherland3 \| Peter M. Potts4 \| Jennifer A. Gill1 1School of Biological Sciences, University of East Anglia, Norwich, UK 2South Iceland Research Centre, University of Iceland, Laugarvatn, Iceland 3Department of Zoology, University of Cambridge, Cambridge, UK 4Farlington Ringing Group, Southampton, UK Received: 5 March 2018 \| Revised: 21 June 2018 \| Accepted: 18 August 2018 Received: 5 March 2018 \| Revised: 21 June 2018 \| Accepted: 18 August 2018 DOI: 10.1002/ece3.4746 Linking warming effects on phenology, demography, and range expansion in a migratory bird population José A. Alves1,2 \| Tómas G. Gunnarsson2 \| William J. Sutherland3 \| Peter M. Potts4 \| Jennifer A. Gill1 Abstract Abstract Phenological changes in response to climate change have been recorded in many taxa, but the population‐level consequences of these changes are largely unknown. If phenological change influences demography, it may underpin the changes in range size and distribution that have been associated with climate change in many species. Over the last century, Icelandic black‐tailed godwits (Limosa limosa islandica) have increased 10‐fold in numbers, and their breeding range has expanded throughout lowland Iceland, but the environmental and demographic drivers of this expansion remain unknown. Here, we explore the potential for climate‐driven shifts in phenol- ogy to influence demography and range expansion. In warmer springs, Icelandic black‐tailed godwits lay their clutches earlier, resulting in advances in hatching dates in those years. Early hatching is beneficial as population‐wide tracking of marked in- dividuals shows that chick recruitment to the adult population is greater for early hatched individuals. Throughout the last century, this population has expanded into progressively colder breeding areas in which hatch dates are later, but temperatures have increased throughout Iceland since the 1960s. Using these established relation- ships between temperature, hatching dates and recruitment, we show that these warming trends have the potential to have fueled substantial increases in recruitment throughout Iceland, and thus to have contributed to local population growth and expansion across the breeding range. The demographic consequences of tempera- ture‐mediated phenological changes, such as the advances in lay dates and increased recruitment associated with early hatching reported here, may therefore be key pro- cesses in driving population size and range changes in response to climate change. K E Y W O R D S Correspondence José A. Alves, School of Biological Sciences, University of East Anglia, Norwich, UK. Email: j.alves@uea.ac.uk Correspondence José A. Alves, School of Biological Sciences, University of East Anglia, Norwich, UK. Email: j.alves@uea.ac.uk Funding information Fundação para a Ciência e a Tecnologia, Grant/Award Number: SFRH/ BPD/91527/2012; Arcadia; Natural Environment Research Council, Grant/ Award Number: NE/H008527/1; Icelandic Centre for Research, Grant/Award Number: 130412-051 climate change, population dynamics, species distribution, temperature, waders Ecology and Evolution. 2019;9:2365–2375. \| 2365 www.ecolevol.org 1 \| INTRODUCTION Understanding biological responses to global climatic and envi- ronmental change is among the most urgent of challenges facing ecologists and conservationists (Lebreton, 2011; Parmesan, 2006). 1 \| INTRODUCTION 1 ecologists and conservationists (Lebreton, 2011; Parmesan, 2006). Changing climatic conditions have been linked to changes in phe- nology (Parmesan & Yohe, 2003; Visser, Holleman, & Gienapp, 2006), distribution (Chen, Hill, Ohlemüller, Roy, & Thomas, 2011; Understanding biological responses to global climatic and envi- ronmental change is among the most urgent of challenges facing \| 2365 www.ecolevol.org \| www.ecolevol.org Ecology and Evolution. 2019;9:2365–2375. 2366 ALVES et al. traits and demographic rates across a population (McLean, Lawson, Leech, & Pol, 2016). Hickling, Roy, Hill, Fox, & Thomas, 2006; Hill et al., 2002), and demography (Both, Bouwhuis, Lessells, & Visser, 2006; Hansen, Aanes, Herfindal, Kohler, & Sæther, 2011; Post & Forchhammer, 2008) across many taxa (Root et al., 2003; Thackeray et al., 2010; Walther et al., 2002), but the mechanistic processes driv- ing these changes in free‐ranging animals, and their population‐ level consequences, remain poorly understood (but see Lane, Kruuk, Charmantier, Murie, & Dobson, 2012; Ozgul et al., 2010). Predicting species responses to future climatic scenarios requires a mechanistic understanding of the ecological, behavioral, and historical factors that influence species demographic and distribu- tional responses to changing environments (Mustin, Sutherland, & Gill, 2007; Norris, 2004). Quantifying the mechanisms through which changing climatic conditions can influence changes in pop- ulation size and distribution is therefore key to predicting future responses to environmental change and identifying associated conservation actions (Beale, Lennon, & Gimona, 2008; Guisan et al., 2013). Among the most rapid and severe changes in climatic conditions to have occurred over the last century are the warming tempera- tures in arctic and subarctic zones (IPCC, 2007; Robinson, 2009). Iceland lies mostly within the subarctic climate zone and has expe- rienced a general increasing trend in temperatures since records began in 1845 (Einarsson, 1984; Jónsson, 2006), with rapid tem- perature increases in the 20th century during which annual mean temperature at the longest running weather station rose ~1.2°C (Jónsson, 2006). Iceland hosts internationally important breed- ing populations of many migratory bird species (Gunnarsson, Gill, Appleton, et al., 2006a), for which changing climatic conditions could have important implications. For example, the breeding and wintering range of Icelandic black‐tailed godwits (Limosa limosa islandica) has expanded over the last century, concomitant with this warming period (Gill et al., 2001; Gunnarsson, Gill, Newton, Potts, & Sutherland, 2005a). FI G U R E 1 Map of Iceland with 200 m asl contour line below which most suitable habitat for breeding Icelandic black‐tailed godwits occurs. Circles show areas occupied by the species before the 1900s (white) or colonized during the 1950s (very light gray), 1960s (light gray), 1970s (gray), 1980s (dark gray), or 1990s (black). All these areas have an operational weather station collecting data for each lowland basin (see Supporting Information Table S3 for details) 1 \| INTRODUCTION In the early 1900s, this species was restricted to the southern lowlands of Iceland but since then it has gradually colonized coastal lowland areas throughout the country (Figure 1) with larger areas closer to occupied sites being colonized first (Gunnarsson, Gill, Petersen, Appleton, & Sutherland, 2005b). The population now numbers ~50,000 individuals, which is likely to be an approximately 10‐fold increase in numbers over the last cen- tury (Gill et al., 2007; Gunnarsson, Gill, Potts, et al., 2005c), but the environmental and demographic changes underpinning this popula- tion increase and range expansion are not known (Gill et al., 2007). Icelandic godwits are long‐lived migratory shorebirds (Alves et al., 2012; Gunnarsson, Gill, Atkinson, et al., 2006b) with a typical lifes- pan of ~15–20 years (Gill et al., 2007), that breed almost exclusively in Iceland and winter in coastal zones of north and west Europe (Gill, Hatton, & Potts, 2002). They have a modal clutch size of four eggs, nesting in lowland wetlands dominated by grasses or by dwarf birch (Betula nana) and sedges (Carex spp.) (Gunnarsson, Gill, Newton, et al., 2005a) and are among the largest nest‐concealing species of the Scolopacidae (Cramp & Simmons, 1983), thus requiring a suitable Phenological changes in response to climate change (e.g., Amano, Smithers, Sparks, & Sutherland, 2010; Gordo, 2007) will only directly affect population demography (Pettorelli, Pelletier, Hardenberg, Festa‐Bianchet, & Côté, 2007) and distribution (Van der Jeugd et al., 2009) if fitness parameters are influenced by changes in the timing of events across the annual cycle (Chuine, 2010; Visser et al., 2006). Phenological mismatches with resource availability have been linked to local population trends in several species (Both et al., 2006; Gaillard et al., 2013; Hansen et al., 2011), but the extent to which these patterns can drive large‐scale population and range expansion or contraction in free‐ranging animals remains poorly understood. In addition, recent studies have found that climate‐ mediated changes in phenology may not always lead to changes in population size (Dunn & Møller, 2014), even when fitness effects are apparent (Reed, Grøtan, Jenouvrier, Sæther, & Visser, 2013a; Reed, Jenouvrier, & Visser, 2013b). Understanding how local climate ef- fects can potentially scale up to population‐level changes requires quantification of the magnitude of climate effects on individual FI G U R E 1 Map of Iceland with 200 m asl contour line below which most suitable habitat for breeding Icelandic black‐tailed godwits occurs. 1 \| INTRODUCTION Circles show areas occupied by the species before the 1900s (white) or colonized during the 1950s (very light gray), 1960s (light gray), 1970s (gray), 1980s (dark gray), or 1990s (black). All these areas have an operational weather station collecting data for each lowland basin (see Supporting Information Table S3 for details) FI G U R E 1 Map of Iceland with 200 m asl contour line below which most suitable habitat for breeding Icelandic black‐tailed godwits occurs. Circles show areas occupied by the species before the 1900s (white) or colonized during the 1950s (very light gray), 1960s (light gray), 1970s (gray), 1980s (dark gray), or 1990s (black). All these areas have an operational weather station collecting data for each lowland basin (see Supporting Information Table S3 for details) FI G U R E 1 Map of Iceland with 200 m asl contour line below which most suitable habitat for breeding Icelandic black‐tailed godwits occurs. Circles show areas occupied by the species before the 1900s (white) or colonized during the 1950s (very light gray), 1960s (light gray), 1970s (gray), 1980s (dark gray), or 1990s (black). All these areas have an operational weather station collecting data for each lowland basin (see Supporting Information Table S3 for details) ALVES et al. 2367 FI G U R E 2 Changes in (a) mean (± SE) annual nest laying date (days from 1st of May); and (b) length of the period from hatching to fledging, of Icelandic black‐tailed godwits in relation to: (a) mean daily temperature in June between 2001 and 2013 (laying date = −5.49 * temperature + 90.7, r2 = 0.75, F(1,6) = 18.32, p = 0.005), and (b) cumulative daily temperature in 2012 (gray) and 2013 (black) during the 21 days post‐hatching (time to fledge = −0.013 * cumulative temperature + 45.91, r2 = 0.40, F(1,16) = 10.69, p = 0.005) vegetation height in order to initiate nesting. As the onset and rate of vegetation growth in subarctic ecosystems (Thorvaldsson, Björnsson, & Hermansson, 2005), as well as the timing of emergence of invertebrate prey for wader chicks (Halldórsson et al., 2013; Tulp & Schekkerman, 2008), are strongly temperature‐dependent, timing of nesting, and chick growth rate are likely to be influenced by local temperatures. 1 \| INTRODUCTION Over eight years during which temperatures in Iceland varied substantially, we quantified (a) the influence of temperature on nest cup vegetation heights, laying dates and the duration of the pre‐ fledging period of Icelandic godwits, and (b) the subsequent influ- ence of hatching dates on recruitment of chicks to the wintering or subsequent breeding population. We then use these relationships to investigate the potential contribution of warming‐derived changes in phenology and recruitment to the rapid population increase, by quantifying (c) variation in spring temperature and hatch dates across breeding areas colonized at different times during the 20th century, and (d) the magnitude of temperature changes and conse- quent estimated rates of change in godwit recruitment within these areas during the period of range expansion. 2.1.1 \| Timing of nesting and vegetation growth FI G U R E 2 Changes in (a) mean (± SE) annual nest laying FI G U R E 2 Changes in (a) mean (± SE) annual nest laying date (days from 1st of May); and (b) length of the period from hatching to fledging, of Icelandic black‐tailed godwits in relation to: (a) mean daily temperature in June between 2001 and 2013 (laying date = −5.49 * temperature + 90.7, r2 = 0.75, F(1,6) = 18.32, p = 0.005), and (b) cumulative daily temperature in 2012 (gray) and 2013 (black) during the 21 days post‐hatching (time to fledge = −0.013 * cumulative temperature + 45.91, r2 = 0.40, F(1,16) = 10.69, p = 0.005) The breeding success of godwits was intensively monitored annu- ally between 2001–2003 and 2005, and 2010–2013, with 11 study sites in South Iceland being surveyed 1–2 times per week during the breeding season (Supporting Information Table S1). For each godwit nest found, the incubation stage of the clutch (all eggs) was measured using standard egg floatation method, allowing the laying date (day when the last egg was laid) and hatch date to be estimated. Categorical classification following (Liebezeit et al., 2007) was adapted to our study species (Supporting Information Table S2): firstly the average length of incubation was determined in successfully hatched nests which were found during egg‐laying (22.7 days ±0.3 SE, n = 7); then two additional floatation levels were added to the five described in Figure 2 of Liebezeit et al. (2007) to allow greater accuracy during the later stages of incuba- tion, based on field observations (J. A. Alves, personal observa- tion). When eggs of the same clutch varied in incubation stage, the middle value between categories (in days) was assigned to the clutch. Every nest was visited regularly, and successful nests were revisited at the estimated hatching date in order to cap- ture and mark chicks and adults with individual combinations of color‐rings. Vegetation height around the rim of the nest cup was quantified when nests were first located, using a tape measure at four equidistant points from the center of the nest, and also at 15 randomly located points in the vicinity of the nests (within 100 m) throughout the breeding season (May to July) in 2011–2013. 2.3 \| Data analyses The influence of temperature on annual variation in mean godwit lay- ing dates across the 11 study areas in South Iceland was explored in regression analyses using (a) mean June temperature (the month in which the majority of godwit clutches are laid; although some clutches were laid during the last week of May, temperatures during late May and June were very strongly correlated (rs = 0.87, n = 8, p < 0.005) in these years) and (b) mean daily temperature during the laying period (from the laying date of the first nest to the laying date of the last nest located) in each year. The former analysis facilitated use of histori- cal mean June temperatures in subsequent analyses while the latter ensured that the patterns were consistent with more highly resolved temperature data which are not available for the historical time series. Mean temperature was chosen as predictor of laying dates as this pa- rameter is strongly related to the growth rates of grasses in Iceland (Thorvaldsson et al., 2005). Temperature data were recorded at the closest weather monitoring station (Eyrarbakki, 63°52′N, 21°09′W; which is within 7.5 km of all the nests found and broods tracked). 2.2.2 within 1–2 days of chick hatching (J. A. Alves, personal observation, 25 June & 3 July 2012). Models of the length of the pre‐fledging period were constructed both including and excluding these two families. Brood mortality was defined as two consecutive failures to relocate tagged families (at 2 and 4 days) after the last confirmed ob- servation (when adults still displayed aggressive defensive behavior). When families were not located at the last known position, scans for tag transmissions were taken at vantage points (minimum of 3) in a triangular shape surrounding the last known position and cover- ing a range of more than 1 km from that position (a distance that exceeds the distance over which godwit broods have been recorded moving; Kentie, Hooijmeijer, Trimbos, Groen, & Piersma, 2013; J. A. Alves, personal observation). Godwit chicks typically fledge at ~25 days (Kentie et al., 2013); however, some families fledged young prior to that age and, for these, visual confirmation of at least one of the fledged young was attained by reading its individual color‐ring combination. To explore the potential influence of temperature changes in recent decades on recruitment rates in the Icelandic godwit population, average monthly temperatures were extracted from the Icelandic Meteorological Office (available at www.vedur.is) for 14 lowland areas around Iceland in which the average year of colonization by godwits during the past century had previously been compiled (Supporting Information Table S3; Figure 1). The average year of colonization has been reconstructed by collating reports and re- cords of new breeding species within each lowland basin provided by local residents which are encouraged by and submitted to the Icelandic Institute of Natural History (see details in Gunnarsson, Gill, Petersen, et al., 2005b; Supporting Information Table S3). None of the weather stations provided temperature data prior to 1949 (Supporting Information Table S3). et al., 2006b), allowing recruitment to the wintering or subsequent breeding population (i.e., any record after the first autumn period) of the color‐marked chicks to be established. As the vast majority of godwit recruits were first recorded within their first 2 years of life (mean no. of years to first observation = 1.5 ± 2.1 SD, N = 394), comparison of chicks that recruited (observed after the first autumn migration period) or did not (never recorded since ringing) was re- stricted to chicks ringed up to and including the summer of 2011 and using sightings up to and including those in summer 2013. More recently ringed individuals (since 2012) that may not yet have been observed in the breeding or nonbreeding sites at the time when this analysis was executed were excluded. TA B LE 1 Results of binomial GLMMs of: (a) the influence of hatching date on the probability of Icelandic black‐tailed godwit chicks recruiting to the wintering population; (b) the effect of laying date on daily nest survival probabilities Estimate SE z value p (a) Intercept 2.53 0.62 4.05 <0.001 Hatch date −0.04 0.01 −4.55 <0.001 (b) Intercept −2.71 0.46 −5.9 <0.001 Lay date −0.01 0.01 −0.55 0.58 Annual variation (in a and b) is included as random factor (see methods for details). Significant effects are highlighted in bold. Annual variation (in a and b) is included as random factor (see methods for details). Significant effects are highlighted in bold. 2.1.2 \| Length of the pre‐fledging period In 2012 and 2013, 32 godwit families were tracked during chick‐ rearing, from hatching to fledging (n = 18) or brood loss (n = 14). The length of the pre‐fledging period could therefore be measured for 18 families (nine in each year). For each family, one of the adults was captured using either a nest‐trap or a hand‐held net‐gun (details in Edwards & Gilchrist, 2011) and fitted with an individual combi- nation of color‐rings and a back‐mounted radio‐tag weighing 2.4 g with a 1.5‐month lifespan (PIP3, Ag393 Biotrack Ltd). The position of each radio‐tagged individual was recorded every two days using a hand‐held Yagi antenna and receiver (Sika, Biotrack Ltd) and a GPS (eTrex Garmin). Exact hatch dates were unknown for the two broods of adults trapped with the net‐gun, but the highly aggressive behavior of the adults (adult godwits stay very close to their chicks and defend them during the pre‐fledging period, Gunnarsson, Gill, Petersen, et al., 2005b) indicated that these had both been captured 2368 ALVES et al. ALVES et al. 3.1 \| Effects of temperature on the phenology of breeding season events response variable. Cumulative temperature was considered a proxy of time available for chicks to forage, as low temperatures often re- quire parental brooding thus reducing foraging time. As cumulative temperatures will inevitably be higher for chicks that take longer to fledge, we restricted the calculation of cumulative temperatures to the first 21 days after hatching (the age at which the earliest brood fledged). Icelandic godwits lay their clutches earlier and rear their chicks faster when temperatures are warmer (Figure 2). In years with warmer mean June temperatures (~11°C, Figure 2a), laying dates are ~11 days earlier than in colder years (~9°C, Figure 2a). These patterns are also appar- ent when using mean daily temperature during the sampled laying pe- riods in each year (measured as the mean daily temperature between the start and end of laying; laying date = −3.07 * temperature + 66.90, r2 = 0.67, F(1,6) = 12.28, p = 0.013). The estimation of laying dates using categorical data for large waders has a reported absolute mean (± SE) deviation ranging from 0.2 (± 0.2) to 2.5 (± 0.5) days (Liebezeit et al., 2007); however, there is no reason to expect systematic an- nual bias in this error and the measured differences in laying dates are considerably larger than the associated error (Figure 2a). Broods that experienced warmer cumulative daily mean temperatures dur- ing the pre‐fledging period also had significantly shorter pre‐fledging periods (Figure 2b). This relationship persisted when the two broods with unknown hatching date were excluded (time to fledge = −0.011 * cumulative temperature + 43.53, r2 = 0.31, F(1,14) = 6.28, p = 0.025). To estimate the seasonal effect of laying date on nest survival, we used a formulation of Mayfield's method (Mayfield, 1961, 1975) as a logistic model and a logit link function (Crawley, 2007) in which success or failure over a given number of days (as a binary variable) was modeled with the number of days over which the nest was monitored as the binomial denominator (Aebischer, 1999; Morrison, Robinson, Leech, Dadam, & Toms, 2014), laying date as a predic- tor and year as a random effect. For predated nests, the number of exposure days was considered to be the mid‐point between the maximum and minimum possible number of exposure days, given the timing of nest visits. 3.1 \| Effects of temperature on the phenology of breeding season events In order to investigate the effect of hatch date on the probabil- ity of recruiting into the wintering population, and as the data were too over‐dispersed for a capture‐mark‐recapture analysis, we con- structed a GLMM with binomial error distribution and a logit link function (Crawley, 2007), with recruited (observed after the first au- tumn migration) or not (not recorded since ringing) as the response variable, hatch date as a fixed factor and year of capture as random factor. Variation in resighting probability of marked juveniles is not included in these analyses but systematic variation in resighting probability in relation to hatch date is unlikely. 2.2.1 \| Determining recruitment rates In addition to the chicks marked at the 11 study sites (see above), Icelandic godwit chicks have been caught, measured, and individu- ally color‐ringed at locations all around Iceland since 1999 (n = 966; yearly average = 69 ± 56.2 SD). The majority of these chicks were captured, marked, and measured (mass to nearest 0.5 g using a spring balance or a digital scale) after leaving the nest and so their age was unknown. As very few chicks are repeatedly recaptured during the pre‐fledging period, we used a previously published growth curve established for this species (Beintema & Visser, 1989) to estimate the age of each chick at capture (and thus hatch date, used in the model below) from their body mass at capture. Although uncertainty in age estimation increases with body mass (Kentie et al., 2013), the vast majority (>75%) of the chicks in this study weighed 150 g or less at ringing. A network of hundreds of volunteer observers regularly report observations of color‐ringed Icelandic godwits from across the migratory range (Alves et al., 2013; Gunnarsson, Gill, Atkinson, To explore the effect of temperature on the length of the chick‐ rearing period, we calculated the daily cumulative temperature sum since hatching for each brood and used this as a predictor in a regres- sion model with the length of the pre‐fledging period (days) as the ALVES et al. 2369 FI G U R E 3 Variation in the probability (solid line ± SE, dashed lines) of Icelandic godwit chicks recruiting to the wintering or subsequent breeding population in relation to their hatch date which temperature data were not available were not included in this analysis. We then used the relationships established between (a) local temperature and hatch dates, and (b) hatch dates and recruitment probability, to estimate the magnitude of change in recruitment probabilities for the breeding areas during the decades following colonization, given average June temperatures in each area in each decade (see Supporting Information for details). These estimates conservatively assume that the relationships between temperature and timing of breeding season events are consistent at the lower temperatures that occur elsewhere in lowland Iceland. All analyses were performed in R 2.15.0 (R Development Core Team, 2011). 2.2.1 \| Determining recruitment rates FI G U R E 3 Variation in the probability (solid line ± SE, dashed lines) of Icelandic godwit chicks recruiting to the wintering or subsequent breeding population in relation to their hatch date \| TA B LE 2 Results of a GLM of variation in mean June temperatures over five decades (1950s–1990s) in 14 breeding areas around Iceland colonized in different years 3.3 \| The influence of temperature, phenology and demography on range expansion df F p Estimate (± SE) Colonization year 1 81.66 <0.001 −0.06 (±0.01) Decade 4 3.88 0.008 Col. year*Decade 53 0.68 0.609 Error 62 Significant effects are highlighted in bold. The sequence of colonization of lowland areas around Iceland by breeding godwits over the last century follows a tempera- ture gradient, with warmer areas being colonized first (Table 2; Figure 4a), and godwit chicks hatch earlier in regions that were colonized first (F4,686 = 12.8, p < 0.001; Figure 4b; Supporting Information Table S3). Following relatively warm average June temperatures in the nine areas around Iceland with available temperature data in the 1950s (and when few sites had been colonized; Figure 1), subsequent temperatures were lower in the 1960s but have generally increased since then (Table 2; Figure 4c). Significant effects are highlighted in bold. Significant effects are highlighted in bold. SE) of chicks hatched early (before day 57), indicating the substantial effect that early hatching can have on the overall numbers of chicks recruiting from warmer than colder years. SE) of chicks hatched early (before day 57), indicating the substantial effect that early hatching can have on the overall numbers of chicks recruiting from warmer than colder years. 3.2 \| Effect of breeding season temperatures and phenology on demography For Icelandic godwit chicks, the probability of successfully recruit- ing to the wintering population is strongly influenced by hatch date (Table 1a). Chicks hatched during the first two weeks of June (before day 47 on Figure 3) have, on average, >60% probability of recruiting, whereas those hatching after 12 July (day 73 on Figure 3) have, on average, <35% probability of recruiting. The benefits of early lay- ing in warmer years could be reduced if hatching success was lower early in the season, but we found no seasonal effect of laying date on daily nest survival (Table 1b). In colder years (2001, 2011), when average June temperatures were below 9.5°C, the mean percent of chicks hatched before day 57 (when recruitment probability exceeds 50%, Figure 3) was 45% (± 1 SE). By contrast, in warmer years (aver- age June temperature≥11°C; 2010, 2012), an average of 64% (± 5 Over the last century, godwits have expanded into progressively colder regions of Iceland. Regional variation in chick hatch dates were explored with an ANOVA test, and rates of spring tempera- ture change during the population expansion in colonized areas were explored in a GLM, with mean decadal June temperature modeled as function of decade (1950s to 1990s), mean year of colonization (from Gunnarsson, Gill, Newton, et al., 2005a), and their interaction. Areas colonized by Icelandic godwits during the past century but for 2370 ALVES et al. 3.3 \| The influence of temperature, phenology and demography on range expansion FI G U R E 4 Variation in mean (± SE): (a) June temperature in 14 lowland areas in Iceland colonized by godwits during the second half of the 20th century (lowland areas colonized in different decades colored as in Figure 1; see Supporting Information Table S3 for details of temperature ranges); (b) hatch dates (ordinal days since 1st of May) of godwit chicks between 1999 and 2011 across regions of Iceland for which temperature and colonization dates are available (number of chicks per region given within bars and bar color reflects the predominant decade of colonization within each region); (c) June temperatures in five decades during the range expansion period (see Table 2 for statistics); and (d) the potential consequences of spatial and annual variation in June temperatures and chick hatch dates for estimated annual recruitment probability in areas colonized in different decades (number of areas with available temperature data shown in parentheses, details in Supporting Information Table S3) during the 1960s (light gray bars), 1970s (gray bars), 1980s (dark gray bars), and 1990s (black bars) across Iceland FI G U R E 4 Variation in mean (± SE): (a) June temperature in 14 lowland areas in Iceland colonized by godwits during the second half of the 20th century (lowland areas colonized in different decades colored as in Figure 1; see Supporting Information Table S3 for details of temperature ranges); (b) hatch dates (ordinal days since 1st of May) of godwit chicks between 1999 and 2011 across regions of Iceland for which temperature and colonization dates are available (number of chicks per region given within bars and bar color reflects the predominant decade of colonization within each region); (c) June temperatures in five decades during the range expansion period (see Table 2 for statistics); and (d) the potential consequences of spatial and annual variation in June temperatures and chick hatch dates for estimated annual recruitment probability in areas colonized in different decades (number of areas with available temperature data shown in parentheses, details in Supporting Information Table S3) during the 1960s (light gray bars), 1970s (gray bars), 1980s (dark gray bars), and 1990s (black bars) across Iceland ALVES et al. ALVES et al. 2371 \| 2 The changes in June temperatures around Iceland since the 1950s (Figure 4a,c) can be used to assess the potential magnitude of temperature‐driven variation in hatch dates and recruitment of Icelandic godwits. 3.3 \| The influence of temperature, phenology and demography on range expansion In every decade, the areas of Iceland that were colonized during the 1950s were warmest and estimated recruit- ment is therefore consistently highest in these areas (Figure 4d). In the 1990s, when all areas had been colonized, recruitment probabil- ity was estimated to be highest in the earlier occupied (warmer) areas and lowest in the most recently colonized (colder) areas (Figure 4d— black bars). Rapid warming between the 1980s and 1990s (Figure 4c) could potentially therefore have driven a widespread increase in re- cruitment rates of up to ~3.0% (Figure 4d) across most of lowland Iceland during this later decade (with the exception of areas colo- nized during the 1960s, for which temperature data in the 1990s are only available for one site). Warming temperatures facilitating earlier nesting and associated increases in chick recruitment therefore have the potential to have contributed quite substantially to the post‐col- onization population growth around Iceland. 4.1 \| Effect of temperature on timing of breeding season events The timing of nest laying and the length of the pre‐fledging pe- riod varied in relation to temperature, with warmer conditions being associated with earlier and shorter breeding periods. In this system, mean laying dates varied by ~11 days between the warm- est (11.2°C, estimated laying = 29.2 ± 2.7 days) and coldest years (9.2ºC, estimated laying = 40.2 ± 2.7 days), and this was the domi- nant driver of annual variation in mean hatching dates. In contrast, the lengths of pre‐fledging period varied by ~3.6 days between the warmest and coldest years. The annual variation in nest laying dates could be also influenced by carry‐over effects of adult pre‐ breeding condition, nesting site availability, or suitability of con- ditions for chick‐rearing. Icelandic godwits are known to migrate with sufficient reserves (Alves et al., 2012) and typically arrive in Iceland 2–3 weeks before nest initiation (Gill et al., 2014). This sub- stantial amount of time to improve body condition prior to nesting may reduce the effects of any carry‐over from winter conditions (Harrison, Blount, Inger, Norris, & Bearhop, 2011). However, faster vegetation growth rates in warmer springs may facilitate earlier nesting because Icelandic godwits are among the largest‐bodied nest‐concealing species of their family (Cramp & Simmons, 1983) and, while average vegetation height in the vicinity of godwit nests on our study areas was ~9–10 cm by mid May, godwit nests have on average a vegetation height of 23.3 cm (±1.2 SE, n = 45). The spring onset and rate of growth of the grasses and sedges in these wet- lands are positively influenced by warm temperatures, particularly by the length of warm periods (Thorvaldsson et al., 2005). Thus, in years with warmer average temperatures, vegetation will reach suitable heights faster, removing potential limitations on nesting site availability (i.e., patches with tall enough vegetation to con- ceal nests), which may allow godwits to nest earlier than in colder years. In addition to suitable vegetation heights for nesting, food resources for chicks are also likely to be more abundant earlier (and generally) in warmer years (Tulp & Schekkerman, 2008), and this association may also contribute to the success of early breeding attempts. 4 \| DISCUSSION Phenological changes in response to changing climatic conditions have been widely reported across many taxa in recent years, but the evidence for population‐level consequences of these shifts for free‐ranging species varies considerably (e.g., Both et al., 2006; Wright et al., 2009). Although climate‐driven changes in phenol- ogy may have little effect on population‐level processes in some cases (Dunn & Møller, 2014; Reed, Grøtan, et al., 2013a), pheno- logical changes that influence demography have the potential to alter population and range size, particularly if these changes facili- tate local population growth and range expansion into previously unoccupied areas in which density‐dependent constraints may be weak (McLean et al., 2016). Icelandic black‐tailed godwits have colonized and become established in breeding locations around lowland Iceland over the last century, concomitant with decades of sustained warming. In south Iceland, earlier nesting occurs in years with warmer temperatures, resulting in advances in hatching dates. As early hatching confers substantial increases in chick recruitment probability, the widespread temperature increases around Iceland may have contributed substantially to the sustained population increase and range expansion of Icelandic godwits during the last century, if the temperature effects on breeding phenology occur throughout the range. As godwits conceal their nests from preda- tors in tall vegetation, more rapid vegetation growth in recent decades in response to warming could have facilitated earlier (and perhaps more successful) nesting across the breeding range. Given the consistently high adult survival rates and philopatry of godwits (Alves et al., 2013; Gill et al., 2001; Gunnarsson, Sutherland, Alves, Potts, & Gill, 2012), increases in recruitment rates as a consequence of earlier nesting are likely to have contributed substantially to the post‐colonization establishment and growth of local populations around Iceland. Phenological changes in response to changing climatic conditions have been widely reported across many taxa in recent years, but the evidence for population‐level consequences of these shifts for free‐ranging species varies considerably (e.g., Both et al., 2006; Wright et al., 2009). Although climate‐driven changes in phenol- ogy may have little effect on population‐level processes in some cases (Dunn & Møller, 2014; Reed, Grøtan, et al., 2013a), pheno- logical changes that influence demography have the potential to alter population and range size, particularly if these changes facili- tate local population growth and range expansion into previously unoccupied areas in which density‐dependent constraints may be weak (McLean et al., 2016). 4.2 \| Cascading effects of temperature‐mediated phenological changes on demography and distribution The ~11‐day delay in hatch dates re- sulting solely from laying dates in warm and cold years equates to a difference in recruitment probability of ~10% (Figure 3), with the additional benefits of earlier fledging in warmer years likely contrib- uting to the higher probability of recruitment. The decline in recruit- ment probability with hatch date means that in cold years, when most nests are laid late, very few chicks are likely to recruit to the adult population. For example, in 2011, the coldest year recorded during our study, only ~10% of 101 ringed chicks recruited into the wintering population. Benefits of early hatching for recruitment are likely to be manifest through advantages associated with suc- cessful and early fledging, such as increased time to improve body condition prior to migration, increased probability of traveling in adult‐dominated migratory flocks, and earlier departure for winter grounds allowing more time in which to select a favorable winter- ing location (Alves et al., 2013; Gunnarsson, 2006). Given that adult survival is relatively high (0.91 ± 0.02 SE) and with little annual vari- ability (Alves et al., 2013), recruitment into the adult population is likely to be a major driver of population growth. Positive associa- tions between temperature and productivity have been reported in migratory common sandpiper Actitis hypoleucos but these appear to have been offset by declines in adult survival rates (Pearce‐Higgins, Yalden, Dougall, & Beale, 2009). Iceland's position in the North Atlantic, between the Atlantic and Arctic oceans, means that the south and west of the country typi- cally experience warmer temperatures than the north and east. Over the last century, the godwit breeding range has expanded from the warmer south and west regions to colonize new sites in the north and east (Gunnarsson, Gill, Petersen, et al., 2005b). Estimated re- cruitment probability is consistently higher in traditionally occupied areas (Figure 4d), primarily as a consequence of the earlier laying and hatching that is possible in these warmer areas (Figure 4b), and potential additional benefits of early fledging. The estimations of recruitment probability assume that the relationships between temperature and timing of breeding season events persist at lower temperatures than those recorded on our study sites. 4.2 \| Cascading effects of temperature‐mediated phenological changes on demography and distribution 4.2 \| Cascading effects of temperature‐mediated phenological changes on demography and distribution Increased recruitment in new areas could also be influenced by increases in food resources or habitat availability, or by reduced lev- els of nest predation. During the early 19th century, both avian and terrestrial nest predator species increased in Iceland, with gull (Larus fuscus and L. argentatus) and owl (Asio flameus) species colonizing the country in ~1920 to 1930 and increasing in numbers until ~1990 to 2010 (Skarphéðinsson, Katrínardóttir, Guðmundsson, & Auhage, 2016), and mink (Mustela vision) spreading around the country after escaping captivity in 1937 (Bonesi & Palazon, 2007). Population trends of native nest predators as raven (Corvus corax) and arctic skua (Stercorarius parasiticus) are unknown, but arctic foxes (Vulpes lagopus) which declined in the 1960s, have increased since to numbers above those recorded in the late 1950s (Unnsteinsdottir, Hersteinsson, Pálsson, & Angerbjörn, 2016). For species breeding at the northern and colder areas of the distribution range, warming temperatures likely relax environmental constraints (e.g., vegetation growth, food abundance) and can positively influence productivity and population growth. The range expansion in this system could have been driven by increased productivity and continuous dispersal from traditionally colonized areas and/or by increased productivity within newly colonized areas. Given the high levels of natal philo- patry in waders, and specifically in this population (Gunnarsson et al., 2012), it seems likely that improved breeding conditions follow- ing colonization of these areas have contributed substantially to the population increase, particularly given the consistently high adult survival rates in this population in recent decades (Alves et al., 2013). Although some recent studies report that climate‐driven changes in phenology might not always lead to population‐level consequences (Dunn & Møller, 2014; Reed, Jenouvrier, et al., 2013b), in Icelandic godwits the links between temperature effects on breeding phenol- ogy and subsequent juvenile recruitment may be apparent because they have occurred during a phase of population expansion and col- onization of previously unoccupied breeding areas, in which density‐ dependent constraints on growth are likely to have been relatively weak (McLean et al., 2016). Overall, the additive effects of an ~11‐day difference in timing of laying, and a ~3.6‐day difference in pre‐fledging period can result in an average difference of up to ~14.6 days at fledging between warm (~11°C) and cold (~9°C) years. 4 \| DISCUSSION Icelandic black‐tailed godwits have colonized and become established in breeding locations around lowland Iceland over the last century, concomitant with decades of sustained warming. In south Iceland, earlier nesting occurs in years with warmer temperatures, resulting in advances in hatching dates. The pre‐fledging period of godwits was slightly longer in colder temperatures. Variation in the length of the pre‐fledging period is a function of chick growth rate, which is also influenced by tempera- ture, with the sum of daily temperature during the pre‐fledging pe- riod likely to provide a proxy for chick foraging time. At low ambient temperatures, wader chicks need to be brooded by their parents for longer (Schekkerman & Boele, 2009), which can limit foraging opportunities and result in depressed growth rates and, in extreme cases, death by starvation (Beintema & Visser, 1989). Greater abun- dance of invertebrate prey resources for chicks in warmer conditions (Halldórsson et al., 2013) is also likely to translate into faster chick growth rates (Eglington et al., 2010). In addition, the trade‐off in al- locating resources between somatic growth and body temperature (Price & Dzialowski, 2018) is likely more biased toward the latter in colder years. 2372 ALVES et al. suggesting a contribution of warming‐driven changes in recruitment to the population expansion. 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Sutherland https://orcid.org/0000-0002-6498-0437 Jennifer A. Gill https://orcid.org/0000-0003-0167-6857 José A. Alves https://orcid.org/0000-0001-7182-0936 Tómas G. Gunnarsson https://orcid.org/0000-0001-7692-0637 William J. Sutherland https://orcid.org/0000-0002-6498-0437 Jennifer A. Gill https://orcid.org/0000-0003-0167-6857 Gill, J. A., Alves, J. A., Sutherland, W. J., Appleton, G. F., Potts, P. M., & Gunnarsson, T. G. (2014). Why is timing of bird migration advancing when individuals are not? Proceedings. Biological sciences/The Royal Society, 281, 20132161. https://doi.org/10.1098/rspb.2013.2161 Gill, J. A., Hatton, L., & Potts, P. M. (2002). Black‐tailed Godwit. In C. V. Wernham, M. P. Toms, J. H. Marchant, J. A. Clark, G. M. Siriwardena, & S. R. Baillie (Eds.), The Migration Atlas: Movements of the birds of Britain and Ireland (pp. 323–325). London, UK: T & A D Poyser. DATA ACCESSIBILITY All data used in this manuscript are presented in the figures and sup- porting information. 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https://openalex.org/W4318979647	https://dt.bsuir.by/jour/article/download/716/266	English	null	E-Commerce of China: its Reasons for Success and Current Development Trends	Cifrovaâ transformaciâ	2,023	cc-by	5,421	http://dx.doi.org/10.35596/1729-7648-2022-28-4-43-52 http://dx.doi.org/10.35596/1729-7648-2022-28-4-43-52 Оригинальная статья Original paper Оригинальная статья Original paper УДК 004; 338.4 © Belarusian State University of Informatics and Radioelectronics, 2022 © Belarusian State University of Informatics and Radioelectronics, 2022 Abstract. E-commerce is one of the highly digitalized industries, which quickly changes the way people buy products and use companies’ services. There are many countries in the world economy now that are trying to adapt informational and communication technologies in various industries to stay competitive and use nowadays’ bene- fits of the Internet, etc. The article considers the e-commerce in China – global leader in the industry. The content of the Chinese e-commerce development foundation, as well as its characteristic features and trends are described. Moreover, recommendations on e-commerce development are given, in particular, for the Republic of Belarus. The novelty of the research conducted lies in the fact that it is the first in the Belarusian practice, which is based on a large array of factual data, and reflects comprehensive analysis of the current state of the Chinese e-commerce, the prerequisites for its rapid development over the past decade and its prospects in the foreseeable future. Keywords: e-commerce, informational and communication technologies, online sale, special economic zone, online store. Conflict of interests. The authors declare no conflict of interests. For citation. Vlasenko A. A., Goloventchik G. G. E-Commerce of China: its Reasons for Success and Current Development Trends. Digital Transformation. 2022; 28 (4): 43–52. itation. Vlasenko A. A., Goloventchik G. G. E-Commerce of China: its Reasons for Success and Curre lopment Trends. Digital Transformation. 2022; 28 (4): 43–52. E-COMMERCE OF CHINA: ITS REASONS FOR SUCCESS AND CURRENT DEVELOPMENT TRENDS ANASTASIA A. VLASENKO, GALINA G. GOLOVENTCHIK Belarusian State University (Minsk, Republic of Belarus) Submitted 01.07.2022 ЭЛЕКТРОННАЯ ТОРГОВЛЯ КИТАЯ: ПРИЧИНЫ УСПЕХА И ТЕКУЩИЕ ПЕРСПЕКТИВЫ РАЗВИТИЯ А. А. ВЛАСЕНКО, Г. Г. ГОЛОВЕНЧИК Белорусский государственный университет (г. Минск, Республика Беларусь) Поступила в редакцию 01.07.2022 © Белорусский государственный университет информатики и радиоэлектроники, 2022 Economic Sciences V. 28, No. 4 (2022) Economic Sciences V. 28, No. 4 (2022) Экономические науки Т. 28, № 4 (2022) Key reasons for rapid e-commerce development in China Nowadays China is an absolute leader on global e-commerce market, however, some decades ago it was not. Key reasons of the Chinese e-commerce success include: government support, special eco- nomic zones existence, investment encouragement, high internet speed, logistics effectiveness, and high number of internet users. So, considering the government support, development of the industry began in 1990 with the development of the Internet on the Chinese territory – the Chinese government prepared “Golden Projects” aimed at implementation of the Internet in China for commercial purposes (clearing systems formation, improvement of data exchange quality, etc.). Thus, in 1994, the Chinese e-commerce industry started its expansion. The first online transaction in China was made in 1998. In 2000, China Elec- tronics Chamber of Commerce was created, and that time appeared first Chinese e-commerce companies.i China is known for its five-year plans of development, and the e-commerce industry there is not an exception. At the first five-year plan for the e-commerce development in 2006–2010, the country popula rized the industry, and in 2007, about 20 % of Chinese internet users have made their purchases in online stores. The second plan for 2011–2015 covered the development of e-commerce applications, institutional and social environment for the industry’s security – technical standards, transactions’ specific in favour of a third party for internet stores were formed. During 2016–2020, China had been working on tradition industries’ digitalization and minimization of state intervention in e-commerce businesses’ activities, only providing fair competition for market actors. The plan for 2021–2025 assumes the e-commerce integration in all economy’s sectors, its expansion in rural areas and the growth of cross-border e-commerce deals [1]. There are also 11 special economic zones for e-commerce development in China [2], and 105 pilot zones for cross-border e-commerce (CBEC). On these zones’ territory various benefits and facilities for the industry exist. In addition, strong basis for the industry’s development formed investment en- couragement in China (for instance, “Go Out Policy”, also known as “Going Global Strategy”). Thus, Chinese foreign investment grew 33 times from 2000 to 2020. In 2020, China invested in other countries the value of 133 billion USD1. The other reason for the e-commerce development is mobile internet connection speed. Конфликт интересов. Авторы заявляют об отсутствии конфликта интересов. Конфликт интересов. Авторы заявляют об отсутствии конфликта интересов. Для цитирования. Власенко А. А., Головенчик Г. Г. Электронная торговля Китая: причины успеха и текущие перспективы развития. Цифровая трансформация. 2022; 28 (4): 43–52. Для цитирования. Власенко А. А., Головенчик Г. Г. Электронная торговля Китая: причины успеха и текущие перспективы развития. Цифровая трансформация. 2022; 28 (4): 43–52. Introduction In recent years the idea of economies’ digital transformation has become a frequent subject of dis- cussion on different levels and in various spheres. Many researchers consider positive effects from using informational and communication technologies in business activities: enhanced data collection and analysis, better customer experience, increased profits and efficiency in general. Some countries have high level of digitalization, while digital development of others is much lower. Although there are many industries affected by digital technologies, one of the main spheres that expands significantly due to digitalization is electronic commerce, i. e. e-commerce. Meanwhile, e-commerce in the country strongly depends on its digitalization level: the higher the level of digitalization, the higher the extent of e-commerce adoption. p From economic perspective, one of the most successful e-commerce industries in global economy that attracts attention of many analysts exists in China. So, its current state, characteristic features, and development trends can be determined. 1 Statista. Available: https://www.statista.com/ (Accessed 15 June 2022). ий государственный университет информатики и радиоэлектроники, 2022 Аннотация. Электронная торговля – одна из самых цифровизированных отраслей в современном мире. Многие страны в мировой экономике адаптируют информационные и коммуникационные технологии в различных отраслях, чтобы оставаться конкурентоспособными и использовать современные преимущест ва интернета и т. д. В статье рассматривается электронная коммерция Китая – мирового лидера в данной отрасли. Раскрыто содержание этапов становления китайской индустрии электронной коммерции, анали- зируются ее характерные особенности и тенденции развития. Приведены общие рекомендации по созда- нию эффективной системы электронной коммерции как для мира в целом, так и для Республики Беларусь в частности. Новизна проведенного исследования заключается в том, что впервые в белорусской практике на основе большого массива фактических данных проведен комплексный анализ современного состояния электронной торговли Китая, предпосылок ее стремительного развития на протяжении последнего десяти- летия и перспектив в обозримом будущем. 43 Цифровая трансформация Т. 28, № 4 (2022) Digital Transformation V. 28, No. 4 (2022) Ключевые слова: электронная торговля, информационно-коммуникационные технологии, онлайн-прода жи, специальная экономическая зона, онлайн-магазин. Chinese e-commerce now: main aspects, features and market players Chinese e-commerce now: main aspects, features and market players According to the latest data of Association of Southeast Asian Nations Plus Three (ASEAN+3), e-commerce transaction value in China, including business-to-business (B2B) and business-to-consu mer (B2C) transactions, in 2020, estimated 5.4 trillion USD, an increase of 8 % from 2019 – its positive growth has been seen from 2011, and this trend persists. In 2025, the value is expected to reach 7.2 tril- lion USD (Fig. 1), increasing from 2020 by 33 %. Fig. 1. China e-commerce transaction value and its growth (trillions of US dollars; percent, year-on-year) [1] Fig. 1. China e-commerce transaction value and its growth g g (trillions of US dollars; percent, year-on-year) [1] Mostly analysed e-commerce industry division assumes 2 segments: B2B and B2C. And nowadays, Asia Pacific region, where China is also located, has the highest level of B2B-transactions: according to the data of Statista on 2021, 80 % of total global online B2B-sales in 2019 were made there (the share has been consistent for about 7 years). In addition, Asia Pacific region, according to eMarketer, amounted to about 60 % of global online B2C-sales – the highest value of B2C-transactions in the world as well (Fig. 2). Fig. 2. Share of Asia Pacific B2C and B2B in global e-commerce sales (percent, year-on-year) Fig. 2. Share of Asia Pacific B2C and B2B in global e-commerce sales (percent, year-on-year) By trading volume, according to the latest UNCTAD’s article considering global e-commerce in 2019, China is the only one country, where both of these segments (B2B and B2C) are almost equal: 41 % of sales are made in B2B e-commerce (Fig. 3), 59 % are made in B2C, so the difference between them is 18 %. Meantime, the difference between these 2 segments in other countries of top-10 e-commerce sales ranking is 44 % (United Kingdom) and higher. Thus, China is the world leader by B2C e-commerce sales as well. China has a leading position in the number of online buyers in the world – almost 40 % of world’s digital buyers (2.14 billion of people globally) are located there. In 2021, in China were about 840 mil- lion of people that shop online, an increase of 8 % from 2020 (Fig. 4). Key reasons for rapid e-commerce development in China China is one of the highest in the world – in 2022, the country is on the 3rd place by the indicator value, and by the fixed internet connection China is on the 5th place from the countries researched. 44 Экономические науки Т. 28, № 4 (2022) Economic Sciences V. 28, No. 4 (2022) Chinese e-commerce now: main aspects, features and market players Herewith, the highest growth in the number of online shoppers in China was seen in 2020 because of the pandemic: more than 100 mil- lion people started to shop online, an increase of 22 % from 2019. Taking into consideration the number of internet users in China and their share in total population of the country (according to Statista, in 2021, there were more than 1 billion people using the Internet in China with the share of 73 % of total population), about 82 % of Chinese internet users shop online, or 64 % of Chinese population are online shoppers. By the share of online buyers in total population there are countries where the numbers are higher. For example, in the Unites States about 80 % of total population are buying online. 45 Digital Transformation V. 28, No. 4 (2022) Цифровая трансформация Т. 28, № 4 (2022) Fig. 3. E-commerce sales: top-10 countries, 20192 H Chi i h d il k Th h f il (B2C) Fig. 4. Number of online shoppers in China (millions of people)1 Fig 3 E l t 10 t i 20192 Fig 3 E commerce sales: top 10 countries 20192 Fig. 3. E-commerce sales: top-10 countries, 20192 Fig. 3. E-commerce sales: top-10 countries, 20192 Fig. 3. E-commerce sales: top-10 countries, 20192 Fig. 4. Number of online shoppers in China (millions of people)1 Fig. 4. Number of online shoppers in China (millions of people)1 However, China is the most penetrated e-commerce retail market. The growth of retail (B2C) e-com- merce sales in China is also one of the highest in the world – in 2021, the sales increased by about 19 %. In addition, China holds more than a half of global retail e-commerce market3. , g Among characteristic features of Chinese e-commerce there are. g Among characteristic features of Chinese e-commerce there are. 1. High level of mobile e-commerce, i. e. m-commerce, in retail sales: share of m-commerce in Chinese retail e-commerce amounted to 83 % in 2021 (Fig. 5), and is supposed to grow to 85 % in 2025, an increase from 2015 of 35 %. Retail m-commerce sales are growing as well: its value could achieve 3.2 trillion USD, increasing by 52 % from 2021 level. 2 Global E-Commerce Jumps to $26.7 Trillion, COVID-19 Boosts Online Retail Sales. Available: https://unctad.org/es/ node/32811 (Accessed 20 June 2022). 3 eMarketer. Available: https://www.emarketer.com/ (Accessed 18 June 2022). 3 eMarketer. Available: https://www.emarketer.com/ (Accessed 18 June 2022). bal E-Commerce Jumps to $26.7 Trillion, COVID-19 Boosts Online Retail Sales. Available: https://unctad.org/es/ 1 (Accessed 20 June 2022). Chinese e-commerce now: main aspects, features and market players One of the reason why m-commerce in China is so popular is the existence of mobile payment applications like WeChat Pay and Alipay of Alibaba, which make online shopping on mobile devices more convenient. Moreover, Chinese e-commerce apps (also known as super apps) usually have whole infrastructures including not only own digital payment systems already mentioned, but also messengers and integrations with social networks. Cost effectiveness of such way of online shopping made it popular as well: the cost of mobile devices and mobile networks has made attractive to invest in them. 46 Экономические науки Т. 28, № 4 (2022) Economic Sciences V. 28, No. 4 (2022) Fig. 5. Retail m-commerce sales in China and their share in total retail e-commerce (trillions of US dollars; percent) Fig. 5. Retail m-commerce sales in China and their share in total retail e-commerce (trillions of US dollars; percent) In addition, a lot of Chinese people go online exactly with the help of mobiles, for instance, smartphones: in 2021, according to the data of Statista on February 2022, about 99.7 % of internet users had used them to access the Internet. China has one of the highest mobile internet connection speed as well, with only the United Arab Emirates and South Korea going ahead. 2. High level of online sales in social commerce: in 2021, the value of social commerce reached 316 billion USD, increasing by 31 % from 2020 – it is about 13 % of total retail e-commerce. Comparing with the United States, in 2021, the Chinese social commerce is 10 times bigger than in the USA, and its share in retail e-commerce is higher as well (Fig. 6). Fig. 6. Social commerce sales in China and the USA, and its share in total retail e-commerce (billions of USD; percent) China USA China USA China USA China USA Fig. 6. Social commerce sales in China and the USA, and its share in total retail e-commerce (billions of USD; percent) The precondition for social commerce development in China was the need to decentralize channels of tradition industries’ sales because of high competition and increase in cost for acquisition of new clients. 4 Livestreaming Driven Social Commerce in China as the Future Growth Engine? Available: https://kathr livestreaming-driven-social-commerce-in-china-as-the-future-growth-engine/ (Accessed 29 June 2022). Chinese e-commerce now: main aspects, features and market players In addition, the main target audience – millennials – expected higher e-commerce adoption and opportunities: digital marketing, innovations, and searching for information, buying and paying for the products without leaving an app.l g In addition, Chinese people often turn to livestreaming, or making purchases while watching influ- encers’ videos on different social platforms. Livestreaming has its roots in teleshopping, but it became more convenient and interactive for internet buyers now. In China such way of interaction with consu mers first became popular in the gaming industry, but then it also changed the way of selling of brands’ products in other spheres. According to eMarketer’s data on May 2021, the number of livestreaming retail e-commerce buyers in China was about 320 million of people in 2021, or about 40 % of all digi tal buyers there3. Ways of livestreaming include: social marketing by key opinion leaders (KOL) and key opinion customers (KOC) – Alibaba and Tencent (QQ, Qzone and WeChat), for instance; social discounters that allow people to make group purchases by lower price (Pinduoduo), etc.4 Other social commerce platforms include: RED, Weibo, and others. p 3. High value of cross-border e-commerce. It became popular due to the government’s support: there are 105 pilot zones for cross-border e-commerce, because of which foreign actors see the potential in entering the market of China. These zones have infrastructure needed for e-commerce development and even offer simplified procedure of cooperation with state structures (customs authorities, for example) on their territory. In 2022, cross-border retail e-commerce can reach about 200 billion USD, increasing by 8 % from 2021 (Fig. 7), and the tendency of cross-border sales growth persists for about 10 years. 47 Digital Transformation V. 28, No. 4 (2022) Цифровая трансформация Т. 28, № 4 (2022) Fig. 7. Cross-border retail e-commerce sales in China and their growth (billions of USD; percent, year-on-year) Fig. 7. Cross-border retail e-commerce sales in China and their growth (billions of USD; percent, year-on-year) Herewith, the gross merchandise value (GMV) of cross-border Chinese e-commerce trade in 2021 reached 2 trillion USD, an increase of about 13 % from the previous year1. The expansion of cross-border transactions in China contributed to the emergence of a new profession on the Chinese market – DaiGou – Chinese individual exporters or group of exporters that live outside China and pur- chase products for Chinese people. 5 Cross-Border E-Commerce in China: Past, Present & Future. Available: https://icsin.org/blogs/2021/08/17/cross-border- e-commerce-in-china-past-present-future/ (Accessed 30 June 2022). Экономические науки Т. 28, № 4 (2022) Экономические науки Т. 28, № 4 (2022) Economic Sciences V. 28, No. 4 (2022) Economic Sciences V. 28, No. 4 (2022) The third largest Chinese e-commerce company is Pinduoduo with GMV of 400 billion USD in 2021, and its share on the market of 15 %. The company facilitates agriculture businesses’ sales, connecting farmers directly with end-consumers. In 2019, Pinduoduo launched the Duo Duo Farm program in order to improve farmers’ knowledge in e-commerce technologies they can use in their business. Within the framework of the program was founded Duo Duo University, where farmers had the opportunity to know how to sell directly to end-consumers, using the Pinduoduo platform. Training courses at the university include e-commerce, online marketing, finance, and other disciplines. Duo Duo Farm helps farmers to cooperate as well. Other large Chinese e-commerce companies are Suning.com (B2C e-commerce platform for household appliances, electronics, cosmetics, products for children etc.) and vip.com (B2C e-commerce with apparel, shoes, food usually on deep discounts).i By number of active buyers, the Alibaba’s platform is the first one on the Chinese e-commerce market throughout the years of 2017–2021. In 2021, about 880 million1 of people made purchases there, increasing from 2020 by 13 % (Fig. 9). With the COVID-19 pandemic the number of such buyers on the platform grew by about 10 %. The second largest company by annual number of digital buyers in 2021 is Pinduoduo. In 2020, the number of active buyers on the Pinduoduo’s platform increased by about 23 %. Meantime, there were 570 million of buyers in 2021 on the JD.com platform, making it the third one by the number of online shoppers in 2021. Fig. 9. Chinese e-commerce companies by number of active users (millions of people) Fig. 9. Chinese e-commerce companies by number of active users (millions of people) Chinese e-commerce now: main aspects, features and market players However, in comparison with the USA and the UK, cross-border e-commerce transactions in China are not so widespread: they amount to about 2 % of total e-commerce, while in the USA and the UK they make up more than 30 %5. Hence, the country is working on CBECs’ promotion. Considering main Chinese e-commerce market players, these one can be named: Alibaba Group (includes Taobao.com, Tmall, Alibaba.com, Aliexpress, and some others) is the biggest e-commerce company in the world with GMV of 1.2 trillion USD in 2021. For comparison, GMV of American Amazon goes hereafter with 600 billion USD in 2021 – 2 times less. In 2022, the market share of Alibaba on the Chinese market is about 46 %3. The company has various operating activities within itself and has rather complex structure, including several companies: Taobao. com that functions as an intermediary in a form of electronic trading platform, and includes Taobao Marketplace (aimed at consumer-to-consumer purchases (C2C)), eTao – electronic searching service. TMall exists for B2C interactions (Taobao Mall earlier) – now the largest CBEC platform in China. Alibaba.com has B2B business model, and Aliexpress, which does not operate in China. The second largest e-commerce Chinese company is JD.com, it is CBEC’s player. Company’s GMV in 2021 reached approximately 40 billion USD. In addition, JD.com has a subsidiary – JD Logistics, which carries out international transportation. The company has an extensive network of warehouses and logistics centres all over the world. The share of JD.com in Chinese e-commerce market is about 18 % (Fig. 8). Fig. 8. Chinese e-commerce companies, share on the market and GMV (percent; trillions of USD) O O Fig. 8. Chinese e-commerce companies, share on the market and GMV (percent; trillions of USD) 5 Cross-Border E-Commerce in China: Past, Present & Future. Available: https://icsin.org/blogs/2021/08/17/cross-border- e-commerce-in-china-past-present-future/ (Accessed 30 June 2022). 5 Cross-Border E-Commerce in China: Past, Present & Future. Available: https://icsin.org/blogs/2021/08/17/cross-border- e-commerce-in-china-past-present-future/ (Accessed 30 June 2022). 48 What comes next with the Chinese e-commerce development? In general, digital payments are usually made with the help of QR-codes, generated either by the buyer, or by the seller, and despite the prevalent role of WeChat and AliPay systems, which shares of Statista’s survey respondents using them are 88 % and 95 % respectively in 20211, other payment services are popular as well: UnionPay with 43 % users, JDPay – 25 %, TenPay – 19 %, etc. In addition, as we can see, some popular among population payment systems are developed by general Chinese e-commerce companies: AliPay – by Alibaba, JDPay – JD.com. TenPay, WeChat are owned by Tencent, also known for its social commerce, as it was mentioned before.i Development trends for digital payments, definitely connected with the e-commerce industry (e-banking, in particular), are the growing financial technologies industry (fintech) and implementation of digital currency [3]. Fintech also contributes to the adoption of Big Data, artificial intelligence, cloud computing, deep learning, robotics process automation (RPA), internet of things (IoT) in businesses, which makes the e-commerce industry more advanced as well. y JD.com already founded JD Finance in 2013, providing wealth management, payment, insurance, financial technology, and other services. Moreover, the company became the first e-commerce platform that started to accept digital yuan, i. e. e-CNY. Since December 2021 to June 2021: there were about 450 thousand of people that had made purchases on the total value of 15.6 million USD in e-CNY. JD.com have also paid some salaries in e-CNY and now is working on enabling usage of digital currency for third-party sellers.i Currently, the fintech industry in China is mainly under the regulation of People’s Bank of China (PBoC) and Cyber Administration Authority of China6. In 2021, new state regulations have shown up: fintech companies in China need to set up holdings now (and it leads to all the financial operations being under one roof), and the companies must be licensed for operating in the sphere. In addition, they must have secure systems for preventing cross-border risks. Moreover, new law on personal data protection (November 2021) makes the regulation of non-bank payment providers more precise what restricts their activities – for instance, companies whether should have their servers in China, or must be approved in Cyber Administration Authority to act abroad7. At first sight, tightened regulation can be seen as a deterrent for the development, but it also stimulates the local fintech companies’ improvement. What comes next with the Chinese e-commerce development? With the popularity of m-commerce in China comes mobile payment systems and platforms, where China has a dominant role in the world as well. Electronic, i. e. digital, payments are highly connected with the e-commerce industry. In 2022, digital payments are projected to reach about 3.5 trillion USD, in 2026 – about 4.9 trillion USD (Fig. 10). Mostly they are made in e-commerce – about 2 trillion USD in 2022, and the value continues to increase. In 2026 it can be 44 % higher than in 2021. Moreover, the penetration rate of digital commerce payments is the highest among these 3 segments: in 2021, the value is 68 %, while in mobile payments it is about 38 %, mobile POS payments – about 1 %. Fig. 10. Transaction value of digital payments in China, by segment (trillions of USD) [3] Fig. 10. Transaction value of digital payments in China, by segment (trillions of USD) [3] 49 Цифровая трансформация Т. 28, № 4 (2022) Цифровая трансформация Т. 28, № 4 (2022) Digital Transformation V. 28, No. 4 (2022) The number of mobile wallet users in the country in 2021 reached 2.5 billion of people (Fig. 11), increasing by 3 % from 2020. And there can be 2.7 billion of them in 2023. The most used mobile wallet is AliPay with the share of about 48 % in total (2021), and WeChat Pay – 40 %. The number of mobile wallet users in the country in 2021 reached 2.5 billion of people (Fig. 11), increasing by 3 % from 2020. And there can be 2.7 billion of them in 2023. The most used mobile wallet is AliPay with the share of about 48 % in total (2021), and WeChat Pay – 40 %. Fig. 11. Mobile wallet users in China (billions of people) Fig. 11. Mobile wallet users in China (billions of people) The only problem with digital payments, and, hence, the precondition for the further development is uneven internet connection: in some parts of China the Internet’s speed is unstable or very poor. 6 Cross Border Balance: Fintech in China. Available: https://fintechmagazine.com/digital-payments/cross-border-balance- fintech-china (Accessed 15 June 2022). 7 China Personal Information Protection Law Has Entered into Force on November 1, 2021. Available: https://www. mondaq.com/china/privacy-protection/1128310/china-personal-information-protection-law-has-entered-into-force-on- november-1-2021 (Accessed 18 June 2022). Экономические науки Т. 28, № 4 (2022) Economic Sciences V. 28, No. 4 (2022) Economic Sciences V. 28, No. 4 (2022) The other trend on the Chinese e-commerce market is so named xiachen (“to sink”), which means marketing and sales’ refocusing on tier cities and provinces – where salaries and income level are lower, and in addition, due to lower cost of people’s lives there, habitants of these regions have more free money they can spend on buying consumer products. y y p y g p Augmented (AR) and virtual reality (VR) technologies are making purchases more interactive as well. AR and VR are used in livestreaming too: animated characters, virtual idols, first common on anime and video sharing platforms, are being integrated in e-commerce now. In addition, their usage reduces the costs and time. Moreover, if the person is sick, virtual streamer can make up for the absence. As for this trend in China now, VR is generating more revenue than AR, but in 2024 the tendency can change. Revenue of both segments is growing dramatically: in 2022, it can increase by about 70 % from 2021 level3 to 18.3 billion USD (Fig. 12), and in 2024 the value can reach 44.7 billion USD, growing by 44 % from its 2022. Fig. 12. Augmented and virtual reality revenues in China (billions of USD) Fig. 12. Augmented and virtual reality revenues in China (billions of USD) Livestreaming8 in social commerce changes from key opinion leaders (KOLs) to key opinion consumers (KOCs). KOLs are experts in the sphere of product they are testing and advertise, or just famous (sometimes the other people are more likely to trust what famous people are saying) – celebrities, models. KOCs are people, whose opinion is considered from perspective of their experience of product using, and the number of their followers is much lower. In addition, followers of KOCs are more loyal. McKinsey notes this trend too: hiring a celebrity for e-commerce marketing campaign costs more money than using micro- and nano-influencers. Moreover, the engagement rate while livestreaming with the help of KOCs are 10 times higher (Instagram). Some general development trends include: omnichannel structure of companies’ communication networks with clients and partners, creating an integrated system that includes both online and offline channels. Personalised and hyper-personalised customer experience with data collection of various users’ information, while they access the e-commerce platform, application or website. 8 It’s Showtime! How Live Commerce is Transforming the Shopping Experience. Available: https://www.mckinsey.com/ business-functions/mckinsey-digital/our-insights/its-showtime-how-live-commerce-is-transforming-the-shopping-experience (Accessed 25 June 2022). Экономические науки Т. 28, № 4 (2022) Advanced tech- nologies of data collection allow offered content to be more relevant to the buyers, increasing the possi- bility of them making a purchase. What comes next with the Chinese e-commerce development? As a result, Chinese people will have an opportunity to use digital financial instruments while buying something on e-commerce platforms. 50 References 1. ASEAN+3 Regional Economic Outlook 2022. Available: https://www.amro-asia.org//wp-content/ uploads/2022/04/AMRO-AREO-2022_AMRO_Full-Final.pdf. p _ _ p 2. FDI China Exclusive: The 21 Free Trade Zones Guide 2022. Available: https://www.fdichina.com/blog/fdi- china-exclusive-the-21-free-trade-zones-guide-2021/. 2 China E-commerce Market Trends 2022. Available: https://tenbagroup.com/12-china-e-commerce-marke ends/#h-2-mobile-payment-platforms. 3. 12 China E-commerce Market Trends 2022. Available: https://tenbagroup.com/12-china-e-commerce-market- trends/#h-2-mobile-payment-platforms. Authors' contribution Vlasenko A. A. conducted the analysis of the Chinese e-commerce industry, summarized the results and gave some practical recommendations. g p Goloventchik G. G. carried out the formulation of the problem for the study. Information about the authors Vlasenko A. A., Master's Student of HSE Graduate School of Business, Moscow, Russian Federation. Goloventchik G. G., Cand. of Sci., Associate Professor at the Department of International Economic Relations of Belarusian State University. Цифровая трансформация Т. 28, № 4 (2022) Successful Chinese experience of e-commerce development and its popularisation in China can be applied in the Republic of Belarus in such areas as: – development of the progressive regulation base and lowering the legislative barriers on the governmental level for cross-border e-commerce development; – encouragement of enterprises for the Internet’s usage in foreign trade; encouragement of enterprises for the Internet s usage in foreign trade; – creation of a national mobile payment system and e-commerce platform; d i f i iff d i lifi i f l d – creation of a national mobile payment system and e-commerce platform;fi – reduction of import tariffs and simplification of customs clearance procedures, etc.; – raising awareness and involvement of the population in e-commerce by popularisin – raising awareness and involvement of the population in e-commerce by popularising the industry in the mass media, explaining the obvious benefits of using e-commerce services; – improving logistics’ efficiency, speed and quality of mobile Internet connection, ensuring maximum cybersecurity; – creation of special economic zones focused both on the development of e-commerce within the country and on cross-border e-commerce, etc. Цифровая трансформация Т. 28, № 4 (2022) Digital Transformation V. 28, No. 4 (2022) taken into account. All Asians, Chinese in particular, usually strongly differ from Europeans and Ame ricans. For instance, considering the Chinese people as a nation, precise planning and high level of their organization is unique. g q 3. However, some general recommendations can be determined. The e-commerce industry deve lopment highly depends on the extent of country’s digitalization, so, the Internet’s speed of good quali ty (both mobile and fixed), its accessibility and stable connection are vital. And from the experience of China, it is seen that it is important to popularise the industry among nation’s population, because the success of the country’s e-commerce comes from people who use its platforms, technologies, in general from digital buyers. Various programs can be conducted aimed at raising awareness: TV shows, media sources’ publications, and national e-commerce companies’ advertisement. g 3. However, some general recommendations can be determined. The e-commerce industry deve lopment highly depends on the extent of country’s digitalization, so, the Internet’s speed of good quali ty (both mobile and fixed), its accessibility and stable connection are vital. And from the experience of China, it is seen that it is important to popularise the industry among nation’s population, because the success of the country’s e-commerce comes from people who use its platforms, technologies, in general from digital buyers. Various programs can be conducted aimed at raising awareness: TV shows, media sources’ publications, and national e-commerce companies’ advertisement. 4. Successful Chinese experience of e-commerce development and its popularisation in China can be applied in the Republic of Belarus in such areas as: 4. Conclusions 1. Undoubtedly, the Chinese e-commerce market expansion can be considered as a successful example for the industry’s planning. But it doesn’t mean the way China regulates its e-commerce market will be suitable for other countries. 2. Firstly, countries should use those ways of industries’ development, which are common for their economies’ types: whether they develop plans each several years, or give more freedom for the private sector’s companies. If the economy is socially oriented, have strong state sector, the Chinese experience can be studied more scrupulous; but if it is not, then the other countries’ practices, which are more fa- miliar to the economy’s type, should be considered instead. Secondly, the nation’s mentality should be 51 Цифровая трансформация Т. 28, № 4 (2022) Address for correspondence 220030, Republic of Belarus, Minsk, Leningradskaya St., 20 Belarusian State University Tel. +375 44 710-69-05 E-mail: galinagoloventchik@mail.ru Goloventchik Galina Gennad’evna 52
https://openalex.org/W2752054090	http://revistaseletronicas.pucrs.br/ojs/index.php/navegacoes/article/download/25252/15874	Portuguese	null	A construção da memória da Guerra Colonial em Os cus de Judas, de Lobo Antunes	Navegações	2,017	cc-by	7,651	Alfeu Sparemberger Universidade Federal de Pelotas Alfeu Sparemberger Universidade Federal de Pelotas Alfeu Sparemberger Universidade Federal de Pelotas Resumo: O presente artigo analisa o romance Os cus de Judas, de autoria do escritor português António Lobo Antunes quanto à sua particularidade em estabelecer a construção de uma memória coletiva sobre a Guerra Colonial. Segundo romance do autor, publicado em 1979 em um contexto pós-colonial apenas quatro anos após a Revolução dos Cravos, a narrativa remete à abordagem da exploração da experiência do autor durante sua participação na Guerra Colonial, em Angola, no início da década de 70. Após a Revolução dos Cravos, em abril de 1974, a sociedade portuguesa tenta apagar o passado traumático ligado à guerra e ao Estado Novo a fim de iniciar um movimento de superação de seu passado problemático e aproximar-se da Europa. A produção ficcional apresenta-se como possibilidade de interpretação da dinâmica política e social existente na construção de um novo Portugal após a Guerra Colonial, a Revolução dos Cravos e a descolonização dos territórios ultramarinos. A literatura assume um papel de destaque no Portugal pós-colonial, pois almeja a construção de uma memória coletiva sobre os últimos capítulos do império português. Palavras-chave: Os cus de Judas; Guerra Colonial; Memória; Pós-colonialismo Abstract: The present article analyzes the novel Os cus de Judas, written by the portuguese writter António Lobo Antunes for their particularity to estabilish the construction of a collective memory about the Colonial War. Second novel of the author published in 1979 in a post-colonial context, just four years after the Carnation Revolution, the narrative refer to approach the exploration of the author’s experience during his participation in the Colonial War in Angola, in the early 1970. After the Carnation Revolution in April 1974, the portuguese society tries to erase the traumatic past linked to the war and the Estado Novo to start a movement of overcoming his troubled past and move closer to Europe. The fictional production is presented as a possible interpretation of the dynamics political and social context in building a new Portugal after the Colonial War, the Carnation Revolution and decolonization of overseas territories. The literature plays an important role in Portugal post-colonial since aims to build a collective memory of the last chapter of the portuguese empire. Ensaios Ensaios Ensaios Exceto onde especificado diferentemente, a matéria publicada neste periódico é licenciada sob forma de uma licença Creative Commons - Atribuição 4.0 Internacional. http://creativecommons.org/licenses/by/4.0/ The construction of the Colonial War memory in Os cus de Judas, of Lobo Antunes The construction of the Colonial War memory in Os cus de Judas Leonardo von Pfeil Rommel Universidade Federal do Rio Grande do Sul Alfeu Sparemberger Universidade Federal de Pelotas Leonardo von Pfeil Rommel Universidade Federal do Rio Grande do Sul Leonardo von Pfeil Rommel Universidade Federal do Rio Grande do Sul Alfeu Sparemberger Universidade Federal de Pelotas Alfeu Sparemberger Universidade Federal de Pelotas Keywords: Os cus de Judas; Colonial War; Memory; Post-colonialism O presente artigo analisa o romance Os cus de Judas, de autoria do escritor português António Lobo Antunes quanto à sua particularidade em estabelecer a construção de uma memória coletiva sobre a Guerra Colonial. Segundo romance do autor, publicado em 1979 em um contexto pós-colonial apenas quatro anos após a Revolução dos Cravos, a narrativa remete à abordagem da exploração da experiência do autor durante sua participação na Guerra Colonial, em Angola, no início da década de 70. Os cus de Judas faz parte do primeiro ciclo da produção literária do escritor português, denominado pela crítica, e pelo próprio Lobo Antunes, de Trilogia da aprendizagem, conjunto de três romances autobiográficos formado por Memória de elefante (1979), Os cus de Judas (1979) e Conhecimento do inferno (1980), que se apoiam na experiência do escritor na Guerra Colonial e tratam do traumático processo de regresso e readaptação dos ex-combatentes no período pós-guerra. 4 Rommel, L. von P., Sparemberger, A. Entre 1961 e 1974 Portugal manteve uma relação extremamente conflituosa com suas colônias africanas. A Guerra Colonial produziu-se em um contexto em que os países africanos buscavam sua independência do colonialismo europeu, que perdurava no continente há séculos. Estima-se que, a fim de combater o movimento de desintegração do corpo físico do império, Portugal tenha enviado para os campos de batalha no continente africano cerca de um milhão de soldados. Sendo assim, a guerra foi responsável por mobilizar uma grande parcela da sociedade portuguesa, que esteve envolvida direta ou indiretamente no conflito e em suas consequências. capaz de fazer frente aos vizinhos europeus, sofre uma ruptura em sua identidade e representação histórica após a Revolução e a descolonização da África. Segundo Costa (2013), a perda dos territórios africanos constituiu-se um traumático abalo na autorepresentação nacional, o que, consequentemente, leva o país a vivenciar um processo de fechamento sobre si próprio, como tentativa de expurgar a memória incômoda da guerra e do fracasso imperialista. Após abril de 1974, a estratégia de silenciamento nacional e a tentativa de apagamento do traumático passado recente, constituem-se uma alternativa de manutenção por parte do governo revolucionário, das condições necessárias para reerguer a nação e mantê-la coesa no objetivo de construção das bases de uma nova História, quando se poderia vislumbrar o futuro e a ultrapassagem do passado imperialista e ditatorial. Alfeu Sparemberger Universidade Federal de Pelotas A Revolução dos Cravos, em abril de 1974, assinala em Portugal o final do período imperial, marcado pela ditadura salazarista e pela Guerra Colonial nos territórios ultramarinos. Após a Revolução, o governo revolucionário imediatamente dá início ao processo de independência e descolonização, almejando, assim, afastar-se do passado imperialista e isolacionista defendido duramente por Salazar durante as quatro décadas em que esteve no poder. Na visão de Costa (2013, p. 141), o esquecimento do passado recente seria “a condição fundamental para que a metáfora de perpétuo movimento da nação pudesse ser novamente reiniciada”, abandonando o passado imperialista e isolacionista construído pela retórica do Estado Novo e aproximando-se, assim, dos vizinhos da Europa, como forma de alterar a imagem de Portugal e defender a sua modernização política e econômica. A Guerra Colonial, a Revolução dos Cravos, a queda do Estado Novo e o processo de descolonização da África, constituem-se uma ruptura na sociedade e na memória coletiva nacional que, durante cerca de cinco séculos, baseou-se em uma visão nacionalista do passado imperialista português. Como aponta Jorge Manuel da Costa (2013), após abril de 1974 Portugal vê-se confrontado com o desmoronar da grandiosidade imperial, conceito muito valorizado pelo discurso oficial do governo ao longo da História. Quanto ao Portugal de Abril e à nova imagem que luta por concretizar, o que se obtém é, no fundo, um modelo identitário que encontra na fuga seletiva ao passado a estratégia preferencial para a manutenção do caráter ideal de que se pretende revestir. Tendencialmente direcionado para o desvio sistemático de situações ou eventos que levantem a ponta do véu traumático e do recente terror do fracasso nacional (o mesmo será dizer, que remetam, ainda que por instantes, o país para um plano de eventual autoquestionação) (COSTA, 2013, p. 3). Liberto de um longo período de relacionamento problemático com a sua memória, instilado ditato- rialmente por uma discursividade eufórica dos feitos bélicos e expansionistas do seu passado histórico, o Portugal de Abril de 74 vê-se confrontado com o desmoronar da grandiosidade imperial que, até esse ponto, havia assinalado o discurso identitário e de memória que oficialmente vigorava no seu espaço interno (COSTA, 2013, p.11). Norberto do Vale Cardoso (2011, p. Navegações, Porto Alegre, v. 10, n. 1, p. 3-11, jan.-jun. 2017 Alfeu Sparemberger Universidade Federal de Pelotas Como aponta ainda a autora, a guerra marca o início do processo de desterritorialização e desmembramento do império colonial português, que o fez regressar para junto da Europa, o que pode ser considerado um fenômeno basicamente novo e traumático para a História de um país mundialmente reconhecido como essencialmente imperialista e colonizador. Segundo Ribeiro (2004, p. 248), o regime salazarista buscava sempre ocultar os acontecimentos da Guerra Colonial, como tentativa de manter o estado de normalidade da população da metrópole na Europa. Ainda, segundo a autora, a consequente ocultação da guerra, mesmo pós- 25 de abril, não se tratava de uma vontade autoritária, “mas sim uma incapacidade de avaliação das condições reais para lidar com tão dolorosa e explosiva herança e imagem de um antigo poder que se queria esquecer”. O silêncio historiográfico, político e social sobre o passado recente, que caracterizou a sociedade portuguesa pós- abril de 1974, deve-se aos naturais mecanismos de recusa, denegação e luto perante o trauma da guerra e da memória opressiva do antigo regime. Em artigo publicado no Diário de Notícias em 1984, dez anos após a Revolução dos Cravos, Eduardo Lourenço demonstra-se surpreso com o fato de a História recente de Portugal, o colonialismo e a Guerra Colonial na África não terem dado origem a manifestações culturais ou reflexões mais aprofundadas nas áreas da historiografia e da política na sociedade pós-25 de abril. Cardoso (2011, p. 172) menciona que o Estado Novo entendia que a Guerra Colonial poderia pôr em risco a paz social, por isso “a guerra se viu transferida para uma ‘lonjura’ como forma de a distanciar da metrópole e reduzir o seu impacto na sociedade portuguesa”, e que a única forma encontrada pelo aparelho estatal de a controlar, foi preenchê-la de um sentido que a englobasse na falsa retórica de missão civilizadora da pátria portuguesa. À aproblematização voluntária do antigo regime sucedeu uma espécie de insólita ocultação acerca dos avatares da última fase da nossa velha – pensar-se-ia capital – aventura colonial. Nem documentários, nem filmes, nem “livros brancos” sobre a nossa história recente em África contribuíram com qualquer explicação ou simples informação sobre o que, para já, conduziu em casa europeia à liquidação de um regime antidemocrático e, fora dela, ao fim de um império (sic) (LOURENÇO, 1984 apud RIBEIRO, 2004, p. 246). Alfeu Sparemberger Universidade Federal de Pelotas 160) destaca que “essa eliminação da História colonial é um mecanismo do novo tempo democrático português para superar o lado negro da sua História”, e que o desejo de reaproximação e integração do país ao continente europeu, por parte do novo governo, seria uma forma de ruptura com o passado ditatorial, isolacionista e opressivo alimentado pelo regime salazarista. Após o esfacelamento do império colonial, a sociedade portuguesa mergulhou em um estado de amnésia coletiva como única forma de contornar os eventos traumáticos ocasionados pela Guerra e pela dinâmica político-social da Revolução e da descolonização. O movimento de apagamento da contemporaneidade, iniciado após abril de 1974, foi responsável por criar uma espécie de estado de exceção, que impedia a criação de uma memória coletiva sobre a contemporaneidade e sobre o processo de construção de uma História pós-colonial. Como aponta Eduardo Lourenço (2013), em seguida à Revolução dos Cravos a sociedade portuguesa tentou destruir a memória da ditadura salazarista, promovendo um processo de distanciamento e deslocação da identificação nacional com a imagem do Estado Novo e do colonialismo na África, condenado diversas vezes pela Organização das Nações Unidas. A imagem do antigo regime expunha as fraquezas de Portugal e impedia a concretização de uma redemocratização e uma Portugal, historicamente possuidor de colônias e territórios ao redor do planeta, país sempre autoconcebido pelo discurso estatal como grande potência imperial, Navegações, Porto Alegre, v. 10, n. 1, p. 3-11, jan.-jun. 2017 5 A construção da memória da Guerra Colonia ... descolonização rápida e exemplar na visão das Forças Armadas após assumirem o poder. portuguesa, pois, após o início dos movimentos de libertação dos países africanos, Portugal viu-se obrigado a combater a si próprio, a fim de que pudesse evitar o desmembramento do corpo político da nação, uma vez que os territórios ultramarinos compunham e participavam ativamente do processo de construção do imaginário e da identidade nacional. A primeira fase da revolução caracterizou-se pela tentativa frenética de deslocar a imagem fascista da realidade nacional presente e passada, de destruir pela raiz o que se supunha mera pintura superficial do país, que, lavado e expurgado dos seus demónios passageiros, poria à mostra as suas virtualidades [...] (sic) (LOURENÇO, 2013, p. 50). Ribeiro (2004) adota uma visão bastante semelhante no que se refere à Guerra Colonial; segundo a autora, a guerra é responsável por efetivar uma espécie de movimento de tensão na identidade nacional. Navegações, Porto Alegre, v. 10, n. 1, p. 3-11, jan.-jun. 2017 Alfeu Sparemberger Universidade Federal de Pelotas Roberto Vecchi (2010) refere que a Guerra Colonial punha em jogo a pseudomemória imperialista construída pelo Estado Novo, e a veiculação de notícias pela imprensa e a sua presença na metrópole europeia apresentavam-se como perigoso obstáculo para a manutenção da conti- nuidade do regime salazarista. Vecchi (2010) salienta, ainda, que nos conflitos no continente africano não estavam em jogo somente os espaços e territórios do império, mas, principalmente, os cinco séculos de História de Portugal. Ainda de acordo com Eduardo Lourenço (1984 apud Ribeiro 2004), em relação ao fenômeno de silenciamento social e cultural sobre o passado recente iniciado a partir do movimento de Revolução e redemocratização em abril de 1974, existia no país uma espécie de “insólita ocultação”, que tornava interditos os capítulos finais do império colonial português, como a queda da ditadura do Estado Novo, a Guerra Colonial e o processo de descolonização dos territórios ultramarinos. (...) em jogo estava algo de mais complexo do que a defesa do espaço colonial: como declamava a retórica do regime salazarista, em jogo estavam cinco séculos da História de Portugal, cinco séculos de colonização ou, como ficou depois da maquilhagem retórica da revisão constitucional de 1951, cinco séculos de relações entre povos e culturas diferentes (VECCHI, 2010, p. 96). Jorge Manuel da Costa (2013, p. 147) afirma que, já no final da década de 80, são ainda extremamente reduzidas as propostas discursivas de abordagem do recente passado imperial português, sendo a sociedade e a cultura ainda dominadas por uma ambiguidade de posicionamentos emanada pela esfera estatal, que entendia a alternativa do esquecimento e da ocultação do passado imperialista e opressivo da nação como uma “arma preferencial para A Guerra Colonial pode ser entendida como uma espécie de estado de exceção na História imperial Navegações, Porto Alegre, v. 10, n. 1, p. 3-11, jan.-jun. 2017 6 Rommel, L. von P., Sparemberger, A. Como se pode observar, num período muito curto de tempo, a realidade portuguesa sofreu profundas mutações – a Revolução, com suas consequências imprevisíveis, imprimirá um dinamismo sem pre- cedentes nas relações sociais e na vida cultural do país. O resultado disso é a sucessão vertiginosa das ideologias, dos modos de comportamento, dos modos de atuação política, o que implicará, às vezes, descompassos entre os acontecimentos e o olhar que tenta registrá-los. [...]. Navegações, Porto Alegre, v. 10, n. 1, p. 3-11, jan.-jun. 2017 Alfeu Sparemberger Universidade Federal de Pelotas Se os jornais – formas não ficcionais de abordar a realidade – se contentam com a fixação do efêmero e a consequente diluição da notícia dentro do dinamismo da história, o romance, que aspira a uma transcendência, a uma atemporalidade, deve se servir de outros mecanismos, que não a pretensa objetividade factual, para não sucumbir ao desenrolar do tempo (GOMES, 1993, p. 84). assegurar a criação ilusória de um perpétuo movimento nacional”. Dado o traumático rompimento da linearidade discursiva da História portuguesa pós-abril de 1974, que esmagou o presente sob o peso de um passado monumentalizado pela memória imperialista, a literatura assumiu-se como um dos únicos discursos capazes de produzir uma reconstituição histórica do traumático passado nacional, marcado pela Guerra Colonial e pela queda do modelo imperialista do Estado Novo. Vecchi (2010) comenta que a literatura surgida após a Revolução dos Cravos, que se destina a tematizar a Guerra Colonial, ganha corpo em um vazio historiográfico, e atende à necessidade social de reler o passado imediato, de ler e escrever a História recente, interdita e silenciada pela repressão estatal. Mediante a mobilização do testemunho e da subjetividade, a literatura assume um papel de combate à amnésia nacional, recuperando o direito social de produzir e comunicar a sua memória individual e coletiva. O discurso ficcional, no Portugal pós-abril de 1974, apresenta-se como possibilidade de análise do contexto social e da dinâmica da evolução do país promovida pela transição do processo revolucionário. O romance surge como uma forma de enfrentamento desse processo dinâmico iniciado com a Revolução, que via na substituição e no esquecimento de um período histórico e de um modelo político-social ultrapassado e opressivo, a única forma de evolução. A ficção se assume como tentativa de resgate dos últimos capítulos do império português, como a Guerra Colonial e a Revolução dos Cravos. A prática da literatura como anamnésia nacional surge copiosamente das estações de abertura política, restabelecimento das liberdades civis, com a explosão da subjetividade da recordação, a necessidade presente de reler o passado imediato, não tanto – ou não apenas – para procurar informações inéditas, visto que directa ou indirectamente os acontecimentos eram de qualquer modo conhecidos, mas para readquirir a sujbectividade, a protagonização de escrever ou de ler em primeira pessoa a história interdita, recuperar o direito de comunicar a memória e a experiência, também singular (VECCHI, 2010, p. 60). Alfeu Sparemberger Universidade Federal de Pelotas que afetaram a sociedade portuguesa durante o período de transição imperial/pós-imperial, auxiliando na construção de uma memória coletiva sobre os últimos capítulos do império português. A participação na guerra serviria como uma espécie de metamorfose, que seria responsável por transformar o ainda jovem e inexperiente médico em um “verdadeiro homem”, a fim de que pudesse se tornar um representante digno e verídico da tradição familiar, portador das virtudes de seus gloriosos antepassados, ironicamente designados pelo narrador como “furibundos generais” participantes de “gloriosos combates de gamão de bilhar”, falecidos muito antes do seu nascimento. Apontado pela crítica como um dos primeiros testemunhos literários sobre a Guerra Colonial na África, Os cus de Judas apresenta-se organizado sob uma estrutura dividida em 23 capítulos, nomeados de acordo com as letras do alfabeto português, de A a Z, cuja organização pode ser considerada uma espécie de “alfabeto da agonia”. De acordo com Seixo (2002, p. 42), a estruturação do romance, segundo a ordem do alfabeto, estabelece “um exame crítico e emocional da guerra em Angola de A a Z”. O embarque do protagonista para a Guerra Colonial no interior de Angola é acompanhado com júbilo e orgulho pelos seus familiares, sendo por ele descrito como um triste e cruel quadro da inoperância e submissão sociais perante os efeitos da opressão da ditadura comandada por Salazar, que revestia a guerra de um caráter de missão humanitária e nacionalista, que visava a extinguir a atividade terrorista nas colônias. Na visão de Norberto Cardoso (2011), o romance, por intermédio de sua violenta narrativa, realiza uma certa reconstrução do mundo português de A a Z, apresentando- se como uma espécie de manual ao avesso, no qual Lobo Antunes executa um processo de requestionamento da vida e da sociedade portuguesa no período pós- Revolução dos Cravos, abordando questões traumáticas para a coletividade nacional, como a violência e os duros efeitos da Guerra sobre os combatentes, a repressão do Estado Novo e o processo de desconstrução do império português. De modo que quando embarquei para Angola, a bordo de um navio cheio de tropas, para me tornar finalmente homem, a tribo, agradecida ao Governo que me possibilitava, grátis, uma tal metamorfose, compareceu em peso no cais, consentindo, num arroubo de fervor patriótico, ser acotovelada por uma multidão agitada e anónima semelhante à do quadro da guilhotina, que ali vinha assistir, impotente, à sua própria morte (ANTUNES, 2009, p. 14). Alfeu Sparemberger Universidade Federal de Pelotas A prática da literatura como anamnésia nacional surge copiosamente das estações de abertura política, restabelecimento das liberdades civis, com a explosão da subjetividade da recordação, a necessidade presente d l d i di ã ã Em algumas circunstâncias históricas, a literatura torna-se instrumento de elaboração e recomposição de traumas e de lutas coletivas, estabelecendo uma íntima associação com a escrita da história. Passa, então, a suprir seus silenciamentos e rupturas em busca da representação, com maior impressão de totalidade, dos acontecimentos traumáticos e interditos de uma determinada coletividade, como foi o caso da Guerra Colonial em Portugal. A abordagem da Guerra Colonial torna a literatura uma espécie de discurso de exceção, responsável por romper com a aparente linearidade e horizontalidade do discurso historiográfico português. Por meio da literatura, acontecimentos traumáticos e interditos pela memória ativa da repressão salazarista são identificados por entre os despojos do desabamento do império colonial português, possibilitando, assim, um espaço de reflexão sobre o Portugal contemporâneo e suas fraturas históricas. [...] existem circunstâncias históricas nas quais a literatura se reveste de funções ulteriores em relação àquelas que tradicionalmente desempenha: torna- se instrumento de elaboração, e de recomposição, diríamos quase que terapêutica de traumas e de lutas colectivas, estabelece uma estreita aliança com a escrita da história e tenta recompor, umas vezes ingenuamente, outras em modo um pouco desencantado, fracturas, descontinuidades com o passado, de cada um e de todos, fornecendo as cifras para a compreensão, possivelmente aquela que melhor dê uma impressão de totalidade ao acontecido (VECCHI, 2010, p. 60). A literatura portuguesa, surgida no momento de transição entre um tempo imperial/pós-imperial, busca efetivar-se como uma espécie de documento, capaz de relatar esta fase de intensas mudanças da sociedade e da cultura nacional. Como assevera Gomes (1993, p. 84), “essa busca de verismo e do histórico fundamenta-se pelo desejo de transformação do romance num documento de uma era de convulsões e de modificações substanciais na sociedade portuguesa”, como a Guerra Colonial e a Revolução dos Cravos. A literatura apresenta-se, assim, como um discurso que possibilita o questionamento da história e dos acontecimentos do Portugal contemporâneo. O discurso artístico visa a recompor e explorar experiências e traumas Navegações, Porto Alegre, v. 10, n. 1, p. 3-11, jan.-jun. 2017 7 A construção da memória da Guerra Colonia ... totalmente dos compromissos, e sua credibilidade, junto aos colegas e pacientes, não é das melhores. Alfeu Sparemberger Universidade Federal de Pelotas Em Os cus de Judas temos como protagonista do romance um médico psiquiatra recém-retornado da Guerra Colonial em Angola, onde exerceu a função de tenente médico do Exército português durante cerca de dois anos. Por meio de uma intensa narrativa em primeira pessoa, o protagonista extravasa suas memórias, angústias e lembranças, muitas vezes fragmentadas, do passado traumático da guerra, tecendo relações com a infância e lançando uma forte crítica à sociedade burguesa e ao sistema imperialista português. No constante processo de rememoração do passado, percebe-se que o protagonista se confronta com as lembranças da infância e com a insistência e pressão dos familiares para que ele cumprisse as normas impostas pela sociedade burguesa. Durante toda a sua formação as suas escolhas nunca foram realmente individuais; tudo foi sempre condicionado e imposto pela família, desde a escolha da profissão da Medicina, que está ligada ao pai, também médico, e também a participação na Guerra Colonial, uma vez que eram sempre muito valorizados os antepassados guerreiros e seus feitos quase que heroicos. Lobo Antunes produz uma narrativa em que, utilizando sua experiência pessoal como combatente em Angola no início da década de 70, constrói, mediante o discurso literário, uma ácida crítica ao regime salazarista do Estado Novo, trazendo para a sociedade o drama dos soldados retornados que, muitas vezes, tinham sua vida pessoal fragmentada, e toda a brutalidade que o sistema colonial impunha ao povo das colônias africanas. A imagem de um ser fragmentado, após cumprir com todas as suas obrigações e perseguir os modelos impostos pela família e sociedade portuguesa, contrasta com outra, de que se estivesse vivendo em Portugal demasiadamente à sombra de um passado opressivo, cujo resultado, ao longo do tempo, encaminhou o país a uma posição periférica e atrasada em razão do fechamento promovido pela máquina fascista do Estado Novo. O protagonista vive às margens da sociedade, uma vez que, ao retornar da guerra, jamais conseguiu restabelecer as suas relações sociais, familiares e profissionais. Encontra- se divorciado da esposa e incompreendido pela família, que sempre esperou que sua participação no Exército o tornasse um homem de verdade, digno de representar toda a tradição ilustre e guerreira da família. Navegações, Porto Alegre, v. 10, n. 1, p. 3-11, jan.-jun. 2017 Alfeu Sparemberger Universidade Federal de Pelotas Esses eram jovens pertencentes a uma geração que foi arrancada de seus sonhos pessoais, afastada dos familiares e de seu país de origem para lutar contra um povo já massacrado pela pobreza e pela desigualdade gerada pelo sistema colonial português, em nome de um ideal ultrapassado, valorizado pelo peso da tradição histórica e totalmente contrário aos direitos básicos da humanidade. Cardoso (2011, p. 221) ressalta ainda, que “a geração que fez a guerra é, assim, vista pela sociedade portuguesa já democratizada como culpada pelas formas mais duras de Portugal ter exercido o colonialismo, sendo muitas vezes associada ao próprio regime”. Em outra passagem, o autor afirma que “essa geração, que vivia já um vácuo de convicções, vê-se atomizada aquando do regresso a casa. Esta seria a dispersão de um capital colectivo temido pelo Estado” (p. 119). De acordo com Cardoso (2011, p. 222), “Judas é um romance que coloca em causa a verdade histórica, instituída por quem não a viveu”, inscrevendo-se como um romance que indaga as várias verdades sobre a guerra, a ditadura e a Revolução. A narrativa é responsável por colocar diretamente em causa a História de Portugal, ao abordar acontecimentos traumáticos para a sociedade, trazendo para debate a guerra e suas consequências. O título do romance, além de remeter à lonjura e ao isolamento geográfico que os combatentes enfrentavam ao serem enviados para a guerra no continente africano, direciona-se também a Judas, personagem bíblico conhecido pela traição, por entregar Jesus aos seus perseguidores, condenando-o à sentença de morte em troca de benefícios irrelevantes. Essa metáfora utilizada por Lobo Antunes transmite a ideia da traição, sendo o soldado português o sujeito que seguiu os ensinamentos de sua pátria, mas que, no decorrer da vida, se encontra marginalizado, entregue a um tempo pós-colonial no qual não é aceito, por ser ele a memória viva de um tempo marcado pelos traumas e pela opressão da guerra e do fascismo do Estado Novo, dos quais ele próprio é igualmente uma vítima. Porque camandro é que não se fala nisto? Alfeu Sparemberger Universidade Federal de Pelotas Quanto ao exercício da medicina, sua rotina desregrada, conduzida pela insônia e pelo constante consumo de álcool, que o ajuda a enfrentar a jornada da agonia da noite, afasta-o Maria José, a mulher com quem o protagonista se encontra em um bar, ouve-lhe com atenção e interesse, mas a seu respeito o leitor não tem acesso, sendo o seu nome mencionado em uma única oportunidade no decorrer de todo o romance. A personagem desempenha uma função Navegações, Porto Alegre, v. 10, n. 1, p. 3-11, jan.-jun. 2017 8 Rommel, L. von P., Sparemberger, A. e o silêncio que o próprio país mantinha diante da Guerra Colonial e do movimento revolucionário. Revela, ainda, a traumática situação dos soldados portugueses que, muitas vezes, regressavam traumatizados dos campos de batalha e acabavam, assim, estigmatizados pela sociedade portuguesa redemocratizada, uma vez que sua imagem encontrava-se ligada à memória da ditadura salazarista. praticamente anônima, servindo de confidente, que ajuda o protagonista na dolorosa travessia da noite, quando cotidianamente é acometido pela depressão e melancolia após a separação da esposa e ao ser assombrado pelas lembranças da morte e do sofrimento na guerra. Cardoso (2011) comenta que o silêncio da interlocu- tora trata-se de uma poderosa forma de discurso sobre a Guerra Colonial e representa a metáfora de uma sociedade assombrada pela memória da repressão salazarista, quando o silêncio era a norma. Segundo o autor, é por meio do seu silêncio que a interlocutora concebe sentido ao discurso do protagonista, sendo, assim, responsável por potencializar o violento relato do narrador. Segundo Cardoso (2011), o sujeito que participou da guerra, ao tomar consciência da traição promovida pela família e pelo Estado português, e mediante os incontornáveis traumas da violência, invariavelmente entra em um processo de autodestruição e gradativo afastamento social e familiar. A geração de soldados retornados da guerra é estigmatizada e excluída pela sociedade e temida pelo Estado, pois seus corpos estão marcados pela experiência destruidora da guerra e pela melancolia e resignação de terem presenciado o absurdo e empobrecedor resultado da valorização e da manutenção do colonialismo na África. Na narrativa de Os cus de Judas, Lobo Antunes contempla o absurdo da guerra, a violência e a dominação imposta não somente aos africanos, mas também aos próprios combatentes portugueses. Navegações, Porto Alegre, v. 10, n. 1, p. 3-11, jan.-jun. 2017 Alfeu Sparemberger Universidade Federal de Pelotas Em certa medida, podemos verificar que essa identidade se liga ao título do seu segundo romance publicado, Os cus de Judas. Referimo-nos aqui à figura de Judas, personagem bíblica à qual se apoia a imagem da “traição”. Daqui retiraremos a ilação de que o soldado português acabou por ser um outro, “um Judas”, aquele que estará numa “porta- giratória”, ou seja, que será ambos os lados e nenhum, algures entre a traição e a exclusão (CARDOSO, 2011, p. 30). A cada ferido de emboscada ou de mina a mesma pergunta aflita me ocorria, a mim, filho da Mocidade Portuguesa, das Novidades e do Debate, sobrinho de catequistas e íntimo da Sagrada Família que nos visitava a domicílio numa redoma de vidro, empurrado para aquele espanto de pólvora numa imensa surpresa: são os guerrilheiros ou Lisboa que nos assassinam, Lisboa, os americanos, os russos, os chineses, o caralho da puta que os pariu combinados para nos foderem os cornos em nome de interesses que me escapam, quem me enfiou sem aviso neste cu de Judas de pó vermelho e de areia, a jogar as damas com o capitão idoso saído de sargento que cheirava a menopausa de escriturário resignado e sofria do azedume crónico da colite, quem me decifra o absurdo disto, as cartas que recebo e me falam de um mundo que a lonjura tornou estrangeiro e irreal, os calendários que risco de cruzes a contar os dias que me separam do regresso e apenas achando à minha frente um túnel infindável de meses, um escuro túnel de meses [...] (ANTUNES, 2010, p. 39-40). Seixo (2002, p. 55) comenta que a questão da identidade do médico protagonista se espraia para questões de “personalidade, de profissão, de família, de identificação amorosa, de terra, de país” e liga-se ao contexto pós-colonial da narrativa. Após o regresso de Angola, o ex-combatente não mais consegue encontrar espaço nesse “novo” tempo em que seu país está inserido, e que ainda busca inserir-se, à custa da ocultação e da superação do passado recente. Ribeiro (2004) salienta que o conceito de traição, aludido pelo título do romance, recai sobre a figura do protagonista de forma dupla, uma vez que ele é alguém que “traiu” o sistema ditatorial, alimentado pelo Estado Novo, ao regressar portador de uma memória incômoda – a perda dos territórios ultramarinos. Alfeu Sparemberger Universidade Federal de Pelotas Começo a pensar que o milhão e quinhentos mil homens que passaram por África não existiram e lhe estou contando uma espécie de romance de mau gosto impossível de acreditar, com que a comovo a fim de conseguir mais depressa (um terço de peleio, um terço de álcool, um terço de ternura) que você veja nascer comigo a manhã na claridade azul pálida que fura as persianas e sobe dos lençóis (ANTUNES, 2010, p. 65). Percebe-se nesta passagem da narrativa que o médico protagonista questiona o fenômeno da amnésia coletiva que tomou conta da sociedade portuguesa após a Revolução dos Cravos. Durante a conversa com sua companheira, ele compara a sua narração dos fatos a um romance criado, em que todo o absurdo da guerra parece ter sido inventado e não passa de uma ficção de mau gosto que ele conta a fim de que consiga atrair sua atenção e compaixão. Cardoso (2011) constata que o soldado antuniano, principalmente em Os cus de Judas, encontra-se em uma posição ambígua ante a sua sociedade, pois ele acaba por ser tomado como um “outro”, alguém que, após cumprir suas funções a mando do Estado, se vê marginalizado, uma vez que não mais encontra espaço no imaginário coletivo. Além de ser ícone de um imperialismo que se prolongou por demasiado tempo, causando graves rupturas na sociedade, ele é portador do estigma da derrota, da perda das colônias. Ele representa os dois lados da moeda – Os cus de Judas assume a forma de uma obra literária que busca trazer à tona e especular o alheamento Navegações, Porto Alegre, v. 10, n. 1, p. 3-11, jan.-jun. 2017 9 A construção da memória da Guerra Colonia ... aquele que defendeu a pátria, mas que ressurge trazendo o sentido da derrota antiépica. portuguesa e simpatizar com os africanos, ter de desempenhar a função de médico. No exercício de sua função, ele é o responsável por, metaforicamente, manter em funcionamento o corpo físico do Estado, representado pela figura dos combatentes, seus companheiros na antagônica experiência da “aprendizagem da agonia”. Cabe a ele o papel de consertar os corpos dilacerados pela violência da guerra, sendo o responsável direto por salvar suas vidas ou assistir seu regresso a Lisboa em caixões de chumbo nos porões dos navios. Portanto, compreender a identidade do “soldado antuniano” será compreender outras identidades dentro da obra do autor. Navegações, Porto Alegre, v. 10, n. 1, p. 3-11, jan.-jun. 2017 Alfeu Sparemberger Universidade Federal de Pelotas apanhado no Mussuma os soldados tiraram o retrato com ela num orgulho de troféu, a guerra tornou-nos em bichos, percebe, bichos cruéis e estúpidos ensinados a matar [...], o mundo-que-o-português-criou são estes luchazes côncavos de fome que não nos entendem a língua, a doença do sono, o paludismo, a amibíase, a miséria [...]. (ANTUNES, 2009, p. 123). ta-se agora como uma cidade vertical, hostil e desprovida de humanidade, uma espécie de labirinto, por onde este Judas, encarcerado entre a vida perdida e o serviço militar cumprido, procura abrigo perante os traumas do passado. Esse retorno, segundo o protagonista, assemelha-se a um “ressuscitar de Lázaro desnorteado, que reaprende penosamente o uso dos objectos e dos sons” (ANTUNES, 2010, p. 195) e que precisa se readaptar à centralidade da vida urbana e às relações familiares e sociais, interrompidas com a fatídica viagem para a guerra na África. Em Lisboa, sua cidade natal, o ambiente torna- se estrangeiro, e na imaginação do médico, habituado à África, lhe parece que os ambientes, tão distintos um do outro, são capazes de se interpenetrar, como se Angola tivesse lhe acompanhado Atlântico adiante. A guerra é responsável por uma espécie de metamorfose, um movimento de desintegração e desumanização, que transforma os combatentes em “animais”, cujos gestos se revestem de crueldade contra o povo africano. O império, o “mundo-que-o-português- criou”, aos olhos do médico protagonista, não passa de uma terra destruída, repleta de fome, doenças e miséria. Os ideais, que historicamente embasaram a expansão portuguesa, são postos à prova na narrativa que averigua o produto final do imperialismo português. Habituara-me demais ao silêncio e à solidão de Angola, e afigurava-se-me inimaginável que o capim não rompesse do alcatrão das avenidas os seus longos dedos verdes acerados pelas primeiras chuvas. Não existia nenhuma máquina de costura ferrugenta e avariada na casa dos meus pais, e o soba do Chiúme não me esperava na sala, a fitar, para lá da estante envidraçada dos livros, a vastidão, húmida de sapos e de lodo, da chana. Idêntico a uma criança quando nasce, contemplava, com órbitas redondas de surpresa, os semáforos, os cinemas, o contorno desequilibrado das praças, as melancólicas esplanadas dos cafés, e tudo se diria possuir, ao meu redor, uma carga de mistério que eu seria sempre incapaz de elucidar. Alfeu Sparemberger Universidade Federal de Pelotas O médico protagonista manifesta, então, de forma ambígua, repulsa e ódio pelo governo de seu país e simpatia pelo ímpeto libertador dos africanos, mas que ao ser submetido à violência da guerra e não mais conseguir reentrar na centralidade europeia, sente-se traído e vitimado pela família e pela máquina estatal, que lhe orientaram e encaminharam para a África. Nesta passagem pode-se perceber o sentimento de inconformidade do protagonista ao se encontrar em uma guerra, na qual diariamente via morrer seus companheiros, muitos deles jovens iguais a ele, a quem o regime ditatorial obrigou a colaborar a fim de manter ilusoriamente a grandiosidade de Portugal. Percebe-se novamente a crítica às origens burguesas que, desde a sua infância, mostrava-se muito presente no seu ambiente familiar. O narrador-personagem não se coloca em nenhum dos espaços, ainda que por simpatia e solidariedade de vitimização pareça estar com os guerrilheiros, mas integrado na estrutura que os combate e, à parte pequenos gestos de raiva e fúria mais ou menos inconsequentes, nada fazendo para a mudar, o conceito de traição recai sobre ele de forma dupla: ele é, depois dos cus de Judas, um traidor (um Judas) em relação aos valores em que tinha sido educado e do Estado que o seu uniforme militar representa, mas apresenta-se sobretudo como vítima de uma traição da família e do Estado que o conduziram a um engano (RIBEIRO, 2004, p. 280). A experiência da guerra apresenta-se decisiva no processo de desestruturação do corpo físico e social do protagonista, que, após o retorno, é impedido, pelos traumas e vivências extremas a que foi submetido no continente africano, de reatar o ritmo de sua vida, que invariavelmente assume um prolongamento do “inferno” e de todo o sofrimento e opressão da guerra. Quem veio aqui não consegue voltar o mesmo, explicava eu ao capitão de óculos moles e dedos membranosos colocando delicadamente no tabuleiro, em gestos de ourives, as peças de xadrez, cada um de nós, os vivos, tem várias pernas a menos, vários braços a menos, vários metros de intestino a menos, quando se amputou a coxa gangrenada ao guerrilheiro do MPLA A posição ambígua do protagonista em relação à guerra e ao seu país destaca-se, também, pelo fato de, apesar de não se identificar com a causa colonialista Navegações, Porto Alegre, v. 10, n. 1, p. 3-11, jan.-jun. 2017 10 Rommel, L. von P., Sparemberger, A. Alfeu Sparemberger Universidade Federal de Pelotas De forma que encolhi a cabeça entre os ombros e curvei as omoplatas como as pessoas sem gabardina perante uma chuva inesperada, oferecendo o mínimo possível do meu corpo a um país que não entendia já, e embarafustei pelo janeiro da cidade. (ANTUNES, 2010, p. 195). Como aponta Ribeiro (2004, p. 270), por meio da guerra o protagonista percebe que aquele mundo- que-o-português-criou, na verdade, não passa de um “espelho grotesco e excessivo da grande mentira da casa portuguesa”. A imagem de grande império nacional, sustentada pela ditadura salazarista, começa a ruir após a Guerra Colonial, que põe em jogo a memória imperial portuguesa e conduz o país ao desmembramento forçado de seus territórios coloniais, outrora centros mantenedores da identidade nacional. A guerra foi responsável por fazer com que o médico protagonista se sentisse “nu de raízes”, repelido tanto pela África, continente a que nunca pertenceu mas que fora obrigado a visitar para combater seu ímpeto libertador, quanto pelo seu país, que, após a esquizofrênica e desumana experiência dos conflitos, já não o aceita, por ser ele o representante do passado ditatorial, estando marcado por um poder estatal que levou até às últimas consequências a manutenção do colonialismo como forma de afirmação econômica e política. A sua casa, ambiente que deveria ser responsável por emanar segurança e conforto, assemelha-se agora a uma “espécie de jazigo, vazio e hirto” (ANTUNES, 2010, p. 125) após o retorno de Angola. Da mesma forma, a cidade é invadida pela guerra e por toda a sua pulsão de morte, desumanizando todas as pessoas, seres e objetos, e tornando-o um sujeito assombrado por fantasmas que insistem em persegui-lo, fazendo com que jamais consiga reconquistar a vida que tinha antes de visitar o “inferno” da guerra e da lá retornar aos pedaços. O medo de voltar ao meu país comprime-me o esófago, porque, entende, deixei de ter lugar fosse onde fosse [...]. Flutuo entre dois continentes que me repelem, nu de raízes, em busca de um espaço branco onde ancorar, e que pode ser, por exemplo, a cordilheira do seu corpo, um recôncavo, uma cova qualquer do seu corpo, para deitar, sabe como é, a minha esperança envergonhada. (ANTUNES, 2010, p. 182). O medo de voltar ao meu país comprime-me o esófago, porque, entende, deixei de ter lugar fosse onde fosse [...]. Navegações, Porto Alegre, v. 10, n. 1, p. 3-11, jan.-jun. 2017 Alfeu Sparemberger Universidade Federal de Pelotas Flutuo entre dois continentes que me repelem, nu de raízes, em busca de um espaço branco onde ancorar, e que pode ser, por exemplo, a cordilheira do seu corpo, um recôncavo, uma cova qualquer do seu corpo, para deitar, sabe como é, a minha esperança envergonhada. (ANTUNES, 2010, p. 182). Conforme Ribeiro (2004, p. 284), “a excessiva e neurótica imagem de África, com os seus cheiros de morte e estropiamento, projecta-se em tudo, bloqueando o futuro”. A morte e a guerra instalam-se, confortavelmente, substituindo a antiga existência do protagonista em sua casa, em sua cidade e em suas relações amorosas. O cotidiano do médico protagonista torna-se agora um “mundo labiríntico e assumidamente marginal” (p. 289), No momento do regresso da África, conforme o protagonista, os soldados retornados assumiram o aspecto de fantasmas que, pouco a pouco, se materializaram, e passaram a ocupar as ruas e os espaços da metrópole, trazendo consigo, na bagagem da memória, o inenarrável e o indizível absurdo da guerra. A Lisboa que o médico reencontra após o regresso definitivo de Angola, apresen- Navegações, Porto Alegre, v. 10, n. 1, p. 3-11, jan.-jun. 2017 11 A construção da memória da Guerra Colonia ... de onde o mundo, tido por ele como “normal” antes da experiência africana, afasta-se progressivamente, deixando-lhe desorientado, morador de uma casa vazia, preenchida tão somente pelos traumas e pela dor que carrega na memória. discurso ficcional, a experiência da Guerra Colonial, que seria fatalmente apagada pelo movimento de evolução do país, é resguardada do esquecimento, simbolizada e transformada em linguagem. Os cus de Judas atua como uma narrativa literária que auxilia na construção de uma memória coletiva sobre a Guerra Colonial, uma vez que o romance aborda um traumático capítulo da sociedade portuguesa. Em Lobo Antunes, a literatura serve como forma de dar vazão e comunicar os traumas da guerra e da opressão gerados sobre toda uma geração de portugueses. No Portugal pós- colonial o discurso literário visa a suprir a impotência do discurso histórico diante da guerra e do processo de redemocratização, evitando, assim, que o passado recente do país caia no esquecimento. O quimbo da Tia Teresa, cercado pelo odor doce dos pés de liamba e de tabaco, é talvez o único sítio que a guerra não logrou invadir do seu cheiro pestilento e cruel. Alfeu Sparemberger Universidade Federal de Pelotas Alastrou por Angola, a terra sacrificada e vermelha de Angola, alcançou Portugal a bordo dos barcos de militares que regressavam, desorientados e tontos, de um inferno de pólvora, insinuou-se na minha humilde cidade que os senhores de Lisboa mascararam de falsas pompas de cartolina, encontrei-a deitada no berço da minha filha como um gato, fitando-me com pupilas de maldade oblíqua, a mirar-me dos lençóis com a turva raiva invejosa [...]. A guerra propagou-se aos sorrisos das mulheres nos bares, sob as ampolas despolidas dos candeeiros que lhes multiplicam de sombras a curva indagadora dos narizes, turvou as bebidas de um gosto azedo de vingança, aguarda-nos no cinema, instalada no nosso lugar, vestida de preto como um notário viúvo a retirar do bolso do casaco o estojo de plástico dos óculos. Está aqui, nesta casa vazia, nos roupeiros desta casa vazia, grávida dos fetos moles da minhas cuecas, no geométrico espaço de trevas que as lâmpadas não alcançam nunca, está aqui e chama-me baixinho com a pálida voz ferida dos camaradas assassinados nas picadas de Ninda e de Chiúme (ANTUNES, 2010, p. 173). Referências ANTUNES, António Lobo. Os cus de Judas. Rio de Janeiro: Objetiva, 2010. CARDOSO, Norberto do Vale. A mão-de-Judas: representações da Guerra Colonial em António Lobo Antunes. Lisboa: Texto Editores, 2011. COSTA, Jorge Manuel de Almeida Gomes da. Para um estudo da memória e identidade portuguesa com António Lobo Antunes. 2013. 241 f. Tese (Doutorado em Letras – Literatura Comparada) – Faculdade de Letras, Centro Regional das Beiras, Universidade Católica Portuguesa, Lisboa, 2013. GOMES, Álvaro Cardoso. A voz itinerante. Ensaio sobre o romance português contemporâneo. São Paulo: Ed. da Universidade de São Paulo, 1993. Pela narrativa de Os cus de Judas, Lobo Antunes aborda a fatídica experiência da Guerra Colonial e seus traumáticos efeitos na sociedade portuguesa. O médico protagonista apresenta-se como o representante de uma geração de portugueses que foi vítima da opressão do regime salazarista, e que foi obrigada a combater em uma guerra a fim de manter a posse dos territórios ultramarinos na África. O romance emerge, assim, como um grito de revolta e de desabafo ante o antigo regime e à sua memória ainda ativa na sociedade no período pós-Revolução dos Cravos. LOURENÇO, Eduardo. O labirinto da saudade. Lisboa: Dom Quixote, 2013. RIBEIRO, Margarida Calafate. Uma história de regressos: império, guerra colonial e pós-colonialismo. Porto: Afronta- mento, 2004. SEIXO, Maria Alzira. Os romances de António Lobo Antunes. Lisboa: Dom Quixote, 2002. VECCHI, Roberto. Excepção atlântica: pensar a literatura da Guerra Colonial. Porto: Afrontamento, 2010. A literatura desempenha um papel fundamental no período pós-1974, pois se apresenta como alternativa para a construção de uma memória coletiva sobre o período de transição pós-colonial em Portugal. Com o Recebido: 12 de setembro de 2016 Aprovado: 29 de maio de 2017 Contato: lvpfeil@hotmail.com berger9889@gmail.com Recebido: 12 de setembro de 2016 Aprovado: 29 de maio de 2017 Contato: lvpfeil@hotmail.com berger9889@gmail.com Navegações, Porto Alegre, v. 10, n. 1, p. 3-11, jan.-jun. 2017
https://openalex.org/W3135193036	https://comptes-rendus.academie-sciences.fr/mecanique/item/10.5802/crmeca.76.pdf	English	null	A contribution to the modelling of creep behaviour of FCC metals	Comptes rendus. Mécanique	2,021	cc-by	4,809	Ahmed Maati∗, a, b, El Hadj Ouakdib, Laurent Tabourotc and Pascale Ballandc a Mechanics Laboratory, Department of Mechanics, Amar Telidji University, Laghouat 03000, Algeria b Laboratory of Physics and Mechanics of Metallic Materials, Setif 1 University, Setif 19000, Algeria c SYMME Laboratory, Univ. Savoie Mont Blanc, FR-74000 Annecy, France c SYMME Laboratory, Univ. Savoie Mont Blanc, FR-74000 Annecy, France E-mails: a.maati@lagh-univ.dz (A. Maati), elouakdi@univ-setif.dz (E. H. Ouakdi), laurent.tabourot@univ-smb.fr (L. Tabourot), pascale.balland@univ-smb.fr (P. Balland) E-mails: a.maati@lagh-univ.dz (A. Maati), elouakdi@univ-setif.dz (E. H. Ouakdi), laurent.tabourot@univ-smb.fr (L. Tabourot), pascale.balland@univ-smb.fr (P. Balland) Abstract. In this paper, a new modelling is proposed to describe the viscoplastic behaviour of face-centred cubic (FCC) metals. Creep tests under various conditions were performed. The material chosen to test the model is Al-1050. The plastic deformation is controlled by intragranular diﬀusion when the test temperature exceeds 0.4Tm. The developed model involves two state variables related to the microstructure: dislocation density and subgrain size. The grain size is assumed to be constant in the intermediate temperature range. Validation tests were proposed to justify the reliability of the developed model in various loading conditions. Keywords. Creep, Subgrain, Strain hardening, Dynamic recovery, Viscoplasticity, Intragranular diﬀusion. Manuscript received 24th October 2020, revised 27th December 2020, accepted 1st February 2021. Manuscript received 24th October 2020, revised 27th December 2020, accepted 1st February 2021. ∗Corresponding author. Comptes Rendus Mécanique Comptes Rendus Mécanique 2021, 349, n1, p. 55-64 https://doi.org/10.5802/crmeca.76 q 2021, 349, n1, p. 55-64 https://doi.org/10.5802/crmeca.76 Synthesis / Synthèse Mécanique This article is licensed under the Creative Commons Attribution 4.0 International License. http://creativecommons.org/licenses/by/4.0/ Les Comptes Rendus. Mécanique sont membres du Centre Mersenne pour l’édition scientiﬁque ouverte www.centre-mersenne.org e-ISSN : 1873-7234 Les Comptes Rendus. Mécanique sont membres du Centre Mersenne pour l’édition scientiﬁque ouverte www.centre-mersenne.org e-ISSN : 1873-7234 Comptes Rendus Mécanique 2021, 349, n1, p. 55-64 https://doi.org/10.5802/crmeca.76 1. Introduction The development of forming operations of sheet metal requires the use of numerical simula- tion tools based on the resolution methods such as ﬁnite element methods. For this purpose, the choice of the accurate modelling of thermomechanical behaviour of the material that has to be introduced in the numerical code is a decisive step. Various models are now available in the literature to describe at low scale the viscoplastic behaviour by taking into account inter- and intragranular eﬀects (model of Pierce and et al. (1983), model of Cuitino Ortiz (1992), model of Teodosiu et al. (1997), etc.). The models are selected so that they present a similar formalism: a ﬂow law plus a hardening law. This makes it possible to use the same numerical scheme to in- tegrate each model [1]. In our case, a viscoplasticity model based on physics is proposed to de- scribe the thermomechanical behaviour during creep tests of face-centred cubic (FCC) metals ISSN (electronic) : 1873-7234 https://comptes-rendus.academie-sciences.fr/mecanique/ 56 Ahmed Maati et al. such as Al-1050. Dislocations are important microstructure features, as their mobility greatly im- pacts the strength and plasticity of crystal materials [2]. Therefore, dislocations and their associ- ated mechanisms for a given microstructure are generally self-suﬃcient to macroscopically ex- plain most of the mechanical properties [3]. Physically, strain hardening and dislocation pattern- ing are strongly interrelated. The microstructure evolution during high-temperature deforma- tion is to be taken into account. Deﬁnitely, the temperature plays an important role in the devel- opment of microstructures by promoting the annihilation of dislocations and thus the dynamic recovery. The evolution of dislocation density and their structuring within the material controls the evolution of ﬂow stress during the plastic deformation. To determine the eﬀects of complex loading, it is wiser to use models whose state variables related to dislocations microstructure are better able to evolve according to the history of the material [4]. In this study, two internal state variables (dislocation density and subgrain size) under diﬀerent loading conditions were inves- tigated. It is important to note that the present article can be considered as an extension of a re- cently published study [5], the latter was devoted to modelling the thermomechanical behaviour of FCC metals (e.g. aluminium 1050A alloy) under tensile tests. All the tests were performed for each imposed temperature and a constant strain rate, the time factor is not taken into account. 1. Introduction The eﬀect of microstructure on the mechanical behaviour during plastic deformation was in- vestigated. Two opposite and simultaneous physical phenomena were generated during plastic deformation: the strain hardening rules that occur because of dislocation multiplication mech- anism within the crystal structure of the metal and the dynamic recovery governed by thermal activation. Dynamic recovery allows dislocations to overcome obstacles to their motion to form a cell structure more homogeneous and less dense [6]. The theory of thermal activation indicates that we will have to apply to the dislocation located at a point, a stress at least equal to the internal stress exerted on the dislocation (σ ≈σµ) [7]. In the intermediate temperature range (T ≥0.4Tm), the process of annihilation and rearrangement of dislocations may give rise to the formation of subgrains or dislocation cells, which contribute to the strength of the metal [8]. In this study, the evolution of internal stress and dislocation microstructures during creep tests under the coupling eﬀect of strain rate and temperature was investigated. New equations were developed to predict the thermomechanical behaviour in creep for FCC metals under various conditions with and without sudden change in load. A set of constitutive equations and model parameters were used to describe the creep behaviour of FCC metals over a wide range of strain rate and temperature. Experimental data derived from literature [9] are used to validate numerical results. Table 1. Numerical values of model constants [5] Table 1. Numerical values of model constants [5] ρs(˙ε,T ) = · 1 bdsKa ¸ +ρ0 (4) ds(˙ε,T ) = ds0 + A µ ˙ε DL ¶−1 n (5) Ka(˙ε,T ) = Ka0 +B µ ˙ε DL ¶−1 n (6) K ′ a = C ·Ka (7) K = 1 1+bdsKaρ0 (8) Ka0 ≈3ds0, (9) Ka0 ≈3ds0, (9) where A,B,C, and n are constants determined from uniaxial tensile test. K is an integration constant. ds0 and Ka0 represent, respectively, the values of ds and Ka for low temperatures and/or high strain rates [11]. ρ0 represents the dislocation density before deformation, we introduced this quantity in order to account properly the initial conditions of the deformation. q y p p y The diﬀusion coeﬃcient DL(T ) is given by the following relation [12]: DL(T ) = 1.7×10−4 exp µ−142 RT ¶ +6×10−7 exp µ−115 RT ¶ . (10) (10) As a reminder, the basic equations (2) to (9) have been developed and demonstrated in the previous publication [5]. In addition, the constants of the model were identiﬁed in this step (Table 1). These values were chosen on the basis that they give a best ﬁt of numerical curves to the experimental data. As a reminder, the basic equations (2) to (9) have been developed and demonstrated in the previous publication [5]. In addition, the constants of the model were identiﬁed in this step (Table 1). These values were chosen on the basis that they give a best ﬁt of numerical curves to the experimental data. 2. Presentation of the thermomechanical model under tensile test For temperatures higher than 0.4Tm, the applied stress depending on the microstructural param- eters is represented by the internal stress according to the following relationship [10]: σ ≈σµ(ε, ˙ε,T ) = α1Gbpρ +α2G s b d +α3G s b D , (1) (1) where α1, α2, and α3 are dimensionless constants. where α1, α2, and α3 are dimensionless constants. The microstructural parameters ρ and d vary signiﬁcantly during plastic deformation and are closely dependant on strain rate and temperature [5]: ρ = ρs(1−K exp(−Kaε)) (2) d = ds(1+2exp(−K ′ aε)) (3) (2) (3) C. R. Mécanique, 2021, 349, n1, 55-64 57 57 Ahmed Maati et al. Table 1. Numerical values of model constants [5] α1 α2 α3 ds0 (µm) n B (m−2/5) D (µm) A (m3/5 ×10−6) ρ0 (µm−2) C b (µm) 0.2 0.07 0.08 0.5 5.0 7500 60 2500 5.0 40 2.86×10−4 Table 1. Numerical values of model constants [5] α1 α2 α3 ds0 (µm) n B (m−2/5) D (µm) A (m3/5 ×10−6) ρ0 (µm−2) C b (µm) 0.2 0.07 0.08 0.5 5.0 7500 60 2500 5.0 40 2.86×10−4 with ρs(˙ε,T ) = · 1 bdsKa ¸ +ρ0 (4) ds(˙ε,T ) = ds0 + A µ ˙ε DL ¶−1 n (5) Ka(˙ε,T ) = Ka0 +B µ ˙ε DL ¶−1 n (6) K ′ a = C ·Ka (7) K = 1 1+bdsKaρ0 (8) Ka0 ≈3ds0, (9) 3. Modelling of creep at constant load The creep process is accompanied by many diﬀerent microstructural rearrangements including dislocation movement, ageing of microstructure, and grain-boundary cavitation [13]. This phe- nomenon is highlighted by tensile test under constant load. The deformation occurs instanta- neously and the behaviour is highly sensitive to strain rate ˙ε. The test reveals the inﬂuence of temperature and applied load on the various stages of creep by taking into account the evolution of two internal parameters (ρ and d). The creep curves can be represented in two types of func- tion ε(t) or ˙ε(ε). For an irreversible deformation process, an incremental relationship involving stress, strain, and strain rate is deﬁned as follows [9]: dσ = θdε+βdln µ ˙ε DL ¶ (11) (11) with with with θ = µ∂σ ∂ε ¶ (˙ε,T ) ; β =   ∂σ ∂ln ³ ˙ε DL ´   ε θ = µ∂σ ∂ε ¶ (˙ε,T ) ; β =   ∂σ ∂ln ³ ˙ε DL ´   ε C. R. Mécanique, 2021, 349, n1, 55-64 C. R. Mécanique, 2021, 349, n1, 55-64 Ahmed Maati et al. 58 Ahmed Maati et al Ahmed Maati et al. 58 θ and β are analytically calculated using the relationships previously described: θ = Kaσρ 2 ·µσρs σρ ¶2 −1 ¸ + CKaσds 2 · 1− µ σd σds ¶2¸ (12) β = − 1 10ds ½ 2σs ·Kaε 2 ¿µσρs σρ ¶2 −1 À −1 ¸ +σd · K ′ aε ¿ 1− µ σd σds ¶2À −1 ¸¾ (13) (12) (13) σρs and σds are used to simplify the expressions of θ and β such as σρs and σds are used to simplify the expressions of θ and β such as σρs = α1Gbpρs; σds = α2G s b ds . The ﬂow stress evolution during creep can also be expressed in exponential form as a function of eﬀective strain: σ = σ0 exp(ε) (14) (14) σ0 is the initial stress corresponding to the beginning of primary stage of creep (at ε = 0). It is deduced from (1) by using the minimum boundary condition: σ0 = α1Gbpρ0 +α2G s b d0 +α3G s b D . (15) (15) This study assumed that the metal does not undergo recrystallization of the structure, then the grain size remains constant (D = D0 = constant). 3. Modelling of creep at constant load The viscoplastic behaviour expressed by the function ˙ε(ε) can therefore be formulated com- bining equations (1) and (15): • Creep at constant load (dσ/dε = σ): • Creep at constant load (dσ/dε = σ): ep at constant load (dσ/dε = σ): d˙ε dε = µσ−θ β ¶ ˙ε (16) (16) • Creep at constant stress (dσ = 0): d˙ε dε = µ−θ β ¶ ˙ε. (17) (17) The numerical integration of relationships (13) and (14) allows us to plot the creep curves ˙ε(ε) at constant load and at constant stress, respectively. However, before making the numerical calculation, we must ﬁrst ﬁnd the initial values dsi and ˙ε0i corresponding just at the beginning of the primary creep. The expression of dsi is obtained directly from the relationship (12). d0 is taken equal to 3 times the value of dsi [14,15]: dsi = α2 2G2b 3(σ0 −σρ0 −σD)2 . (18) (18) The expression of the initial strain rate (˙ε0i) corresponding to subgrain size dsi can be determined from the relationship (2): µ A ¶5 The expression of the initial strain rate (˙ε0i) corresponding to subgrain size dsi can be determined from the relationship (2): 5 ˙ε0 = µ A dsi −ds0 ¶5 DL. (19) (19) To ensure the consistency of our model, several tests under various conditions were performed by comparing the calculated results with the experimental ones. A computer program (Fortran in our case) is used to plot numerical graphs from the diﬀerent relationships of the model. Figure 1, for example, describes the creep curves at constant load for diﬀerent conditions of solicitation. The three stages of creep deformation are well described. It is checked that high initial stress levels and high temperatures accelerate the creep process. NB/In the calculated curves, the same conditions of solicitation as those of the experimental tests are imposed. C. R. Mécanique, 2021, 349, n1, 55-64 59 Ahmed Maati et al. Figure 1. Creep curves for Al-1050 sheet under diﬀerent conditions of solicitation. Figure 1. Creep curves for Al-1050 sheet under diﬀerent conditions of solicitation. Several experimental works [16,17] showed that during creep, an abrupt change of the initial stress amplitude (σ0) induces a signiﬁcant change in the material microstructure. Various tests were carried out in this context. 3. Modelling of creep at constant load At a given temperature (T ≥0.4Tm) and for a deﬁned level of plastic deformation ε∗, an abrupt change of σ0 causes a noticeable change in the microstructure via the internal parameters ρ and d. The variation in ﬂow stress ∆σ due to the jump in load between two amounts (F1 = σ01 ×S1 →F2 = σ02 ×S2) can be determined as follows: σ(ε) = ½σ1 = σ01 exp(ε),ε < ε∗ σ2 = σ02 exp(ε),ε ≥ε∗ ¾ (20) ∆σ = (σ02 −σ01)expε∗. (21) (20) (21) According to experimental and numerical works studying the physicomechanical behaviour of metals, creep test with sudden change in load (or temperature) leads to a rapid change in the material microstructure. This can be explained by the appearance of a transient during the jump: the subgrain size evolves rapidly to reach its new stationary value (ds2), while the dislocation density ρ has a negligible change as long as the deformation is almost constant. For this reason, only the variation of the parameter d during the jump was considered. Two steps to consider: Two steps to consider: • Just before the jump (ε < ε∗): • Just before the jump (ε < ε∗): ½d1 = ds1[1+2exp(−K ′ a1ε∗)] ρ1 = ρ∗ 1 ¾ (22) • Just after the jump (ε > ε∗): ½d2 = ds2[1+2exp(−K ′ a2ε∗)]¾ (23) ½d1 = ds1[1+2exp(−K ′ a1ε∗)] ρ1 = ρ∗ 1 ¾ (22) (22) • Just after the jump (ε > ε∗): ½d2 = ds2[1+2exp(−K ′ a2ε∗)] ρ2 = ρ∗ 2 ¾ , (23) (23) where ρ∗ 1 and ρ∗ 2 represent the dislocation density just before and after the jump, respectively. Considering that the dislocation density remains constant during sudden change in load, that here ρ∗ 1 and ρ∗ 2 represent the dislocation density just before and after the jump, respectivel where ρ∗ 1 and ρ∗ 2 represent the dislocation density just before and after the jump, respectively. Considering that the dislocation density remains constant during sudden change in load, that is, ρ∗ 1 = ρ∗ 2 and using the relationship (1), we can deduce the physical expression of ∆σ: where ρ∗ 1 and ρ∗ 2 represent the dislocation density just before and after the jump, respectively. 3. Modelling of creep at constant load Considering that the dislocation density remains constant during sudden change in load, that is, ρ∗ 1 = ρ∗ 2 and using the relationship (1), we can deduce the physical expression of ∆σ: Considering that the dislocation density remains constant during sudden change in load, that is, ρ∗ 1 = ρ∗ 2 and using the relationship (1), we can deduce the physical expression of ∆σ: ∆σ = α2G p b Ã 1 p d2 − 1 p d1 ! . (24) (24) C. R. Mécanique, 2021, 349, n1, 55-64 60 Ahmed Maati et al. Figure 2. Creep test at constant load with a sudden decrease in the load. Figure 2. Creep test at constant load with a sudden decrease in the load. In order to simplify the mathematical expression of the relationship (21), we can replace the instantaneous subgrain size by its stationary value ds, that is: In order to simplify the mathematical expression of the relationship (21), we can replace the instantaneous subgrain size by its stationary value ds, that is: • Creep under constant load: 1 2 • Creep under constant load: • Creep under constant load: ds2 = " (σ02 −σ01)expε∗ α2G p b + µ 1 ds1 ¶ 1 2 #−2 (25) (25) • Creep under constant stress: ds2 = " (σ02 −σ01) α2G p b + µ 1 ds1 ¶ 1 2 #−2 . (26) (26) In order to check the reliability of the proposed model, the calculated results are compared with relevant experimental results in the literature. Two diﬀerent tests were carried out; one with a sudden decrease in load (Figure 2), while the other is performed by a sudden increase in load (Figure 3). As shown in both ﬁgures, a good ﬁt is observed between experimental and calculated curves. These ﬁgures show two types of behaviour: continuous creep (without jump) and discontinuous creep (with jump). After certain period of time, the discontinuous curve tends towards a steady state and con- verges towards the continuous curve. Note that, the transient reﬂect the profound evolution of the internal variable (d) that should be progressive (not sudden) between two stationary values ds1 and ds2. Eventually, the curve after the jump tends towards the steady state and converges towards the curve illustrating the test without the jump. C. R. Mécanique, 2021, 349, n1, 55-64 3. Modelling of creep at constant load Therefore, we can observe that the abrupt decrease in the load causes a rapid increase in creep rate, while increasing the load causes a rapid decrease in creep rate followed by accelerated growth in its last stage (tertiary creep). A comparative study was carried out between the two types of creep: at constant load and at constant stress. For this purpose, we have chosen one of the experimental cases available in 61 61 Ahmed Maati et al. Figure 3. Creep test at constant load with a sudden increase in the load. Figure 4. Comparison between the creep curve at constant load and creep curve at con- stant stress. the literature (σ0 = 27.65 MPa and T = 480 K). Figure 4 illustrates the two types of curves clearly diﬀerentiated: in the ﬁrst case, a signiﬁcant acceleration of creep in the tertiary stage is observed this is due to the increased inﬂow stress, while in the second case, a steady state creep is observed and the tertiary stage has not been reached yet, it indicates a thermomechanical balance between the two simultaneous and antagonistic phenomena: strain hardening and dynamic recovery. Figure 3. Creep test at constant load with a sudden increase in the load. Figure 3. Creep test at constant load with a sudden increase in the load. Figure 4. Comparison between the creep curve at constant load and creep curve at con- stant stress. Figure 4. Comparison between the creep curve at constant load and creep curve at con- stant stress. the literature (σ0 = 27.65 MPa and T = 480 K). Figure 4 illustrates the two types of curves clearly diﬀerentiated: in the ﬁrst case, a signiﬁcant acceleration of creep in the tertiary stage is observed, this is due to the increased inﬂow stress, while in the second case, a steady state creep is observed and the tertiary stage has not been reached yet, it indicates a thermomechanical balance between the two simultaneous and antagonistic phenomena: strain hardening and dynamic recovery. the literature (σ0 = 27.65 MPa and T = 480 K). 3. Modelling of creep at constant load Figure 4 illustrates the two types of curves clearly diﬀerentiated: in the ﬁrst case, a signiﬁcant acceleration of creep in the tertiary stage is observed, this is due to the increased inﬂow stress, while in the second case, a steady state creep is observed and the tertiary stage has not been reached yet, it indicates a thermomechanical balance between the two simultaneous and antagonistic phenomena: strain hardening and dynamic recovery. the literature (σ0 = 27.65 MPa and T = 480 K). Figure 4 illustrates the two types of curves clearly diﬀerentiated: in the ﬁrst case, a signiﬁcant acceleration of creep in the tertiary stage is observed, this is due to the increased inﬂow stress, while in the second case, a steady state creep is observed and the tertiary stage has not been reached yet, it indicates a thermomechanical balance between the two simultaneous and antagonistic phenomena: strain hardening and dynamic recovery. C. R. Mécanique, 2021, 349, n1, 55-64 62 Ahmed Maati et al. Figure 5. Evolution of the subgrain size with temperature and/or strain rate. Figure 5. Evolution of the subgrain size with temperature and/or strain rate. Furthermore, the steady state creep behaviour of Al-1050 alloy can be described by (24) derived directly from (1) using the maximum boundary condition: σs = α1Gbpρs +α2G s b ds +α3G s b D . (27) (27) The proposed model still allows us to describe other changes in the material microstructure; for example, Figure 5 shows the evolution of the average subgrain size ds as a function of the term (˙ε/DL), which includes the combined eﬀect of strain rate and temperature (replaces the physical parameter of Zener–Hollomon Z). The experimental curve is plotted for diﬀerent values of temperature and strain rate. In the intermediate temperature range (293 K ≤T ≤540 K), a better agreement is observed between calculated results and experimental data. C. R. Mécanique, 2021, 349, n1, 55-64 Nomenclature σ (MPa) Applied stress F (N) Applied load to the specimen S (mm2) Specimen cross section θ (MPa) Strain hardening rate β Strain rate sensitivity ρ (µm−2) Dislocation density ρs (µm−2) Saturation value of the variable ρ d (µm) Subgrain size or dislocation cell size ds (µm) Saturation value of the variable d D (µm) Grain size σρ (MPa) Internal stress depending on the variable ρ σd (MPa) Internal stress depending on the variable d σD (MPa) Internal stress depending on the variable D G (MPa) Shear modulus ˙ε (s−1) Strain rate T (K) Deformation temperature b (µm) Burgers vector magnitude ka Annihilation factor k′ a Rate constant DL (m2·s−1) Diﬀusion coeﬃcient R (J·mol−1·K−1) The gas constant Tm (K) Metal melting temperature References [1] P. Balland, L. Tabourot, M. Fivel, “Comparison of physically based constitutive laws used for numerical simulations of plasticity of metals”, J. Phys. IV 11 (2001), p. 381-388. [1] P. Balland, L. Tabourot, M. Fivel, “Comparison of physically based constitutive laws used for numerical simulations of plasticity of metals”, J. Phys. IV 11 (2001), p. 381-388. “ l d b d l l l f h l d h h [2] G. Monnet, L. Vincent, B. Devincre, “Dislocation-dynamics based crystal plasticity law for the low- a temperature deformation regimes of bcc crystal”, Acta Mater. 61 (2013), p. 6178-6190. [3] L. Tabourot, A. Maati, P. Balland, M. Vautrot, “Inﬂuence of heterogeneities and of their distribution on the elastoplas- tic”, in International Symposium on Plasticity, Montego Bay, Jamaica, January 2015. [4] E. Nes, “Modelling the evolution in microstructure and properties during plastic deformation of f.c.c metals and alloys — an approach towards a uniﬁed model”, Mater. Sci. Eng. A 322 (2002), no. 1, p. 176-193. [4] E. Nes, “Modelling the evolution in microstructure and properties during plastic deformation of f alloys — an approach towards a uniﬁed model”, Mater. Sci. Eng. A 322 (2002), no. 1, p. 176-193. y pp g p [5] A. Maati, E. H. Ouakdi, L. Tabourot, P. Balland, M. Demouche, “Modelling of the thermomechanical behaviour of FCC metals under various conditions”, Ann. Chim. Sci. Matér. 42 (2018), no. 1, p. 115-127. 5] A. Maati, E. H. Ouakdi, L. Tabourot, P. Balland, M. Demouche, “Modelling of the thermomechanical behavio FCC metals under various conditions”, Ann. Chim. Sci. Matér. 42 (2018), no. 1, p. 115-127. 6] F. Zerilli, “Dislocation mechanics-based constitutive equations”, Metall. Mater. Trans. 35 (2004), p. 2547-2555. “ d f h d ﬀ h h l b h [7] M. C. Cai, “A constitutive description of the strain rate and temperature eﬀects on the mechanical behaviour of materials”, Mech. Mater. 42 (2010), p. 774-781. [7] M. C. Cai, “A constitutive description of the strain rate and temperature eﬀects on the mechanical behaviour of materials”, Mech. Mater. 42 (2010), p. 774-781. [8] H. Fengbo, T. B. Tang, K. Hongchao, L. Jinshan, “Eﬀects of subgrain size and static recrystallization on the mechanical performance of polycrystalline material: a microstructure-based crystal plasticity ﬁnite element analysis”, Prog. Nat. Sci.: Mater. Int. 25 (2015), p. 58-65. [8] H. Fengbo, T. B. Tang, K. Hongchao, L. 4. Conclusion The proposed study is an extension of preliminary study which has already been published. The encouraging results obtained previously motivated us to develop the proposed model in order to extend it to other complex mechanical behaviour. Understanding response of the material to the combined eﬀect of temperature and strain rate is useful in designing failure resistant systems. Also, understanding the dependence of deformation mechanisms at microscopic scale on the macroscopic material behaviour is more relevant than empirical-based models. The proposed model shows, in a wide range of temperatures and strain rates, the eﬀect of the microstructure parameters on creep behaviour of Al-1050 alloy. This approach can be generalized to metals having FCC crystalline structure. The diﬀerent ﬁgures depict a good agreement between calculated results and experimental data. In addition, this study was carried out on the two types of creep: at constant load and at constant stress. Besides, to assess the reliability of the model, we conducted tests with and without load jump. The study also shows that the ﬂow stress is governed simultaneously by the strain hardening rate (θ) and the strain rate sensitivity (β). The proposed Ahmed Maati et al. 63 63 Ahmed Maati et al. 63 methodology has enabled to achieve an adequate description of the thermomechanical response of the material studied during creep tests. Computer programs were carried out to represent the diﬀerent results. methodology has enabled to achieve an adequate description of the thermomechanical response of the material studied during creep tests. Computer programs were carried out to represent the diﬀerent results. Finally, this numerical modelling can contribute to simulate several phenomena that result from hot forming, for example, stress relaxation, necking, and thinning behaviour), predict the creep-damage behaviour of metals, extend the study to other types of structure (cc for example) etc. C. R. Mécanique, 2021, 349, n1, 55-64 References Jinshan, “Eﬀects of subgrain size and static recrystallization on the mechanical performance of polycrystalline material: a microstructure-based crystal plasticity ﬁnite element analysis”, Prog. Nat. Sci.: Mater. Int. 25 (2015), p. 58-65. C. R. Mécanique, 2021, 349, n1, 55-64 64 Ahmed Maati et al. [9] E. H. Ouakdi, R. Louahdi, G. Ferron, “A physical model describing the viscoplastic behaviour of aluminum under tension and under creep for T > 0.4Tm”, J. Mater. Sci. Lett. 15 (1996), p. 1555-1557. 0] J. R. Klepaczko, “Proceedings of the 8th. RISO Int. Symp. on Metallurgy and Materials Science”, Riso, Denmark, [10] J. R. Klepaczko, Proceedings of the 8th. RISO Int. Symp. on Metallurgy and Materials Science , Riso, Denmark, 1987. [11] E. H. Ouakdi, R. Louahdi, “A constitutive model describing the necking behaviour of aluminum during tension and creep”, J. Mater. Sci. Lett. 17 (1998), p. 193-196. 1] E. H. Ouakdi, R. Louahdi, “A constitutive model describing the necking behaviour of aluminum during tensio creep”, J. Mater. Sci. Lett. 17 (1998), p. 193-196. [12] H. Luthy, A. K. Miller, O. D. Sherby, “The stress and temperarture dependance of steady state ﬂow at intermediate temperatures for pure polycristalline aluminum”, Acta Metall. 28 (1980), p. 169-178. [13] L. Donghuan, L. Haisheng, L. Yinghua, “Numerical simulation of creep damage and life prediction of superalloy turbine blade”, Math. Probl. Eng. 1 (2015), p. 1-10. [14] T. Furu, R. Orsund, E. Nes, “Subgrain growth in heavily deformed aluminium—experimental investigation and modelling treatment”, Acta Metall. Mater. 43 (1995), no. 6, p. 2209-2232. [15] J. Gil Sevillano, P. van Houtte, E. Aernoudt, “Large strain work hardening and textures”, Prog. Mater. Sci. 2 p. 69-134. [16] D. M. Dimiduk, “Microstructure-property-design relationships in the simulation era: an introduction”, in Computa- tional Methods for Microstructure-Property Relationships, Springer, Boston, MA, 2011. f p y p p g [17] E. H. Ouakdi, “Modélisation physique du comportement plastique de l’aluminium a moyenne température, appli- cation a l’étude de la striction”, 1988, Doctoral Thesis, Metz University. [17] E. H. Ouakdi, “Modélisation physique du comportement plastique de l’ cation a l’étude de la striction”, 1988, Doctoral Thesis, Metz University.
https://openalex.org/W4315483950	https://www.shs-conferences.org/articles/shsconf/pdf/2023/02/shsconf_ssphe2023_01013.pdf	English	null	Research on the impact mechanism of major public health emergency on China's economic growth	SHS web of conferences	2,023	cc-by	4,082	Research on the impact mechanism of major public health emergency on China's economic growth Fang Zheng 1,2* and Xiaowen Yu2 1School of Modern financial, Jiaxingnanhu University, Jiaxing 314001, China 2School of Economics, Zhejiang Gongshang University, Hangzhou 310016, China Fang Zheng 1,2* and Xiaowen Yu2 1School of Modern financial, Jiaxingnanhu University, Jiaxing 314001, China 2School of Economics, Zhejiang Gongshang University, Hangzhou 310016, China Abstract. Under different transmission mechanisms, major public health emergencies have different effects on economic growth. Based on the specific economic impact of COVID-19, a literature analysis method was used to systematically explain the economic impact of different channels and specific transmission mechanisms caused by major public health emergencies. Major public health emergencies caused serious damage to life and health, and had a huge impact on the entire economic environment. However, different economic sectors have different levels and channels of impact from the pandemic. The existing literature has analysed the various impact mechanisms of major public health emergencies on the economic development process from many angles, but there are still problems that need in-depth discussion and supplementary research. SHS Web of Conferences 153, 01013 (2023) SSPHE2022 SHS Web of Conferences 153, 01013 (2023) SSPHE2022 https://doi.org/10.1051/shsconf/202315301013 © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). * Corresponding author: zhengfang0576@126.com 2.1 Macroeconomic level In the short term, public emergencies have an impact on the development of an economy. The pandemic could reduce GDP by 1% and 4.25% under both moderate and severe scenarios according to the CBO（Congressional Budget Office）. The World Bank estimates that the outbreak of the pandemic will cause a loss of 0.8% to 12% of global GDP, and some countries or regions may even be as high as 50%. Since the outbreak of COVID-19, the impact of the black Swan incident on the economy and society has attracted extensive attention from domestic and foreign scholars, who have analysed the impact of public health emergencies on the macro economy from different perspectives and paths. Studies have shown that the COVID-19 pandemic has had varying degrees of impact on economic growth, and the government's response to the pandemic largely determines the severity. In the long run, most scholars show that public health emergencies will not destroy the foundation, structure and other factors on which economic development depends, and will not have too much negative impact on the economy [1]. However, there is also literature confirming that exogenous shocks may have long-term negative impacts on the development of a city. 2.2 Medium level Different industries and regions show significant differences when reflecting the impact of public emergencies on them [2]. Some industries with relatively concentrated personnel contact but not particularly important to residents' life will be directly impacted, but also provide development opportunities for some industries, such as the new digital industry. In addition, areas with higher levels of economic development and richer medical and health resources are less negatively affected. Sudden shocks have different effects on the capital- labor ratio of different sectors. If an emergency reduces the capital-labor ratio of a sector, the output growth rate of the sector will decrease [3]. After the outbreak of COVID-19, the stock trend of medical devices and medical supplies is a typical response to major public health emergencies. In addition, the internal characteristics of the economy will be affected by the external environment, which is mainly manifested in the different responses of the economy to the heterogeneous shock [4]. 1 Introduction Public health emergencies can be classified into grades I, II, III and IV according to their nature, degree of harm, and scope. Grade I is a particularly major public health emergency. Major emergencies refer to emergencies with a high degree of social harm and a large scope of influence. On January 20, 2020, China's National Health Commission (NHC) listed COVID-19 as a Class B statutory notifiable infectious disease and managed it as class A, making COVID-19 a typical major public health emergency. The occurrence of public health emergencies often has different degrees of negative impact on the economic activities of different subjects, making them deviate from the normal operation track, causing various short-term shocks and long-term impacts. Judging from the frequency of individual losses and major disasters, in the past 20 years, especially since 2003, public health emergencies have caused no less damage to cities than natural disasters such as earthquakes and floods, resulting in staggering economic losses and casualties. Due to the impact of COVID-19, the growth rate of GDP in 2020 was only 2.3%, and the number of new jobs and the growth rate of industrial added value both dropped by varying degrees. The occurrence of public health emergencies usually causes various short-term shocks and potential long-term impacts on the daily activities of micro-subjects and the macroeconomic operations of specific regions and SHS Web of Conferences 153, 01013 (2023) SSPHE2022 https://doi.org/10.1051/shsconf/202315301013 even the entire country. The research on sudden shock in existing literature mainly focuses on the level of micro consumer psychology, medium level and industry level. even the entire country. The research on sudden shock in existing literature mainly focuses on the level of micro consumer psychology, medium level and industry level. 3.1 Channel transmission mechanism Public health emergencies will disrupt the normal order of politics, economy, production and life, and have a certain impact on the consumption and psychology of enterprises and residents. The government will also invest a huge amount of human and material resources for post-disaster reconstruction, which will have a certain impact on government expenditure and government trust. Different from other natural disasters, public health emergencies do not directly damage physical property such as infrastructure, so the micro losses all come from the health maintenance and behavior change of people. At the macro level, macro- economic activities in a country or a certain region cannot be carried out normally due to the pandemic, resulting in losses to specific industries and the overall economy, which will eventually affect the overall economic system. y y Barro was the first economist to study health human capital at the macro level [6]. He constructed a three-sector economy including material capital, health human capital and education human capital, focusing on how health human capital can promote economic growth by affecting labor productivity [7]. Public health emergencies can have a certain degree of negative impact on the health of the workforce in the short term, but in the long run, they can help change public health habits and improve public health. With the widespread prevalence of unhealthy lifestyles and the rapid spread of young and middle-aged people, the prevalence of some infectious diseases is being affected both by the ageing of the population and by the younger onset of the disease, with implications for labor productivity. The relationship between healthy human capital and economic growth is complicated. Some scholars believe that healthy human capital can promote economic growth more effectively, while others believe that health investment may have both positive and negative effects on economic growth [8]. Socioeconomic conditions have a significant impact on residents' immediate response and post-disaster reconstruction, and those with lower incomes are more disadvantaged in comparison. Compared with the period of economic contraction, the pandemic will be more severely impacted during the economic expansion, that is, the period of high economic growth is also a relatively fragile period, and the impact will be more severe. Toya and Skidmore [9] took the annual disaster data of 151 countries from 1960 to 2003 as samples, and established regression models respectively with the data of death toll and economic loss caused by disasters. 2.3 Micro level Different families have different psychological expectations for the same public health incident, leading to different economic decisions. Because of the increased uncertainty, households are likely to respond to the crisis by severely restricting consumption levels, but there is also the possibility of desperate consumption, leading to higher consumption levels in the short term. In addition, the damage to social trust caused by public crisis incidents will destroy the public's trust in society and government, affect the operation of social order, and reduce the public's sense of security and happiness. For enterprises, major public health emergencies not only directly affect their daily production activities through the demand channel, but also have a significant impact on the supply of factors of production. Especially in the context of globalization, the COVID-19 situation is severe and complex, and the secondary crisis brought by it will further hinder the development of enterprises [5]. 2 SHS Web of Conferences 153, 01013 (2023) SSPHE2022 https://doi.org/10.1051/shsconf/202315301013 3 Discussion on transmission mechanism of impact of public health emergencies on economic growth The impact of public health emergencies on China's macro, medium and micro levels has a great negative economic impact, but the specific transmission mechanism of the impact on economic growth is not clear. Following the clue that public health emergencies affect economic growth and relying on the driving factors of economic growth, this paper constructs the transmission mechanism related to economic growth, refines the two paths of element transmission mechanism and channel transmission mechanism, and discusses the impact of public health emergencies on economic growth. 3.1 Channel transmission mechanism The results showed that the increase of income level could reduce the loss caused by natural disasters, and at the same time, the education level and the degree of external openness were relatively high. A better financial system and a smaller government also played a positive role in reducing disaster losses. Air transport and tourism are two important fields that are very sensitive to public emergencies, which will bring great impact both from the actual loss level and from the psychological level [10]. Emergencies will also affect the investment field, and different investment fields will be affected differently. For example, the performance of the real estate 3 SHS Web of Conferences 153, 01013 (2023) SSPHE2022 https://doi.org/10.1051/shsconf/202315301013 market deserves special attention [11]. Empirical results showed that the SARS outbreak caused a 1.6% drop in average prices for all properties. Property prices in cholera communities remain significantly different 160 years later. In addition, public health emergencies can also affect the import and export sector. With the deepening of global economy, economic cooperation among countries is becoming more and more complex. The outbreak of this health crisis increases the risk of "rupture" in the global industrial chain, which is undoubtedly frost on the snow for industries that rely on import and export. Governments at all levels will properly allocate pandemic prevention funds based on economic characteristics, turn the crisis into an opportunity, and realize strategic transformation of enterprises and industrial optimization and upgrading in the process of resuming work and production, thus promoting stable economic development and minimizing disaster losses [12]. Adequate financial resources, efficient organizations, advanced technology, and coordinated action are all crucial for governments to reduce the impact of public health emergencies. The ability to rebuild after disasters depends as much on technical and organizational constraints as on government finances [13]. Public emergencies will lead to the increase of local social demand and the impact of government expenditure. Major public emergencies will not only suddenly magnify the demand of industries related to the resumption of work and production, but also lead to an increase in government expenditure [14]. The damage to social trust caused by public health emergencies will destroy the public's trust in the society and the government, affect the orderly operation of the society, and reduce the public's happiness and security. 3.2 Factor transmission mechanism The mainstream framework of economic growth was focused on from capital accumulation, human capital and exogenous technological progress, to endogenous technological progress and policies that affect technological progress, and to spatial factors that affect long-term economic growth. The impact of major public health emergencies on economic growth is also transmitted through factors of production [16]. Due to the significant improvement of globalization and urbanization rate, the popularization of global transportation network and social network has greatly promoted the large-scale and rapid flow of personnel and factors within cities, between cities, between regions and across countries. This paper will sort out the transmission mechanism of public health emergencies affecting economic growth for different factors of production. Capital factor is the first factor that affects economic growth. Rapid economic development depends on the rapid growth of the capital stock [17]. Public health shocks affect physical capital accumulation by crowding out other fiscal expenditures or affecting investment, and then impact economic growth [18]. In addition, as uncertainty increases, investors such as individuals and businesses will reduce their investment activities when they do not receive sufficient market signals. But in the long term, the impact of health emergency shocked on capital investment is uncertain. The impact of major public health emergencies just reflects the shortcomings of local governments in social governance, health facilities and emergency response capacity. Therefore, the impact of public health emergencies may lead to a rapid increase in capital accumulation in some areas, shaping the external environment for economic development and leading economic development. Human capital factor is the second factor that affects economic growth. From the perspective of labor supply, the impact of public health emergencies on human capital is reflected in two aspects: first, the sudden impact may affect the number of workers, the carrier of human capital. When the pandemic breaks out, a large number of people will be quarantined or treated. Even healthy workers who have not been infected will be forced to travel less because of the pandemic, further reducing the supply of workers. Second, sudden shocks may have an impact on education. According to UNESCO (United Nations Educational, Scientific, and Cultural Organization), as of 17 March 2020, 850 million children and adolescents around the world have been suspended from school because of COVID-19, about half of the global total, and this number is expected to continue to grow [19]. 3.1 Channel transmission mechanism Public health emergencies have a significant impact on the overall social structure and promote social reform and improvement of security system. For example, due to institutional changes during SARS, the speed of economic, political, medical and social security system reform was improved, laying a stable institutional foundation for China's long-term economic development. Public health emergencies will be accompanied by the conflict between public interests and private rights, which reflects the existence of institutional problems. The COVID - 19 outbreak hit in the highlight of our country's institutional advantages at the same time, also exposed in public health pandemic prevention and control in our country, although our country has preliminarily established the emergent public health incident emergency management system, but the reality of different institutions perform effect is poor, such as rural schools of emergent public health emergency system can't effectively play a role. The fundamental reason lies in the existence of some institutional regulatory barriers. g y Trust is an interactive social relation, which exists in many aspects such as economic relation, political relation and social relation, and has great capital value. When the pandemic occurs, the public will be dissatisfied with individual local governments. Especially when the pandemic begins to spread, the local governments' response is not timely enough, the information disclosure is not accurate enough, and the spread of various false information is constantly consuming the trust capital among economic entities. The weakening of social trust hinders the circulation of social resources, which leads to the failure of the social system to play its due social function and is not conducive to the establishment of government credibility. The lower the level of trust, the greater the damage to the economy and society. The government's discursive response has a significant positive impact on the public cognitive bias, thus aggravating the degree of public cognitive bias. However, the government's action response has a significant negative impact on public cognitive bias, which can significantly reduce the degree of public cognitive bias. The practice shows that the substantial punishment to the trusted person can often achieve more positive trust repair effect [15]. There is a positive correlation between government self-inspection and regulation and social trust after public health emergencies. 4 4 4 https://doi.org/10.1051/shsconf/202315301013 SHS Web of Conferences 153, 01013 (2023) SSPHE2022 4 The regulatory role and means of government emergency response In the face of any crisis, government has two equally important responsibilities: Solve the problem at hand and prevent it from happening again. The government plays an extremely important role in dealing with public emergencies. The current global spread of COVID-19 is a public crisis that needs to be addressed urgently. With the rapid development of economy and society, various uncertain factors have soared, and economic and social levels have become complex and diverse. Especially in recent years, public security incidents have occurred continuously, such as social security accidents. In the Internet era, the speed of information dissemination is accelerated, causing a certain degree of influence on social emotions. With the devolution, the governance of the local government plays a leading role in dealing with public crisis, required the government to improve the ability of disaster warning beforehand, rapid response ability and disaster recovery ability, to the fastest speed to eliminate the crisis, ensure the safety of residents' lives and property to the maximum extent and economic and social activities to the normal track. After the outbreak of COVID-19, more and more scholars began to realize the decisive role of government as a main body in resolving major urban crises. On the one hand, the improvement of government governance capacity can effectively deal with the direct impact of sudden public health crisis, significant intervention on public health can effectively control the spread of COVID-19. Strict lockdown, social isolation, faster detection speed, government stimulus policies and faster intervention and implementation can effectively suppress the spread of COVID-19 and to counter its negative impact on the economy and society. The speed and intensity of the government's response to the pandemic determines the negative impact of public health emergencies on the economy and society. On the other hand, there could be indirect beneficial economic effects through the channel of reducing the number of confirmed cases. Ashraf used daily data of 77 countries from January 22, 2020 to April 17, 2020 to analyse the impact of government intervention on the stock market to test the expected economic impact of government actions, and found that social policies announced by the government (such as home quarantine, closing of public places, etc.) had a direct negative impact on the stock market. Preferential policies have brought positive market reactions to a large extent. Feng also verified that effective government intervention reduced the fluctuation of exchange rate market. 3.2 Factor transmission mechanism In addition, public health emergencies restrict the daily operation of enterprises, and enterprises tend to hire temporary workers to replace regular employees to reduce production costs, which will lead to a large number of unemployed labor force and increase social instability factors [20]. Technological factor is the third factor that affects economic growth. Sudden shocks may promote or hinder technological progress or change the external environment of technological innovation. For developing countries, sudden shocks have a negative impact on technology spillover in foreign trade. In the face of the impact of emergencies, the importance of technology is increasingly obvious. The tech sector has been less affected than labor-intensive industries, and the pandemic has even created opportunities for early-stage growth tracks, especially for technology-based industries such as online education, telecommuting and internet health care, which are in the early stages of business development. In the long term, the pandemic will inevitably force industrial transformation and upgrading to optimize China's industrial structure and promote long-term economic development. Space factor is the fourth factor that affects economic growth. Public health emergency impacts not only directly affect industrial and regional development, but also further affect economic growth through spatial spillover, it is mainly reflected in the restricted flow of personnel, reduced timeliness of logistics, and sharp decline in demand. To be specific, different local governments have adopted different levels of emergency and pandemic 5 SHS Web of Conferences 153, 01013 (2023) SSPHE2022 https://doi.org/10.1051/shsconf/202315301013 prevention measures, which has increased the cost of inter-regional movement and greatly reduced logistics and transportation capacity. The flow of products and factors is crucial for the survival of enterprises. However, in the face of public health emergencies, the policies of provinces and countries are different, and cross-border transportation has a great impact on enterprises, which also brings great pressure for survival. When a region suffers sudden shocks, it will further cause changes in the scale of consumption and investment in related regions, leading to further economic decline. No matter the spread of the pandemic, government prevention and control policies or economic and social risks, there are strong spatial spillover effects at multiple scales [21]. 5 Conclusions In this paper, the transmission mechanism of major public health emergencies on economic growth is sorted out, and the research is carried out from channel and factor perspective. There are still problems that need further discussion and supplementary research in the future: First, the existing research about the impact of public health emergency on economic growth focus mostly around the whole macro economy, and the research of micro family and industry medium subject related study is less. Therefore, micro theories can be introduced to further analyse the economic consequences of public health emergencies. Second, from the perspective of the impact mechanism of public health emergency on economic growth, in the past were independent incidents, making it difficult to conduct comparative analysis and research on policy intervention. In the context of rapid population movement and pandemic spread across the world, there is room for comparison and quantitative policy exploration in the emergency management capacity and policies of different local governments. Therefore, it is particularly important and urgent to study the transmission mechanism of public health emergencies from the dimension of dynamic tracking. Third, the government's emergency response measures play a leading role in dealing with the pandemic, which can reduce and eliminate the crisis in the fastest speed. Disaster warning ability, rapid response ability and post-disaster recovery ability are closely related to the efficiency of inter-departmental linkage and coordination, and ultimately restore the economic and social operation to the normal track. 4 The regulatory role and means of government emergency response Up to now, the whole country is in the stage of normal pandemic prevention and control. The situation abroad is not optimistic, and there are still small-scale outbreaks in China. Most virologists in the world believe that the virus will not disappear in a short period of time. In addition, to maximize the integration of all levels of government resources and strength, in the limited and the shortest possible time to effectively cope with public health emergencies, and establish a good cooperation mechanism, emergency management as a whole the related materials reserves, do zone linkage, maximum limit to control the outbreak scale minimum range, reduce the negative impact on the economic and social disease. 6 6 https://doi.org/10.1051/shsconf/202315301013 SHS Web of Conferences 153, 01013 (2023) SSPHE2022 15. R.E. Hall, C.I. Jones. QJE, 114, 83-116 (1999) 15. R.E. Hall, C.I. Jones. QJE, 114, 83-116 (1999) 16. A.B. Krueger, M. Lindahl. JEL, 39, 1101-1136 (2001) References 1. A. Ambrus, E. Field, R. Gonzalez. AER, 110, 475-525 (2020) 2. A. P. Loayza, B.A. Loiselle. J Mammal, 90, 732-742 (2009) 3. L. Brown, T. Greenbaum. Urban Stud., 54, 1347-1366 (2017) 4. Z. Eckstein, D. Tsiddon. J. Monetary Econ., 51, 971-1002 (2004) 4. Z. Eckstein, D. Tsiddon. J. Monetary Econ., 51, 971-1002 (2004 5. N.Dennis. Science (2020) 5. N.Dennis. Science (2020) 6. R.J. Barro. J. Econ. Growth, 1, 1-27 (1996) 7. A. Van Zon, J. Muysken. J Health Econ., 20, 169-185 (2001) 8. M. Masozera, M. Bailey, C. Kerchner. Ecol. Econ.,63, 299-306 (2007) 9. H. Toya, M. Skidmore. Econ Lett, 94, 0-25 (2007) 10. W. Enders, A. Sachsida, T. Sandler. PRQ, 59, 517-531 (2006) 11. W. Grace. JUE, 63, 74-95 (2008) 12. S. Hallegatte, H. Jean-Charles, P. Dumas. Ecol. Econ., 62, 330-340 (2007) 13. N. Harp, M. McKenna, N. Shute, et al. JL Med.&Ethics, 33, 64 (2005) 14. P. Slovic, E. Peters. Curr. Dir. Psychol. Sci., 15, 322-325 (2006) 16. A.B. Krueger, M. Lindahl. JEL, 39, 1101-1136 (2001) 17. R. Bachmann, C. Bayer. J Monetary Eco., 60, 704-719 (2013) 18. D. Almond. JPE, 114, 672-712 (2006) 19. R. Valletta, L. Bengali. [R]. FRBSF Economic Letter, 24 (2013) 20. J.C. Cuaresma. WBER, 24, 280-302 (2010) 21. B. Gates. NEJM, 382, 1677-1679 (2020) 7
https://openalex.org/W3095744674	https://www.dora.lib4ri.ch/psi/islandora/object/psi%3A36359/datastream/PDF/K%C3%BCssau-2020-Functional_characterization_of_the_N-acetylmuramyl-L-alanine-%28published_version%29.pdf	English	null	Functional Characterization of the N-Acetylmuramyl-l-Alanine Amidase, Ami1, from Mycobacterium abscessus	Cells	2,020	cc-by	17,486	Functional Characterization of the N-Acetylmuramyl-l-Alanine Amidase, Ami1, from Mycobacterium abscessus Tanja Küssau 1, Niël Van Wyk 1,† , Matt D. Johansen 1,‡ , Husam M. A. B. Alsarraf 1,2 , Aymeric Neyret 3, Claire Hamela 1, Kasper K. Sørensen 4, Mikkel B. Thygesen 4 , Claire Beauvineau 5, Laurent Kremer 1,6,* and Mickaël Blaise 1,* 1 Institut de Recherche en Infectiologie de Montpellier (IRIM), Université de Montpellier, CNRS UMR 9004, CEDEX 5, 34293 Montpellier, France; tanja.kuessau@gmail.com (T.K.); nielvw@gmail.com (N.V.W.); Matt.Johansen@uts.edu.au (M.D.J.); husam.alsarraf@irim.cnrs.fr (H.M.A.B.A.); claire.hamela@irim.cnrs.fr (C.H.) 2 Department of Molecular Biology and Genetics, Aarhus University, 8000 Aarhus C, Denmark 3 CEMIPAI CNRS UM UMS3725 CEDEX 5 34293 M ll F @ f p gy , y, , 3 CEMIPAI CNRS UM UMS3725, CEDEX 5, 34293 Montpellier, France; aymeric.neyret@irim.cnrs.fr 4 Department of Chemistry, Faculty of Science, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark; kso@chem.ku.dk (K.K.S.); mbth@chem.ku.dk (M.B.T.) 5 Chemical Library Institut Curie/CNRS, CNRS UMR9187, INSERM U1196 and CNRS UMR3666, INSERM U1193, Université Paris-Saclay, F-91405 Orsay, France; Claire.Beauvineau@curie.fr 6 INSERM, IRIM, 34293 Montpellier, France CEMIPAI CNRS UM UMS3725, CEDEX 5, 34293 Montpellier, France; aymeric.neyret@irim.cnrs.fr 4 Department of Chemistry, Faculty of Science, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark; kso@chem.ku.dk (K.K.S.); mbth@chem.ku.dk (M.B.T.) 5 Chemical Library Institut Curie/CNRS, CNRS UMR9187, INSERM U1196 and CNRS UMR3666, INSERM U1193, Université Paris-Saclay, F-91405 Orsay, France; Claire.Beauvineau@curie.fr CEMIPAI CNRS UM UMS3725, CEDEX 5, 34293 Montpellier, France; aymeric.neyret@irim.cnrs.fr 4 Department of Chemistry, Faculty of Science, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark; kso@chem.ku.dk (K.K.S.); mbth@chem.ku.dk (M.B.T.) g 5 Chemical Library Institut Curie/CNRS, CNRS UMR9187, INSERM U1196 and CNRS UMR3666, INSERM U1193, Université Paris-Saclay, F-91405 Orsay, France; Claire.Beauvineau@curie.fr 6 INSERM, IRIM, 34293 Montpellier, France p * Correspondence: laurent.kremer@irim.cnrs.fr (L.K.); mickael.blaise@irim.cnrs.fr (M.B.); Tel.: +33-(0)-434-359-447 (L.K. & M.B.) † Present address: Institut für Mikrobiologie und Biochemie, Hochschule Geisenheim University, 65366 Geisenheim, Germany. ‡ Present address: Centre for Inﬂammation, Faculty of Science, Centenary Institute and University of Technology Sydney Sydney NSW 2006 Australia ‡ Present address: Centre for Inﬂammation, Faculty of Science, Centenary Institute and University of Technology Sydney, Sydney, NSW 2006, Australia. ‡ , y , y y Technology Sydney, Sydney, NSW 2006, Australia. Technology Sydney, Sydney, NSW 2006, Australia. cells cells 1. Introduction Peptidoglycan (PG), composed of glycan chains cross-linked by short peptides, surrounds most bacteria and protects them against environmental and osmotic stress. PG consists of polymers of the N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) disaccharide units. The three-dimensional PG network is formed by the cross-linking of the peptide stems attached to the MurNAc moiety [1]. Although contributing to the rigidity of the cell envelope, PG remains a highly-dynamic structure participating in important physiological processes, such as bacterial elongation and separation during cell division [2]. During these dynamic phases, PG is remodeled and the balance between PG synthesis and hydrolysis is tightly regulated [3]. PG remodeling is performed by numerous autolysins acting on speciﬁc substrates and at diﬀerent stages during bacterial growth. While glycosidases, such as muraminidases and glucosaminidases act to hydrolyze the glycosidic bonds between the carbohydrate units, peptidases, such as N-acetylmuramyl-l-alanine amidases, l,d-endopeptidases, d,l-endopeptidases, l,d-carboxypeptidases, and dd-carboxypeptidases, hydrolyze the amide bonds of the peptide stem at speciﬁc positions [4]. In mycobacteria, PG diﬀers from Gram-positive or Gram-negative bacteria as it is covalently linked to the atypical mycobacterial envelope [5]. d,l-endopeptidases belonging to the papain-like peptidase superfamily, possessing an NlpC/P60 catalytic domain [6], have been widely studied in mycobacteria. Among them, RipA has been shown to interact with the resuscitation-promoting factor B (RpfB), a lytic transglycosylase, and the bifunctional PG-synthesizing enzyme, penicillin-binding protein 1 (PBP1) [7]. Rpf-interacting protein A (RipA) activation also requires the Mycobacterium acid resistance protease (MarP) during acid stress [8]. These studies emphasize the coordination of the PG hydrolytic enzymes, which are tightly regulated during the mycobacterial life cycle. Until recently the role of the N-acetylmuramyl-l-alanine amidases (amidases) has been poorly documented in mycobacteria. The genomes of Mycobacterium smegmatis and Mycobacterium tuberculosis encode four potential amidases. CwlM (Rv3915), also known as Ami2, is annotated as a PG amidase but does not appear to be catalytically active [9]. However, it plays an important role in modulating the activity of the UDP-N-acetylglucosamine 1-carboxyvinyltransferase MurA, a key player of PG biosynthesis [9]. Rv3811/MSMEG_6406, designated Ami3, is not essential for mycobacterial growth, but overexpression of Ami3, due to its inherent PG hydrolytic activity, is lethal for mycobacteria [10]. Ami3 activity is therefore tightly modulated by proteolysis mediated by the HtrA protease [10]. Received: 6 October 2020; Accepted: 29 October 2020; Published: 4 November 2020 Abstract: Peptidoglycan (PG) is made of a polymer of disaccharides organized as a three-dimensional mesh-like network connected together by peptidic cross-links. PG is a dynamic structure that is essential for resistance to environmental stressors. Remodeling of PG occurs throughout the bacterial life cycle, particularly during bacterial division and separation into daughter cells. Numerous autolysins with various substrate speciﬁcities participate in PG remodeling. Expression of these enzymes must be tightly regulated, as an excess of hydrolytic activity can be detrimental for the bacteria. In non-tuberculous mycobacteria such as Mycobacterium abscessus, the function of PG-modifying enzymes has been poorly investigated. In this study, we characterized the function of the PG amidase, Ami1 from M. abscessus. An ami1 deletion mutant was generated and the phenotypes of the mutant were evaluated with respect to susceptibility to antibiotics and virulence in human macrophages and zebraﬁsh. The capacity of puriﬁed Ami1 to hydrolyze muramyl-dipeptide was demonstrated in vitro. In addition, the screening of a 9200 compounds library led to the selection of three compounds inhibiting Ami1 in vitro. We also report the structural characterization of Ami1 which, combined with in silico docking studies, allows us to propose a mode of action for these inhibitors. Keywords: N-acetylmuramyl-l-alanine amidase; peptidoglycan; drug screening; x-ray crystallography; Mycobacterium abscessus Cells 2020, 9, 2410; doi:10.3390/cells9112410 www.mdpi.com/journal/cells Cells 2020, 9, 2410 2 of 25 1. Introduction MSMEG_6281 (Ami1Msm), containing an amidase_3 domain, is dispensable for bacterial growth but ami1 deletion mutants present an atypical lateral branching and a defect in cell separation and septal PG turnover [11]. This mutant is also more susceptible to antibiotics due to increased cell wall permeability; a phenotype subsequently conﬁrmed in an independent study which also reported the attenuation of the virulence the ami1 mutant in a mouse model of infection [12]. While cell division was impaired in the ami1Msm knockout strain, this phenotype could be rescued by complementation with the orthologue gene, Rv3717, from M. tuberculosis (ami1Mtb), suggesting a similar mode of action in both species [13]. Furthermore, ami1Msm has also been shown to be important for bioﬁlm formation [14]. In addition, mycobacterial Ami1 has been biochemically and structurally characterized. Puriﬁed Ami1Mtb hydrolyzes PG fragments in vitro in a zinc-dependent manner and its crystal structure revealed a typical amidase_3 signature [15,16]. However, the functional role of Ami1 in mycobacteria appears more complex than originally thought, for several reasons: (1) a single ami1 deletion mutant or multiple deletions of ami1 and ami3 or ami4 do not display any growth defect in M. smegmatis [10], suggesting that amidases have redundant functions as reported in other bacteria, such as in Escherichia coli [17–19] and (2) Ami1 shares overlapping functions with the endopeptidase RipA, as Ami1 rescued RipA’s activity during cell division but not for persistence and chronic infection in a mice infection model [20]. The PG remodeling machinery of M. abscessus, a rapidly growing non-tuberculous mycobacterium, has been poorly explored. The M. abscessus complex [21] comprises three diﬀerent subspecies—M. abscessus sensu stricto (hereafter designated M. abscessus), M. abscessus subsp. bolletii, 3 of 25 Cells 2020, 9, 2410 and M. abscessus subsp. massiliense. Infections caused by this complex are increasing globally and are notoriously diﬃcult to treat, particularly due to the extreme resistance of M. abscessus to most antibiotic classes [21]. The two M. abscessus morphotypes, (R) rough and (S) smooth, display similar antibiotic susceptibility proﬁles but, largely diﬀer in their virulence proﬁle as emphasized in the embryonic zebraﬁsh model [22]. The S variant is considered as the environmental form that colonizes the airways and that can transition to the more virulent R form [21]. 2.1. Generation of Unmarked Deletion Mutant Deletion of MAB_0318c (ami1Mab) in the wild-type strain M. abscessus CIP104536T was carried out using the double homologous recombination unmarked strategy, reported previously [26]. The DNA sequences located upstream, left arm (LA), and downstream, right arm (RA), of the ami1 gene were PCR-ampliﬁed and subsequently cloned after an overlapping PCR reaction into the pUX1-katG using the following primers, Forward: Mab0318_pUX1katG_LA_F and Reverse: Mab0318_pUX1katG_LA_R and Forward: Mab0318_pUX1katG_RA_F and Reverse: Mab0318_pUX1katG_RA_R (Supplementary Table S1). p pp y The resulting plasmid was electroporated in the smooth (S) and rough (R) variants of M. abscessus and the red ﬂuorescent, kanamycin (KAN)-resistant colonies which underwent the ﬁrst homologous recombination event were selected on Middlebrook 7H10 (Sigma-Aldrich, Saint-Quentin Fallavier, France) agar supplemented with 10% oleic acid-albumin-dextrose-catalase (OADC) in the presence of 200 µg/mL KAN. Cultures of selected colonies were then subjected to a counter-selection on 7H10 agar plates supplemented with OADC containing 50 µg/mL isoniazid (INH) to promote a second homologous recombination and removal of the KAN cassette. Non-ﬂuorescent, INH-resistant colonies were picked up and further genotyped for ami1Mab deletion following PCR using the Mab0318_pUX1katG_LA_F and Mab0318_pUX1katG_RA_R primers (Supplementary Table S1). Proper deletion was further conﬁrmed by DNA sequencing of the genomic region surrounding the site of gene deletion. For functional complementation of the ami1Mab deletion mutant, the genomic sequence of ami1Mab was fused at its 3′end in frame with the sequence encoding the Streptag-II, using the following primers, Forward: Mab0318_pMV306hsp_EcoRI_F and Reverse: Mab0318_SalI_C-termStrepTag (Supplementary Table S1) and subsequently cloned into the pMV306 plasmid possessing a KAN-resistance cassette and resulting in the creation of pMV306::ami1Mab. The ami1Mab deletion mutants were transformed with pMV306::ami1Mab and selected onto 7H10 agar supplemented with OADC and KAN (200µg/mL). 1. Introduction The presence or absence of surface-exposed glycopeptidolipids (GPL) in both the S and R morphotypes, respectively, has been shown to correlate with virulence in several animal models as well as in humans [23–25]. Herein, we evaluated the impact of ami1Mab deletion with respect to growth and bacterial division in both S and R morphotypes of M. abscessus and assayed their capacity to infect human macrophages and zebraﬁsh larvae. We characterized the hydrolytic properties of puriﬁed Ami1Mab and determined its crystal structure at high resolution. We also report a thermal-shift assay screening approach that led to the discovery of three Ami1Mab inhibitors. 2.4. Single-Cell Preparation for M. abscessus The various strains expressing tdTomato were grown in 50 mL 7H9 supplemented with 10% OADC, 0.025% tyloxapol and appropriate antibiotics (750 µg/mL hygromycin and 200 µg/mL KAN for complemented strains) for 3 days at 37 ◦C under agitation. After bacteria were centrifuged for 15 min at 3000× g, the pellet was resuspended in 1 mL of 7H9 and transferred into 1.5 mL Eppendorf tubes. To dissociate bacterial aggregates and generate single-cell bacteria, the samples were syringed through a 26G needle 20 times followed by a 15 s sonication step, resuspended with 7H9 media and passed through a 5 µm ﬁlter. Bacterial suspensions were centrifuged for 15 min at 3000× g and the pellet resuspended in 1 mL of 7H. The OD was measured and 10 µL aliquots were prepared in 1.5 mL Eppendorf tubes and stored at −80 ◦C. 2.3. Mycobacterial Culture Conditions The MIC were determined according to the Clinical & Laboratory Standards Institute (CLSI) guidelines [27]. M. abscessus strains were grown in Middlebrook 7H9 broth (BD Difco) supplemented with 10% OADC and 0.025% tyloxapol (Sigma-Aldrich, Saint-Quentin Fallavier, France) at 37 ◦C under agitation (unless otherwise stated) in the presence of antibiotics, when required (750 µg/mL hygromycin for all tdTomato-expressing strains, and 200 µg/mL kanamycin for complemented strains). On plates, strains were grown on Middlebrook 7H10 agar (BD Difco) supplemented with 10% OADC enrichment in the presence of antibiotics when required and grown at 37 ◦C. 2.2. Cloning for Recombinant Protein Expression The genomic sequence encoding ami1Mab depleted from its secretion peptide signal was PCR-ampliﬁed from M. abscessus genomic DNA with the following primers, Forward: Mab0318_pET151_F and Reverse: Mab0318_pET151_R (Supplementary Table S1) and cloned in a ligation-free independent manner into the directional Champion™pET151 TOPO™ (Thermo Fisher Scientiﬁc, Villebon-sur-Yvette, France) using manufacturer’s instructions. The genomic sequence of ami1Msm without its peptide signal of secretion was PCR-ampliﬁed from M. smegmatis mc2155 genomic DNA with the Forward and Reverse primers, Smeg6281_pET30_F 4 of 25 Cells 2020, 9, 2410 and Smeg6281_pET30_R (Supplementary Table S1) and was cloned into the pET-30 ek/LIC plasmid between the KpnI and EcoRI restriction sites in frame with the His- and S-tags followed by a Tobacco etch virus (TEV) protease cleavage site. The genomic sequence of ami1Mtb without its peptide signal of secretion was PCR-ampliﬁed from M. tuberculosis genomic DNA with the Forward Mtb_3717_F and Reverse Mtb_3717_R primers (Supplementary Table S1) and was cloned in a ligation-free independent manner into the pET-30 ek/LIC plasmid in frame with the His- and S-tags followed by a Tobacco etch virus (TEV) protease cleavage site. 2.5. Protein Expression and Puriﬁcation The E. coli BL21(DE3) strain resistant to phage T1 (New England Biolabs, Evry, France) containing the pRARE2 plasmid was transformed with the pET-151::ami1Mab, pET-30::ami1Msm, or pET-30::ami1Mtb constructs. Bacterial cultures were performed in Luria-Bertani broth (LB) medium containing 200 µg/mL ampicillin or 50 µg/mL KAN and 30 µg/mL chloramphenicol at 37 ◦C. When an OD at 600 nm of 0.6 to 1 was reached, the cultures underwent a 30 min cold shock in icy water before adding 1 mM isopropyl-β-d-1-thiogalactopyranoside (IPTG). Cultures were then grown for 20 h at 16 ◦C and then spun-down (8400× g, 4 ◦C, 20 min). Bacterial pellets were resuspended in lysis buﬀer (50 mM Tris-HCl pH 8, 400 mM NaCl, 20 mM imidazole, 1 mM benzamidine, and 5 mM β-mercaptoethanol) and the cells were disrupted by sonication prior to clariﬁcation by an additional centrifugation step (27,000× g, 4 ◦C, 45 min). Proteins were then puriﬁed by a ﬁrst ion metal aﬃnity chromatography (IMAC) step (Ni-nitrilotriacetic acid sepharose, Cytiva, Europe, GmbH). To remove the N-terminal tags, the eluted proteins were mixed in a 1:50 (w:w) ratio with TEV protease and dialyzed overnight at 4 ◦C against 50 mM Tris-HCl pH 8, 200 mM NaCl, and 5 mM β-mercaptoethanol. The dialyzed proteins were loaded onto a second IMAC, the non-tagged proteins were eluted in the ﬂow-through, while tags, uncleaved protein, and TEV protease bound the column. Proteins were concentrated using a 10 kDa cut-oﬀcentricon (Sartorius) and subjected to a ﬁnal step of puriﬁcation on size-exclusion chromatography, Supderdex 75 10/300 GL using an elution buﬀer containing 50 mM Tris-HCl pH 8, 200 mM NaCl, and 5 mM β-mercaptoethanol for Ami1Mab and Ami1Mtb or the same buﬀer but at pH 7.4 for Ami1Msm. The proteins prepared in these conditions were for crystallization purpose. Ami1Mab and Ami1Mtb used for the thin-layer chromatography (TLC)-based activity assay were 5 of 25 Cells 2020, 9, 2410 puriﬁed under similar conditions, except the last step of puriﬁcation on SEC was performed in 1 x Phosphate-Buﬀered-Saline (PBS) buﬀer. 2.8. Drug Susceptibility Testing and MIC Determination Minimum inhibitory concentrations (MICs) were determined in cation-adjusted Mueller–Hinton broth (Sigma-Aldrich, Saint Quentin Fallavier, France) using the microdilution method. Brieﬂy, 2 µL of the drug was added to 198 µL bacterial suspension and 2 times serial dilutions were performed with a ﬁnal volume of 100 µL per well (5 × 106 colony forming unit CFU/mL). The MIC was deﬁned as the minimal drug concentration required to inhibit growth and was recorded by visual inspection after 4 days of incubation at 30 ◦C. All experiments were completed in technical replicates and performed in three independent replicates. 2.6. Crystallization, Data Collection, and Structure Reﬁnement Crystals were grown in sitting drops in MR Crystallization Plates™(Hampton Research) at 18 ◦C by mixing 2 µL of protein solution with 2 µL of reservoir solution. Ami1Mab crystals were obtained at a protein concentration of 10 mg/mL and crystallized in 0.1 M Bis-Tris pH 5.5, 1.6 M ammonium sulfate, and 1% (w/v) polyethylene glycol (PEG). Ami1Mab plus dipeptide crystals were obtained in similar conditions and in presence of 5 mM of the ligand. Crystals were grown for several months and cryo-protected by a brief soaking step into a solution made of 0.1 M Bis-Tris pH 5.5, 1.6 M ammonium sulfate, 1% (w/v) PEG 3350, and 10–15% glycerol prior being cryo-cooled in liquid nitrogen. Ami1Msm crystals were obtained at a concentration of 13 mg/mL in the presence of 5 mM muramyl-dipeptide (MDP) and in a reservoir solution made of 0.1 M Bis-Tris pH 5.5, and 17% (w/v) PEG. Crystals were cryo-protected by a brief soaking step into a solution made of 0.1 M Bis-Tris pH 5.5, 30% (w/v) PEG 3350, and 10% PEG 400 prior being cryo-cooled in liquid nitrogen. X-ray diﬀraction data were collected at the Swiss-light source on the X06DA-PXIII beamline and at the European Synchrotron Radiation Facility on the ID30B beamline. The Ami1Mab structure was solved by molecular replacement using the crystal structure of the Ami1Mtb structure (PDB: 4LQ6) as a search request. The Ami1Mab dipeptide and Ami1Msm structures were solved similarly but using the Ami1Mab structure as a search model. Autobuild from the Phenix (version 1.18.2) software suite [28] was used to automatically improve the models which were then manually rebuilt and reﬁned using the Coot (version 0.89) [29] and Phenix software suites. The coordinates and structures factors have been deposited at the protein data bank under accession numbers 7AGL for Ami1Mab, 7AGO for Ami1Mab plus dipeptide, and 7AGM for Ami1Msm. 2.7. In Silico Docking The three-dimensional structure of Ami1Mab bound to dipeptide was depleted from its ligand and was used for in silico docking. The docking search was performed with the PyRx software (version 0.9.4) [30] running AutoDock Vina [31]. All the amino acid side chains were kept as rigid. Docking was carried out using the following grid parameters: center X = 0.59, Y = 16.38, and Z = −8.5 and dimensions (Å) X = 40, Y = 44, and Z =28 and an exhaustiveness of 8. 2.8. Drug Susceptibility Testing and MIC Determination 2.9. Macrophage Experiments THP-1 macrophages were grown in Roswell Park Memorial Institute (RPMI) medium (BD Gibco) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich, Saint Quentin Fallavier, France) (RPMIFBS) and incubated at 37 ◦C with 5% CO2. THP-1 monocytes were diﬀerentiated into macrophages in the presence of 20 ng/mL phorbol myristate acetate (PMA) in 24-well ﬂat-bottom tissue culture microplates (105 cells/mL) and incubated for 48 h at 37 ◦C with 5% CO2. Macrophages were infected with M. abscessus strains at a multiplicity of infection (MOI) of 2:1 (bacteria:macrophage) in 500 µL of RPMIFBS for 2–4 h at 37 ◦C with 5% CO2. Following infection, the bacterial inoculum was removed and cells were carefully washed three times with PBS and then incubated with fresh RPMIFBS supplemented 6 of 25 Cells 2020, 9, 2410 with 250 µg/mL amikacin for 2 h to kill extracellular bacteria. Macrophages were then washed three times with PBS prior to the addition of 500 µL RPMIFBS supplemented with 50 µg/mL amikacin to prevent extracellular growth. At various time points, macrophages were washed three times with PBS and lysed with 100 µL of 1% Triton X-100. Serial dilutions were plated on LB agar to count the colony-forming units. 2.10. Synthesis of Peptidoglycan Dipeptide, l-Alanyl-d-Isoglutamine (DP) The dipeptide was prepared by Fmoc solid-phase peptide synthesis and was performed on a Biotage® Initiator+ AlstraTM microwave-assisted, automated peptide synthesizer from Biotage AB, Uppsala, Sweden. Amino acids, reagents, and solvents were purchased from Iris Biotech GmbH, Marktredwitz, Germany. Peptide couplings were carried out using 5 equivalents of amino acid, 5 equivalents of 1-hydroxybenzotriazole (HOBt), 4.75 equivalents of N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethanaminium hexaﬂuorophosphate N-oxide (HBTU), and 9.75 equivalents of N,N-diisopropylethylamine (DIEA) in N-methylpyrrolidone (NMP). A coupling time of 10 min at 75 ◦C, and subsequent deprotection with piperidine in N,N-dimethylformamide (DMF) (2:3) for 3 min, followed by piperidine-DMF (1:4) for 10 min was used. After each coupling and deprotection step, a washing procedure with 2x NMP, 1x dichloromethane, then 2 x NMP was used. The synthesis was carried out on a 0.1 mmol scale on 0.25 mmol/g Tenta Gel S Rink resins from Rapp Polymere GmbH, Tuebingen, Germany. Peptide coupling of Fmoc-d-Glu(tBu)-OH was followed by Fmoc-d-Ala-OH. The peptide was cleaved from the resin with triﬂuoroacetic acid–water–triethylsilane 95:3:2 for two hours. Because of the high polarity of the product, puriﬁcation was performed in reversed-phase mode on an Isolera™system (Biotage AB, Uppsala, Sweden) with a SNAP Bio C18 300 Å column (Biotage AB, Uppsala, Sweden), with water–acetonitrile containing 0.1% TFA, and using a gradient of acetonitrile from 5% to 30%. Peptide purity was evaluated by analytical HPLC (Dionex Ultimate 3000 system from Thermo Scientiﬁc, Waltham, MA, USA) on an analytical C18 column (Gemini NX 110 Å, 5 µm, 4.6 × 50 mm from Phenomenex, Torrance, CA, USA) using a linear gradient ﬂow from 2% to 25% acetonitrile, containing 0.1% formic acid, giving a purity >98%, and identiﬁcation was carried out by electrospray ionization mass spectrometry using an Impact HD UHR-QTOF mass spectrometer from Bruker Corp., Billerica, MA, USA. MS (ESI): calculated for C8H15N3O4: 217.1063; found: [M+H]+ 218.1132, and [M+Na]+ 240.0956. 162.2 [M+H]+ 2 13 T i i El t Mi 2.13. Transmission Electron Microscopy 2.13. Transmission Electron Microscopy 2.13. Transmission Electron Microscopy 50 mL cultures were grown in 7H9 (supplemented with 10% OADC and 0.025% tyloxapol) and appropriate antibiotics to an OD600 = 0. Bacteria were harvested by centrifugation at 4000 g for 15 min and washed twice with 1 mL sterile PBS. Cells were fixed in 2.5% glutaraldehyde in PHEM (60mM Pipes, 25mM Hepes, 10mM EGTA, 2mM MgCl2, pH 6.9) buffer for 1 h at room temperature, post-fixed in OsO4 1%/K4Fe(CN)6 0.8% 1 h at room temperature then dehydrated in successive ethanol bathes (50/70/90/100%). The samples were then infiltrated with propylene oxide/epon 812 mixes, embedded in epon 812 and polymerized at 60 °C for 24 h. Then, 70nm ultrathin sections were cut with a PowerTome XL ultramicrotome (RMC, Tucson, Arizona,USA). Cuts were stained with 0.2% OTE (Oolong Tea Extract)/lead citrate and observed on a Tecnai G2 F20 (200 kV, FEG) TEM at 2.13. Transmission Electron Microscopy 50 mL cultures were grown in 7H9 (supplemented with 10% OADC and 0.025% tyloxapol) and appropriate antibiotics to an OD600 = 0. Bacteria were harvested by centrifugation at 4000 g for 15 min and washed twice with 1 mL sterile PBS. Cells were fixed in 2.5% glutaraldehyde in PHEM (60mM Pipes, 25mM Hepes, 10mM EGTA, 2mM MgCl2, pH 6.9) buffer for 1 h at room temperature, post-fixed in OsO4 1%/K4Fe(CN)6 0.8% 1 h at room temperature then dehydrated in successive ethanol bathes (50/70/90/100%). The samples were then infiltrated with propylene oxide/epon 812 mixes, embedded in epon 812 and polymerized at 60 °C for 24 h. Then, 70nm ultrathin sections were cut with a PowerTome XL ultramicrotome (RMC, Tucson, Arizona,USA). Cuts were stained with 0.2% OTE (Oolong Tea Extract)/lead citrate and observed on a Tecnai G2 F20 (200 kV, FEG) TEM at the latfo Plateau de Mi o o ie Ele t o i ue COMET INM Platfo Mo t ellie RIO 50 mL cultures were grown in 7H9 (supplemented with 10% OADC and 0.025% tyloxapol) and appropriate antibiotics to an OD600 = 0. Bacteria were harvested by centrifugation at 4000 g for 15 min and washed twice with 1 mL sterile PBS. Cells were fixed in 2.5% glutaraldehyde in PHEM (60mM Pipes, 25mM Hepes, 10mM EGTA, 2mM MgCl2, pH 6.9) buffer for 1 h at room temperature, post-fixed in OsO4 1%/K4Fe(CN)6 0.8% 1 h at room temperature then dehydrated in successive ethanol bathes (50/70/90/100%). the platform Plateau de Microsco Imaging p Imaging. Imaging. 2.14. Zebraﬁsh Infection Experiments I agi g 2.14. Zebrafish Infection Experiments Experiments were done with M. abscessus strains expressing tdTomato, obtained after transformation with the replicative plasmid pTEC27 [22]. Embryos were dechorionated using pronase (1 mg/mL final concentration) for 5 min at room temperature, followed by extensive washes in zebrafish water. At 30 h post-fertilization, embryos were anesthetized with 160 μg/mL tricaine (Sigma-Aldrich, Saint Quentin Fallavier, France) followed by caudal vein microinjection of 2–3 nL of bacterial suspension (approximately 100 CFU/nL). The size of the inoculum was verified a posteriori 2.14. Zebrafish Infection Experiments Experiments were done with M. abscessus strains expressing tdTomato, obtained after transformation with the replicative plasmid pTEC27 [22]. Embryos were dechorionated using pronase (1 mg/mL final concentration) for 5 min at room temperature, followed by extensive washes in zebrafish water. At 30 h post-fertilization, embryos were anesthetized with 160 μg/mL tricaine (Sigma-Aldrich, Saint Quentin Fallavier, France) followed by caudal vein microinjection of 2–3 nL of bacterial suspension (approximately 100 CFU/nL). The size of the inoculum was verified a posteriori b i j ti 2 L f h b t i l i i t il PBS d l t d 7H10 + OADC T it 2.14. Zebrafish Infection Experiments Experiments were done with M. abscessus strains expressing tdTomato, obtained after transformation with the replicative plasmid pTEC27 [22]. Embryos were dechorionated using pronase (1 mg/mL final concentration) for 5 min at room temperature, followed by extensive washes in zebrafish water. At 30 h post-fertilization, embryos were anesthetized with 160 μg/mL tricaine (Sigma-Aldrich, Saint Quentin Fallavier, France) followed by caudal vein microinjection of 2–3 nL of bacterial suspension (approximately 100 CFU/nL). The size of the inoculum was verified a posteriori by injecting 2 nL of each bacterial suspension in sterile PBS and plated on 7H10 + OADC. To monitor Experiments were done with M. abscessus strains expressing tdTomato, obtained after transformation with the replicative plasmid pTEC27 [22]. Embryos were dechorionated using pronase (1 mg/mL ﬁnal concentration) for 5 min at room temperature, followed by extensive washes in zebraﬁsh water. At 30 h post-fertilization, embryos were anesthetized with 160 µg/mL tricaine (Sigma-Aldrich, Saint Quentin Fallavier, France) followed by caudal vein microinjection of 2–3 nL of bacterial suspension (approximately 100 CFU/nL). The size of the inoculum was veriﬁed a posteriori by injecting 2 nL of each bacterial suspension in sterile PBS and plated on 7H10 + OADC. 2.11. Chemical Library C7H7N5—Theoretical molecular weight M = 161.1640 g/mol 2-(1H-tetrazol-5-yl)aniline (inhibitor 3) 1H NMR (300 MHz, DMSO-d6) δ 9.09 (s, 3H), 7.84 (dd, J = 7.9, 1.2 Hz, 1H), 7.37 – 7.18 (m, 1H), 7.00 (d, J = 8.0 Hz, 1H), 6.80 (t, J = 7.2 Hz, 1H), LRMS (ESI-MS) m/z = g g 2-(1H-tetrazol-5-yl)aniline (inhibitor 3) 1H NMR (300 MHz, DMSO-d6) δ 9.09 (s, 3H), 7.84 (dd, J = 7.9, 1.2 Hz, 1H), 7.37 – 7.18 (m, 1H), 7.00 (d, J = 8.0 Hz, 1H), 6.80 (t, J = 7.2 Hz, 1H), LRMS (ESI-MS) m/z = 162 2 [M+H]+ 2-(1H-tetrazol-5-yl)aniline (inhibitor 3) 1H NMR (300 MHz, DMSO-d6) δ 9.09 (s, 3H), 7.84 (dd, J = 7.9, 1.2 Hz, 1H), 7.37 – 7.18 (m, 1H), 7.00 (d, J = 8.0 Hz, 1H), 6.80 (t, J = 7.2 Hz, 1H), LRMS (ESI-MS) m/z = 162.2 [M+H]+ 2-(1H-tetrazol-5-yl)aniline (inhibitor 3) 1H NMR (300 MHz, DMSO-d6) δ 9.09 (s, 3H), 7.84 (dd, J = 7.9, 1.2 Hz, 1H), 7.37 – 7.18 (m, 1H), 7.00 (d, J = 8.0 Hz, 1H), 6.80 (t, J = 7.2 Hz, 1H), LRMS (ESI-MS) m/z = 162.2 [M+H]+. 162.2 [M+H]+ 2 13 T i i El t Mi 2.13. Transmission Electron Microscopy 2.13. Transmission Electron Microscopy The samples were then infiltrated with propylene oxide/epon 812 mixes, embedded in epon 812 and polymerized at 60 °C for 24 h. Then, 70nm ultrathin sections were cut with a PowerTome XL ultramicrotome (RMC, Tucson, Arizona,USA). Cuts were stained with 0.2% OTE (Oolong Tea Extract)/lead citrate and observed on a Tecnai G2 F20 (200 kV, FEG) TEM at the platform Plateau de Microscopie Electronique COMET, INM, Platform Montpellier RIO 50 mL cultures were grown in 7H9 (supplemented with 10% OADC and 0.025% tyloxapol) and appropriate antibiotics to an OD600 = 0. Bacteria were harvested by centrifugation at 4000× g for 15 min and washed twice with 1 mL sterile PBS. Cells were ﬁxed in 2.5% glutaraldehyde in PHEM (60 mM Pipes, 25 mM Hepes, 10 mM EGTA, 2 mM MgCl2, pH 6.9) buﬀer for 1 h at room temperature, post-ﬁxed in OsO4 1%/K4Fe(CN)6 0.8% 1 h at room temperature then dehydrated in successive ethanol bathes (50/70/90/100%). The samples were then inﬁltrated with propylene oxide/epon 812 mixes, embedded in epon 812 and polymerized at 60 ◦C for 24 h. Then, 70 nm ultrathin sections were cut with a PowerTome XL ultramicrotome (RMC, Tucson, AZ, USA). Cuts were stained with 0.2% OTE (Oolong Tea Extract)/lead citrate and observed on a Tecnai G2 F20 (200 kV, FEG) TEM at the platform Plateau de Microscopie Electronique COMET, INM, Platform Montpellier RIO Imaging. 2.11. Chemical Library The Institut Curie-CNRS library (9200 compounds, 2013 version) was dissolved in anhydrous dimethyl sulfoxide (DMSO, Carl Roth, Karlsruhe, Germany) and stored in a 10 mg/mL stock solution. 2.12. General Procedures Relative to the Chemistry of Ami1Mab Inhibitors Proton (1H) NMR spectra were recorded on a Bruker Avance 300 (300 MHz for 1H), using Tetramethylsilane as an internal standard. Deuterated DMSO-d6 was purchased from Euriso-Top (Gif-Sur-Yvette, France). Chemical shifts are given in parts per million (ppm) (δ relative to the residual solvent peak for 1H). The following abbreviations are used: singlet (s), doublet (d), triplet (t), and multiplet (m). The instrument used was an Alliance Waters system [Alliance Waters 2695 (pump) and Waters 2998 (Photodiode Array detector)] and the column was a Waters XBridge C-18, 3.5 µm particle size (3.0 mm × 100 mm) (Waters, Saint-Quentin-en-Yvelines, France). Electrospray ionization mass spectrometry was recorded on a micromass ZQ 2000 (Waters, Saint-Quentin-en-Yvelines, France). Compounds 1 to 3 (purity >90% by liquid chromatography-mass spectrometry) came from the Institute Curie/CNRS chemical library (Orsay, France). 7 of 25 7 of 26 7 of 26 Cells 2020, 9, 2410 Cells 2020, 9, x Cells 2020, 9, x etical molecular weight etical molecular weight C8H4ClNO3—Theoretical molecular weight M = 197.5753 g/mol C8H4ClNO3—Theoretical molecular weight M = 197.5753 g/mol -c]pyridine-3-carboxylic acid (inhibitor 1) 1H NMR (300 MHz, DM C8H4ClNO3—Theoretical molecular weight M = 197.5753 g/mol C8H4ClNO3—Theoretical molecular weight M = 197.5753 g/mol -c]pyridine-3-carboxylic acid (inhibitor 1) 1H NMR (300 MHz DM 4-chlorofuro [3,2-c]pyridine-3-carboxylic acid (inhibitor 1) 1H NMR (300 MHz, DMSO-d6) δ 8.81 (s, 1H), 8.38 (d, J = 5.7 Hz, 1H), 7.85 (d, J = 5.7 Hz, 1H), LRMS (ESI-MS) m/z = 198.0 + 200.0 [M+H]+. 4-chlorofuro [3,2-c]pyridine-3-carboxylic acid (inhibitor 1) 1H NMR (300 MHz, DMSO-d6) δ 8.81 (s, 1H), 8.38 (d, J = 5.7 Hz, 1H), 7.85 (d, J = 5.7 Hz, 1H), LRMS (ESI-MS) m/z = 198.0 + 200.0 [M+H]+. 4-chlorofuro [3,2-c]pyridine-3-carboxylic acid (inhibitor 1) 1H NMR (300 MHz, DMSO-d6) δ 8.81 (s, 1H), 8.38 (d, J = 5.7 Hz, 1H), 7.85 (d, J = 5.7 Hz, 1H), LRMS (ESI-MS) m/z = 198.0 + 200.0 [M+H]+. [ , ]py y ( ) ( , ) ( , 1H), 8.38 (d, J = 5.7 Hz, 1H), 7.85 (d, J = 5.7 Hz, 1H), LRMS (ESI-MS) m/z = 198.0 + 200.0 [M+H]+. 2.11. Chemical Library LRMS (ESI-MS) m/z = 202.0 + 204.0 [M+H]+ 2-chloropyridine-3,4-dicarboxylic acid (inhibitor 2) 1H NMR (300 MHz, DMSO-d6) δ 14.07 (s, 2H), 8.62 (d, J = 5.0 Hz, 1H), 7.82 (d, J = 5.0 Hz, 1H). LRMS (ESI-MS) m/z = 202.0 + 204.0 [M+H]+. 2-chloropyridine-3,4-dicarboxylic acid (inhibitor 2) 1H NMR (300 MHz, DMSO-d6) δ 14.07 (s, 2H), 8.62 (d, J = 5.0 Hz, 1H), 7.82 (d, J = 5.0 Hz, 1H). LRMS (ESI-MS) m/z = 202.0 + 204.0 [M+H]+ , 1H), 7.82 (d, J = 5.0 Hz, 1H). LRMS (ESI-MS) m/z = 202.0 + 204.0 [M+H]+ C7H7N5—Theoretical molecular weight M = 161 1640 g/mol C7H7N5—Theoretical molecular weight M = 161.1640 g/mol C7H7N5 Theoretical molecular weight M = 161 1640 g/mol C7H7N5—Theoretical molecular weight M = 161.1640 g/mol C7H7N5—Theoretical molecular weight M = 161.1640 g/mol 2-(1H-tetrazol-5-yl)aniline (inhibitor 3) 1H NMR (300 MHz, DMSO-d6) δ 9.09 (s, 3H), 7.84 (dd, J = 7.9, 1.2 Hz, 1H), 7.37 – 7.18 (m, 1H), 7.00 (d, J = 8.0 Hz, 1H), 6.80 (t, J = 7.2 Hz, 1H), LRMS (ESI-MS) m/z = g g 2-(1H-tetrazol-5-yl)aniline (inhibitor 3) 1H NMR (300 MHz, DMSO-d6) δ 9.09 (s, 3H), 7.84 (dd, J = 7.9, 1.2 Hz, 1H), 7.37 – 7.18 (m, 1H), 7.00 (d, J = 8.0 Hz, 1H), 6.80 (t, J = 7.2 Hz, 1H), LRMS (ESI-MS) m/z = 162 2 [M+H]+ 2-(1H-tetrazol-5-yl)aniline (inhibitor 3) 1H NMR (300 MHz, DMSO-d6) δ 9.09 (s, 3H), 7.84 (dd, J = 7.9, 1.2 Hz, 1H), 7.37 – 7.18 (m, 1H), 7.00 (d, J = 8.0 Hz, 1H), 6.80 (t, J = 7.2 Hz, 1H), LRMS (ESI-MS) m/z = 162.2 [M+H]+ 2-(1H-tetrazol-5-yl)aniline (inhibitor 3) 1H NMR (300 MHz, DMSO-d6) δ 9.09 (s, 3H), 7.84 (dd, J = 7.9, 1.2 Hz, 1H), 7.37 – 7.18 (m, 1H), 7.00 (d, J = 8.0 Hz, 1H), 6.80 (t, J = 7.2 Hz, 1H), LRMS (ESI-MS) m/z = 162.2 [M+H]+. 2.11. Chemical Library 4-chlorofuro [3,2-c]pyridine-3-carboxylic acid (inhibitor 1) 1H NMR (300 MHz, DMSO-d6) δ 8.81 (s, 1H), 8.38 (d, J = 5.7 Hz, 1H), 7.85 (d, J = 5.7 Hz, 1H), LRMS (ESI-MS) m/z = 198.0 + 200.0 [M+H]+. 4-chlorofuro [3,2-c]pyridine-3-carboxylic acid (inhibitor 1) 1H NMR (300 MHz, DMSO-d6) δ 8.81 (s, 1H), 8.38 (d, J = 5.7 Hz, 1H), 7.85 (d, J = 5.7 Hz, 1H), LRMS (ESI-MS) m/z = 198.0 + 200.0 [M+H]+. 4-chlorofuro [3,2-c]pyridine-3-carboxylic acid (inhibitor 1) 1H NMR (300 MHz, DMSO-d6) δ 8.81 (s, 1H), 8.38 (d, J = 5.7 Hz, 1H), 7.85 (d, J = 5.7 Hz, 1H), LRMS (ESI-MS) m/z = 198.0 + 200.0 [M+H]+. [ ]py y ( ) ( ) ( 1H), 8.38 (d, J = 5.7 Hz, 1H), 7.85 (d, J = 5.7 Hz, 1H), LRMS (ESI-MS) m/z = 198.0 + 200.0 [M+H]+. ti l l l i ht ti l l l i ht C7H4ClNO4 Theoretical molecular weight M= 201 5640 g/mol C7H4ClNO4—Theoretical molecular weight, M= 201.5640 g/mol C7H4ClNO4—Theoretical molecular weight, M= 201.5640 g/mol C7H4ClNO4 Theoretical molecular weight, M 201.5640 g/mol 2-chloropyridine-3,4-dicarboxylic acid (inhibitor 2) 1H NMR (300 MHz, DMSO-d6) δ 14.07 (s, 2H), 8 62 (d J = 5 0 Hz 1H) 7 82 (d J = 5 0 Hz 1H) LRMS (ESI MS) m/z = 202 0 + 204 0 [M+H]+ 2-chloropyridine-3,4-dicarboxylic acid (inhibitor 2) 1H NMR (300 MHz, DMSO-d6) δ 14.07 (s, 2H), 8.62 (d, J = 5.0 Hz, 1H), 7.82 (d, J = 5.0 Hz, 1H). LRMS (ESI-MS) m/z = 202.0 + 204.0 [M+H]+ 2-chloropyridine-3,4-dicarboxylic acid (inhibitor 2) 1H NMR (300 MHz, DMSO-d6) δ 14.07 (s, 2H), 8.62 (d, J = 5.0 Hz, 1H), 7.82 (d, J = 5.0 Hz, 1H). LRMS (ESI-MS) m/z = 202.0 + 204.0 [M+H]+. 2-chloropyridine-3,4-dicarboxylic acid (inhibitor 2) 1H NMR (300 MHz, DMSO-d6) δ 14.07 (s, 2H), 8.62 (d, J = 5.0 Hz, 1H), 7.82 (d, J = 5.0 Hz, 1H). LRMS (ESI-MS) m/z = 202.0 + 204.0 [M+H]+ g , g/ 2-chloropyridine-3,4-dicarboxylic acid (inhibitor 2) 1H NMR (300 MHz, DMSO-d6) δ 14.07 (s, 2H), 8 62 (d J = 5 0 Hz 1H) 7 82 (d J = 5 0 Hz 1H) LRMS (ESI MS) m/z = 202 0 + 204 0 [M+H]+ 2-chloropyridine-3,4-dicarboxylic acid (inhibitor 2) 1H NMR (300 MHz, DMSO-d6) δ 14.07 (s, 2H), 8.62 (d, J = 5.0 Hz, 1H), 7.82 (d, J = 5.0 Hz, 1H). 2.17. Muramyl-Dipeptide Hydrolysis Assay The reaction mixture was composed of 0.5 µL of Ami1 at diﬀerent concentrations, 0.5 µL of MDP (InvivoGen, Toulouse, France) at 10 mg/mL and 4 µL of 1X PBS, and was incubated for 30 min at 37 ◦C. The whole reaction volume was spotted at the bottom of a silica gel TLC plate (10 × 10 or 20 × 20 cm) in 1 µL steps. Migration of the reaction products was done in a glass chamber using butanol–ethanol–water (ratio 50:32:18, v/v/v) as a mobile phase. After approximately 1 h, plates were removed, allowed to dry, sprayed with 2% ninhydrin solution (diluted in ethanol) and charred. MDP, the dipeptide, and N-acetylmuramic acid (Sigma-Aldrich, Saint-Quentin Fallavier, France) were used as internal standards. 2.16. Statistical Analyses Statistical analyses were performed on Prism 5.0 (Graphpad, La Jolla, CA, USA) and detailed in each ﬁgure legend. Macrophage experiments were analyzed using Student’s unpaired two-tailed t-tests. For zebraﬁsh embryo survival curve analysis, log-rank (Mantel–Cox) statistical tests were used. 2.18. Compounds Screening by Thermal-Shift Assay The procedure was largely based on the protocol described by Niesen et al., [33]. Thirty-nine microliters of puriﬁed Ami1Mab at a concentration of 80 µM were dispensed in 96-well PCR plates. One microliter of each compound at 10 mg/mL was added in each well. As a non-treated control and to determine Ami1Mab melting temperature (Tm), 1 µL of DMSO was added to one well with Ami1Mab. Thereafter, 40 µL of the ﬂuorescent dye, SYPRO orange (Sigma-Aldrich, Saint-Quentin Fallavier, France), at a concentration of 10X (stock solution 5000X in 100% DMSO) was added. Plates were sealed and placed in a preheated qPCR machine (Agilent Mx3005) equipped with a Cy3 ﬁlter (excitation 545, emission 568). The heat cycle program was as followed: a 10 min step at 25 ◦C followed by an incremental of 1◦/min step until the temperature reached 95 ◦C. After the run, the melting curves of the protein in each well were plotted using Graphpad Prism software. Curves were ﬁtted using Boltzmann sigmoidal functions and the Tm was subsequently determined. A Tm value approximately of 2 ◦C to 5 ◦C higher than the DMSO control was considered as strong binding to the protein. the platform Plateau de Microsco Imaging p Imaging. Imaging. 2.14. Zebraﬁsh Infection Experiments To monitor embryo survival, infected larvae were transferred into 24-well plates (three embryos per well) and incubated at 28.5 ◦C. 8 of 25 Cells 2020, 9, 2410 Embryos were monitored daily and dead embryos were determined based on the absence of a heartbeat and removed from the well. Embryos were monitored daily and dead embryos were determined based on the absence of a heartbeat and removed from the well. 2.15. Zebraﬁsh Breeding and Ethics Zebraﬁsh experiments were completed in accordance with the guidelines deﬁned by the European Union for the use of laboratory animals. All animal experimentations were approved by the Direction Sanitaire et Vétérinaire de l’Hérault et Comité d’Ethique pour l’Expérimentation Animale de la région Languedoc Roussillon under the reference CEEA-LR-1145. Adult zebraﬁsh were housed at the Centre National de la Recherche Scientiﬁque, Montpellier, France. Zebraﬁsh embryos were obtained by natural spawning and maintained at 28.5 ◦C in 60 µg/mL ocean salts. Zebraﬁsh experiments were performed using the golden mutant line [32]. 3.1. Phenotypic Analysis of ami1 Deletion Mutant in the R and S Morphotypes 3.1. Phenotypic Analysis of ami1 Deletion Mutant in the R and S Morphotypes 3.1.1. ami1Mab Is Dispensable for M. abscessus Growth in Vitro To investigate the role of Ami1Mab, a ∆ami1Mab deletion mutant was generated based on the development of a recent recombineering technique allowing the production of unmarked deletion mutants in M. abscessus [26]. Gene deletion was generated in both the S and R backgrounds. Proper gene inactivation was conﬁrmed by PCR and sequencing analysis (Figure 1A). 9 of 25 Cells 2020, 9, 2410 ls 2020, 9, 2410 9 o Cells 2020, 9, x 9 of 26 Figure 1. Generation and phenotypic analyses of Δami1Mab. (A) Schematic representation of the genomic region around ami1Mab (MAB_0318c) in the parental (WT) and Δami1Mab strains of Mycobacterium abscessus. The size of the PCR amplicons used for genotyping the Δami1Mab mutants is indicated. (B) PCR profile confirming the proper deletion of ami1Mab in the mutant strains. The PCR products of 1.6 kb were amplified from Δami1Mab genomic DNA from rough (R) and smooth (S) variants while a band at 2.4 kb is expected for the parental (WT) strain. (C) In vitro growth curves at Figure 1. Generation and phenotypic analyses of ∆ami1Mab. (A) Schematic representation of the genomic region around ami1Mab (MAB_0318c) in the parental (WT) and ∆ami1Mab strains of Mycobacterium abscessus. The size of the PCR amplicons used for genotyping the ∆ami1Mab mutants is indicated. (B) PCR proﬁle conﬁrming the proper deletion of ami1Mab in the mutant strains. The PCR products of 1.6 kb were ampliﬁed from ∆ami1Mab genomic DNA from rough (R) and smooth (S) variants while a band at 2.4 kb is expected for the parental (WT) strain. (C) In vitro growth curves at 37 ◦C of the M. abscessus (Mab) WT, ∆ami1Mab, and complemented strains, in the R and S variants, respectively. Bacteria were grown in 7H9 broth supplemented with 10% oleic acid-albumin-dextrose-catalase (OADC) and 0.025% tyloxapol. The experiment is one representative of three replicates. (D) Transmission electron microscopy (TEM) of the M. abscessus WT, ∆ami1Mab, and complemented strain in the S morphotype. Septa are indicated by black arrows. (E) Cell length measurements from the TEM data. 3.1. Phenotypic Analysis of ami1 Deletion Mutant in the R and S Morphotypes Cells 2020, 9, 2410 10 of 25 The size of the amplicon ∆ami1 mutants relative to the wild-type strain conﬁrms the proper deletion of the gene in both the S and R morphotypes (Figure 1B). Complemented deletion strains were generated by cloning ami1Mab into the integrative pMV306 [34], yielding pMV306::ami1Mab and allowing gene expression under the control of the strong hsp60 promoter. Growth curves of the ∆ami1Mab and the corresponding complemented strains were similar to those of their wild-type progenitors, suggesting that the inactivation of ami1 does not aﬀect the growth of M. abscessus in broth medium (Figure 1C). Overall, these results indicate that ami1Mab is dispensable for M. abscessus growth in vitro. 3.1. Phenotypic Analysis of ami1 Deletion Mutant in the R and S Morphotypes Mean cell length = 1.27 µm for Mab S WT (n = 518); mean cell length = 1.34 µm for ∆ami1Mab (n = 557); mean cell length = 1.27 µm from the complemented strain (n = 576), the scale bar represents 500 nm. For statistical analysis, an unpaired Cells 2020, 9, x 9 of 26 Figure 1. Generation and phenotypic analyses of Δami1Mab. (A) Schematic representation of the genomic region around ami1Mab (MAB_0318c) in the parental (WT) and Δami1Mab strains of Mycobacterium abscessus. The size of the PCR amplicons used for genotyping the Δami1Mab mutants is indicated. (B) PCR profile confirming the proper deletion of ami1Mab in the mutant strains. The PCR products of 1.6 kb were amplified from Δami1Mab genomic DNA from rough (R) and smooth (S) variants while a band at 2.4 kb is expected for the parental (WT) strain. (C) In vitro growth curves at Figure 1. Generation and phenotypic analyses of ∆ami1Mab. (A) Schematic representation of the genomic region around ami1Mab (MAB_0318c) in the parental (WT) and ∆ami1Mab strains of Mycobacterium abscessus. The size of the PCR amplicons used for genotyping the ∆ami1Mab mutants is indicated. (B) PCR proﬁle conﬁrming the proper deletion of ami1Mab in the mutant strains. The PCR products of 1.6 kb were ampliﬁed from ∆ami1Mab genomic DNA from rough (R) and smooth (S) variants while a band at 2.4 kb is expected for the parental (WT) strain. (C) In vitro growth curves at 37 ◦C of the M. abscessus (Mab) WT, ∆ami1Mab, and complemented strains, in the R and S variants, respectively. Bacteria were grown in 7H9 broth supplemented with 10% oleic acid-albumin-dextrose-catalase (OADC) and 0.025% tyloxapol. The experiment is one representative of three replicates. (D) Transmission electron microscopy (TEM) of the M. abscessus WT, ∆ami1Mab, and complemented strain in the S morphotype. Septa are indicated by black arrows. (E) Cell length measurements from the TEM data. Mean cell length = 1.27 µm for Mab S WT (n = 518); mean cell length = 1.34 µm for ∆ami1Mab (n = 557); mean cell length = 1.27 µm from the complemented strain (n = 576), the scale bar represents 500 nm. For statistical analysis, an unpaired t-test with Welch’s correction was performed. p = 0.0035 for WT vs. ∆ami1Mab and p = 0.0053 for ∆ami1Mab vs. ∆ami1Mab complemented (compl.) n.s. stands non statistically signiﬁcant and ** stands for p < 0.01. MIC in µg/mL of the diﬀerent antibiotics were determined as described in the Materials and Methods, Section 2.8. 3.1.2. ami1Mab Deletion Mutants Do Not Present Division Defects It was previously reported that 20% of the M. smegmatis ami1 deletion mutants harbored an atypical cell division [11], although this observation was neither conﬁrmed in an independent study [10] nor in a M. tuberculosis ami1 deletion mutant [20]. We, therefore, investigated the shape and morphology of the M. abscessus S ∆ami1 mutants by transmission electron microscopy (TEM) (Figure 1D). The R variant was not included in this analysis because of its high propensity to aggregate, making it diﬃcult to obtain homogeneous bacterial suspensions, therefore rendering TEM processing very challenging. Analysis of approximately 500 individual WT, ∆ami1Mab, and ∆ami1Mab complemented cells failed to show morphological or cell division defects. Chain formation, typifying bacteria unable to divide properly, was not observed in ∆ami1Mab, thus diﬀering from the chaining phenotype observed in Helicobacter pylori [35] or E. coli [17] strains in which single or multiple amidase-encoding genes were invalidated. Analysis of the size of a large number of single bacilli showed a slight but statistically-signiﬁcant average shorter cell length for the WT (1.27 µm ± 0.018) and complemented strains (1.27 µm ± 0.017) as compared with the ∆ami1Mab mutant (1.34 µm ± 0.017) (Figure 1E). Cells 2020, 9, 2410 Cells 2020, 9, 2410 11 of 25 The wild-type sensitivity to these drugs was restored upon complementation. In contrast, the MIC of antibiotics that do not target PG (amikacin, clofazimine, and tigecycline) remained unaﬀected (Table 1). The wild-type sensitivity to these drugs was restored upon complementation. In contrast, the MIC of antibiotics that do not target PG (amikacin, clofazimine, and tigecycline) remained unaﬀected (Table 1). Taken together, these results suggest that although ∆ami1Mab mutants do not present major growth and cell division defects, they display a slightly-increased susceptibility proﬁle to SDS and to β-lactam antibiotics, suggestive of a cell wall alteration. 3.2. Analysis of the Virulence Phenotype of the M. abscessus ami1 Deletion Mutant 3.2. Analysis of the Virulence Phenotype of the M. abscessus ami1 Deletion Mutant 3.2.1. ∆ami1Mab Is Not Impaired in Uptake and Persistence in Human Macrophages 3.1.3. ∆ami1Mab Is More Sensitive to β-Lactam Antibiotics and Detergent 3.1.3. ∆ami1Mab Is More Sensitive to β-Lactam Antibiotics and Detergent We showed previously that inactivation of a dehydratase involved in cell wall biosynthesis was associated with increased susceptibility to detergents in M. abscessus [36]. In this study, we assessed whether ami1 deletion aﬀects cell wall integrity and thus susceptibility to sodium dodecyl sulfate (SDS) on solid medium. A moderate increase in sensitivity to SDS treatment at a concentration of 0.0025% was observed in ∆ami1 as compared to the WT strain, particularly visible at the 10−2 dilution (Supplementary Figure S1). The eﬀect was more pronounced in the presence of 0.0037% SDS for the R variant. This low detergent sensitivity was partially restored upon complementation with ami1Mab (Supplementary Figure S1). As the ∆ami1 M. smegmatis and M. tuberculosis were more susceptible to antibiotics [11,20], we next inquired whether a similar antibiotic susceptibility proﬁle is seen in ∆ami1Mab. The MIC of several β-lactam antibiotics (imipenem, cefuroxime, and cefamandole), known to target PG, were two times lower in the ∆ami1Mab strains compared to their respective parental progenitors (Table 1). Table 1. MIC determination M. abscessus S WT M. abscessus S ∆ami1 M. abscessus S ∆ami1 Compl. Imipenem 4 2 4 Cefoxitine 16 16 16 Cefuroxime 187–375 93 187 Cefamandole 2000 500–1000 2000 Amikacin 16 16 16 Clofazimine 0.78 0.78 0.78 Tigecycline 4 4 4 MIC in µg/mL of the diﬀerent antibiotics were determined as described in the Materials and Methods, Secti Table 1. MIC determination 3.2.1. ∆ami1Mab Is Not Impaired in Uptake and Persistence in Human Macrophages abscessus S and R variants, respectively. The M. abscessus Figure 2. Assessment of ∆ami1Mab in human macrophage and zebraﬁsh larvae. (A) M. abscessus-infected THP-1 macrophages with amikacin maintained at 50 µg/mL in the RPMI medium were lysed from day 0 to day 5 for CFU enumeration. Bacteria were plated at day 0 (2–4 h phagocytosis followed by a 2 h amikacin treatment at 250 µg/mL) day 1, 3 and day 5. Histograms and error bars represent means and standard deviations calculated from three independent experiments. For statistical analysis, the unpaired t-test with Welch’s correction was applied. , , , and ** stand for p < 0.1, p < 0.01, p < 0.001, and p < 0.0001, respectively. Left and right panels are for M. abscessus S and R variants, respectively. The M. abscessus S and R wild-type strains used as controls were not transformed with pMV306. (B) Survival curves of zebraﬁsh embryos infected with the diﬀerent M. abscessus strains. The data shown are corresponding to a pool of data from three independent experiments. For each experiment, 20 embryos were injected with 200–300 CFUs, respectively. Left and right panels are Figure 2. Assessment of Δami1Mab in human macrophage and zebrafish larvae. (A) M. abscessus-infected THP-1 macrophages with amikacin maintained at 50 μg/mL in the RPMI medium were lysed from day 0 to day 5 for CFU enumeration. Bacteria were plated at day 0 (2–4 h phagocytosis followed by a 2 h amikacin treatment at 250 μg/mL) day 1, 3 and day 5. Histograms and error bars represent means and standard deviations calculated from three independent experiments. For statistical analysis, the unpaired t-test with Welch’s correction was applied. , , , and ** stand for p < 0.1, p < 0.01, p < 0.001, and p < 0.0001, respectively. Left and right panels are for M. abscessus S and R variants, respectively. The M. abscessus S and R wild-type strains used as controls were not transformed with pMV306. (B) Survival curves of zebrafish embryos infected with the different M. abscessus strains. The data shown are corresponding to a pool of data from three independent experiments. For each experiment, 20 embryos were injected with 200–300 CFUs, respectively. Left and right panels are for M. abscessus S and R variants, respectively. The M. abscessus S and R wild-type strains used as controls were transformed with pTEC27 but not transformed with Figure 2. 3.2.1. ∆ami1Mab Is Not Impaired in Uptake and Persistence in Human Macrophages The recent study by Healy et al., emphasized the importance of Ami1 for M. tuberculosis virulence, particularly for the successful establishment of chronic infection in mice [20]. Because immunocompetent mice rapidly clear the infection with M. abscessus, this model is not permissive to the establishment of a chronic infection [23]. We thus analyzed the virulence of the ∆ami1Mab mutants in the human THP-1 macrophages. Both S and R ∆ami1Mab strains were internalized by macrophages similarly to the parental strains, as judged from the CFU determination at day 0 (Figure 2A). The capacity of the mutants to replicate inside macrophages was also slightly modiﬁed as CFU counts between day 1 and day 5 post-infection modestly increased as compared to the CFU of the corresponding WT strains (Figure 2A). Unexpectedly, the complemented strains overexpressing Ami1 showed an enhanced capacity to invade macrophages (up to 1-Log and 0.5-Log higher for the R and S variants at day 0, respectively) as compared to corresponding WT and mutant strains. However, the diﬀerence was less prominent at day 5 post-infection (Figure 2A). These results suggest that ami1Mab is not required for macrophage internalization and intracellular replication but that overexpression of Ami1Mab confers an intracellular survival advantage to the bacilli. 12 of 25 Cells 2020, 9, 2410 Figure 2. Assessment of Δami1Mab in human macrophage and zebrafish larvae. (A) M. abscessus-infected THP-1 macrophages with amikacin maintained at 50 μg/mL in the RPMI medium were lysed from day 0 to day 5 for CFU enumeration. Bacteria were plated at day 0 (2–4 h phagocytosis followed by a 2 h amikacin treatment at 250 μg/mL) day 1, 3 and day 5. Histograms and error bars represent means and standard deviations calculated from three independent experiments. For statistical analysis, the unpaired t-test with Welch’s correction was applied. , , , and ** stand for p < 0.1, p < 0.01, p < 0.001, and p < 0.0001, respectively. Left and right panels are for M. abscessus S and R variants, respectively. The M. abscessus S and R wild-type strains used as controls were not transformed with pMV306. (B) Survival curves of zebrafish embryos infected with the different M. abscessus strains. The data shown are corresponding to a pool of data from three independent experiments. For each experiment, 20 embryos were injected with 200–300 CFUs, respectively. Left and right panels are for M. abscessus S and R variants, respectively. The M. 3.2.1. ∆ami1Mab Is Not Impaired in Uptake and Persistence in Human Macrophages abscessus S and R wild-type strains used as controls were transformed with pTEC27 but not transformed with Figure 2. Assessment of ∆ami1Mab in human macrophage and zebraﬁsh larvae. (A) M. abscessus-infected THP-1 macrophages with amikacin maintained at 50 µg/mL in the RPMI medium were lysed from day 0 to day 5 for CFU enumeration. Bacteria were plated at day 0 (2–4 h phagocytosis followed by a 2 h amikacin treatment at 250 µg/mL) day 1, 3 and day 5. Histograms and error bars represent means and standard deviations calculated from three independent experiments. For statistical analysis, the unpaired t-test with Welch’s correction was applied. , , , and ** stand for p < 0.1, p < 0.01, p < 0.001, and p < 0.0001, respectively. Left and right panels are for M. abscessus S and R variants, respectively. The M. abscessus S and R wild-type strains used as controls were not transformed with pMV306. (B) Survival curves of zebraﬁsh embryos infected with the diﬀerent M. abscessus strains. The data shown are corresponding to a pool of data from three independent experiments. For each experiment, 20 embryos were injected with 200–300 CFUs, respectively. Left and right panels are for M. abscessus S and R variants, respectively. The M. abscessus S and R wild-type strains used as Figure 2. Assessment of Δami1Mab in human macrophage and zebrafish larvae. (A) M. abscessus-infected THP-1 macrophages with amikacin maintained at 50 μg/mL in the RPMI medium were lysed from day 0 to day 5 for CFU enumeration. Bacteria were plated at day 0 (2–4 h phagocytosis followed by a 2 h amikacin treatment at 250 μg/mL) day 1, 3 and day 5. Histograms and error bars represent means and standard deviations calculated from three independent experiments. For statistical analysis, the unpaired t-test with Welch’s correction was applied. , , , and ** stand for p < 0.1, p < 0.01, p < 0.001, and p < 0.0001, respectively. Left and right panels are for M. abscessus S and R variants, respectively. The M. abscessus S and R wild-type strains used as controls were not transformed with pMV306. (B) Survival curves of zebrafish embryos infected with the different M. abscessus strains. The data shown are corresponding to a pool of data from three independent experiments. For each experiment, 20 embryos were injected with 200–300 CFUs, respectively. Left and right panels are for M. 3.2.2. Virulence of ∆ami1Mab Is Not Aﬀected in the Zebraﬁsh Model of Infection 3.2.2. Virulence of ∆ami1Mab Is Not Aﬀected in the Zebraﬁsh Model of Infection Next, we assessed the virulence phenotype of ∆ami1Mab strains in zebraﬁsh embryos, which are particularly suitable to study the chronology of M. abscessus infection [22]. WT, ∆ami1Mab, and complemented strains were ﬁrst transformed with pTEC27 encoding tdTomato, allowing infection to be monitored in real-time by ﬂuorescence microscopy. Bacterial suspensions (200–300 CFU) were microinjected in the caudal vein of zebraﬁsh embryos at 30 h post-fertilization [22]. As reported previously, the S variant is predominantly avirulent in the embryos, with only 20% of embryo death at 12 days post-infection [22,37]. The survival curves of the embryos infected with ∆ami1Mab and the complemented strains were comparable to that of the WT (Figure 2B, left panel). In contrast, the R strains appeared more virulent with 70% larval death at 12 dpi, in agreement with previous reports [22] (Figure 2B, right panel). A slight but not statistically-signiﬁcant killing delay was observed for the ∆ami1Mab- complemented strain (Figure 2B, right panel). These ﬁndings were corroborated with whole-embryo imaging, which failed to show infection foci in the embryos infected with the diﬀerent S-derivative strains (Figure 2C, left panel), while robust infection occurred in the embryos challenged with the R derivative strains, typiﬁed by the presence of massive infection and important bacterial burdens within the central nervous system (Figure 2C, right panel). Together, these results suggest that Ami1Mab is not required for M. abscessus virulence in zebraﬁsh. 3.3. Biochemical and Structural Characterization of Ami1Mab 3.3.1. Ami1Mab Is an Active PG Hydrolase 3.3. Biochemical and Structural Characterization of Ami1Mab 3.2.1. ∆ami1Mab Is Not Impaired in Uptake and Persistence in Human Macrophages Assessment of ∆ami1Mab in human macrophage and zebraﬁsh larvae. (A) M. abscessus-infected THP-1 macrophages with amikacin maintained at 50 µg/mL in the RPMI medium were lysed from day 0 to day 5 for CFU enumeration. Bacteria were plated at day 0 (2–4 h phagocytosis followed by a 2 h amikacin treatment at 250 µg/mL) day 1, 3 and day 5. Histograms and error bars represent means and standard deviations calculated from three independent experiments. For statistical analysis, the unpaired t-test with Welch’s correction was applied. , , , and ** stand for p < 0.1, p < 0.01, p < 0.001, and p < 0.0001, respectively. Left and right panels are for M. abscessus S and R variants, respectively. The M. abscessus S and R wild-type strains used as controls were not transformed with pMV306. (B) Survival curves of zebraﬁsh embryos infected with the diﬀerent M. abscessus strains. The data shown are corresponding to a pool of data from three independent experiments. For each experiment, 20 embryos were injected with 200–300 CFUs, respectively. Left and right panels are for M. abscessus S and R variants, respectively. The M. abscessus S and R wild-type strains used as controls were transformed with pTEC27 but not transformed with pMV306. (C) Representative images of infected zebraﬁsh embryos at 5 days post-infection. Scale bar represents 1 mm. Infection foci are displayed in red. Cells 2020, 9, 2410 13 of 25 13 of 25 3.3.1. Ami1Mab Is an Active PG Hydrolase Activity of both Ami1Mab and Ami1Mtb is inhibited Figure 3. Biochemical characterization of Ami1Mab. (A) Ami1Mab elution proﬁle on Superdex 75 10/300 GL size-exclusion chromatography column. The denaturating polyacrylamide electrophoresis gel attests to the high protein purity after three chromatography steps. (B) Schematic of the muramyl-dipeptide hydrolysis assay. Ami1Mab cleaves muramyl-dipeptide (MDP) to yield N-acetylmuramic acid (MurNAc) and l-Ala-d-IsoGln (dipeptide). (C) The thin-layer chromatography (TLC)-based activity assay revealed a correlation between the disappearance of MDP and the concentration of Ami1Mab. Pure MDP, MurNAc, and dipeptide (DP) were loaded on the right part of the TLC plate as migration controls. TLC was revealed by ninhydrin and charring. (D) The enzymatic activity of Ami1 is Zn2+-dependent. Activity of both Ami1Mab and Ami1Mtb is inhibited after incubation with EDTA. 3 3 2 Th l Shif B d D S i Id iﬁd Th P I hibi f A i1 14 of 25 Cells 2020, 9, 2410 14 of 25 Cells 2020, 9, x 14 of 26 Cells 2020, 9, 2410 Cells 2020, 9, x Figure 3. Biochemical characterization of Ami1Mab. (A) Ami1Mab elution profile on Superdex 75 10/300 GL size-exclusion chromatography column. The denaturating polyacrylamide electrophoresis ge attests to the high protein purity after three chromatography steps. (B) Schematic of the muramyl-dipeptide hydrolysis assay. Ami1Mab cleaves muramyl-dipeptide (MDP) to yield N-acetylmuramic acid (MurNAc) and L-Ala-D-IsoGln (dipeptide). (C) The thin-layer chromatography (TLC)-based activity assay revealed a correlation between the disappearance of MDP and the concentration of Ami1Mab. Pure MDP, MurNAc, and dipeptide (DP) were loaded on the right part of the TLC plate as migration controls. TLC was revealed by ninhydrin and charring. (D) h f A d d A f b h A d A h b d Figure 3. Biochemical characterization of Ami1Mab. (A) Ami1Mab elution proﬁle on Superdex 75 10/300 GL size-exclusion chromatography column. The denaturating polyacrylamide electrophoresis gel attests to the high protein purity after three chromatography steps. (B) Schematic of the muramyl-dipeptide hydrolysis assay. Ami1Mab cleaves muramyl-dipeptide (MDP) to yield N-acetylmuramic acid (MurNAc) and l-Ala-d-IsoGln (dipeptide). (C) The thin-layer chromatography (TLC)-based activity assay revealed a correlation between the disappearance of MDP and the concentration of Ami1Mab. Pure MDP, MurNAc, and dipeptide (DP) were loaded on the right part of the TLC plate as migration controls. TLC was revealed by ninhydrin and charring. (D) The enzymatic activity of Ami1 is Zn2+-dependent. 3.3.1. Ami1Mab Is an Active PG Hydrolase Activity of both Ami1Mab and Ami1Mtb is inhibited after incubation with EDTA. 3.3.1. Ami1Mab Is an Active PG Hydrolase The biological function of Ami1 in M. smegmatis and M. tuberculosis underscores its requirement for growth, cell division, and virulence. Ami1 appears, therefore, as a potential drug target to exploit in mycobacteria. The macrophage infection results highlight the intracellular advantage conferred by overexpression of Ami1Mab in M. abscessus. Comforted by these studies, we initiated biochemical studies, searched for speciﬁc inhibitors, and characterized the structure of Ami1Mab. Ami1Mab was puriﬁed without its ﬁrst 40 amino acids in N-terminus, predicted to correspond to the signal peptide required for secretion. It was puriﬁed as a recombinant protein expressed in E. coli following a three-step puriﬁcation procedure, yielding a highly pure and homogeneous protein preparation (Figure 3A). The hydrolytic activity of pure Ami1Mab was next monitored by thin-layer chromatography (TLC) and using the commercial N-acetylmuramyl-l-alanyl-d-isoglutamine substrate, also known as muramyl-dipeptide (MDP). If the amidase is active on MDP, N-acetylmuramic acid and l-Ala-d-IsoGln are expected to be released (Figure 3B). Substrate and reaction products could be visualized following separation by TLC, spraying with ninhydrin, and charring. We ﬁrst monitored the hydrolysis of MDP using a range of protein concentrations (from 2 to 32 µM). MDP was rapidly hydrolyzed by Ami1Mab, indicating that it is an active PG hydrolase that cleaves the amide bond between the N-acetylmuramic acid and l-Ala (Figure 3C). Since Zn2+ is an essential divalent ion for PG amidases, we next assessed the dependence of Ami1Mab for this metal ion. Incubating Ami1Mab with the chelating agent EDTA prior to the reaction almost completely abolished the enzymatic activity (Figure 3D). These results demonstrate that, like Ami1Mtb (Figure 3D), [15,16] Zn2+ is required for catalysis of Ami1Mab. Cells 2020, 9, 2410 14 of 25 Cells 2020, 9, x 14 of 26 Figure 3. Biochemical characterization of Ami1Mab. (A) Ami1Mab elution profile on Superdex 75 10/300 GL size-exclusion chromatography column. The denaturating polyacrylamide electrophoresis gel attests to the high protein purity after three chromatography steps. (B) Schematic of the muramyl-dipeptide hydrolysis assay. Ami1Mab cleaves muramyl-dipeptide (MDP) to yield N-acetylmuramic acid (MurNAc) and L-Ala-D-IsoGln (dipeptide). (C) The thin-layer chromatography (TLC)-based activity assay revealed a correlation between the disappearance of MDP and the concentration of Ami1Mab. Pure MDP, MurNAc, and dipeptide (DP) were loaded on the right part of the TLC plate as migration controls. TLC was revealed by ninhydrin and charring. (D) The enzymatic activity of Ami1 is Zn2+-dependent. The enzymatic activity of Ami1 is Zn2+-dependent. Activity of both Ami1Mab and Ami1Mtb is after incubation with EDTA. 3.3.2. Thermal-Shift-Based Drug Screening Identiﬁed Three Potent Inhibitors of Ami1Mab The hydrolytic activity of pure Ami1Mab was next monitored by thin-layer chromatography (TLC) and using the commercial N-acetylmuramyl-L-alanyl-D-isoglutamine substrate, also known as muramyl-dipeptide (MDP). If the amidase is active on MDP, N-acetylmuramic acid and Despite being a robust assay to monitor PG hydrolytic activity, the TLC-based assay is not compatible with a large screening program to identify speciﬁc inhibitors of amidases. To overcome this hurdle, we employed thermal-shift assay (TSA) [38,39] to screen a large chemical library. This technique 15 of 25 Cells 2020, 9, 2410 is based on the principle that if a small molecule binds to the protein, the melting temperature (Tm) of the protein is modiﬁed. The Tm can easily be followed by adding a ﬂuorescent dye, SYPRO orange, that binds to hydrophobic patches of a protein that are exposed after melting. Monitoring of the signal can be performed by standard real time-PCR. It is noteworthy that this method does not necessarily select for direct enzyme inhibitors but identiﬁes molecules that can stabilize or destabilize the protein of interest. A TSA approach was developed in a 96-well plate format to screen the 9200 compounds of the Curie-CNRS library. The entire screen consisted of 115 plates, each containing 80 chemicals as well as a non-treated control and Ami1Mab plus 5% dimethyl sulfoxide (DMSO). Compound selection was based on Tm values ranging from 2 to 5 ◦C higher than the Tm of Ami1Mab plus DMSO control. Application of this threshold allowed the identiﬁcation of 654 compounds. All selected compounds were then assessed individually (at a concentration of 0.125 mg/mL) for their ability to inhibit Ami1Mab activity using the TLC-based assay. This narrowed down our initial screen to 54 compounds. A third round of selection was next applied, keeping only compounds inhibiting the enzyme activity at a concentration below 1 mM. This led to the selection of only three potential hits. 3.3.3. Biochemical Characterization of Ami1Mab Inhibitors 3.3.3. Biochemical Characterization of Ami1Mab Inhibitors Among these three hits, two belong to the pyridine chemical group while the third one is a tetrazole-derived compound. Compound 1 is a 4-chlorofuro [3,2-c]pyridine-3-carboxylic acid while compound 2 is a 2-(1H-1,2,3,4-tetrazol-5-yl)aniline and compound 3 is a 2-chloropyridine-3,4-dicarboxylic acid (Figure 4A). The validity of these hits was subsequently reassessed by TSA with respect to their capacity to increase the Tm of Ami1Mab. The three compounds increased the Tm of Ami1Mab by 5.5, 2.4, and 3.5 ◦C, respectively, when compared with Ami1Mab incubated with DMSO (Tm= 45 ◦C) (Figure 4B). The capacity of the three compounds to inhibit the MDP hydrolytic activity of Ami1Mab was determined in a concentration-dependent manner using the TLC-based activity assay. Compound 1 displayed the highest inhibitory eﬀect, starting at 150–200 µM, followed by compound 3 between 250–500 µM, and compound 2, which showed a moderate inhibitory activity starting at 800 µM (Figure 4C). Since two of the inhibitors possess carboxylic groups, we addressed whether the inhibitory eﬀect was not due to a pH change during the reaction. The Ami1Mab activity was thus tested under various acidic conditions, resulting in an active protein until pH 2 was reached. In parallel, products and substrates of the reaction were separated by TLC under the same pH range to ensure that the observed inhibition was not due to a TLC migration artifact dependent on the pH (Figure 4D). These biochemical data indicate that all three compounds inhibit the Ami1Mab activity in vitro. To enlighten the mode of inhibition of these compounds, structural studies of Ami1Mab were undertaken. 16 of 25 6 of 26 16 of 25 6 of 26 Cells 2020, 9, 2410 Cells 2020, 9, x Figure 4. Characterization of Ami1Mab inhibitors. (A) Chemical structure of the three compounds selected by thermal-shift assay. Chloride atom is in green, carbon in black, oxygen in red and nitrogen in blue (B) Melting curves of Ami1Mab in the presence of compound 1 (blue) at 244 μM, compound 2 (red) at 879 μM, and compound 3 (blue) at 845 μM as compared with protein incubated with 5% DMSO (black). (C) Assessment of Ami1Mab inhibition by compounds 1, 2, and 3 by TLC. (D) Evaluation of the Ami1Mab activity over an acidic pH range. Figure 4. Characterization of Ami1Mab inhibitors. (A) Chemical structure of the three compounds selected by thermal-shift assay. 3.3.3. Biochemical Characterization of Ami1Mab Inhibitors Chloride atom is in green, carbon in black, oxygen in red and nitrogen in blue (B) Melting curves of Ami1Mab in the presence of compound 1 (blue) at 244 µM, compound 2 (red) at 879 µM, and compound 3 (blue) at 845 µM as compared with protein incubated with 5% DMSO (black). (C) Assessment of Ami1Mab inhibition by compounds 1, 2, and 3 by TLC. (D) Evaluation of the Ami1Mab activity over an acidic pH range. Figure 4. Characterization of Ami1Mab inhibitors. (A) Chemical structure of the three compounds selected by thermal-shift assay. Chloride atom is in green, carbon in black, oxygen in red and nitrogen in blue (B) Melting curves of Ami1Mab in the presence of compound 1 (blue) at 244 μM, compound 2 (red) at 879 μM, and compound 3 (blue) at 845 μM as compared with protein incubated with 5% DMSO (black). (C) Assessment of Ami1Mab inhibition by compounds 1, 2, and 3 by TLC. (D) Evaluation of the Ami1Mab activity over an acidic pH range. Figure 4. Characterization of Ami1Mab inhibitors. (A) Chemical structure of the three compounds selected by thermal-shift assay. Chloride atom is in green, carbon in black, oxygen in red and nitrogen in blue (B) Melting curves of Ami1Mab in the presence of compound 1 (blue) at 244 µM, compound 2 (red) at 879 µM, and compound 3 (blue) at 845 µM as compared with protein incubated with 5% DMSO (black). (C) Assessment of Ami1Mab inhibition by compounds 1, 2, and 3 by TLC. (D) Evaluation of the Ami1Mab activity over an acidic pH range. Figure 4. Characterization of Ami1Mab inhibitors. (A) Chemical structure of the three compounds selected by thermal-shift assay. Chloride atom is in green, carbon in black, oxygen in red and nitrogen in blue (B) Melting curves of Ami1Mab in the presence of compound 1 (blue) at 244 μM, compound 2 (red) at 879 μM, and compound 3 (blue) at 845 μM as compared with protein incubated with 5% DMSO (black). (C) Assessment of Ami1Mab inhibition by compounds 1, 2, and 3 by TLC. (D) Evaluation of the Ami1Mab activity over an acidic pH range. Figure 4. Characterization of Ami1Mab inhibitors. (A) Chemical structure of the three compounds selected by thermal-shift assay. 3.3.4. Biochemical Characterization of Ami1Mab Inhibitors 3.3.4. Biochemical Characterization of Ami1Mab Inhibitors 3.3.4. Biochemical Characterization of Ami1Mab Inhibitors The crystal structure of Ami1Mab was solved at a high resolution in its free form and also bound to l-Ala-d-IsoGln, one of the end-product of the reaction (Table 2). In addition, to have a complete structural view and to compare the Ami1 structures from M. tuberculosis, M. smegmatis, and M. abscessus, we have also solved the crystal structure of Ami1Msm at a high resolution (Table 2). Ami1Mab and Ami1Msm displayed an overall globular structure comprising a central β-sheet (β1, 4, 5, 6, 7, 8) surrounded by strands β2 and β3 forming an antiparallel β-sheet, six α-helices, and one 310 helix (η1) (Figure 5A). The catalytic zinc is coordinated by the highly-conserved triad which constitutes the amidase_3 motif signature, namely H25, E60, and H115 in Ami1Mab and H51, E86, and H141 in Ami1Msm (Figure 5A, left and middle panels). Ami1Mab shares 68% and 65% primary sequence identity with Ami1Msm and Ami1Mtb, respectively, while Ami1Msm and Ami1Mtb possess 79% identity. All three structures of the apo forms are very similar since the superposition of Ami1Mab with Ami1Msm and Ami1Mtb (PDB id: 4LQ6) leads to r.m.s.d. values of 0.46Å and 0.55Å, respectively, while Ami1Msm and AmiMtb display a r.m.s.d. value of 0.51Å. The co-crystal structure of Ami1Mab bound to l-Ala-d-IsoGln was also solved at a high resolution. The Fo-Fc simulated-annealed OMIT map attests to the presence of the dipeptide in the active site of the protein (Figure 5B). This structure represents the enzyme state after the hydrolysis of MDP. The dipeptide binds close to the Zn2+ ion (Figure 5C). The l-Ala residue of the dipeptide is tightly bound through water-mediated H-bonding established with the side chains of Asp117 and Arg173, itself stabilized by the side chain of Asp175. Glu190 interacts with the dipeptide ligand and this residue was shown to be critical for the in vitro and in vivo activity of Ami1Mtb [15,16,20]. The carboxylic group of d-IsoGln is contacted by a salt-bridge interaction with Lys45 as well as by the main chain of Ala177 and Gly178. The side chain of Gln48 ﬁnalizes the interaction with the amide group of d-IsoGln. All these interactions are conserved in the dipeptide-bound Ami1Mtb structure (PDB id: 4M6G) (Figure 5D) and presumably also in Ami1Msm since all the residues interacting with the ligand in Ami1Mab and Ami1Mtb are conserved in Ami1Msm (Figure 5E). 3.3.3. Biochemical Characterization of Ami1Mab Inhibitors Chloride atom is in green, carbon in black, oxygen in red and nitrogen in blue (B) Melting curves of Ami1Mab in the presence of compound 1 (blue) at 244 µM, compound 2 (red) at 879 µM, and compound 3 (blue) at 845 µM as compared with protein incubated with 5% DMSO (black). (C) Assessment of Ami1Mab inhibition by compounds 1, 2, and 3 by TLC. (D) Evaluation of the Ami1Mab activity over an acidic pH range. Cells 2020, 9, 2410 17 of 25 17 of 25 Overall, these ﬁndings demonstrate that Ami1Mab is a functional PG hydrolase that shares a very strong structural conservation with Ami1Mab and Ami1Mtb. Values in parentheses are for the last resolution shell. Values in parentheses are for the last resolution shell. 3.3.4. Biochemical Characterization of Ami1Mab Inhibitors We noticed, however, a slight diﬀerence in ligand binding in the structures of Ami1Mab and Ami1Mtb bound to l-Ala-d-IsoGln. In the latter, the NH2 group of the Ala residue of the dipeptide is close to Zn2+ and only interacting with Glu200 (Figure 5C,D). In Ami1Mab, the amine group is interacting with the side chains of Asp117, Glu190 and, Arg173 (Figure 5C). Overall, these ﬁndings demonstrate that Ami1Mab is a functional PG hydrolase that shares a very strong structural conservation with Ami1Mab and Ami1Mtb. Cells 2020, 9, 2410 18 of 25 Table 2. Crystallographic data collection and reﬁnement statistics. Ami1Mab Ami1Mab l-Ala-d-IsoGln Ami1Msm PDB accession 7AGL 7AGO 7AGM Beamlime ESRF-ID30B SLS-PXIII-X06DA SLS-PXIII-S06DA Wavelength 0.979 1 0.979 Resolution range 38.4–1.6 (1.65–1.6) 42.8–1.7 (1.76–1.7) 46.7–1.35 (1.39–1.35) Space group P 41 21 2 P 41 21 2 P 1 21 1 Unit cell 85.92 85.92 73.66 90 90 90 85.74 85.74 75.63 90 90 90 41.85 68.84 63.72 90 91.866 90 Total reﬂections 228,475 (22,850) 824,584 (80,940) 526,861 (52,052) Multiplicity 6.4 (6.3) 26.1 (26.1) 6.7 (6.6) Completeness (%) 96.7 (99.2) 99.8 (100) 99.9 (99.9) Mean I/sigma(I) 11.1 (1.3) 18.8 (2.4) 16.0 (1.3) Wilson B-factor 22.4 20.9 15.4 R-meas 0.094 (1.25) 0.131 (1.29) 0.069 (1.47) CC1/2 0.99 (0.48) 0.99 (0.85) 0.99 (0.61) Reﬂections used in reﬁnement 35,744 (3601) 31,542 (3107) 79,191 (7884) Reﬂections used for R-free 2000 (201) 2000 (197) 3960 (394) R-work 0.168 (0.294) 0.162 (0.256) 0.155 (0.285) R-free 0.195 (0.329) 0.186 (0.307) 0.180 (0.319) Number of non-hydrogen atoms 1831 1857 3672 Macromolecules 1598 1628 3218 Ligands 1 15 2 Solvent 232 242 452 Protein residues 212 214 434 RMS (bonds, Å) 0.007 0.006 0.005 RMS (angles, ◦) 0.89 0.79 0.77 Ramachandran favored (%) 97.14 97.17 97.91 Ramachandran allowed (%) 2.86 2.83 2.09 Ramachandran outliers (%) 0.00 0.00 0.00 Rotamer outliers (%) 1.18 1.18 0.30 Clashscore 0.63 1.55 1.72 Average B-factor (Å2) 27.6 22.6 23.2 Macromolecules 25.8 20.5 21.6 Ligands 20.4 33.4 18.3 Solvent 40.1 35.8 34.3 Values in parentheses are for the last resolution shell. 19 of 25 19 of 25 Cells 2020, 9, 2410 Cells 2020, 9, x 19 of Figure 5. Structural comparisons of the Ami1Mab, Ami1Mtb, and Ami1Msm. (A) Overall crystal structure of Ami1Mab (left), Ami1Msm (middle) and Ami1Mtb (PDB id: 4LQ6) (right). Each structure is shown as a cartoon representation and is colored differently and according to its secondary structures. 3.3.4. Biochemical Characterization of Ami1Mab Inhibitors α, β, and η indicate alpha-helices, beta-strands, and 310 helices, respectively. The catalytic zinc ion (Zn2+) appears as a wheat-colored sphere. (B) Fo-Fc simulated-annealed OMIT map. The electron density map as seen in the Ami1Mab:dipeptide co-crystal structure and surrounding L-Ala-D-IsoGln (yellow sticks) is displayed as a grey mesh and contoured at 3 σ level. (C) Close up of the active site of Ami1Mab as seen in the Ami1Mab: dipeptide co-crystal structure. L-Ala-D-IsoGln (yellow sticks) tightly interacts by H-bonding and salt-bridge as illustrated by the dashed lines. Water molecules appear as green spheres. (D) The figure shows the molecular interactions seen in the co-crystal structure of Ami1Mtb bound to dipeptide (PDB id: 4M6G). (E) Active site of the Ami1Msm. Figure 5. Structural comparisons of the Ami1Mab, Ami1Mtb, and Ami1Msm. (A) Overall crystal structure of Ami1Mab (left), Ami1Msm (middle) and Ami1Mtb (PDB id: 4LQ6) (right). Each structure is shown as a cartoon representation and is colored diﬀerently and according to its secondary structures. α, β, and η indicate alpha-helices, beta-strands, and 310 helices, respectively. The catalytic zinc ion (Zn2+) appears as a wheat-colored sphere. (B) Fo-Fc simulated-annealed OMIT map. The electron density map as seen in the Ami1Mab:dipeptide co-crystal structure and surrounding l-Ala-d-IsoGln (yellow sticks) is displayed as a grey mesh and contoured at 3 σ level. (C) Close up of the active site of Ami1Mab as seen in the Ami1Mab: dipeptide co-crystal structure. l-Ala-d-IsoGln (yellow sticks) tightly interacts by H-bonding and salt-bridge as illustrated by the dashed lines. Water molecules appear as green spheres (D) The ﬁgure shows the molecular interactions seen in the co-crystal structure of Ami1Mtb bound to dipeptide (PDB id: 4M6G). (E) Active site of the Ami1Msm. Figure 5. Structural comparisons of the Ami1Mab, Ami1Mtb, and Ami1Msm. (A) Overall crystal structure of Ami1Mab (left), Ami1Msm (middle) and Ami1Mtb (PDB id: 4LQ6) (right). Each structure is shown as a cartoon representation and is colored differently and according to its secondary structures. α, β, and η indicate alpha-helices, beta-strands, and 310 helices, respectively. The catalytic zinc ion (Zn2+) appears as a wheat-colored sphere. (B) Fo-Fc simulated-annealed OMIT map. The electron density map as seen in the Ami1Mab:dipeptide co-crystal structure and surrounding L-Ala-D-IsoGln (yellow sticks) is displayed as a grey mesh and contoured at 3 σ level. (C) Close up of the active site of Ami1Mab as seen in the Ami1Mab: dipeptide co-crystal structure. 3.3.4. Biochemical Characterization of Ami1Mab Inhibitors L-Ala-D-IsoGln (yellow sticks) tightly interacts by H-bonding and salt-bridge as illustrated by the dashed lines. Water molecules appear as green spheres. (D) The figure shows the molecular interactions seen in the co-crystal structure of Ami1Mtb bound to dipeptide (PDB id: 4M6G). (E) Active site of the Ami1Msm. Figure 5. Structural comparisons of the Ami1Mab, Ami1Mtb, and Ami1Msm. (A) Overall crystal structure of Ami1Mab (left), Ami1Msm (middle) and Ami1Mtb (PDB id: 4LQ6) (right). Each structure is shown as a cartoon representation and is colored diﬀerently and according to its secondary structures. α, β, and η indicate alpha-helices, beta-strands, and 310 helices, respectively. The catalytic zinc ion (Zn2+) appears as a wheat-colored sphere. (B) Fo-Fc simulated-annealed OMIT map. The electron density map as seen in the Ami1Mab:dipeptide co-crystal structure and surrounding l-Ala-d-IsoGln (yellow sticks) is displayed as a grey mesh and contoured at 3 σ level. (C) Close up of the active site of Ami1Mab as seen in the Ami1Mab: dipeptide co-crystal structure. l-Ala-d-IsoGln (yellow sticks) tightly interacts by H-bonding and salt-bridge as illustrated by the dashed lines. Water molecules appear as green spheres. (D) The ﬁgure shows the molecular interactions seen in the co-crystal structure of Ami1Mtb bound to dipeptide (PDB id: 4M6G). (E) Active site of the Ami1Msm. Cells 2020, 9, 2410 Overall, th t t t 20 of 25 es a very 3.3.5. In Silico Docking Allows Predicting That Ami1Mab Inhibitors May Compete with Substrate Binding 3.3.5. In Silico Docking Allows Predicting That Ami1Mab Inhibitors May Compete with S Binding 3.3.5. In Silico Docking Allows Predicting That Ami1Mab Inhibitors May Compete with Substrate Binding 3.3.5. In Silico Docking Allows Predicting That Ami1Mab Inhibitors May Compete with S Binding We did not succeed in solving the co-crystal structure of Ami1Mab bound to any of the three selected inhibitors. The relatively-low potency of the inhibitors is in favor of a low aﬃnity of these inhibitors for the amidase and might explain our vain attempt at obtaining the co-structures. As an alternative, in silico docking was performed to gain insight into their potential mechanism of inhibition. To facilitate the search for the potential inhibitor’s binding-site, the Ami1Mab crystal structure was probed with the CASTp server to identify cavities in the protein structure [40]. This analysis led to the identiﬁcation of a single cavity of a volume of 72 Å2 comprising the active site of Ami1Mab. 3.3.4. Biochemical Characterization of Ami1Mab Inhibitors The figure depicts the best docking poses of the three compounds. All three inhibitors appear able to bind in the Ami1Mab active site with similar Figure 6. In silico docking of the three inhibitors. The ﬁgure depicts the best docking poses of the three compounds. All three inhibitors appear able to bind in the Ami1Mab active site with similar binding energies. Inhibitors are displayed in stick representation. Nitrogen atoms are in blue, oxygen in red, hydrogen in white and carbon atoms in yellow for the ligand and grey for the protein. Inhibitor 1 (∆G◦= −6.5 kcal/mol) appears to be able to occupy the active site by interacting with the main chain of Ala177 and Gly178 through its carboxylic group and a weaker H-bonding with the Lys45 side chain. The nitrogen of the pyridine moiety is interacting with the side chain of Asp22, His25, Gln48, and His115 interact as well by Van der Waals interactions. Inhibitor 2 (∆G◦= −6.0 kcal/mol) interacts by H-bonding with Lys45 and Gln48 as well as with His25 and His115 by weaker contacts. Inhibitor 3 could be docked with a ∆G◦= −6.4 kcal/mol, where multiple interactions involve the two carboxylic groups. The carboxylate in position 3 interacts with the side chains of Gln48, Arg99, and the main chain of His25, while the carboxylate in position 4 is able to contact the side chains of Asp22, Ser113, and Lys186. Ile96 side chain is in close vicinity with the chloride in position 2. The proposed docking poses strongly suggest that the three inhibitors are able to occupy the active site in a manner similar to the dipeptide seen in the crystal structure. The carboxylate group of inhibitor 1 is interacting with the same residues Lys45, Ala177, and Gly178 involved in binding of the d-IsoGln carboxylate. The docking study supports the view that these inhibitors might act by competing with the binding of the PG peptide stem. 3.3.4. Biochemical Characterization of Ami1Mab Inhibitors In silico docking by deﬁning a search region in this area was performed with the AutoDock Vina software [31]. The three inhibitors could be docked into the Ami1Mab active site with similar binding energy (∆G◦) (Figure 6). We did not succeed in solving the co-crystal structure of Ami1Mab bound to any of the three selected inhibitors. The relatively-low potency of the inhibitors is in favor of a low affinity of these inhibitors for the amidase and might explain our vain attempt at obtaining the co-structures. As an alternative, in silico docking was performed to gain insight into their potential mechanism of inhibition. To facilitate the search for the potential inhibitor’s binding-site, the Ami1Mab crystal structure was probed with the CASTp server to identify cavities in the protein structure [40]. This analysis led to the identification of a single cavity of a volume of 72 Å2 comprising the active site of Ami1Mab. In silico docking by defining a search region in this area was performed with the AutoDock Vina software [31]. The three inhibitors could be docked into the Ami1Mab active site with similar binding energy (ΔG°) (Figure 6). Figure 6. In silico docking of the three inhibitors. The figure depicts the best docking poses of the three compounds. All three inhibitors appear able to bind in the Ami1Mab active site with similar Figure 6. In silico docking of the three inhibitors. The ﬁgure depicts the best docking poses of the three compounds. All three inhibitors appear able to bind in the Ami1Mab active site with similar binding energies. Inhibitors are displayed in stick representation. Nitrogen atoms are in blue, oxygen in red, hydrogen in white and carbon atoms in yellow for the ligand and grey for the protein. Figure 6. In silico docking of the three inhibitors. The figure depicts the best docking poses of the three compounds. All three inhibitors appear able to bind in the Ami1Mab active site with similar Figure 6. In silico docking of the three inhibitors. The ﬁgure depicts the best docking poses of the three compounds. All three inhibitors appear able to bind in the Ami1Mab active site with similar binding energies. Inhibitors are displayed in stick representation. Nitrogen atoms are in blue, oxygen in red, hydrogen in white and carbon atoms in yellow for the ligand and grey for the protein. Figure 6. In silico docking of the three inhibitors. 4. Discussion The PG biosynthesis pathway has long been overlooked as an interesting drug target for subsequent drug developments against mycobacterial diseases, largely due to the fact that most β-lactam antibiotics inhibiting transpeptidation show poor activity against mycobacteria. This is explained by the presence of speciﬁc secreted β-lactamases, which neutralize these antibiotics. However, targeting PG biosynthesis in mycobacteria has recently regained interest after the discovery 21 of 25 Cells 2020, 9, 2410 of β-lactamases inhibitors which, together with β-lactam antibiotics, can successfully inhibit growth of M. tuberculosis [41] and M. abscessus [42,43]. PG remodeling in mycobacteria and particularly PG hydrolytic enzymes has been more thoroughly investigated in recent years [44]. The NlpC/P60 endopeptidases RipA, RipB, RipC, and RipD have been widely scrutinized [7,20,45–50]. The functional redundancy and the non-essential individual roles of RipA and RipB hydrolyzing the PG peptide stem between the 2nd and 3rd amino acid were established both in M. smegmatis [50] and in M. tuberculosis [20]. Redundancy of PG amidases has also been proposed since single or multiple deletion of amide-encoding genes (ami2, ami3, and ami4) in M. smegmatis is not detrimental for growth [10]. The major septal PG hydrolase RipA can sustain the amidase Ami1 PG hydrolytic activity in M. tuberculosis [20]. However, despite the fact that mycobacterial amidases share redundant functions and that ami1 is not essential for growth or cell division of M. tuberculosis, Ami1 is required for the persistence of the bacilli within the host [20]. This contrasts with studies reporting that Ami1 is essential for cell separation and that inactivation of the corresponding gene is associated with atypical cell division phenotypes [11,13]. Apart from M. smegmatis, little is known about PG-hydrolyzing enzymes in other non-tuberculous mycobacteria. To ﬁll this gap, we investigated the role of ami1 in the human pathogen, M. abscessus. We showed that ami1Mab, as reported in M. smegmatis and M. tuberculosis, is dispensable for cell growth in the R and S variants. Consistent with observations in M. tuberculosis, ami1Mab is also not essential for cell division in M. abscessus. We also showed that ami1Mab is neither required for invasion nor for persistence in macrophages while overexpression of ami1Mab partially increased the uptake and intracellular survival of the bacilli in THP-1 macrophages. In M. smegmatis, deletion of ami1 increases type 3-3 crosslinks as compared to the WT strain [11]. In M. 4. Discussion Author Contributions: Conceptualization, T.K., N.V.W., M.D.J., K.K.S., M.B.T., C.B., L.K., and M.B.; methodology, T.K., N.V.W., M.D.J., K.K.S., M.B.T., C.B., H.M.A.B.A., L.K., and M.B.; validation, T.K., N.V.W., M.D.J., K.K.S., M.B.T., C.B., L.K., and M.B.; formal analysis, T.K., N.V.W., M.D.J., K.K.S., M.B.T., C.B., L.K., and M.B.; investigation, T.K., N.V.W., M.D.J., K.K.S., M.B.T., C.B., A.N., H.M.A.B.A., C.H., and M.B.; writing—original draft preparation, M.B.; writing—review and editing, N.V.W., M.D.J., L.K., and M.B.; supervision, L.K. and M.B.; project administration, L.K. and M.B.; funding acquisition, L.K. and M.B. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Fondation pour la Recherche Médicale (FRM) (grant number DEQ20150331719 to L.K.). T.K. was supported by a Ph.D. fellowship from Ministère de l’Enseignement Supérieur, de la Recherche et de l’Innovation (MESRI). N.V.W. was supported by a postdoctoral fellowship from the Infectiopôle Sud. Acknowledgments: We thank the staﬀat the Swiss Light Source and European Synchrotron Radiation Facility beamlines for support during data collection, W. Daher for preparing THP-1 macrophage cultures, A. Viljoen for fruitful discussions, and F. Roquet-Banères for technical support and help with MIC determination. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. 4. Discussion abscessus, changes in PG cross-link ratio and type of crosslinking is correlated with the growing phase. PG of exponentially-growing M. abscessus has more cross-links characterized by an increasing ratio of 3-3 cross-links [51]. As the deletion of ami1 in M. smegmatis increases the 3-3 crosslinks ratio [11] one could propose that overexpression of ami1Mab might trigger a decrease in 3-3 and consequently an increase in 4-3-type cross-links, which may confer, for yet unidentiﬁed reasons, intracellular advantage. This, however, remains to be further investigated in future studies. The evidence of ami1Mab being involved in PG remodeling was also supported by a slight increase in the susceptibility to β-lactam antibiotics in ∆ami1Mab. MICs for non-targeting PG antibiotics were not modiﬁed, suggesting that ami1 deletion triggers speciﬁc cell wall modiﬁcation in PG rather than modiﬁcations of the permeability of the cell wall. The cell wall impairment in ∆ami1Mab is also supported by a modest increased sensitivity to detergent. Infections studies in zebraﬁsh embryos and macrophages suggest that ∆ami1Mab is as virulent as its WT progenitor. In light of the recent studies related to Ami1 from M. tuberculosis and M. smegmatis, this work on Ami1Mab emphasizes functional diﬀerences regarding the contribution of Ami1 from the three species regarding growth, invasion, and persistence. These ﬁndings are particularly unexpected considering that all three mycobacteria possess the four amidase-encoding genes and that the corresponding orthologues share a highly-conserved primary sequence, suggesting that these functional diﬀerences are unlikely connected to their enzymatic activity per se. Super-resolution microscopy analyses unraveled diﬀerences in the spatiotemporal synthesis of PG in M. smegmatis and M. tuberculosis [52], which may explain the functional divergence of Ami1 between the diﬀerent mycobacterial species. Growth conditions and bacterial environment also appear crucial in modulating PG remodeling enzymes as reported for ripA deletion mutant which is only impaired in cell division under acid stress conditions (8, 37). A possible explanation for the diﬀerence of Ami1 contribution may arise from the respective lifestyles of the environmental M. abscessus as opposed to the human-speciﬁc M. tuberculosis pathogen. In this context, it is noteworthy that the obligate intracellular Mycobacterium leprae, carrying a reduced genome, encodes for only two amidases, namely Ami1 (ML2131) and Ami2 (CwlM, ML2704). This supports the view that Ami1 and Ami2 are required for the intracellular mycobacterial lifestyle and were conserved in M. leprae due to the selection pressure. 4. Discussion Additionally, transcriptomic analyses showed Cells 2020, 9, 2410 22 of 25 up- and down-regulation of multiple genes in M. abscessus subspecies massiliense when comparing planktonic, intra-macrophage, and intra-amoebal growth [53]. These included genes from the PG metabolic pathway found to be down-regulated in macrophages as compared with planktonic growth conditions [53]. Biochemical and structural characterizations of Ami1Mab unambiguously demonstrate that it is an active PG amidase, very closely related to Ami1Msm and Ami1Mtb. A noticeable diﬀerence in dipeptide binding was observed between Ami1Mab and Ami1Mtb. This could be explained by the fact that the Ami1Mab structure was co-crystallized with the dipeptide while the structure of Ami1Mtb which was generated by crystallization with MDP, likely hydrolyzed during the crystallization process and only the dipeptide part was visible in the structure. In the Ami1Mab co-structure, the peptide is further away from the catalytic Zn2+. This state might, therefore, represent a post-hydrolysis state i.e., a state where the product is about to be released from the enzyme. This study describes the ﬁrst inhibitors of this class of enzymes, which may represent a ﬁrst step towards the design of more potent inhibitors. Further work through structure–activity relationship approaches is expected to improve the properties of these inhibitors. In summary, this work represents the ﬁrst study describing a PG hydrolytic enzyme in M. abscessus. Further work is needed to (i) explore the role of Ami1 in M. abscessus and its intricate relationships with other amidases/endopeptidases such as RipA and (ii) study its regulation under various stress conditions. Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4409/9/11/2410/s1, Table S1: Primers sequences; Figure S1: Detergent susceptibility assay. Author Contributions: Conceptualization, T.K., N.V.W., M.D.J., K.K.S., M.B.T., C.B., L.K., and M.B.; methodology, T.K., N.V.W., M.D.J., K.K.S., M.B.T., C.B., H.M.A.B.A., L.K., and M.B.; validation, T.K., N.V.W., M.D.J., K.K.S., M.B.T., C.B., L.K., and M.B.; formal analysis, T.K., N.V.W., M.D.J., K.K.S., M.B.T., C.B., L.K., and M.B.; investigation, T.K., N.V.W., M.D.J., K.K.S., M.B.T., C.B., A.N., H.M.A.B.A., C.H., and M.B.; writing—original draft preparation, M.B.; writing—review and editing, N.V.W., M.D.J., L.K., and M.B.; supervision, L.K. and M.B.; project administration, L.K. and M.B.; funding acquisition, L.K. and M.B. All authors have read and agreed to the published version of the manuscript. References 1. Meroueh, S.O.; Bencze, K.Z.; Hesek, D.; Lee, M.; Fisher, J.F.; Stemmler, T.L.; Mobashery, S. Three-dimensional structure of the bacterial cell wall peptidoglycan. Proc. Natl. Acad. Sci. USA 2006, 103, 4404–4409. [CrossRef] 2. Typas, A.; Banzhaf, M.; Gross, C.A.; Vollmer, W. 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https://openalex.org/W2795181852	http://www.scielo.br/pdf/csp/v34n4/1678-4464-csp-34-04-e00142617.pdf, https://www.scielo.br/j/csp/a/gdwmZwGHLwkPhX6wKBXk44B/?lang=pt&format=pdf	es		Alimentação escolar e constituição de identidades dos escolares: da merenda para pobres ao direito à alimentação	Cadernos de Saúde Pública	2,018	cc-by	8,305	ENSAIO ESSAY Alimentação escolar e constituição de identidades dos escolares: da merenda para pobres ao direito à alimentação School feeding and the establishment of school identities: from meals for the poor to the right to food Alimentación escolar y constitución de identidades entre escolares: de la merienda para pobres al derecho a la alimentación Edleuza Oliveira Silva 1,2 Lígia Amparo-Santos 2 Micheli Dantas Soares 1 doi: 10.1590/0102-311X00142617 Resumo Este ensaio busca problematizar a alimentação escolar enquanto prática que contribui para a constituição de identidades escolares. Parte-se de uma revisão bibliográfica, não sistemática, de publicações sobre alimentação escolar e identidades no contexto das escolas públicas brasileiras. Discute-se, inicialmente, a persistência de discursos e práticas de caráter assistencialista que reduziam a alimentação escolar a uma comida para pobres, questão observada nos estudos. Os significados desse caráter assistencialista concorrem para o entendimento de que ele parece funcionar como um mecanismo de poder capaz de inscrever, nos escolares, uma identidade de pobreza e inferioridade. Esse entendimento é situado nas relações de poder existentes na escola, no exercício do poder disciplinar e seu potencial de produzir identidades, assim como nas práticas de resistência decorrentes desses poderes no contexto da alimentação escolar. Considera-se ainda que os escolares sejam agentes de seus próprios processos identitários, haja vista que, em sua relação com a alimentação escolar, evidenciam-se, além de processos de sujeição, também processos de resistência e de construção ativa de identidade alimentar, nos quais agregam o tradicional e o moderno, o local e o global, dentre outros aspectos. Mesmo que ambiguidades sejam percebidas nesse cenário, entende-se que são indícios de mudanças no paradigma do pensar e fazer a alimentação escolar, trazendo elementos para problematizá-la: de um lado, ainda como um dispositivo de manutenção das desigualdades sociais e, de outro, esforços e ações para propiciá-la como um direito e promotora de identidades emancipatórias. Correspondência E. O. Silva Universidade Federal do Recôncavo da Bahia. Av. Carlos Amaral 1015, Santo Antônio de Jesus, BA 44574-490, Brasil. edleuza@ufrb.edu.br 1 Universidade Federal do Recôncavo da Bahia, Santo Antônio de Jesus, Brasil. 2 Universidade Federal da Bahia, Salvador, Brasil. Alimentação Escolar; Comportamento Alimentar; Segurança Alimentar e Nutricional; Assistência Social Este é um artigo publicado em acesso aberto (Open Access) sob a licença Creative Commons Attribution, que permite uso, distribuição e reprodução em qualquer meio, sem restrições, desde que o trabalho original seja corretamente citado. Cad. Saúde Pública 2018; 34(4):e00142617 2 Silva EO et al. Introdução O termo “alimentação escolar” tem sido utilizado para denominar o conjunto de refeições ofertadas pelo Programa Nacional de Alimentação Escolar (PNAE). Desse modo, a alimentação escolar constitui uma prática alimentar relevante nas escolas públicas brasileiras de ensino fundamental e médio. O ato de comer na escola se realiza em meio a outras práticas que compõem o espaço escolar, configurando-se em experiências e processos que influenciam na construção de hábitos alimentares e de identidades de crianças e adolescentes. O PNAE tem como objetivo contribuir para o desenvolvimento biopsicossocial, o rendimento escolar e a formação de hábitos alimentares saudáveis dos escolares, por meio de ações de educação alimentar e nutricional e oferta de refeições que atendam às suas necessidades nutricionais durante a sua permanência na escola 1. O orçamento do programa, em 2010, foi de três bilhões de Reais para uma cobertura de 47 milhões de estudantes 2, constituindo uma das principais políticas de alimentação e nutrição no Brasil. Todavia, sua longa trajetória foi constituída em meio a diversas e divergentes intencionalidades. Santos et al. 3, ao percorrer o histórico do PNAE, marcou seus principais percursos. O programa foi criado em 1950, sob o nome de Campanha Nacional de Merenda Escolar (CNME), em um contexto de programas de combate à desnutrição, tomando uma conotação de um programa de suplementação alimentar para escolares de baixa renda. Agrega-se o fato de ter sido implementado, desde o início, sob a égide dos interesses da indústria de alimentos, inicialmente, servindo de escoamento dos excedentes da produção americana e, em seguida, com uma forte ligação com a indústria de alimentos brasileira. Tal situação permitiu a predominância, durante décadas, de alimentos formulados nos cardápios escolares. Na década de 1970, durante o II Programa Nacional de Alimentação e Nutrição (PRONAN II), a CNME foi renomeada como Programa Nacional de Merenda Escolar, sendo um dos programas mais abrangentes. No entanto, sua gestão era centralizada e burocrática. Já na década de 1980, iniciase um processo de descentralização, conferindo maior autonomia aos municípios. Somente a partir da década de 1990, a elaboração de refeições mais compatíveis com os hábitos alimentares regionais foi normatizada, iniciando um estímulo à aquisição de alimentos básicos e in natura de produtores locais e o enfrentamento do domínio dos alimentos formulados 3. Esses percursos sinalizam uma transição de uma cultura assistencialista e clientelista em torno da alimentação escolar e também de ações para transformá-la em direito. O início do século XXI marca um novo cenário político no Brasil, no qual, a Segurança Alimentar e Nutricional foi alçada como prioridade de governo. Em 2003, o Conselho Nacional de Segurança Alimentar é reativado 4, sendo um órgão articulador entre governo e sociedade civil, possibilitando interlocução e legitimidade aos movimentos sociais e à militância política de intelectuais e pesquisadores na luta pelo Direito Humano à Alimentação Adequada e Saudável (DHAA) para todo brasileiro. Como importantes marcos políticos, destacam-se a promulgação, em 2006, da Lei Orgânica de segurança alimentar nutricional e a criação do Sistema Nacional de Segurança Alimentar e Nutricional (SISAN), no qual, o PNAE integra como uma das estratégias de garantir o DHAA nas escolas 5. E, em 2010, finalmente, a alimentação inscreve-se como um direito social no Brasil, por meio da Emenda Constitucional no 64/2010 6, a ser garantido por um conjunto de ações intersetoriais que compõem o SISAN. O PNAE também ganha um marco regulatório em 2009, sendo estruturado como uma política de segurança alimentar nutricional 1, iniciando um importante processo de mudança de paradigma. Atualmente, o PNAE é uma política reconhecida mundialmente, sendo referência para implantação de programas de alimentação escolar sustentáveis em outros países e, portanto, não resta dúvida de que os avanços ocorridos concorrem para melhores condições de aprendizagem e de permanência dos alunos na escola. Entretanto, ainda que se leve, em consideração, a conquista da alimentação escolar alçada como direito humano, importa refletir sobre as ambiguidades que têm marcado as práticas em torno da alimentação escolar no cotidiano das escolas brasileiras, nesse contexto de mudança. Isso porque se tem observado a coexistência de esforços para propiciar uma alimentação digna com discursos e práticas de caráter assistencialista que associa os escolares que consomem a alimentação escolar a uma identidade de pobre e necessitado 7,8,9,10,11. Cad. Saúde Pública 2018; 34(4):e00142617 ALIMENTAÇÃO ESCOLAR E CONSTITUIÇÃO DE IDENTIDADES DOS ESCOLARES Parte-se do entendimento de que as práticas alimentares participam dos processos identitários, pois a alimentação assume uma posição central no aprendizado e na formação social, por sua natureza vital, rotineira, geradora de sociabilidades e possibilidades de escolhas 12. Para Poulain 13, um alimento é incorporado tanto no plano físico quanto no simbólico, ou seja, não cumpre apenas a função de nutrir mediante suas propriedades sensoriais e nutricionais, mas produz prazer, promove prestígio, inclusão e distinção social, mediante suas propriedades simbólicas, e ainda, por outro lado, segundo Contreras & Arnaiz 14 pode produzir sofrimento, privação e humilhação. Ou seja, as práticas alimentares não são apenas expressões ou recursos de afirmação de identidades, mas se inscrevem nos próprios processos identitários 13,15. A discussão sobre processos identitários adquire maior relevo na sociedade contemporânea, em virtude das mudanças resultantes da crise das estruturas sociais, das tradições e dos paradigmas 16,17,18. A identidade diz respeito a processos de individuação e identificação, resultantes de interações sociais e relações de poder entre indivíduos, situações e contextos 18,19, ao passo que também abarca a construção de vínculos e necessidades de reconhecimento e visibilidade 20. Assim, no âmbito da discussão deste ensaio, pretende-se construir um diálogo entre duas categorias complexas de estudo – processos identitários e práticas alimentares em torno da alimentação escolar. Nesse sentido, não se pode escapar da discussão dos processos identitários do escolar como, por exemplo, a identidade de ser aluno, que, por seu turno, implica no desejo pela escolarização e de pertencimento à comunidade escolar, o que não é dado apenas pela condição de aluno, mas envolve submissão às normas e aos padrões escolares 21,22. Por outro lado, processos identitários envolvem práticas e interações permeadas por relações de poder que podem significar a construção de identidades desejadas e valorizadas, assim como de identidades inferiorizadas que limitam ou privam o acesso a bens sociais que, por sua vez, levam a processos de resistência e luta por outras identidades 16,18,19. Assim, este ensaio busca problematizar a alimentação escolar enquanto prática que contribui para a constituição de identidades escolares. Para tanto, foram feitas uma busca e análise de publicações sobre alimentação escolar no contexto das escolas públicas brasileiras, além de estudos sobre práticas alimentares e identidade. Assim, realizou-se uma revisão bibliográfica, não sistemática, sem recorte temporal, por meio das bases de dados Scopus, Web of Science, PubMed, LILACS e SciELO, bem como em outras bases disponíveis como o Catálogo de Teses e Dissertações da Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Capes), além de livros que tratassem sobre o tema. Foram utilizados como termos de busca: “school feeding”, “school eating”, “school food”, “school lunch”, “school meal” (“refeição escolar”, “merenda escolar”, “alimentação escolar”) e “identity” (“identidade”) em suas possíveis combinações. Foram usados como critério de seleção, a partir da leitura de títulos e resumos, estudos empíricos que incluíram, em seus objetivos ou resultados, a análise de práticas, percepções, representações e significados sobre a alimentação escolar, seja de estudantes ou de outros atores da comunidade escolar ou da gestão do PNAE. Foram selecionadas sete publicações brasileiras 7,8,9,10,11,23,24, às quais foram agregadas mais nove, identificadas a partir das próprias referências dessas publicações e também de buscas exploratórias, que, embora não mencionassem a temática de identidades, discutiram significados e práticas sobre a alimentação escolar que dialogavam com a constituição de identidades escolares 25,26,27,28,29,30,31,32,33. A busca também permitiu identificar seis estudos sobre refeições escolares em outros países que permitiram um diálogo com o contexto do PNAE 20,34,35,36,37,38. Outros estudos sobre identidade 16,18,19,39,40, PNAE 2,3,41, práticas alimentares e identidade 13,15, práticas escolares e identidade 21,22,42,43, também foram utilizados, assim como documentos e páginas eletrônicas do governo brasileiro. Entendendo que o escopo do tema incide sobre práticas alimentares de escolares no contexto do PNAE, cabe fazer breves considerações sobre os 16 estudos selecionados que apresentaram elementos capazes de dialogar com a temática de processos identitários de escolares. Os estudos, ainda que com objetivos distintos, permitiram delinear um conjunto de discursos e práticas de alunos, merendeiras, professores, diretores e nutricionistas que revelaram significados e representações sociais sobre a alimentação escolar praticada em instituições públicas da Educação Básica. Os contextos foram escolas públicas da capital e interior do Rio de Janeiro 8,11,33, da capital e interior de São Paulo 23,26, da capital Cad. Saúde Pública 2018; 34(4):e00142617 3 4 Silva EO et al. do Ceará 25, da capital e interior da Bahia 7,9,10,24,27,28,29,30, da capital de Goiás 32 e do interior dos estados do Pará, Piauí, Goiás, Minas Gerais e Santa Catarina 31. Os estudos se concentram no período de 2005-2016, convergindo com o recente momento de reestruturação do PNAE e criação da política de segurança alimentar e nutricional. Discute-se sobre as relações de poder no espaço escolar e seu potencial de influenciar na construção de identidades escolares, e, em seguida, em plano interpretativo, apresenta-se uma reflexão sobre processos identitários que podem ocorrer em torno da alimentação escolar praticada na escola, que foram denominados de: processos de sujeição, de resistência e de identificação. Reitera-se que os 16 estudos não são representativos da realidade da alimentação escolar no Brasil, entretanto, na medida em que trazem questões sobre alimentação escolar e identidades, que encontram ressonância com a trajetória da política de alimentação escolar, apontam questões sociais significativas que demandam reflexão. Discursos e práticas em torno da alimentação escolar praticada no cotidiano da escola A alimentação escolar, como realização do DHAA nas escolas, materializa-se, entre outras ações, pela garantia da oferta de refeições produzidas a partir dos princípios da alimentação saudável, valorizando costumes regionais e uso de alimentos oriundos da agricultura familiar; pelas ações de educação alimentar e nutricional e pela criação de Conselhos de Alimentação Escolar estaduais e municipais que possibilitam a participação da sociedade civil na política do PNAE 1. Embora seja uma árdua conquista, o arcabouço jurídico-normativo e resoluções operacionais não têm sido suficientes para sua realização e apropriação plena como direito. Entende-se que, no decorrer de décadas de existência do programa, formou-se uma cultura clientelista e assistencialista. A transformação dessa realidade para o desenvolvimento de uma cultura de direitos a ser desenvolvida pela política de segurança alimentar e nutricional fundamentada no DHAA requer a instituição de novas práticas e novas institucionalidades 44. Assim, o momento presente traduz-se em um período de transição marcado por esforços, avanços, resistências e ambiguidades em torno da alimentação escolar praticada no cotidiano das escolas. Nesse contexto, percebe-se que o assistencialismo, enquanto prática e discurso, ainda se perpetua no âmbito da alimentação escolar, entendendo-se que se imbrica com questões ideológicas, econômicas e políticas em torno do PNAE, e também da escola pública, ao longo de sua história. Destaca-se que os próprios autores dos estudos 7,8,9,10,11,23,25,26,27,28,29,32 interpretaram que os discursos dos entrevistados, sejam alunos, professores, merendeiras, diretores e nutricionistas, expressavam representações de conotação assistencialista sobre a alimentação escolar. Como também identificaram, a partir da observação do cotidiano da escola, a existência de práticas assistencialistas. Ressalta-se que esses estudos se situam em um pequeno e recente intervalo de tempo (2005-2016), portanto, representa o momento de mudança, visto que, somente a partir do ano de 2009, o PNAE é reestruturado, normativa e operacionalmente, como parte da política de segurança alimentar e nutricional, ainda que lutas e avanços tenham ocorrido ao longo de sua trajetória. A interpretação dos autores foi explicada a partir de diversos significados sobre o caráter assistencialista que recai sobre a alimentação escolar, a saber: produz um habitus – no sentido bourdieusiano, estruturas estruturadas e estruturantes 45 – com poder de orientar as práticas relacionadas ao comer na escola, à organização do trabalho pedagógico e à jornada escolar 25; reduz a alimentação escolar a uma suplementação nutricional 8,23,25,26,28,32; traduz a alimentação escolar como uma doação aos estudantes mais pobres, estabelecendo uma relação de poder sobre quem a recebe 11,27,28; dificulta o reconhecimento da alimentação escolar como direito de todos os escolares, não apenas para os mais pobres, contribuindo para situações de privação, discriminação e exclusão 10,11,25; dificulta a participação de pais e escolares na definição do cardápio e de reconhecer suas preferências e gostos 8,25,26. Esses significados instigam a interrogar se esse caráter assistencialista funciona como um mecanismo de poder capaz de inscrever, nos escolares, uma identidade de pobre e necessitado. Conquanto, importa distinguir assistência de assistencialismo. Considera-se que ações assistenciais sempre serão necessárias quando há violação ou privação de direitos. A assistência, vale ressaltar, está relacionada à obrigação estatal de prover direitos, mediante políticas públicas que assegurem a dignidade humana. Cad. Saúde Pública 2018; 34(4):e00142617 ALIMENTAÇÃO ESCOLAR E CONSTITUIÇÃO DE IDENTIDADES DOS ESCOLARES Ao mesmo tempo, a assistência se constitui em uma parceria entre poderes públicos e comunidade, possibilitando participação e recursos para a emancipação. Por sua vez, o assistencialismo distorce a ação assistencial, insinuando uma condição de retribuição à ação proporcionada. E, por isso, constitui-se em uma prática de dominação, produzindo corpos dóceis e manipuláveis 46. No âmbito das escolas, as relações e mecanismos de poder estão atrelados a vários tipos de hierarquias, as quais possuem o poder de influenciar as escolhas, as opiniões e a própria percepção dos estudantes sobre si próprios e, portanto, influenciar em seus processos identitários e nas suas percepções sobre a alimentação escolar. Assim, professores e funcionários, em suas interações cotidianas com crianças e adolescentes, desenvolvem, entre si, relações que podem ser constituídas de cuidados, afetos, admiração ou de dominação, influenciando processos identitários e formativos, isto é, em crenças, valores, hábitos, comportamentos e práticas. Nesse contexto escolar, os professores representam um sistema de poder – o sistema educacional, o conhecimento – e, portanto, são agentes com poder de definir e determinar identidades 43,47. Por exemplo, quando professores expressam representações sobre a alimentação escolar, associando seu consumo à condição de pobreza, podem estar contribuindo para legitimar uma identidade estigmatizada, desvalorizada e não desejada 11, visto que a identidade de ser pobre está associada a estigma, exclusão e culpabilização dos próprios sujeitos por sua condição de pobreza 48. Influências como essas podem contribuir para inscrever, em crianças e adolescentes, a naturalização de identidades de sujeição às estruturas de poder e de submissão às condições de pobreza e desigualdade. Entretanto, crianças e adolescentes não são passivos em suas relações com outros sujeitos e estruturas sociais, eles são agentes de seus próprios processos identitários e práticas alimentares. Por exemplo, no ambiente familiar, crianças de camadas populares participam das escolhas relacionadas às compras de gêneros alimentícios da família 49. No ambiente escolar, mesmo os estudantes mais pobres podem não aceitar a alimentação ofertada, conforme relata um dos estudos, no qual se observou que o consumo da alimentação escolar não dependia apenas da renda familiar, visto que, no estrato mais pobre, a aceitação estava em torno de 68,7% e, no estrato de maior renda, de 44,2% 31. Tal fato corrobora com a reflexão sobre a interação dos processos identitários dos alunos em sua relação com a alimentação escolar. Outros estudos relatam ações de rejeição, brincadeiras e desperdício com as refeições servidas, além de sentimento de indignação por não participarem da elaboração dos cardápios 10,27,28,29. São situações que podem significar resistência ao uso da alimentação escolar como um instrumento de dominação e controle sobre os escolares, de legitimação de identidades subalternas. Essas práticas e discursos sinalizam que a alimentação escolar, ao se materializar em refeições que compõem o cotidiano da escola, além de cumprir funções de alimentar e nutrir, comunica significados que constituem, de modo diverso, as identidades dos que dela participam. Alimentação escolar e constituição de identidades de sujeição, de resistência e de identificação Admite-se que processos identitários, sejam de escolares ou de qualquer outra categoria, não sejam fáceis de serem identificados e caracterizados, pois identidades são múltiplas, transitórias e contingentes. Reitera-se que identidade diz respeito à necessidade de reconhecimento e pertencimento social, ou seja, construção de vínculo entre indivíduo e sociedade 18, e, por isso, para crianças e adolescentes, a escola representa um espaço social importante para essa construção. Entretanto, esse espaço pode contribuir na construção de identidades emancipadoras ou apenas formar corpos dóceis para uso das estruturas de poder. Assim, cabe analisar como a alimentação escolar tem contribuído para esses processos. A partir do contexto de esforços de consecução da alimentação escolar como direito, mas permeado ainda por discursos e práticas assistencialistas, tenta-se elucidar processos de sujeição, de resistência e de identificação dos estudantes em relação à alimentação escolar praticada na escola. Mesmo com a melhoria dos níveis de segurança alimentar e nutricional da população brasileira, mesmo que a Organização das Nações Unidas tenha declarado que o Brasil saiu do Mapa Mundial da Fome em 2014 50, escolares das camadas populares ainda convivem com os constrangimentos de Cad. Saúde Pública 2018; 34(4):e00142617 5 6 Silva EO et al. ordem socioeconômica, instando-os a processos de sujeição tais como, se um aluno estiver faminto na escola e se deparar com uma preparação da alimentação escolar que não lhe agrada, a fome poderá impeli-lo a comer mesmo assim 30. Sendo assim, importa discutir como se dão esses processos de sujeição. Em um contexto mais geral, alguns estudos relatam práticas variadas dos alunos em relação à alimentação escolar: uma parte consome apenas a alimentação fornecida pela escola; outros comem esporadicamente, dependendo das suas preferências e das condições em que a refeição é ofertada; outros recusam e nunca comem 7,26,27,29. Ressalta-se que escolhas e práticas também variam de acordo com a situação socioeconômica dos alunos, variações que Oliveira & Santos 29 chamaram de “escala dos níveis de pobreza”: aqueles que tinham dinheiro para comprar uma merenda; aqueles que levavam merenda de casa; aqueles que consumiam a merenda escolar. Tais situações colocam a alimentação escolar também como um dispositivo de produção da diferença, construto inerente a processos identitários, mas, nesse caso, ela parece contribuir para classificar e inferiorizar aqueles que a consomem, haja vista relatos de situações em que escolares comem a alimentação escolar mesmo sem gostar, em um silêncio que comunica um constrangimento frente à impossibilidade de recusar, por não terem alternativas para se alimentar 28, situações também interpretadas com um consumo satisfeito e resignado 29, ou como um esforço de vencer a vergonha e o limiar do que não era aceito 30. Somam-se a esses relatos de constrangimento, as situações de privação, quando o aluno prefere ficar sem comer nada a ser identificado como pobre e necessitado se consumir a alimentação escolar 11. Essas são situações que podem ser entendidas como efeitos do uso da alimentação escolar como dispositivo de poder, revelado também nas representações sociais sobre a alimentação escolar presentes nos discursos de professores e merendeiras, tais como “obrigação; necessidade; o governo dá, e o aluno tem que receber; não precisa ter gosto” 28 (p. 42). Por isso, importa pensar que repercussões têm essas representações nas identidades dos escolares, pois parecem constituir processos de sujeição vivenciados cotidianamente ao longo de sua trajetória escolar, misturada aos esforços de ter sucesso em seus processos de escolarização e reconhecimento social, que, por sua vez, lhes colocam permanentemente em condições de submissão às normas da escola ou de subversão. São situações que integram uma teia de relações de poder inerente à escola, por ser ela mesma uma instituição de exercício do poder disciplinar que alcança a todos que a constituem. Conforme Foucault 51, o poder disciplinar diz respeito às formas de controle dos sujeitos por meio da disciplina, ou seja, mecanismos que, ao mesmo tempo, tornam-os obedientes e úteis às estruturas de poder. A saber, de um lado, os professores encarregados em fazer cumprir um currículo por meio de práticas escolares que produzam uma escolarização padronizada, definida por instâncias supranacionais que conjugam políticas educacionais com políticas econômicas 42, de outro, os escolares em intenso processo de construção de identidades, buscando reconhecimento e construção de vínculo social. Ambos, professores e escolares, constituindo-se agentes que interagem em um espaço social que lhes permite, ou não, o uso de recursos para atingir os seus objetivos. Nesse contexto, a alimentação escolar, além de um direito, constitui-se um recurso de poder, porém, à mercê de apropriações ou desapropriações moduladas por quem tem mais poder. Entretanto, onde há poder, há resistência 51. Processos de resistência em torno da alimentação escolar estão associados a reações a significados que desvalorizam tanto a alimentação escolar como o aluno que a consome. E situações concretas que afrontam a dignidade humana, tais como refeições produzidas e distribuídas em condições higiênicas inadequadas, também produzem processos de resistência. Como exemplos, temos o uso de colher e prato plásticos para escolares, sejam crianças ou adolescentes, submetendo-os a comportamentos infantilizados, ou ainda a ausência de refeitórios, obrigando os escolares a comerem em pé, andando ou sentados no chão. Atrelam-se a essas questões do comer na escola, os constrangimentos da sua estrutura física, como a existência ainda de escolas com muros altos e portões de estrutura compacta que as equiparam a presídios, além de outras situações descritas em vários estudos 25,28,29,30. Assim, processos de resistência se constituem como reação às representações sociais da alimentação escolar como uma comida para pobres, traduzida em práticas e estruturas 27, abarcando também a própria escola como uma estrutura de reprodução de identidades subalternas. Pode-se inferir também, como processos de resistência, as práticas de trazer alimentos para a escola, comprar alimentos da cantina ou de vendedores próximos ao entorno da escola ou ficar sem Cad. Saúde Pública 2018; 34(4):e00142617 ALIMENTAÇÃO ESCOLAR E CONSTITUIÇÃO DE IDENTIDADES DOS ESCOLARES comer 7,10,25,26. Essas situações trazem, para discussão, o direito à escolha no contexto do DHAA na escola. Estudos mostram se tratar de uma equação difícil de solucionar entre permitir opções e, ao mesmo tempo, não produzir discriminações. A exemplo do contexto estadunidense, foi visto que tanto a restrição de opções no próprio cardápio do Programa Nacional de Almoço Escolar dos Estados Unidos (National School Lunch Program – NSLP) quanto a existência de alternativas alimentares, como cantinas particulares no interior da escola, têm contribuído para processos de estigmatização de escolares 37,38. E por sua vez, produzem práticas de resistências, como descreve Nukaga 34, em um estudo com escolares imigrantes de origem coreana em duas escolas públicas americanas, situadas em um bairro de classe média, em que eles traziam refeições de casa com características étnicas e ainda realizavam trocas entre si. O autor interpretou essas práticas como formas de construção de identidades e marcação das diferenças em meio à homogeneização cultural produzida pela oferta de refeições padronizadas pelo NSLP, uma política que também possui uma relevância social, mas tem sido questionada em suas práticas no cotidiano escolar 37. Retomando ao contexto das escolas públicas brasileiras, outros processos de resistência são observados diante de situação de rejeição a certas preparações como o mingau e a sopa, provocada não apenas pelo sabor, aparência, horário, mas também porque os escolares as associam à alimentação de bebês e idosos 10, um comportamento que pode significar marcação da diferença e empenho dos escolares na construção de identidades compatíveis com sua idade, com seu grupo social. Outros pratos regionais, como arroz-doce e o mungunzá, podem estar associados a um possível baixo status social 10. Assim, se a alimentação escolar é composta de alimentos de baixo status social pode comunicar uma identidade de pobreza a quem a consome 8. Enfim, as recusas, a compra de outras merendas, o desperdício e até mesmo ficar sem comer podem representar processos de resistência ao uso da alimentação escolar como dispositivo de dominação e necessitam ser analisados à luz de uma cultura de direitos, considerando, nesse bojo, a dinâmica da cultura alimentar. Salienta-se que a cultura alimentar de uma dada comunidade é um processo dinâmico de trocas entre o local e o global e, por conseguinte, afetada por processos de homogeneização da cultura alimentar promovida pela globalização. E, nesse contexto, hábitos alimentares regionais, por vezes, são considerados anacrônicos, enquanto que certo conjunto de produtos industrializados e globais é associado à modernidade, à juventude, dessa forma, conferindo baixo status social aos alimentos regionais. Todavia, como efeito reverso da própria globalização da alimentação, surgem movimentos de resistência e de interesse em preservar e fomentar a agricultura local e valorizar a cultura alimentar regional 9. Nessa esteira, destaca-se que, em meio a processos de sujeição e resistências, também se pode perceber a contribuição da alimentação escolar na formação de uma cultura identitária alimentar, haja vista que os esforços em ofertar alimentos saudáveis e regionais pelo PNAE representam um avanço importante em comparação às décadas iniciais do programa, em que predominavam alimentos industrializados e “estranhos” à cultura alimentar 9. Nesse contexto, é que se destacam, sob o ponto de vista da política pública, dispositivos no arcabouço jurídico-normativo que contribuem para a formação de uma cultura identitária. A Lei no 11.947/2009 1 estabelece que os cardápios da alimentação escolar devam ser elaborados com gêneros alimentícios básicos e que, no mínimo 30%, sejam adquiridos da agricultura familiar e de suas organizações, respeitando não só as referências nutricionais, mas também a cultura alimentar da localidade 1. Com efeito, em 2010, 47,4% dos municípios brasileiros compraram alimentos da agricultura familiar, alcançando uma média de 22,7% de recursos aplicados nessa aquisição, considerada significativa para o primeiro ano em que a lei passou a vigorar 41. Destaca-se também que a Resolução no 38/2009 52 torna obrigatório servir frutas e verduras no cardápio semanal da alimentação escolar. As aquisições provenientes da agricultura familiar buscam associar-se aos hábitos alimentares dos brasileiros, em suas diversas matizes, conforme a região, pois é ela que produz e contribui com um volume significativo dos alimentos básicos como a farinha de mandioca, o feijão, o milho, o café, o arroz, o leite, as carnes e o trigo 41. Cabe salientar que o incentivo à compra de gêneros da agricultura familiar ainda propicia a inclusão de alimentos não habituais produzidos na região como, por exemplo, o iogurte, considerado, ao mesmo tempo, saudável e símbolo da modernidade e, ainda que não faça parte do hábito alimentar das classes populares, possui boa aceitação pelos escolares 10, possivelmente pelo status social conferido pela publicidade da agroindústria. Cad. Saúde Pública 2018; 34(4):e00142617 7 8 Silva EO et al. Nesse contexto, chama a atenção a preferência de escolares por preparações salgadas, incluindo comidas típicas à base de arroz e feijão, relatadas em um estudo que envolveu 55 municípios baianos 27. Assim, ainda que os estudantes considerem estranho servirem preparações com características de almoço ou jantar na hora da merenda, 10 horas da manhã e 3 horas da tarde 7, possivelmente, a preferência por essas preparações está associada à cultura alimentar local e nacional, construída desde a infância, que ainda persiste, mesmo com a inclusão de novos alimentos da modernidade. Considera-se ainda que a mistura feijão com arroz constitui um símbolo identitário da cultura alimentar brasileira 40, é uma comida conhecida, familiar, consagrada pela cultura e que resulta em convergência do prazer sensorial com o prazer de comer a mesma comida, partilhando a mesma identidade. Todavia, para um escolar, comer uma comida de almoço ou jantar na hora da merenda também pode representar ainda situações de pobreza, em que se aceita a merenda como um mini almoço ou mini jantar. Pode-se também considerar que essa aceitação é favorecida pelas mudanças da alimentação na contemporaneidade, a exemplo da flexibilização das refeições, inclusive dos horários 13, situação que pode ser vista com naturalidade por adolescentes. Assim, as refeições do PNAE podem produzir reações diversas, em um momento, produzir o estranhamento, que pode levar à rejeição, em outro, a aceitação pela familiaridade e identificação com a preparação, propiciando a comensalidade e o desfrute de uma comida típica. Em outra direção, escolares de dois municípios baianos preferem mais refeições do tipo biscoito com suco 10,30. Nesse caso, pode-se observar uma aproximação das características da refeição ao que os escolares consideram, de fato, uma merenda, rápida e leve, que não atrapalhe brincar durante o recreio 7,10. Isso se deve ao fato de que preferências alimentares dos escolares podem estar associadas à publicidade de um conjunto de alimentos reconhecidos como marca identitária de um estilo de “ser jovem”, denominados de “besteiras”, que incluem preponderantemente produtos industrializados como biscoitos, refrigerantes, hambúrguer, cachorro-quente, pizza, sorvete, salgados, doces, chocolate, batata frita, achocolatados, nuggets, entre outros 53. Ainda se deve levar em conta que vários produtos industrializados já fazem parte do hábito alimentar do brasileiro, a exemplo dos biscoitos doces e salgados 54,55, como resultado do desenvolvimento das indústrias de alimentos ocorrido na primeira metade do século XX. Diferentemente do início do PNAE, nos anos 1950, em que predominavam os formulados oriundos da indústria americana, na atualidade, os alimentos industrializados não causam estranhamento. Ao contrário, são símbolos de modernidade e praticidade, amplamente promovidos pela publicidade, conferindo status social. Santos & Soares 56 apontam a necessidade de buscar compreender como se dá a construção de significados em torno dos produtos alimentícios e suas marcas, assim como das estratégias de identificação dos sujeitos a elas. Nessa direção, Andrade 24 observou que as escolhas alimentares de escolares adolescentes de uma escola pública em Salvador, Bahia, são determinadas pela fome, prazer e gosto, mas fortemente associadas às necessidades de visibilidade, de convívio social e de pertencimento. Semelhantemente, ainda que seja outra realidade, mas considera-se haver similaridades nos processos identitários de escolares, Stead et al. 35 apontaram que as escolhas alimentares de escolares adolescentes de duas escolas públicas inglesas levavam, em conta, o status social e a popularidade das marcas dos alimentos, e escolhas que representem danos à própria imagem perante seus grupos sociais podem ser prejudiciais ao escolar. Em uma direção semelhante, Vargas et al. 33, em um estudo de intervenção visando à prevenção de obesidade em escolares de duas escolas públicas em Niterói, Rio de Janeiro, relataram que alunos que referiram gostar de frutas e verduras foram veementemente contestados por outros colegas que enfatizavam suas preferências por alimentos tidos como não saudáveis. O estudo sinaliza interfaces entre necessidades de pertencimento social, construção identitária, publicidade e formação de hábitos alimentares de adolescentes. Isso instiga a refletir sobre o poder da publicidade na formação das práticas alimentares e como isso é traduzido na relação dos estudantes com a alimentação escolar. Assim, preferências por preparações regionais e também por alimentos propagados pela mídia não necessariamente expressam ambiguidades nos processos identitários dos escolares, e sim uma aderência às tendências das práticas alimentares na modernidade, caracterizada, entre outras coisas, pela flexibilização, e que, de certa forma, a alimentação escolar contribui nesse processo. Ressalta-se que a coexistência de identificação com alimentos da cultura alimentar nacional e com alimentos que ultrapassam as fronteiras culturais Cad. Saúde Pública 2018; 34(4):e00142617 ALIMENTAÇÃO ESCOLAR E CONSTITUIÇÃO DE IDENTIDADES DOS ESCOLARES se nota em outros contextos escolares com programas de alimentação escolar. Theodore et al. 36, ao estudarem, na Cidade do México, representações sobre a alimentação em escolas beneficiárias de um programa de refeições escolares, identificaram que os escolares, ao mesmo tempo, revelavam um caráter identitário mediante o gosto e o prazer pelas comidas tradicionais como tacos, tortilhas e tamales, como também pelo consumo de alimentos globalizados como a pizza e o refrigerante. Assim, as ambiguidades percebidas na relação dos escolares com a alimentação escolar são indícios de mudanças, traduzidos em processos de identificações, expressadas pelo consumo de preparações de suas preferências, reunindo o regional e o global, em processos de sujeições ante a necessidade de se alimentar frente à restrição de alternativas e em processos de resistências que comunicam o desejo de “ser mais”, como dizia Paulo Freire 57, ao se referir ao processo de enfrentamento social e político da condição de pobreza como uma relação de opressor-oprimido. Considerações finais A discussão da alimentação escolar como um elemento constitutivo de identidades escolares foi motivada pela problemática levantada em alguns estudos sobre a persistência de discursos e práticas de caráter assistencialista que reduziam a alimentação escolar à comida para pobres. A alimentação escolar, ao ser instituída em meio a políticas de alimentação e nutrição como uma ação para minimizar a desnutrição, foi reduzida, por muito tempo, a uma ação assistencial focalizada para grupos vulneráveis socialmente, traduzidos, no senso comum, como uma comida para alunos pobres e necessitados. Todavia, sua instituição como um direito de todos os escolares, fruto de lutas e movimentos sociais redundaram em sua implementação como uma estratégia de segurança alimentar e nutricional fundamentada no DHAA. Entretanto, a alimentação escolar se dá em um espaço de relações de poder historicamente legitimadas, também produtor de práticas e identidades, onde se percebe uma ressonância das práticas escolares com a prática da alimentação escolar. Ou seja, o papel ambíguo da escola, enquanto dispositivo de poder e sujeição e, ao mesmo tempo, de possibilidade de construção de identidades emancipatórias, também influencia na dinâmica da alimentação escolar como constitutiva de identidades. Por sua vez, as ambiguidades percebidas, tanto na execução da alimentação escolar como nas práticas alimentares dos estudantes, dão indícios de mudanças no paradigma do pensar e fazer a alimentação escolar. Nesse contexto, emergem elementos para problematizar a alimentação escolar: de um lado, a existência de práticas que ainda a constituem como um dispositivo de conservação social e manutenção das desigualdades sociais, no sentido bourdieusiano e foucaultiano, e, de outro, esforços para propiciá-la como um direito possibilitam outros processos identitários para além da sujeição. Portanto, a alimentação escolar, apesar da persistência do assistencialismo, pode representar, para os escolares das classes populares, uma possibilidade de projetar outras identidades que não a subalterna, como processos de resistência às práticas de dominação e controle e processos de construção de identidade alimentar que agreguem criticamente o tradicional e o moderno, o local e o global. Compreender a alimentação escolar como um constitutivo de identidades escolares pode instrumentalizar os sujeitos para questionar as identidades que as estruturas de poder querem legitimar e questionar o sistema de representação que lhes dá sustentação, como sinaliza Silva 47, além de contribuir para a construção de conhecimentos que possam instrumentalizar tanto o planejamento da política do PNAE como também os escolares e todos da comunidade escolar. Convém ressaltar que as práticas alimentares, incluindo a alimentação escolar, sendo constitutivas dos processos identitários, implicam em interações que também afetam a saúde do corpo, requerendo estudos que elucidem as interfaces entre práticas alimentares, identidades e saúde. Cad. Saúde Pública 2018; 34(4):e00142617 9 10 Silva EO et al. Colaboradores Referências E. O. Silva e L. Amparo-Santos contribuíram na concepção, delineamento e escrita do ensaio. M. D. Soares colaborou no delineamento e revisão crítica. 1. Agradecimentos 2. 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A feira livre sob um olhar etnográfico. In: Freitas MCS, Fontes GAV, Oliveira N, organizadoras. Escritas e narrativas sobre alimentação e cultura. Salvador: EDUFBA; 2008. p. 129-48. 49. Soares MD, Trad LAB. Cenas e sentidos do cuidado alimentar de crianças no mundo cotidiano. In: Freitas MCS, Amparo-Santos L, Fontes GAV, Ramos L, organizadoras. Narrativas sobre cuidado alimentar e o comer na escola. Salvador: EDUFBA; 2016. p. 13-36. 50. Portal Brasil. Relatório indica que Brasil saiu do Mapa Mundial da Fome. http://www.brasil.gov.br/governo/2014/09/relatorio-indicaque-brasil-saiu-do-mapa-mundial-da-fomeem-2014 (acessado em 05/Jul/2017). Cad. Saúde Pública 2018; 34(4):e00142617 51. Foucault M. Microfísica do poder. 16a Ed. Rio de Janeiro: Edições Graal; 2001. 52. Ministério da Educação. Resolução/CD/ FNDE no 38, de 16 de julho de 2009. Dispõe sobre o atendimento da alimentação escolar aos alunos da educação básica no Programa Nacional de Alimentação Escolar (PNAE). Diário Oficial da União 2009; 17 jun. 53. Serra GMA, Araújo IS, Santos EM. Comer com os olhos: discursos televisivos e produção de sentidos na promoção da saúde nutricional de adolescentes. RECIIS (Online) 2012; 6:1-14. 54. Souza AM, Pereira RA, Yokoo EM, Levy RB, Sichieri R. Alimentos mais consumidos no Brasil: Inquérito Nacional de Alimentação 2008-2009. Rev Saúde Pública 2013; 47 Suppl 1:190s-9s. 55. Levy RB, Castro IRR, Cardoso LO, Tavares LF, Sardinha LMV, Gomes FS, et al. Consumo e comportamento alimentar entre adolescentes brasileiros: Pesquisa Nacional de Saúde do Escolar (PeNSE), 2009. Ciênc Saúde Coletiva 2010; 15 Suppl 2:3085-97. 56. Santos LAS, Soares MD. “Abrindo a felicidade”: interações entre os discursos publicitário e saudável no campo da alimentação e nutrição. In: Ferreira FR, Freitas RF, Prado SD, Carvalho MCVS, organizadores. Consumo, comunicação e arte. Curitiba: Editora CRV; 2015. p. 259-74. 57. Freire P. Pedagogia do oprimido. São Paulo: Paz e Terra; 1997. ALIMENTAÇÃO ESCOLAR E CONSTITUIÇÃO DE IDENTIDADES DOS ESCOLARES Abstract Resumen This essay aims to analyze school feeding as a practice contributing to the establishment of school identities. The point of departure is a nonsystematic review of publications on school feeding and identities in Brazil’s public schools. The discussion begins with the persistence of paternalistic discourses and practices that reduce school feeding to food for the poor, observed in the studies. The meanings in this paternalistic approach suggest that it appears to function as a power mechanism to brand the schoolchildren with an identify of poverty and inferiority. This understanding is situated in the prevailing power relations in schools, in the exercise of disciplinary power and its potential to produce identities, as well as the practices of resistance resulting from such power in the school feeding context. The schoolchildren are also agents of their own identity processes, considering that their relations with school feeding involve processes not only of subordination but also of resistance and active identity-building, combining the traditional with the modern, the local with the global, among other aspects. Even the ambiguities in this scenario are signs of a paradigm shift in the planning and practice of school feeding, raising elements to analyze it: on the one hand, as a device for the maintenance of social inequalities, and on the other, efforts and actions to support school feeding as an essential right and factor for emancipatory identities. Este estudio busca problematizar la alimentación escolar, como práctica que contribuye a la configuración de identidades escolares. Se parte de una revisión bibliográfica, no sistemática, de publicaciones sobre alimentación escolar e identidades en el contexto de las escuelas públicas brasileñas. Se discute, inicialmente, la persistencia de discursos y prácticas de carácter asistencialista, que reducían la alimentación escolar a una comida para pobres, cuestión observada en diversos estudios. El concepto de ese carácter asistencialista concurre hacia la comprensión del fenómeno y su interpretación, como un mecanismo de poder capaz de adscribir a los escolares una identidad de pobreza e inferioridad. Esta interpretación está imbricada en las relaciones de poder existentes en la escuela, en el ejercicio del poder disciplinario y su potencial de producir identidades, así como en las prácticas de resistencia, provenientes de esos poderes en el contexto de la alimentación escolar. Se considera incluso que los escolares sean agentes de sus propios procesos identitarios, a la vista que, en su relación con la alimentación escolar, se evidencian, además de procesos de subordinación, también procesos de resistencia y de construcción activa de identidad alimentaria, en los que se agrega lo tradicional y lo moderno, lo local y lo global, entre otros aspectos. A pesar de que se perciban ambigüedades en este escenario, se entiende que son indicios de transformaciones en el paradigma de la conceptualización y puesta en práctica de la alimentación escolar, llevando consigo elementos para problematizarla: por un lado, todavía, como dispositivo de soporte de las desigualdades sociales y, por otro, los esfuerzos y acciones para propiciarla como un derecho y como promotora de identidades emancipadoras. School Feeding; Feeding Behavior; Food and Nutrition Security; Social Assistance Alimentación Escolar; Conducta Alimentaria; Seguridad Alimentaria y Nutricional; Asistencia Social Recebido em 17/Ago/2017 Versão final reapresentada em 12/Jan/2018 Aprovado em 02/Fev/2018 Cad. Saúde Pública 2018; 34(4):e00142617 13
https://openalex.org/W4395089667	https://jurnal.minartis.com/index.php/jkomdis/article/download/1579/1370	Indonesian	null	Peminat Radio dan Podcast Kalangan Generasi Z Saat Berkendara	Jurnal Ilmu Komunikasi dan Media Sosial	2,024	cc-by	10,797	Abstrak Kemajuan teknologi telah menjadi perhatian semua manusia, konvergensi media mempercepat transformasi pasar, penciptaan produk dan layanan inovatif untuk penikmat yang semakin tanggap dan cerdas. Dari Konvergensi Media inilah penelitian dilakukan untuk menyelidiki bagaimana dampak konvergensi media di bidang Audio visual terhadap minat dengar Generasi Z seperti audio on-demand, platform streaming, dan monetisasi podcast terhadap industri radio dan podcast secara keseluruhan. Penelitian perubahan minat dengar Generasi Z ini mengeksplorasi perubahan minat dengar Radio daripada Podcast dan menilai faktor-faktor apa saja yang menjadi pendukung perubahan minat dengar tersebut. Penelitian dilakukan untuk memahami bagaimana perkembangan teknologi seperti platform audio digital, dan integrasi platform media sosial mempengaruhi distribusi konten audio seperti podcast. Mengeksplorasi perilaku konsumen, kebiasaan mendengarkan, dan preferensi audiens terutama dalam kalangan Generasi Z terkait dengan radio dan podcast. Bagaimana faktor-faktor seperti jenis konten, gaya penyampaian, dan kecepatan akses mempengaruhi minat mendengarkan podcast dan stasiun radio online. Menjelajahi sejauh mana ketersediaan konten audio digital seperti podcast dan radio online mempengaruhi kepuasan pendengar. mempelajari faktor-faktor yang mempengaruhi pemilihan konten, kesesuaian dengan minat audiens, dan bagaimana respons pendengar terhadap konten yang disajikan serta kecenderungan komoditas dengan tujuan yang berbeda. Keywords : Komunikasi, Teknologi dan Masyarakat, Generasi Z, Radio, Podcast, Konvergensi Media. This work is licensed under Creative Commons Attribution License 4.0 CC-BY International license Konvergensi Radio Terhadap Podcast di Kalangan Generasi Z Konvergensi Radio Terhadap Podcast di Kalangan Generasi Z Nindyo Andayaning Pandusaputri1, Rachmat Bintang Ramadhan Mokodompit2, Irwansyah3 Mahasiswa Magister Ilmu Komunikasi, Universitas Pelita Harapan, Jakarta, Indonesia, 01689230026@student.uph.edu Mahasiswa Magister Ilmu Komunikasi, Universitas Pelita Harapan, Jakarta, Indonesia, 01674230001@student.uph.edu Staf Pengajar Ilmu Komunikasi FISIP UI dan MIKOM FISIP UPH, Jakarta, Indonesia, dr.irwansyah.ma@gmail.com Submitted: 16-03-2024, Reviewed: 17-03-2024, Accepted 20-01-2024 Abstract Technological advancement has become a concern for all humans, media convergence accelerates market transformation, the creation of innovative products and services for an increasingly perceptive and discerning audience. It is from this Media Convergence that research was conducted to investigate how the impact of media convergence in the Audio visual field on Generation Z listening interest such as audio on-demand, streaming platforms, and podcast monetisation on the radio and podcast industry as a whole. This Generation Z listening interest change research explores the changes in listening interest in Radio rather than Podcasts and assesses what factors support the changes in listening interest. Research was conducted to understand how technological developments such as digital audio platforms, and the integration of social media platforms affect the distribution of audio content such as podcasts. Exploring consumer behaviour, listening habits, and audience preferences especially within Generation Z in relation to radio and podcasts. How factors such as content type, delivery style, and speed of access affect interest in listening to podcasts and online radio stations. Explore the extent to which the availability of digital audio content such as podcasts and online radio affects listener satisfaction. study the factors that influence content selection, suitability to audience interests, and how listeners respond to the content presented as well as commodity tendencies with different purposes. Keywords : Komunikasi, Teknologi dan Masyarakat, Generasi Z, Radio, Podcast, Konvergensi Media. Jurnal Ilmu Komunikasi Dan Media Sosial (JKOMDIS) Vol. 4 No. 1 Edisi Januari -April 2024 Hal. 179-192 DOI : https://doi.org/10.47233/jkomdis.v3i3.1579 Jurnal Ilmu Komunikasi Dan Media Sosial (JKOMDIS) Vol. 4 No. 1 Edisi Januari -April 2024 Hal. 179-192 DOI : https://doi.org/10.47233/jkomdis.v3i3.1579 ISSN : 2807-6087 Jurnal Ilmu Komunikasi Dan Media Sosial (JKOMDIS) Vol.04 No. 01 Edisi Januari - April 2024 This work is licensed under Creative Commons Attribution License 4.0 CC-BY International license PENDAHULUAN Untuk memahami lebih dalam tentang faktor-faktor yang mempengaruhi perubahan minat pendengar dari radio ke podcast merupakan topik utama dalam penelitian ini. Bagaimana identifikasi preferensi? bagaimana kebiasaan mendengarkan Generasi Z? dan apa motivasi pendengar dalam memilih antara radio dan podcast? serta bagaimana perkembangan teknologi dan tren terkini dalam industri media audio? Dengan memahami pergeseran minat pendengar, dapat diidentifikasi bagaimana teknologi seperti platform audio digital, aplikasi streaming, dan 179 Jurnal Ilmu Komunikasi Dan Media Sosial (JKOMDIS) Vol.04 No. 01 Edisi Januari - April 2024 Jurnal Ilmu Komunikasi Dan Media Sosial (JKOMDIS) Vol. 4 No. 1 Edisi Januari -April 2024 Hal. 179-192 DOI : https://doi.org/10.47233/jkomdis.v3i3.1579 ISSN : 2807-6087 kemajuan podcast memengaruhi cara pendengar mengkonsumsi konten audio (Ronan, 2016). Dengan memahami perubahan minat pendengar tersebut, produsen konten dan penyiar radio/podcast dapat mengadaptasi strategi konten, mencari ciri khas dan menginovasi layanan mereka untuk lebih sesuai dengan kebutuhan dan preferensi pendengar (Aziz, M.A, 2021). Tujuan Penelitian ini adalah untuk mengetahui dampak dari konvergensi media dalam ranah Radio terhadap Podcast, apa saja faktor perubahan minat Generasi Z dalam mendangarkan radio dan podcast, kemudian apakah alternatif media selain radio dan podcast, serta bagaimana cara memahami dan beradaptasi dalam peralihan minat dengar tersebut. Peralihan minat dari radio dan podcast pada Generasi Z dapat disebabkan oleh beberapa faktor yang mana dari faktor-faktor tersebut menimbulkan efek terhadap Generasi sebelum Generasi Z yang cenderung lebih memilih radio daripada podcast. Faktor dan alasan itulah yang akan dibahas lebih lanjut untuk menghasilkan analisis bagaimana peralihan minat dengan radio dan podcast pada Generasi Z menggunakan metode analisis social construction of technology (Pinch & Bijker, 1984). Commented [RM1]: menyambung ke paragraf berikut tentang scot METODE PENELITIAN Diagram Sistematis Literatur Reviu (SLR) Identifikasi Pencarian Penyaringan Kelayakan Ekstraksi Data Penilaian Kualitas Sintesis Data Hasil dan Kesimpulan Pelaporan Pembaruan Co Ekstraksi Data Pembaruan Pelaporan Dengan metode SLR ini diharapkan dapat menghasilkan Informasi berkualitas, pemahaman yang mendalam, mengidentifikasi kesenjangan pengetahuan, serta penelitian yang lebih terfokus, selain itu dapat mendukung pengambilan kesimpulan dari penelitian. Dengan metode SLR ini diharapkan dapat menghasilkan Informasi berkualitas, pemahaman yang mendalam, mengidentifikasi kesenjangan pengetahuan, serta penelitian yang lebih terfokus, selain itu dapat mendukung pengambilan kesimpulan dari penelitian. METODE PENELITIAN Commented [NP2R1]: ini ternyata bukujadi gue ga nemu doina, cara masukin citationnya gmn? Metode tinjauan literatur dipilih sebagai pendekatan penelitian dalam studi ini (Snyder, 2019). Standar PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) 2020 ditaati dalam tinjauan sistematis ini, yang menjamin penyajian temuan studi yang menyeluruh dan terbuka. Penelitian literatur mengkaji dan memahami berbagai teori dan konsep terkait yang diambil dari literatur yang ada, terutama makalah yang telah diterbitkan dalam berbagai publikasi ilmiah. Gagasan dan teori yang akan menjadi dasar penelitian ini dibangun di atas fondasi yang kuat yang disediakan oleh evaluasi literatur ini. Tinjauan literatur merupakan tahap penting dalam penelitian, terutama penelitian akademis yang mencoba mengembangkan aspek teoritis dan praktis dari topik yang diteliti, seperti yang dijelaskan oleh Sujarweni (2014). Proses menemukan, menilai, dan menginterpretasikan semua penelitian yang saat ini tersedia dan berkaitan dengan perumusan masalah atau area topik yang sedang dipertimbangkan dikenal sebagai tinjauan literatur sistematis, atau SLR. Proses menemukan, mengevaluasi, dan menginterpretasikan semua data penelitian yang tersedia dengan tujuan menawarkan jawaban atas pertanyaan tertentu dikenal sebagai tinjauan sistematic literature review (SLR). Tujuan dari penelitian SLR adalah untuk menemukan, menilai, dan memahami semua temuan penelitian yang relevan. Analisis semua temuan studi terkait yang berkaitan dengan topik, isu, atau fenomena penelitian tertentu. Pertanyaan subjek tertentu atau kejadian yang mengkhawatirkan (Kitchenham & Brereton, 2009). Jurnal Ilmu Komunikasi Dan Media Sosial (JKOMDIS) Vol.04 No. 01 Edisi Januari - April 2024 180 (Kitchenham & Brereton, 2009). Diagram Sistematis Literatur Reviu (SLR) Dengan metode SLR ini diharapkan dapat menghasilkan Informasi berkualitas, pemahaman yang mendalam, mengidentifikasi kesenjangan pengetahuan, serta penelitian yang lebih terfokus, selain itu dapat mendukung pengambilan kesimpulan dari penelitian. HASIL DAN PEMBAHASAN Jurnal ini mengeksplorasi konstruksi sosial dari teknologi, menganalisis data dari 31 jurnal penelitian sebelumnya, menyoroti sesuai judul penelitian yang dampaknya terhadap kehidupan sosial, terutama dalam Konvergensi Radio di Kalangan Generasi Z. Analisis Literatur Reviu dari 31 Jurnal No. Nama Pengarang Q1 Q2 Q3 Q4 Identifikasi Pencarian Penyaringan Kelayakan Ekstraksi Data Penilaian Kualitas Sintesis Data Hasil dan Kesimpulan Pelaporan Pembaruan Commented [RM3]: Pake B.Indo ato B.Ing yg benar? (Kitchenham & Brereton, 2009). Diagram Sistematis Literatur Reviu (SLR) Identifikasi Pencarian Penyaringan Kelayakan Ekstraksi Data Penilaian Kualitas Sintesis Data Hasil dan Kesimpulan Pelaporan Pembaruan Co (Kitchenham & Brereton, 2009). HASIL DAN PEMBAHASAN HASIL DAN PEMBAHASAN Jurnal ini mengeksplorasi konstruksi sosial dari teknologi, menganalisis data dari 31 jurnal penelitian sebelumnya, menyoroti sesuai judul penelitian yang dampaknya terhadap kehidupan sosial, terutama dalam Konvergensi Radio di Kalangan Generasi Z. 180 Jurnal Ilmu Komunikasi Dan Media Sosial (JKOMDIS) Vol. 4 No. 1 Edisi Januari -April 2024 Hal. 179-192 DOI : https://doi.org/10.47233/jkomdis.v3i3.1579 ISSN : 2807-6087 1. The Social Construction of Facts and Artefacts (Pinch, T., & Bijker, W., 1984) Y Y U U 2. A Systematic Review of Systematic Review Process Research in Software Engineering (Kitchenham & Brereton, 2009) U Y Y N 3. Literature review as a research methodology: An overview and guidelines (Snyder, H, 2019) Y U N Y 4. Metodologi Penelitian (Sujawerni, V. W., 2014) Y Y U U 5. (Daramola's, 2003) U Y Y N 6. The Interpersonal Communication Book (DeVito, 2009) Y U U Y 7. (Tomecek, 2009) U Y Y N 8. Handbook of Public Policy Analysis: Theory, Politics, and Methods (Fischer et al, 2007) Y Y U U 9. The Nature of Human Communication (Hewitt, 2011) U N N Y 10. (Viceno, 2004) Y U Y U 11. Management and Socio-Humanities Intercultural Adaptation: The Case Of International Student Exchange (Flanja dan Gaz, 2011) N U Y U 12. Social Psychology of the Digital Age: The Interpersonal Neuroscience of Mediated Communication (Spape & Rajava, 2014) Y U N U 13. The Emergence of a Hazardous Concept (Leo Marx, 1997) Y Y U Y 14. The Culture of Technology (Pacey,1983) N U N Y 15. Understanding Media The Extensions of Man (McLuhan, 1964) Y Y U N 16. McLuhan and Media Ecology (Levinson, 1999) U Y Y U 17. Understanding New Media (Siapera, 2017) N Y Y U 18. The Language of New Media (Manovich, 2001) Y Y Y U 19. Mediamorphosis Understanding New Media (Fidler, 1997) N U Y U 20. Meaning in Technology (Pacey, 1999) Y Y U U 21. Memahami Masyarakat Dan Perspektifnya (Prasetyo, 2020) Y Y Y U 22. Theories Of The Information Society (Webster & Frank, 2014) Y U Y U 23. Industry4.0 vs Society5.0 (Suherman, 2020) U N U Y 24. Relevansi Industri 4.0 dan Society 5.0 Terhadap Pendidikan Di Indonesia (Teknowijoyo, 2022) Y Y U N 25. Everything You’ve Wanted to Know About Gen Z But Afraid to Ask. (Brown, 2020) U Y U U 26. Ketika ahli etologi menemukan bahwa isyarat non-verbal yang terkait dengan kawin, bermain, berburu, dan mencari makan pada hewan disebut perilaku sosial, istilah "makhluk sosial" tidak menjadi inti dari kemanusiaan (Onyeator & Okpara, 2019). Dengan demikian, konsep makhluk sosial berkaitan dengan evolusi masyarakat. Menurut mara, tidak Diagram Hasil Kajian Literatur Dari hasil 31 penelitian terdahulu, dapat disimpulkan bahwa berdasar rumusan masalah mengenai kesesuaian konsep SCOT dengan judul terdapat 15 penelitian yang sudah sesuai, 5 penelitian yang belum sesuai, dan 10 penelitian yang tidak memiliki kejelasan. Selain itu, kepaduan antara metodenya dari penelitian terdahulu dapat dihasilkan sebanyak 16 penelitian yang sudah sesuai, 5 penelitian belum sesuai, dan 9 penelitian yang tidak memiliki kejelasan. Selanjutnya, berdasar indikator mengenai bahan dasar penelitian terdapat 13 penelitian yang sudah sesuai, 4 penelitian yang belum sesuai, dan 14 penelitian yang tidak memiliki kejelasan selanjutnya yang terakhir terkait nilai kritis yang terdapat dalam penelitian, dihasilkan bahwa 15 penelitian memiliki kritisisasi terhadap judul, 10 penelitian belum memiliki nilai kritis, dan 6 penelitian lainnya belum memiliki kejelasan. Untuk mempermudah pemahaman lebih lanjut, dijelaskan melalui lima penelitian yang dianggap lebih relevan untuk bahan pembanding dalam kajian literatur. Untuk menunjukkan bahwa fokus penelitian sesuai dengan teori SCOT, mereviu dari penelitian menggunakan konsep SCOT. Komunikasi, Teknologi dan Masyarakat Diagram Hasil Kajian Literatur Dari hasil 31 penelitian terdahulu, dapat disimpulkan bahwa berdasar rumusan masalah mengenai kesesuaian konsep SCOT dengan judul terdapat 15 penelitian yang sudah sesuai, 5 penelitian yang belum sesuai, dan 10 penelitian yang tidak memiliki kejelasan. Selain itu, kepaduan antara metodenya dari penelitian terdahulu dapat dihasilkan sebanyak 16 penelitian yang sudah sesuai, 5 penelitian belum sesuai, dan 9 penelitian yang tidak memiliki kejelasan. Selanjutnya, berdasar indikator mengenai bahan dasar penelitian terdapat 13 penelitian yang sudah sesuai, 4 penelitian yang belum sesuai, dan 14 penelitian yang tidak memiliki kejelasan selanjutnya yang terakhir terkait nilai kritis yang terdapat dalam penelitian, dihasilkan bahwa 15 penelitian memiliki kritisisasi terhadap judul, 10 penelitian belum memiliki nilai kritis, dan 6 penelitian lainnya belum memiliki kejelasan. Untuk mempermudah pemahaman lebih lanjut, dijelaskan melalui lima penelitian yang dianggap lebih relevan untuk bahan pembanding dalam kajian literatur. Untuk menunjukkan bahwa fokus penelitian sesuai dengan teori SCOT, mereviu dari penelitian menggunakan konsep SCOT. Jurnal Ilmu Komunikasi Dan Media Sosial (JKOMDIS) Vol.04 No. 01 Edisi Januari - April 2024 HASIL DAN PEMBAHASAN Generation Z and its implications for companies (Francis & Hoefel, 2018) Y N Y U 27. Generation And Their Acceptance of Technology (Linnes & Metcalf, 2017) N U Y Y 28. Eksistensi Jurnalisme Di Era Media Sosial (Hamna, 2017) Y N U Y 29. Attenuations in Wireless Radio Communication (Ado, 2020) U N Y Y 30. Science and the Internet: Introduction (Tokar, 2012) U Y U N 31. Sensus Penduduk Indonesia (BPS, 2020) U Y U Y ISSN : 2807-6087 Jurnal Ilmu Komunikasi Dan Media Sosial (JKOMDIS) Vol.04 No. 01 Edisi Januari - April 2024 181 Keterangan : Q1 : Penyebaran konsep SCOT sesuai dengan judul penelitian Q2 : Penyebaran konsep SCOT berdasarkan metode penelitian yang digunakan Q3 : Apakah Teori SCOT dapat digunakan sebagai bahan dasar penelitian Q4 : Apakah terdapat nilai kritis dalam penelitian Y : Yes 181 Jurnal Ilmu Komunikasi Dan Media Sosial (JKOMDIS) Vol. 4 No. 1 Edisi Januari -April 2024 Hal. 179-192 DOI : https://doi.org/10.47233/jkomdis.v3i3.1579 Jurnal Ilmu Komunikasi Dan Media Sosial (JKOMDIS) Vol. 4 No. 1 Edisi Januari -April 2024 Hal. 179-192 DOI : https://doi.org/10.47233/jkomdis.v3i3.1579 ISSN : 2807-6087 N : No U : Unclear P i Pentingnya tinjauan dan studi literatur ditekankan dalam jurnal ini sebagai pendekatan metodis untuk memahami dan merangkai gagasan tentang konstruksi sosial dalam teknologi dan media baru. Penyusunan kerangka konsep dan teori teori yang akan digunakan dalam penelitian ini juga dibantu oleh tinjauan pustaka. Untuk mengeksplorasi implikasi teori SCOT pada penggemar radio dan podcast Generasi Z penelitian ini memiliki dasar yang kuat berkat teknik studi literatur. Diagram Hasil Kajian Literatur Dari hasil 31 penelitian terdahulu, dapat disimpulkan bahwa berdasar rumusan masalah mengenai kesesuaian konsep SCOT dengan judul terdapat 15 penelitian yang sudah sesuai, 5 penelitian yang belum sesuai, dan 10 penelitian yang tidak memiliki kejelasan. Selain itu, kepaduan antara metodenya dari penelitian terdahulu dapat dihasilkan sebanyak 16 penelitian yang sudah sesuai, 5 penelitian belum sesuai, dan 9 penelitian yang tidak memiliki kejelasan. Selanjutnya, berdasar indikator mengenai bahan dasar penelitian terdapat 13 penelitian yang sudah sesuai, 4 penelitian yang belum sesuai, dan 14 penelitian yang tidak memiliki kejelasan selanjutnya yang terakhir terkait nilai kritis yang terdapat dalam penelitian, dihasilkan bahwa 15 penelitian memiliki kritisisasi terhadap judul, 10 penelitian belum memiliki nilai kritis, dan 6 penelitian lainnya belum memiliki kejelasan. Untuk mempermudah pemahaman lebih lanjut, dijelaskan melalui lima penelitian yang dianggap lebih relevan untuk bahan pembanding dalam kajian literatur. Untuk menunjukkan bahwa fokus penelitian sesuai dengan teori SCOT, mereviu dari penelitian menggunakan konsep SCOT. Komunikasi, Teknologi dan Masyarakat Komunikasi digunakan oleh manusia untuk melakukan interaksi., komunikasi ini juga dikenal sebagai human communication. Kemampuan manusia untuk mengkomunikasikan apa yang mereka butuhkan, inginkan, dan ketertarikan adalah bagian dari komunikasi manusia (Meyer, 2010). Manusia juga mengkomunikasikan perasaan dan emosi mereka, seperti gembira, takut, benci, empati, marah, dan cinta, melalui gerak tubuh, ucapan, isyarat, dan tulisan. Interaksi sosial yang terjadi antara orang melalui pertukaran pesan terus menjadi definisi komunikasi manusia seperti pada Maurice (2007) “Describe human communication as the social interaction that takes place between people when they receive messages”, yang berarti bahwa komunikasi manusia itu sendiri adalah interaksi sosial yang terjadi antara orang melalui pertukaran pesan. “Through mutual understanding, human communication strengthens feelings of togetherness among people” (DeVito, 2009). Komunikasi berfungsi sebagai jalur interaksi individu untuk membangun hubungan. Komunikasi manusia, melalui kesepahaman antara satu sama lain, dan meningkatkan rasa satu sama lain. Kemampuan untuk berpikir dan berkomunikasi adalah dua ciri yang membedakan manusia dari hewan. hanya eksistensi manusia yang membentuk pemahaman persona, tetapi juga tindakan dan interaksi dengan mereka (Fischer et al., 2007). Aristoteles mengatakan bahwa mengetahui kebajikan seperti moralitas, kasih sayang, kerja sama, kreativitas, antusiasme, pengampunan, kebenaran, keadilan, cinta, kemurahan hati, kesetiaan, moderasi, kesopanan, kesabaran, kebijaksanaan, pengertian, dan toleransi adalah dasar untuk memahami kemanusiaan. Karena sebagian besar ditunjukan dengan mengekspresikannya dalam interaksi tatap muka antar individu(Jaeger & Robinson, 1961). “Humans communicate with one another in interpersonal, group, and public contexts as well as introspectively in intrapersonal contexts” (Hewitt, 2011). Komunikasi manusia biasanya dimulai dari orang-orang yang berinteraksi dalam kelompok sosial kecil, contohnya adalah komunikasi keluarga. Komunikasi paling mendasar yang terjadi pada manusia tidak terbatas pada interaksi tatap muka dengan anggota keluarga, saudara kandung, orang tua, anak. Individu dalam kelompok sosial kecil ini juga belajar berkomunikasi, bertukar pesan, dan memperoleh rasa nilai melalui interaksi dengan anggota keluarga lainnya (Flanja & Gâz, 2011). Dengan bertemu secara fisik untuk berbagi makna dan pesan, manusia Psikolog sosial berpendapat bahwa persepsi pesan, kognisi, pengambilan keputusan, dan tindakan dipengaruhi oleh kehadiran fisik orang lain selama interaksi (Spapé & Ravaja, 2014). Namun, di era modern, kehadiran fisik manusia semakin dimediasi oleh teknologi yang membantu menjadi suatu perangkat komunikasi, yang tidak hanya mengubah proses komunikasi tetapi juga perilaku manusia. Teknologi merupakan suatu hal yang mana dapat merujuk pada sistem sosio teknis yang luas sistem sosio teknis yang luas, seperti Web, artefak tertentu, standar, rutinitas, dan kepercayaan yang membentuk sistem, seperti komputer, internet protokol, rutinitas e-mail, dan keyakinan tentang keandalan informasi online (Marx, 1997). Arnold Pacey menekankan dalam bukunya "Meaning in technology" bagaimana hubungan, tujuan, dan aspirasi manusia muncul dalam konteks teknologi. Pacey menyatakan bahwa manusia menggunakan mesin dan teknologi lainnya dalam cara yang sama seperti mereka menggunakan musik dan alat musik. Dia percaya bahwa pengalaman musik terkait dengan cara manusia bekerja dengan alat atau tangan. Untuk mendukung teori tersebut, ada contoh buruh pertanian yang meningkatkan produktivitas mereka dengan bernyanyi atau bekerja di iringi irama drum. Selain itu, dalam buku- buku tersebut ditegaskan bahwa ada hubungan antara penggunaan alat tangan, bersepeda, atau bermain piano dengan keterampilan tubuh tertentu dan irama otot yang digerakkan. Teknologi sebagai “penerapan pengetahuan ilmiah dan pengetahuan terorganisir lainnya untuk praktis dengan sistem yang teratur yang melibatkan manusia dan mesin (Pacey, 1983). McLuhan menegaskan bahwa teknologi komunikasi memiliki pengaruh yang besar terhadap manusia dan budayanya. Jurnal Ilmu Komunikasi Dan Media Sosial (JKOMDIS) Vol.04 No. 01 Edisi Januari - April 2024 Komunikasi, Teknologi dan Masyarakat Komunikasi digunakan oleh manusia untuk melakukan interaksi., komunikasi ini juga dikenal sebagai human communication. Kemampuan manusia untuk mengkomunikasikan apa yang mereka butuhkan, inginkan, dan ketertarikan adalah bagian dari komunikasi manusia (Meyer, 2010). Manusia juga mengkomunikasikan perasaan dan emosi mereka, seperti gembira, takut, benci, empati, marah, dan cinta, melalui gerak tubuh, ucapan, isyarat, dan tulisan. Interaksi sosial yang terjadi antara orang melalui pertukaran pesan terus menjadi definisi komunikasi manusia seperti pada Maurice (2007) “Describe human communication as the social interaction that takes place between people when they receive messages”, yang berarti bahwa komunikasi manusia itu sendiri adalah interaksi sosial yang terjadi antara orang melalui pertukaran pesan. “Through mutual understanding, human communication strengthens feelings of togetherness among people” (DeVito, 2009). Komunikasi berfungsi sebagai jalur interaksi individu untuk membangun hubungan. Komunikasi manusia, melalui kesepahaman antara satu sama lain, dan meningkatkan rasa satu sama lain. Kemampuan untuk berpikir dan berkomunikasi adalah dua ciri yang membedakan manusia dari hewan. Ketika ahli etologi menemukan bahwa isyarat non-verbal yang terkait dengan kawin, bermain, berburu, dan mencari makan pada hewan disebut perilaku sosial, istilah "makhluk sosial" tidak menjadi inti dari kemanusiaan (Onyeator & Okpara, 2019). Dengan demikian, konsep makhluk sosial berkaitan dengan evolusi masyarakat. Menurut mara, tidak Jurnal Ilmu Komunikasi Dan Media Sosial (JKOMDIS) Vol.04 No. 01 Edisi Januari - April 2024 182 Jurnal Ilmu Komunikasi Dan Media Sosial (JKOMDIS) Vol. 4 No. 1 Edisi Januari -April 2024 Hal. 179-192 DOI : https://doi.org/10.47233/jkomdis.v3i3.1579 ISSN : 2807-6087 Pemahaman manusia dalam skala besar di revolusi oleh teknologi, dan pergeseran teknologi baru memerlukan dalam hal cara pandang terhadap manusia. McLuhan percaya bahwa komputer yang digabungkan dengan sistem digital, atau mesin pintar elektronik, akan dapat mereproduksi dirinya sendiri tanpa memerlukan interaksi manusia (Levinson, 1999; McLuhan, 1994). Dalam hal pergeseran ke media elektronik, khususnya Teknologi Informasi, manusia mengalami realitas ganda. Orang dewasa tidak lagi bekerja dalam sistem mekanisasi dan spesialisasi, melainkan memiliki perspektif yang masih penuh dengan seluk-beluk mekanisasi dan spesialisasi. Anak- anak, di sisi lain, hidup di dunia sekolah yang kurang akan informasi dengan aturan dan bentuk pembelajaran yang sangat terstruktur, sementara pada saat yang sama mereka hidup di dunia elektronik yang penuh dengan informasi dan hiburan. Media baru saat ini telah melintasi setiap sektor yang ada di dunia kontemporer dan oleh karena itu menjadi sangat penting untuk meneliti makna dan kemungkinannya dipahami untuk mengukur situasi di seluruh dunia (Siapera, 2017). Media baru mengacu pada media digital atau yang terkini meliputi internet, media sosial, smartphone, streaming video, konsol game, virtual reality dll. (Manovich, 2001). Terutama berkaitan dengan penyebaran informasi dan komunikasi melalui digital platform. Namun yang membedakan media digital dengan media konvensional adalah interaktifnya, di mana konsumen dapat berpartisipasi aktif dalam pembuatan dan berbagi konten, daripada sekadar mengonsumsinya. g y Perubahan teknologi komunikasi dan teknologi media media yang digunakan oleh manusia menghasilkan perkembangan teknologi alternatif media komunikasi dan memadukan berbagai serpihan pikiran dan visi untuk dipahami atau dapat disebut dengan mediamorphosis (Fidler, 1997). Mendorong untuk mengetahui dan memahami betul semua bentuk dan system terkait masa lalu, sekarang dan masa yang akan datang. Buku "Meaning in Technology" menekankan betapa pentingnya pengalaman seseorang dengan teknologi. Tujuan utamanya adalah mengakui bahwa pengalaman individu harus dihargai tanpa dianggap "hanya subyektif". Diskusi diatur berdasarkan dimensi praktik atau tingkat pengalaman. Terdiri dari dua pembahasan, pada bagian pertama buku ini membahas pengalaman pribadi dengan teknologi, sedangkan bagian kedua membahas bagaimana teknologi digunakan dalam hubungannya dengan alam dan masyarakat (Pacey, 1999). Jurnal Ilmu Komunikasi Dan Media Sosial (JKOMDIS) Vol.04 No. 01 Edisi Januari - April 2024 183 Jurnal Ilmu Komunikasi Dan Media Sosial (JKOMDIS) Vol. 4 No. 1 Edisi Januari -April 2024 Hal. 179-192 DOI : https://doi.org/10.47233/jkomdis.v3i3.1579 ISSN : 2807-6087 Berdasarkan penjelasan di atas, sangat penting untuk memahami bahwa masyarakat terdiri dari sekelompok orang yang hidup berdampingan untuk memenuhi kebutuhan dan mengikuti aturan, konvensi, dan tatanan kehidupan setempat. Berasal dari bahasa Arab "Kata masyarakat yang berarti musyarak, atau "masyarakat", dengan kata lain “teman” dalam English Society dan Latin societas. Kumpulan individu yang hidup dan tinggal bersama tanpa dibatasi oleh negara, lingkungan, atau faktor lainnya disebut sebagai masyarakat. Di sisi lain, kumpulan kecil orang yang dibatasi oleh kebangsaan, wilayah, atau kelas disebut sebagai masyarakat. Sekelompok individu yang bersatu untuk mencapai tujuan bersama adalah definisi lain dari masyarakat. Sederhananya, masyarakat adalah sebuah kumpulan individu yang saling berkaitan dengan berinteraksi atau terlibat satu sama lain karena pengalaman bersama-sama. Manusia merespons lingkungannya melalui pikiran, perasaan, dan keinginan mereka, yang membentuk masyarakat (Prasetyo, 2020). Masyarakat saat ini mengarah kepada masyarakat dengan sumber informasi dalam era digital. Berbagai aktivitas masyarakat seperti sosial, politik, ekonomi, kultural di dunia nyata termuat dalam informasi secara digital. Era informasi yang terjadi saat ini dapat dikatakan bahwa ini merupakan migrasi kehidupan masyarakat secara masif dan sedang berlangsung yaitu dari informasi secara konvensional menuju ke informasi secara modern yaitu digital (Piliang, 2012). Lima definisi masyarakat yang dapat dibedakan, yaitu setiap definisi menyajikan kriteria dalam mengidentifikasikan terkait kebaruan diantaranya; teknologi, ekonomi, pekerjaan, spasial, dan budaya. Hal diatas tidak harus saling dipisahkan sesuai teori oleh beberapa ahli, bahwa satu atau faktor lainnya akan menyajikan skenarionya masing-masing (Webster & Frank, 2014). y g g Revolusi Industri 4.0 melahirkan integrasi terhadap teknologi dan perkembangan masyarakat. Masyarakat 5.0 adalah sebuah konsep dimana masyarakat yang menempatkan manusia sebagai suatu pusat atau penggabungan kemajuan teknologi digital dengan penyelesaian masalah sosial melalui suatu integrasi antara ruang maya dan ruang nyata. Konsep di atas dikenalkan pada awalnya oleh Jepang yang memungkinkan penggunaan teknologi modern dengan teknologi kecerdasan buatan atau Artificial Intelligence (AI), robot, dan Internet of Things atau IoT dalam memenuhi kebutuhan masyarakat. Bertujuan untuk menghadirkan masyarakat yang dapat menikmati sebuah kehidupan yang lebih mudah dan praktis serta membawa kemajuan. Pada tanggal 21 Januari 2019 adalah waktu di mana Masyarakat 5.0 atau dikenal sebagai Society 5.0 diresmikan. Hal ini sebagai wujud untuk menanggapi dampak negatif yang muncul akibat perkembangan Revolusi Industri 4.0, dimana hal ini dikhawatirkan dapat memberikan dampak merugikan bagi masyarakat (Suherman, 2020). Dalam membahas masyarakat 5.0 tidak lupa pula untuk mengetahui tiga nilai utama yaitu fokus kepada manusia, bagaimana keberlanjutannya, serta ketahanan terhadap tatanan kehidupan masyarakat. Dengan mengutamakan nilai kemanusiaan serta menempatkan manusia sebagai pusat suatu kehidupan bermasyarakat maka dalam hal memproduksi kebutuhan atau mengerjakan kepentingan masyarakat bukan berfokus terhadap suatu kemajuan teknologi saja. Karena hal ini menjadi suatu yang penting untuk membawa peran suatu kehidupan manusia dengan perkembangan industri menjadi perkembangan yang mementingkan manusia sebagai hal paling penting. Teknologi diciptakan untuk memenuhi kebutuhan manusia, dapat memudahkan manusia dalam melakukan pekerjaanya. Dalam hal pekerjaan, era masyarakat 5.0 ini bertujuan bahwa perkembangan teknologi di era revolusi industri dapat membuat lingkungan kerja yang efektif, mudah, aman, dan inklusif yang harus dibangun untuk menjaga kesehatan fisik manusia, mental serta kesejahteraan manusia dalam bekerja. Menjadi hal yang paling utama adalah untuk melindungi hak-hak dasar masyarakat seperti martabat, provasi, serta bersifat otonom bagi manusia (Teknowijoyo, 2022). Jurnal Ilmu Komunikasi Dan Media Sosial (JKOMDIS) Vol.04 No. 01 Edisi Januari - April 2024 Generasi Z Generasi Z lebih menyukai konten yang berbentuk visual, seperti gambar dan video, daripada hanya teks. Hal ini mungkin disebabkan oleh perkembangan media sosial yang menyajikan konten secara visual. Selain itu, generasi ini juga cenderung mencari informasi secara cepat dan tidak suka membaca teks yang panjang. Hal ini dapat menciptakan perbedaan dalam hal pendekatan komunikasi dan gaya hidup dibandingkan dengan generasi sebelumnya. Faktor-faktor ini bersama-sama membentuk identitas dan karakteristik khas generasi Z yang membedakannya dari generasi-generasi sebelumnya. Generasi Z Masyarakat Indonesia saat ini didominasi dengan kelompok usia dalam kategori Gen Z, dilihat dari demografi penduduk jumlah penduduk Gen Z sebanyak 27,94% dari total penduduk Indonesia (BPS, 2020). Lebih dari 20% tenaga kerja organisasi akan berasal dari Gen Z, menurut Deloitte (2017). Meskipun demikian, perhatian organisasi terhadap Generasi Y atau milenial, atau generasi milenial, tampaknya tidak terfokus saat berbicara tentang kehadiran Gen Z di dunia kerja. Seperti yang didefinisikan oleh beberapa penelitian sebelumnya(Francis & Hoefel, 2018; Linnes & Metcalf, 2017), Gen Z juga disebut sebagai "generasi pascamilenial". Terdapat empat komponen utama yang mempengaruhi perilaku Generasi Z. Prinsip dasar dari semuanya adalah bahwa Generasi Z adalah generasi yang mencari kebenaran. Pertama Gen Z dikenal sebagai "the undefined ID" karena mereka sangat terbuka untuk menghargai keunikan setiap orang dan menghargai ekspresi setiap orang tanpa melabelinya. Kedua, "The communa holic" adalah sebutan untuk Gen Z, mereka adalah generasi yang sangat terbuka dan ingin berkontribusi kepada sebanyak mungkin komunitas dengan menggunakan teknologi canggih. Ketiga, Gen Z dikenal sebagai "sang pendialog" mereka adalah generasi yang merasa bahwa berbicara dan menyelesaikan perselisihan melalui komunikasi adalah hal yang sangat penting untuk membuat perubahan. Selain itu, Generasi Z menerima semua sudut pandang dan suka berinteraksi dengan berbagai macam orang dan kelompok. Generasi Z dikenal sebagai "generasi yang realistis" Jurnal Ilmu Komunikasi Dan Media Sosial (JKOMDIS) Vol.04 No. 01 Edisi Januari - April 2024 184 Jurnal Ilmu Komunikasi Dan Media Sosial (JKOMDIS) Vol. 4 No. 1 Edisi Januari -April 2024 Hal. 179-192 DOI : https://doi.org/10.47233/jkomdis.v3i3.1579 ISSN : 2807-6087 karena, dibandingkan dengan generasi sebelumnya, mereka lebih cenderung membuat keputusan yang analitis dan pragmatis. Gen Z lebih suka bertanggung jawab atas keputusan mereka sendiri karena mereka suka belajar secara mandiri dan mencari pengetahuan. Gen Z menyadari betapa pentingnya memiliki stabilitas kehidupan sejalan dengan kemudahan dan perkembangan teknologi. Hal ini beriringan dalam kenyataan yang menunjukkan dimana Gen Y dan Baby Boomer cenderung lebih idealis, dan kurang melek terhadap perkembangan teknologi terutama dalam hal fleksibilitas. Saat ini, penggunaan teknologi untuk membantu pembelajaran sudah cukup lazim. Salah satu contohnya adalah pembelajaran online. Literasi teknis, literasi data, dan literasi manusia adalah tiga kemampuan literasi yang dibutuhkan untuk melakukan pembelajaran online ini. Dalam ranah pendidikan saat ini, Generasi Z menggunakan berbagai macam strategi pembelajaran, termasuk pembelajaran online. Bagaimana Generasi Z Indonesia dapat menjadi lebih melek melalui penerapan teknologi. Ada beberapa faktor yang membuat generasi Z berbeda dari generasi sebelumnya menurut (Twenge & Campbell, 2008)antara lain: 1. Jurnal Ilmu Komunikasi Dan Media Sosial (JKOMDIS) Vol.04 No. 01 Edisi Januari - April 2024 Generasi Z Teknologi Generasi Z tumbuh di era digital dengan akses mudah ke internet dan perangkat teknologi canggih. Mereka terbiasa dengan multitasking, mengandalkan teknologi untuk segala hal, tumbuh dengan mengalami perkembangan pesat teknologi dan media sosial. Hal ini membuat mereka menjadi sangat terbiasa menggunakan teknologi dalam berkomunikasi. dan lebih terampil dalam penggunaan media sosial. 1. Teknologi Generasi Z tumbuh di era digital dengan akses mudah ke internet dan perangkat teknologi canggih. Mereka terbiasa dengan multitasking, mengandalkan teknologi untuk segala hal, tumbuh dengan mengalami perkembangan pesat teknologi dan media sosial. Hal ini membuat mereka menjadi sangat terbiasa menggunakan teknologi dalam berkomunikasi. dan lebih terampil dalam penggunaan media sosial. 2. Nilai dan Sikap Generasi Z cenderung lebih inklusif, terbuka terhadap keberagaman, dan memiliki sikap yang progresif terhadap isu-isu sosial seperti lingkungan, kesetaraan gender, dan hak asasi manusia. Mereka juga sering dianggap sebagai generasi yang lebih kolaboratif dan memperhatikan keberlanjutan. 3. Pendidikan Generasi Z memiliki jaringan yang lebih lebar terhadap informasi dan pendidikan melalui internet, yang memungkinkan mereka untuk belajar di luar kelas dan mengembangkan minat serta keterampilan mereka sendiri. Generasi Z memiliki jaringan yang lebih lebar terhadap informasi dan pendidikan melalui internet, yang memungkinkan mereka untuk belajar di luar kelas dan mengembangkan minat serta keterampilan mereka sendiri. 4. Lingkungan Sosial Generasi Z tumbuh dalam dunia yang lebih terhubung secara global, memungkinkan mereka untuk berinteraksi dengan orang dari berbagai budaya dan negara dengan lebih mudah. Hal ini juga mempengaruhi pandangan mereka terhadap nilai-nilai global. 4. Lingkungan Sosial Generasi Z tumbuh dalam dunia yang lebih terhubung secara global, memungkinkan mereka untuk berinteraksi dengan orang dari berbagai budaya dan negara dengan lebih mudah. Hal ini juga mempengaruhi pandangan mereka terhadap nilai-nilai global. 5. Ketergantungan pada media sosial Generasi Z cenderung lebih terpapar dengan media sosial dan teknologi, yang dapat mempengaruhi cara mereka berkomunikasi, bersosialisasi, dan mengonsumsi informasi. Generasi Z lebih menyukai konten yang berbentuk visual, seperti gambar dan video, daripada hanya teks. Hal ini mungkin disebabkan oleh perkembangan media sosial yang menyajikan konten secara visual. Selain itu, generasi ini juga cenderung mencari informasi secara cepat dan tidak suka membaca teks yang panjang. Hal ini dapat menciptakan perbedaan dalam hal pendekatan komunikasi dan gaya hidup dibandingkan dengan generasi sebelumnya. 5. Ketergantungan pada media sosial Generasi Z cenderung lebih terpapar dengan media sosial dan teknologi, yang dapat mempengaruhi cara mereka berkomunikasi, bersosialisasi, dan mengonsumsi informasi. Interaktivitas Konvergensi media telah memberikan ruang bagi interaktivitas yang lebih besar antara penyiar dan pendengar. Baik radio maupun podcast sekarang dapat mendukung fitur interaktif seperti polling, panggilan langsung, atau komentar online yang memungkinkan pendengar untuk terlibat secara langsung dalam konten yang disajikan. Konvergensi media telah memberikan ruang bagi interaktivitas yang lebih besar antara penyiar dan pendengar. Baik radio maupun podcast sekarang dapat mendukung fitur interaktif seperti polling, panggilan langsung, atau komentar online yang memungkinkan pendengar untuk terlibat secara langsung dalam konten yang disajikan Konvergensi media telah memberikan ruang bagi interaktivitas yang lebih besar antara penyiar dan pendengar. Baik radio maupun podcast sekarang dapat mendukung fitur interaktif seperti polling, panggilan langsung, atau komentar online yang memungkinkan pendengar untuk terlibat secara langsung dalam konten yang disajikan komentar online yang memungkinkan pendengar untuk terlibat secara langsung dalam konten yang disajikan. 4. Fleksibilitas Konsumsi 4. Fleksibilitas Konsumsi Dengan konvergensi media, pendengar sekarang memiliki fleksibilitas yang lebih besar dalam konsumsi konten audio. Mereka dapat mendengarkan siaran radio secara langsung atau mengunduh episode podcast untuk didengarkan kapan pun mereka mau, sesuai dengan preferensi dan jadwal mereka. 5. Kemungkinan Kolaborasi Konvergensi media membuka peluang untuk kolaborasi antara radio dan podcast, di mana stasiun radio dapat bekerja sama dengan podcaster untuk menciptakan konten yang lebih bervariasi dan menarik untuk pendengar. 6. Pembaharuan Format Konvergensi media juga mendorong radio dan podcast untuk terus berinovasi dalam pembaharuan format konten agar tetap relevan dan kompetitif. Hal ini membawa keberagaman dalam konten audio yang ditawarkan kepada pendengar. Dengan pengaruh konvergensi media ini, radio dan podcast telah saling mempengaruhi dan memperkaya satu sama lain dalam hal produksi, distribusi, dan konsumsi konten audio. Hal ini membuka peluang baru dan menantang kedua media untuk terus berkembang dan menyesuaikan diri dengan perubahan tren industri media secara keseluruhan. jenis media yang tadinya berbeda, seperti radio, televisi, komputer, dan surat kabar, menjadi satu media yang kohesif. Menurut Hamna (2017), penyatuan akan beberapa media sebagai kolaborasi antarmedia. Teknologi yang maju kemudian memungkinkan media konvergensi untuk memanfaatkan sifat-sifat dan fungsi-fungsi yang berbeda serta menawarkan kepada khalayak baru. Pendekatan konvergensi juga membantu mengidentifikasi perbedaan persepsi media konvergensi dan media tradisional yang harmonis. Hal ini dihasilkan melalui pasukan pengintegrasian media yang melibatkan media logis, teknis, budaya, dan energi yang berbeda. Hal ini menawarkan nilai jual tambahan terkait kompleksitas dan kemudahan transportasi. Konsep mobil baru dan media yang berhubungan dengan khalayak, seperti Wiki dan Podcast, telah memperluas peran media konvergensi di berbagai sektor. Dibalik perkembangan teknologi yang cepat dan berbagai tokoh yang berpengaruh di balik teknologi-restu, menuntut penerapan konsekuensi media sebagai kehadiran teknologi yang semakin agresif di berbagai sektor. Negara dan warga menjadi lebih kritikal dan cerdas terhadap media dalam melacak pergerakan zaman, yang merupakan hasil alami dari konvergensi medianya sendiri yang memperkuatnya. Perkembangan konvergensi teknologi juga telah menghasilkan banyak manfaat bagi pengguna dan konsumen akhir. Misalnya, dengan adanya kemajuan teknologi smartphone dan konvergensi media, seseorang sekarang dapat mengakses berbagai layanan melalui ponsel mereka, termasuk perdagangan elektronik, transaksi perbankan, dan layanan pemesanan dan pengiriman makanan. Kemampuan untuk mengakses berbagai layanan ini secara terintegrasi melalui satu perangkat telah menyederhanakan kehidupan sehari-hari dan memberikan lebih banyak pilihan kepada konsumen. Konvergensi media, yaitu penyatuan antara media konvensional seperti radio dengan media digital seperti podcast (Muslimah, 2022), telah memberikan dampak signifikan terhadap kedua media tersebut. Beberapa dampak konvergensi media terhadap radio dan podcast antara lain: g p p 1. Penyebaran Konten Konvergensi media telah memungkinkan konten dari radio untuk dapat didistribusikan secara digital melalui podcast. Hal ini membuka peluang baru bagi stasiun radio untuk menjangkau pendengar mereka di platform digital, sehingga memperluas cakupan dan audience base mereka. y Konvergensi media telah memungkinkan konten dari radio untuk dapat didistribusikan secara digital melalui podcast. Hal ini membuka peluang baru bagi stasiun radio untuk menjangkau pendengar mereka di platform digital, sehingga memperluas cakupan dan audience base mereka. igital, sehingga memperluas cakupan dan audience base mereka. 2. Kemajuan Teknologi Kemajuan teknologi dalam konvergensi media telah membawa perubahan signifikan dalam cara konten audio diproduksi, didistribusikan, dan dikonsumsi. Radio dan podcast sekarang bisa memanfaatkan teknologi yang sama, seperti streaming online, aplikasi mobile, dan algoritma personalisasi untuk menyesuaikan konten dengan preferensi pendengar. 3. Jurnal Ilmu Komunikasi Dan Media Sosial (JKOMDIS) Vol.04 No. 01 Edisi Januari - April 2024 Konvergensi Media Kecenderungan barang dengan fungsi terpisah yang kemudian menjadi satu untuk menghasilkan satu barang atau jasa yang multi guna dikenal sebagai konvergensi teknologi, dan ini merupakan fenomena yang berkembang dan mengubah lanskap komersial. Kecepatan konvergensi yang dikejar adalah mengubah pasar dan menghasilkan barang dan jasa mutakhir untuk basis pelanggan yang menjadi semakin cerdik dan pintar. Selain itu, meskipun banyak yang mengasosiasikan konvergensi teknologi dengan media dan komunikasi, gagasan itu sendiri jauh melampaui hal ini dalam konteks wilayah yang belum dipetakan yang selalu dieksplorasi. Semua sektor telah berkembang dan berekspansi pada tingkat yang fenomenal, memperkenalkan barang dan jasa mutakhir ke pasar. Berbagai variabel, seperti permintaan dan keinginan konsumen, tren, dan meningkatnya minat serta dukungan terhadap peralatan, telah berkontribusi terhadap konvergensi, meskipun demikian penyebab utama yang mendasarinya adalah persaingan dan penciptaan atau daya cipta. Konvergensi dapat juga disebut sebagai penggabungan berbagai Jurnal Ilmu Komunikasi Dan Media Sosial (JKOMDIS) Vol.04 No. 01 Edisi Januari - April 2024 185 Jurnal Ilmu Komunikasi Dan Media Sosial (JKOMDIS) Vol. 4 No. 1 Edisi Januari -April 2024 Hal. 179-192 DOI : https://doi.org/10.47233/jkomdis.v3i3.1579 ISSN : 2807-6087 Podcast Podcast adalah jenis media suara yang dapat diakses secara online dan memungkinkan pendengar mendengarkan berbagai jenis konten seperti berita, cerita, hiburan, pendidikan, dan lain-lain dengan mudah kapan pun dan di manapun melalui perangkat elektronik seperti smartphone, laptop, atau tablet (Nugroho & Irwansyah, 2021). Dalam review literatur ini, membahas sejarah perkembangan podcast, penggunaan dalam pendidikan, dan dampak podcast terhadap perilaku dan pengetahuan pendengar. Pada tahun 2004, Adam Curry, mantan VJ MTV, dan Dave Winer, perancang perangkat komputer, mengembangkan teknologi bernama "Really Simple Syndication" (RSS), yang memungkinkan pengguna mengunduh dan mendengarkan konten audio secara otomatis. Ini menandai awal podcast, yang membuka era baru dalam media bercerita. Podcast adalah evolusi dari gaya komunikasi audio tersebut. Menurut (Geoghegan & Klass, 2007), podcasting bermula dari audioblog, yang merupakan kumpulan berbagai aktivitas audio di situs web yang mulai berkembang pada tahun 2001 bersamaan dengan diperkenalkannya RSS feed. Munculnya umpan RSS. Podcast sebagian besar dipandang sebagai hasil dari blogging, karena menggunakan metode yang sama untuk mendistribusikan materi kepada pemirsa. Podcasting mengacu pada penyebaran informasi audio atau video melalui internet. Konten dapat ditransmisikan ke PC atau pemutar audio portabel. Podcasting sangat menarik karena memungkinkan individu untuk berpartisipasi secara aktif, mengartikulasikan pemikiran mereka, berbagi konsep, dan mempromosikan barang dagangan mereka. Kemudian, pada tahun 2005, Apple meluncurkan iTunes 4.9, yang memiliki fitur podcast yang memungkinkan pengguna mengunduh dan mengorganisasi konten podcast dengan lebih mudah . Ini semakin mempopulerkan podcast dan membuat Apple menjadi salah satu platform terbesar untuk podcast (Sullivan, 2019). Biasanya pendengar podcast memiliki kecenderungan untuk mendengarkan podcast secara konstan dan lebih cenderung untuk memiliki sikap yang lebih positif terhadap topik yang dibahas dalam podcast tersebut. Hal ini menunjukkan bahwa pendengar podcast tidak hanya mendengarkan untuk hiburan semata, tetapi juga memiliki efek yang lebih dalam terhadap pengetahuan dan perilaku mereka. Selain itu, podcast juga dapat mempengaruhi perilaku konsumsi pendengar terhadap suatu produk atau layanan. Sebagian besar pendengar podcast lebih cenderung untuk membeli produk yang direkomendasikan oleh pembuat podcast yang mereka dengarkan. Hal ini menunjukkan bahwa podcast dapat menjadi media yang efektif untuk pemasaran produk atau layanan (McNamara & Min, 2024). Radio Transmisi radio mulai berkembang pesat pada tahun 1920-an, Pada tahun 1946, muncul teknologi pendukung radio seperti transistor dan FM (Frequency Modulation) yang memungkinkan sinyal radio menjadi lebih jernih dan dapat diakses di lebih banyak tempat. Pada tahun 1954, muncul radio portable yang kemudian menjadi populer di kalangan remaja. Ekspansi mencapai puncaknya pada tahun 1926, ketika sejumlah stasiun besar, termasuk National Jurnal Ilmu Komunikasi Dan Media Sosial (JKOMDIS) Vol.04 No. 01 Edisi Januari - April 2024 186 Jurnal Ilmu Komunikasi Dan Media Sosial (JKOMDIS) Vol. 4 No. 1 Edisi Januari -April 2024 Hal. 179-192 DOI : https://doi.org/10.47233/jkomdis.v3i3.1579 ISSN : 2807-6087 Broadcasting Corporation (NBC) hadir. Kemudian disusul dengan kemunculan delapan stasiun termasuk Columbia Broadcasting System (CBS). Menurut Ado (2020) radio adalah perangkat yang berkomunikasi dengan perangkat radio lain melalui gelombang elektromagnetik. Radio dapat digunakan untuk komunikasi antara komputer dan manusia. Instrumen apa pun yang memiliki kemampuan untuk menghasilkan atau bereaksi terhadap gelombang radio dianggap sebagai radio. Radio telah berkembang menjadi salah satu instrumen paling penting dalam peradaban kontemporer dan memungkinkan untuk berkomunikasi. Data diubah menjadi gelombang radio oleh pemancar radio sehingga dapat dikirim ke dalam dan ke luar Bumi. Dengan memanfaatkan gelombang radio pada posisi yang berbeda, penerima radio menciptakan informasi baru. Berikut ini adalah fungsi komunikasi radio dua arah, hanya pemancar dan penerima yang beroperasi dalam dua arah. Seorang penemu yang menciptakan teknik ini pada tahun 1903 untuk mengubah huruf- huruf mesin ketik menjadi gelombang radio. Itu adalah contoh bagaimana ilmu pengetahuan direfleksikan secara online (Tokar et al., 2012). ( ) Radio siaran adalah media elektronik perantara, yang beradaptasi dengan berbagai situasi karena kemajuan teknologi komunikasi. Karakteristiknya yang unik, seperti penyampaian konten program yang lambat, menjadikannya alat yang berharga. Konvergensi media juga mencakup kemampuan media konvensional, seperti halnya transmisi radio yang bersifat satu arah, menjadi multi-saluran. Hal ini dimungkinkan oleh kemajuan teknologi dan hal tersebut merupakan karakteristik konvergensi media. Saat ini, radio tetap menjadi salah satu media yang paling populer untuk mendapatkan informasi, hiburan, dan musik. Meskipun telah ada banyak variasi media baru yang menawarkan akses ke informasi dan hiburan, radio masih memiliki kekuatan yang khas dan kemampuan untuk menyebarkan informasi kepada pendengarnya. Dengan perkembangan teknologi yang terus berjalan, radio akan terus mengalami evolusi dan terus menjadi media yang penting dalam kehidupan manusia. Jurnal Ilmu Komunikasi Dan Media Sosial (JKOMDIS) Vol.04 No. 01 Edisi Januari - April 2024 DISKUSI Dengan demikian, konvergensi media pada radio terkait minat mendengar Generasi Z terhadap podcast bisa menjadi kesempatan yang baik bagi industri radio untuk terus berkembang dan bertahan di tengah persaingan yang semakin ketat. Minat mendengarkan memainkan peran penting dalam keputusan Generasi Z untuk beralih dari radio ke podcast. Generasi Z biasanya memiliki minat yang tinggi dalam eksplorasi konten audio yang sesuai dengan minat dan preferensi mereka. Peralihan minat dari radio ke podcast pada Generasi Z dapat disebabkan oleh beberapa faktor, antara lain: 1. Kemudahan Akses Generasi Z tumbuh di era digital di mana akses informasi sangat mudah. Mereka dapat dengan mudah mengunduh atau streaming podcast favorit mereka melalui berbagai platform digital, sehingga lebih praktis daripada menunggu siaran radio tertentu. Generasi Z tumbuh di era digital di mana akses informasi sangat mudah. Mereka dapat dengan mudah mengunduh atau streaming podcast favorit mereka melalui berbagai platform digital, sehingga lebih praktis daripada menunggu siaran radio tertentu. y g Podcast seringkali menawarkan konten yang lebih personal dan relevan bagi pendengarnya. Generasi Z terutama menyukai konten yang sesuai dengan minat dan kebutuhan mereka, sehingga mereka cenderung lebih tertarik pada podcast yang dapat menyesuaikan konten dengan preferensi mereka. 3. Pilihan Variasi Konten 3. Pilihan Variasi Konten Podcast memiliki berbagai macam topik dan genre yang beragam, sehingga Generasi Z memiliki pilihan yang lebih banyak untuk memilih konten yang mereka sukai. Sebaliknya, siaran radio mungkin memiliki keterbatasan dalam variasi konten yang disajikan. Podcast memiliki berbagai macam topik dan genre yang beragam, sehingga Generasi Z memiliki pilihan yang lebih banyak untuk memilih konten yang mereka sukai. Sebaliknya, siaran radio mungkin memiliki keterbatasan dalam variasi konten yang disajikan dalam variasi konten yang disajikan. 4. Kemampuan Menyesuaikan Jadwal Podcast memungkinkan pendengar untuk mendengarkan kapan pun mereka inginkan, sehingga cocok dengan kemampuan multitasking dan mobilitas Generasi Z. Mereka dapat mendengarkan podcast saat dalam perjalanan, saat berolahraga, atau di waktu luang lainnya. 5. Interaksi yang Lebih Aktif Melalui podcast, Generasi Z memiliki kesempatan untuk berpartisipasi dalam mendiskusikan konten, memberikan komentar, atau bahkan membuat podcast mereka sendiri. Hal ini memberikan pengalaman interaktif yang tidak selalu bisa didapatkan dari siaran radio konvensional. 5. Interaksi yang Lebih Aktif Melalui podcast, Generasi Z memiliki kesempatan untuk berpartisipasi dalam mendiskusikan konten, memberikan komentar, atau bahkan membuat podcast mereka sendiri. Hal ini memberikan pengalaman interaktif yang tidak selalu bisa didapatkan dari siaran radio konvensional. DISKUSI Konvergensi media pada radio terkait minat mendengarkan Generasi Z terhadap podcast berdampak positif bagi industri radio. Dengan konvergensi media, radio dapat menghadirkan konten podcast yang menarik dan relevan bagi Generasi Z yang lebih suka mendengarkan podcast daripada siaran radio konvensional. Ini bisa meningkatkan minat mendengarkan radio pada generasi yang lebih muda. Manfaat dari konvergensi media ini antara lain 187 Jurnal Ilmu Komunikasi Dan Media Sosial (JKOMDIS) Vol.04 No. 01 Edisi Januari - April 2024 Jurnal Ilmu Komunikasi Dan Media Sosial (JKOMDIS) Vol. 4 No. 1 Edisi Januari -April 2024 Hal. 179-192 DOI : https://doi.org/10.47233/jkomdis.v3i3.1579 ISSN : 2807-6087 meningkatkan variasi konten yang ditawarkan radio, menciptakan pengalaman mendengar yang lebih personal, dan memperluas audiens radio dengan menjangkau generasi yang lebih muda. Radio juga dapat memanfaatkan platform digital untuk mempromosikan podcast mereka dan menjangkau lebih banyak pendengar. Selain itu, dengan konvergensi media, radio dapat memberikan konten yang lebih beragam dan sesuai dengan minat Generasi Z, sehingga dapat lebih merelevansikan diri dengan kebutuhan para pendengarnya. Hal ini dapat membantu radio dalam mendapatkan loyalitas dari generasi yang lebih muda dan memperluas basis pendengar mereka secara keseluruhan. Dengan memanfaatkan konvergensi media dan memperkenalkan podcast yang menarik, radio bisa tetap relevan dan bersaing di era digital ini. Dengan demikian, konvergensi media pada radio terkait minat mendengar Generasi Z terhadap podcast bisa menjadi kesempatan yang baik bagi industri radio untuk terus berkembang dan bertahan di tengah persaingan yang semakin ketat. Minat mendengarkan memainkan peran penting dalam keputusan Generasi Z untuk beralih dari radio ke podcast. Generasi Z biasanya memiliki minat yang tinggi dalam eksplorasi konten audio yang sesuai dengan minat dan preferensi mereka. Peralihan minat dari radio ke podcast pada Generasi Z dapat disebabkan oleh beberapa faktor, antara lain: meningkatkan variasi konten yang ditawarkan radio, menciptakan pengalaman mendengar yang lebih personal, dan memperluas audiens radio dengan menjangkau generasi yang lebih muda. Radio juga dapat memanfaatkan platform digital untuk mempromosikan podcast mereka dan menjangkau lebih banyak pendengar. Selain itu, dengan konvergensi media, radio dapat memberikan konten yang lebih beragam dan sesuai dengan minat Generasi Z, sehingga dapat lebih merelevansikan diri dengan kebutuhan para pendengarnya. Hal ini dapat membantu radio dalam mendapatkan loyalitas dari generasi yang lebih muda dan memperluas basis pendengar mereka secara keseluruhan. Dengan memanfaatkan konvergensi media dan memperkenalkan podcast yang menarik, radio bisa tetap relevan dan bersaing di era digital ini. DISKUSI Peralihan minat dari radio ke podcast pada Generasi Z adalah refleksi dari perubahan paradigma dalam konsumsi media mereka. Sebagai media baru, podcast memiliki empat keunggulan dibanding media komunikasi tradisional lain seperti radio. Pertama, podcast memungkinkan pendengar mendengarkan konten podcast sesuai permintaan, kapan saja dan di mana saja mereka menginginkannya. Kedua, produksi untuk podcast relatif murah. Radio pun akhirnya mengikuti tren ini dengan menyajikan podcast mereka sendiri atau memperluas konten digital mereka untuk tetap relevan dan menarik bagi audiens yang lebih muda. Ada beberapa konvergensi media lain selain radio dan podcast yang juga dapat menjadi alternatif yang menarik bagi Generasi Z untuk menikmati konten audio. Beberapa di antaranya termasuk : No. Jenis Platform Penjelasan 1. Music Streaming Services Layanan streaming musik seperti Spotify, Apple Music, atau Deezer juga dapat menjadi opsi bagi Generasi Z. Mereka dapat menikmati musik favorit mereka, mendengarkan podcast, atau menikmati audiobook dalam satu platform yang sama (Barata & Coelho, 2021). 2. Live Streaming Live streaming audio, seperti acara radio online atau sesi live podcast, juga dapat menjadi alternatif menarik. Generasi Z dapat terlibat dalam interaksi real-time dengan pembuat konten atau host acara mereka sukai (Diwi et al., 2015). Jurnal Ilmu Komunikasi Dan Media Sosial (JKOMDIS) Vol 04 No 01 Edisi Januari April 2024 188 No. Jenis Platform Penjelasan 1. Music Streaming Services Layanan streaming musik seperti Spotify, Apple Music, atau Deezer juga dapat menjadi opsi bagi Generasi Z. Mereka dapat menikmati musik favorit mereka, mendengarkan podcast, atau menikmati audiobook dalam satu platform yang sama (Barata & Coelho, 2021). 2. Live Streaming Live streaming audio, seperti acara radio online atau sesi live podcast, juga dapat menjadi alternatif menarik. Generasi Z dapat terlibat dalam interaksi real-time dengan pembuat konten atau host acara mereka sukai (Diwi et al., 2015). 188 Jurnal Ilmu Komunikasi Dan Media Sosial (JKOMDIS) Vol. 4 No. 1 Edisi Januari -April 2024 Hal. 179-192 DOI : https://doi.org/10.47233/jkomdis.v3i3.1579 ISSN : 2807-6 3. Voice Assistants Perangkat voice assistant seperti Amazon Alexa atau Google Home juga menyediakan akses ke berbagai jenis konten audio, mulai dari musik, berita, hingga podcast. Generasi Z dapat dengan mudah mengakses konten audio favorit mereka melalui perintah suara (Arifin & Lennerfors, 2022). 4. Audio Social Media Platform media sosial seperti Clubhouse menyediakan format audio untuk berinteraksi dan berdiskusi dengan orang lain secara langsung. Generasi Z dapat menikmati konten audio secara interaktif dengan relawan bergabung dalam berbagai ruang obrolan. 5. DISKUSI Audiobook Audiobook merupakan format audio yang berisi narasi buku atau cerita. Generasi Z yang suka mendengarkan cerita atau informasi dapat menemukan audiobook sebagai alternatif yang menarik. Platform seperti Audible dan Storytel menyediakan berbagai judul audiobook yang bisa dinikmati (Whittingham et al., 2013). ISSN : 2807-6087 Dengan kemajuan teknologi dan variasi platform yang tersedia, Generasi Z memiliki banyak pilihan alternatif untuk menikmati konten audio sesuai dengan minat dan preferensi mereka. Hal ini menunjukkan konvergensi media yang terus berkembang dan memungkinkan akses lebih luas terhadap konten audio yang bervariasi. Dengan perbandingan Generasi Z dan Generasi-generasi sebelumnya, terlihat bahwa Generasi Z memiliki kecenderungan yang lebih besar untuk mendengarkan podcast sebagai alternatif mendengarkan radio konvensional. Mereka menghargai kemudahan akses, pilihan konten yang beragam, dan interaksi yang lebih aktif dalam menikmati konten audio. Generasi sebelum Generasi Z cenderung lebih memilih radio daripada podcast (Yuniati & Puspitasari, 2019) karena beberapa alasan diantaranya : 1. Historis dan Keterbiasaan Generasi sebelum Generasi Z tumbuh dan dibesarkan di era sebelum kemajuan teknologi digital. Radio telah lama menjadi salah satu sumber hiburan dan informasi utama bagi generasi sebelumnya, sehingga mereka lebih terbiasa dengan radio dan telah membangun hubungan emosional dengan medium tersebut. 2. Keterbatasan Teknologi Pada masa lalu, teknologi belum sekompleks sekarang dan radio adalah salah satu cara utama untuk mengakses informasi dan hiburan secara cepat dan mudah. Belum ada banyak pilihan alternatif seperti yang tersedia sekarang, sehingga radio menjadi pilihan utama untuk mendapatkan konten audio. 3. Sifat Pasif Mendengarkan Radio umumnya merupakan medium yang lebih pasif, di mana pendengar hanya perlu mendengarkan tanpa harus berinteraksi terlalu banyak. Generasi sebelum Generasi Z mungkin lebih suka gaya mendengarkan yang lebih pasif daripada generasi muda yang lebih suka interaktif dan terlibat dalam konten yang mereka konsumsi. 4. Keterbatasan Pilihan pada masa lalu, radio adalah salah satu cara utama untuk mengakses musik, berita, dan program hiburan. Keterbatasan pilihan media pada waktu itu membuat radio menjadi pilihan utama alih-alih podcast atau platform on-demand lainnya yang baru muncul belakangan ini. 5. Keberlanjutan Tradisi Beberapa generasi sebelum Generasi Z mungkin melestarikan kegiatan mendengarkan radio sebagai bagian dari kebiasaan dan tradisi yang telah berlangsung lama dalam keluarga atau masyarakat mereka. Alasan-alasan di atas menjelaskan mengapa generasi sebelum Generasi Z cenderung memilih radio daripada podcast, karena keterbiasaan historis, keterbatasan teknologi, sifat pasif mendengarkan, keterbatasan pilihan, dan keberlanjutan tradisi yang mempengaruhi preferensi mereka terhadap medium radio. DISKUSI Sayangnya, tidak ada data statik khusus yang bisa disediakan dalam konteks ini secara langsung. Data statistik yang spesifik tentang minat Generasi Z untuk mendengarkan podcast dapat bervariasi tergantung pada sumber data dan penelitian yang dilakukan. Namun, berdasarkan tren yang mengarah pada preferensi media Generasi Z dan adopsi teknologi digital, dapat disimpulkan 189 Jurnal Ilmu Komunikasi Dan Media Sosial (JKOMDIS) Vol.04 No. 01 Edisi Januari - April 2024 189 Jurnal Ilmu Komunikasi Dan Media Sosial (JKOMDIS) Vol. 4 No. 1 Edisi Januari -April 2024 Hal. 179-192 DOI : https://doi.org/10.47233/jkomdis.v3i3.1579 ISSN : 2807-6087 bahwa minat Generasi Z terhadap mendengarkan podcast cenderung lebih tinggi dibandingkan dengan generasi sebelumnya. Berdasarkan pengamatan dan studi pasar terbaru, beberapa temuan umum yang mungkin mendukung klaim bahwa Generasi Z memiliki minat berbeda terhadap podcast antara lain peningkatan Penggunaan Platform Audio Digital Generasi Z cenderung lebih aktif menggunakan platform audio digital seperti Spotify, Apple Podcasts, Google Podcasts, dan platform podcast lainnya untuk mendengarkan konten audio dibandingkan dengan generasi sebelumnya. Konten yang Bersifat Edukatif dan Menghibur diamana Generasi Z lebih menyukai konten podcast yang bersifat edukatif, menghibur, atau memberikan wawasan yang bermanfaat dalam kehidupan sehari-hari mereka. Podcast- podcast seperti itu mungkin lebih diminati oleh generasi ini. Meningkatnya Jumlah dan Ragam Podcast, Seiring dengan pertumbuhan industri podcast, ada banyak pilihan podcast dengan berbagai topik yang tersedia bagi pendengar. Generasi Z yang terbiasa dengan eksplorasi konten digital dapat lebih bersemangat untuk mencari dan mendengarkan podcast yang sesuai dengan minat mereka. Interaksinya dengan Content Creators, Generasi Z cenderung lebih terhubung secara emosional dengan content creators atau podcaster yang menghasilkan konten yang mereka nikmati. Hubungan ini dapat memotivasi mereka untuk terus mendengarkan podcast tersebut. Fleksibilitas dan Kemudahan Akses mendengarkan podcast secara on-demand di berbagai perangkat seperti ponsel pintar atau komputer juga merupakan faktor yang mungkin meningkatkan minat Generasi Z terhadap podcast. Meskipun tidak ada data statistik tertentu yang dapat disediakan, pola dan tren yang lebih besar dalam perilaku konsumsi media Generasi Z dapat memberikan indikasi bahwa mereka cenderung memiliki minat yang lebih tinggi dalam mendengarkan podcast daripada generasi sebelumnya. Jika Anda membutuhkan data statistik yang lebih spesifik, disarankan untuk mencari sumber data yang dapat memberikan informasi yang lebih terperinci terkait minat Generasi Z terhadap podcast. Beberapa cara yang dapat dilakukan untuk memahami dan mengadaptasi perubahan minat pendengar adalah: 1. Pengamatan Pendengar Salah satu langkah yang efisien dan efektif untuk memahami minat pendengar yaitu dengan menjalankan pengamatan. KESIMPULAN Minat pendengar radio terhadap podcast di kalangan Generasi Z dapat berdampak positif bagi industri radio. Radio dapat menghadirkan konten podcast yang menarik dan relevan bagi Generasi Z, membantu radio dalam mendapatkan loyalitas dari generasi yang lebih muda dan memperluas audiens radio dengan menjangkau generasi yang lebih muda. Dengan memanfaatkan konvergensi media dan memperkenalkan podcast yang menarik, radio bisa tetap relevan dan bersaing di era digital ini. Peralihan minat dari radio ke podcast pada Generasi Z dapat disebabkan oleh beberapa faktor, antara lain: Kemudahan Akses, Konten yang personal dan relevan, Pilihan Variasi Konten, Kemampuan Menyesuaikan Jadwal, Interaksi yang Lebih Aktif, Audiobook, Jenis Platform, Penjelasan, Music Streaming Services, Live Streaming, Voice Assistants, dan Audio Social Media. Dengan kemajuan teknologi dan variasi platform, Generasi Z memiliki banyak pilihan alternatif untuk menikmati konten audio sesuai dengan minat dan preferensi. Hal ini menunjukkan konvergensi media terus berkembang dan memungkinkan akses lebih luas terhadap konten audio yang bervariasi. Generasi Z tumbuh dan dibesarkan di era sebelum kemajuan teknologi digital. Radio telah lama menjadi sumber hiburan dan informasi utama bagi generasi sebelumnya, membangun hubungan emosional dengan medium tersebut. Keterbatasan teknologi, sifat pasif mendengarkan, keterbatasan pilihan, dan keberlanjutan tradisi mempengaruhi preferensi terhadap radio. Generasi Z memilih radio dari podcast, karena keterbiasaan historis, keterbatasan teknologi, sifat pasif mendengarkan, keterbatasan pilihan, dan keberlanjutan tradisi mempengaruhi preferensi. Data statistik yang spesifik tentang minat Generasi Z untuk mendengarkan podcast dapat bervariasi tergantung pada sumber data dan penelitian yang dilakukan. Berdasarkan pengamatan dan studi pasar terbaru, beberapa temuan umum mungkin mendukung klaim bahwa Generasi Z memiliki minat berbeda terhadap podcast antara lain peningkatan Platform Audio Digital. Generasi Z lebih menyukai konten podcast yang bersifat edukatif, menghibur, atau memberikan wawasan yang bermanfaat dalam kehidupan sehari-hari. Interaksinya dengan Content Creators, Generasi Z cenderung lebih terhubung secara emosional dengan content creators atau podcaster yang menghasilkan konten yang mereka. Fleksibilitas dan Kemudahan Akses mendengarkan podcast secara on-demand di berbagai perangkat seperti ponsel pintar atau komputer juga merupakan faktor yang mungkin meningkatkan minat Generasi Z terhadap podcast. Beberapa cara yang dapat dilakukan untuk memahami dan mengadaptasi perbuhan minat pendengar adalah pengamatan, analisis data pendengar, mengikuti judul topik yang populer, dan mengikuti kemampuan yang bersifat dan kemampuan yang bersifat. DISKUSI Pengamatan ini dapat dilakukan secara online atau melalui media sosial. Pertanyaan dalam pengamatan dapat mencakup preferensi musik, topik yang diminati, acara yang paling sering didengar, dan faktor-faktor yang membuat mereka tetap loyal menjadi pendengar setia. 2. Analisis Data Pendengar Dengan memanfaatkan data pendengar yang ada, produsen konten dapat memahami preferensi dan perilaku pendengar. Data seperti umur, jenis kelamin, lokasi, dan platform yang paling digunakan dapat memberikan informasi berharga tentang audiens. 3. Mengikuti Judul Topik yang Populer Selalu up-to-date dengan topik dan tren yang populer dapat membantu meningkatkan minat pendengar. Produsen konten dapat memantau perkembangan di media sosial, berita, dan dalam industri terkait untuk mengetahui topik yang paling menarik dan relevan untuk didiskusikan. 4. Melibatkan Pendengar dalam Pembuatan Konten Mengundang pendengar untuk berpartisipasi dalam acara atau pembuatan konten dapat membantu produsen konten memahami minat dan preferensi pendengar. Hal ini juga menciptakan interaksi yang lebih dekat antara produsen konten dan pendengar, yang membantu dalam memperkuat loyalitas para pendengar. 5. Berkomunikasi dengan Pendengar Selalu membuka saluran komunikasi dengan pendengar dapat memberikan produsen konten informasi yang berharga tentang perubahan minat dan preferensi pendengar. Dengan berkomentar dan membalas tanggapan pendengar, produsen konten dapat menghasilkan keterkaitan yang lebih dekat dengan penerima atau pendengar dan mengetahui apa yang mereka inginkan. Dengan memahami dan mengadaptasi perubahan minat pendengar, produsen konten dan penyiar radio/podcast dapat menciptakan konten yang lebih menarik, relevan, dan berdampak pada pendengar. Hal ini juga dapat membantu dalam mempertahankan dan menarik pendengar baru. Selain itu, produsen konten juga dapat memanfaatkan inovasi teknologi dan media yang semakin berkembang untuk menjangkau dan menyesuaikan dengan minat pendengar yang terus berubah. 190 Jurnal Ilmu Komunikasi Dan Media Sosial (JKOMDIS) Vol.04 No. 01 Edisi Januari - April 2024 190 Jurnal Ilmu Komunikasi Dan Media Sosial (JKOMDIS) Vol. 4 No. 1 Edisi Januari -April 2024 Hal. 179-192 DOI : https://doi.org/10.47233/jkomdis.v3i3.1579 ISSN : 2807-6087 UCAPAN TERIMA KASIH Kami mengucapkan Terima Kasih kepada Dosen mata kuliah Komunikasi dan Teknologi Masyarakat Bapak Dr. Irwansyah atas bimbingan dan wawasannya yang berharga selama ini, dan kepada para pustakawan serta sumber informasi yang telah mendukung penyelesaian artikel ini. Kami mengucapkan Terima Kasih kepada Dosen mata kuliah Komunikasi dan Teknologi Masyarakat Bapak Dr Irwansyah atas bimbingan dan wawasannya yang berharga selama ini dan kepada para pustakawan serta sumber DAFTAR PUSTAKA [1] Ado, A. M. (2020). 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https://openalex.org/W3165417123	https://tckh.daihoctantrao.edu.vn/index.php/sjttu/article/download/178/109	Vietnamese	null	Bức tranh ngôn ngữ về thế giới - đặc trưng văn hoá - tư duy cộng đồng ngôn ngữ	Khoa học	2,021	cc-by-sa	2,789	1. Đặt vấn đề Khái niệm “bức tranh ngôn ngữ về thế giới” bắt nguồn một mặt từ tư tưởng của V.Humboldt và các nhà Tân Humboldt, mặt khác từ các tư tưởng của các nhà Ngôn ngữ học dân tộc Mĩ mà phần nào là từ giả thuyết tương đối về ngôn ngữ của Sapir - Whorf. Khái niệm này ngày càng được nhiều nhà khoa học trong và ngoài nước quan tâm, nhất là khi Ngôn ngữ học tri nhận phát triển. Mặc dù có những điểm chung nhưng mỗi ngôn ngữ tự nhiên có bức tranh ngôn ngữ về thế giới của riêng mình. Dẫn quan điểm của Iu.D. Aprexjan, Nguyễn Đức Tồn khẳng định: “Từ quan điểm của Ngôn ngữ học tri nhận, phương thức ý niệm hoá, hay còn gọi là cách nhìn thế giới, một phần có tính phổ quát, một phần có tính đặc thù dân tộc, nên những người nói những thứ tiếng khác nhau có thể nhìn thấy thế giới hơi khác nhau thông qua lăng kính ngôn ngữ của mình”. [59, trang 93] Ngôn ngữ học trong nước phải kể đến các nhà khoa học như Lý Toàn Thắng, Trần Văn Cơ, Nguyễn Đức Tồn, Lê Quang Thiêm... Theo quan niệm phổ biến của các nhà Ngôn ngữ học tri nhận trong và ngoài nước, có thể nhận định: Bức tranh ngôn ngữ về thế giới được hình thành trong nhận thức hằng ngày của một cộng đồng ngôn ngữ là tổng thể hình ảnh về thế giới được phản ánh trong ngôn ngữ, là phương thức tiếp nhận và cấu trúc hoá thế giới, là sự ý niệm hoá thực tế. Với quan niệm về bức tranh ngôn ngữ về thế giới như cách nhìn riêng đặc thù cho cộng đồng ngôn ngữ, chúng ta hoàn toàn có thể tiếp cận các vấn đề ngôn ngữ trong mối quan hệ với đặc trưng văn hoá - tư duy cộng đồng. Một trong những bình diện quan trọng để tìm hiểu đặc trưng văn hoá - tư duy cộng đồng từ ngôn ngữ là bình diện từ vựng. Cụ thể, xem xét cách nhìn của cộng đồng từ các góc độ: TAP CHÍ KHOA HỌC ĐẠI HỌC TÂN TRÀO ISSN: 2354 - 1431 ngôn ngữ và là thông tin về thế giới ẩn chứa trong hệ thống nghĩa của từ”. [81, trang 68] No.06_September 2017\|Số 06 - Tháng 9 năm 2017\|p.5-7 No.06_September 2017\|Số 06 - Tháng 9 năm 2017\|p.5-7 No.06_September 2017\|Số 06 - Tháng 9 năm 2017\|p.5-7 TAP CHÍ KHOA HỌC ĐẠI HỌC TÂN TRÀO ISSN: 2354 - 1431 http://tckh.daihoctantrao.edu.vn/ Bức tranh ngôn ngữ về thế giới - đặc trưng văn hoá - tư duy cộng đồng ngôn ngữ Đỗ Việt Hùng a a Trường Đại học Sư phạm Hà Nội Article info Abstract Recieved: 02/6/2017 Accepted: 03/8/2017 The linguistic worldview is formed in the daily perception of a language community. It is the overall image of the world which is reflected in language, it is the mode of accessing and the worldwide structure, it is the practical conceptualization. In the relations with cultural characteristics – community thinking, it can be considered from many sides, especially the world's division of a community through vocabulary of language. Statistics, comparisons, comparisons of identifying words have reflected the world’s division of a community. Dividing the world into meaning pieces of words has created the diversity of thinking and community culture. On the one hand, it has had a great influence on the formation of language; on the other hand it has brought the richness of thinking and culture which also makes the distinctions in the lexical meaning of words. Keywords: Linguistic worldview; Culture – thinking; World’s division; Vocabulary. 2. Nội dung vấn đề So sánh lượng từ ngữ của các ngôn ngữ, như vậy, sẽ có được những hiểu biết về sự chia cắt thế giới của cộng đồng ngôn ngữ, từ đó nhận ra những dấu hiệu đặc trưng văn hóa - tư duy của tộc người. Điều này khá quan trọng đối với thực tiễn nghiên cứu ngôn ngữ văn hóa ở một đất nước đa dân tộc như nước ta. Chẳng hạn người Việt chỉ có một từ duy nhất để chỉ thế giới tinh thần, đó là linh hồn Nhưng với người Thái thì khác bắt nguồn từquan Một trong những phương pháp quan trọng trong nghiên cứu mức độ “chia cắt” thế giới là lí thuyết xác lập ô trống. Phương pháp này dựa trên sự đối chiếu - so sánh các ngôn ngữ ở nhiều bình diện khác nhau, trong đó có so sánh vốn từ vựng giữa các ngôn ngữ theo mô hình ma trận để xác lập các ô trống ở mỗi ngôn ngữ (ô trống là ô không có từ ngữ tương ứng về nghĩa với từ ngữ trong ngôn ngữ được đối chiếu - so sánh). Nguyễn Đức Tồn và các học trò đã sử dụng phương pháp này để tiến hành đối chiếu các từ ngữ chỉ bộ phận cơ thể người, tên gọi động vật và tên gọi thực vật. Kết quả của các nghiên cứu này như sau: - Số lượng bộ phận cơ thể người được “chia cắt” định danh trong tiếng Việt là 289 và trong tiếng Nga là 227. Để định danh những bộ phận cơ thể người, tiếng Việt sử dụng 397 tên gọi, tiếng Nga sử dụng 251 (Dẫn theo [3], trang 227). - Về tên gọi động vật, theo thống kê của Nguyễn Thuý Khanh, tiếng Việt có 623 tên gọi, còn tiếng Nga có 394 tên gọi (Dẫn theo [3], trang 229). - Về tên gọi thực vật, theo thống kê của Cao Thị Thu, tiếng Việt có 657 tên gọi. (Dẫn theo [3], trang 230). Những số liệu đồng đại về số lượng từ ngữ ở các ngôn ngữ cho phép có được những nhận xét chính xác và thú vị về đặc điểm nhận thức thế giới, mức độ “chia cắt” thế giới của mỗi cộng đồng ngôn ngữ. 2. Nội dung vấn đề (i) Sự chia cắt thế giới của cộng đồng thể hiện qua vốn từ của ngôn ngữ; Phần lớn các nhà Ngôn ngữ học tri nhận đều nhận định rằng bức tranh ngôn ngữ về thế giới gắn với nhận thức về thế giới của cộng đồng ngôn ngữ được hình thành trong lịch sử và được truyền từ thế hệ này sang thế hệ khác. Popora Z.D. và Sternin I.A. định nghĩa: “Bức tranh ngôn ngữ về thế giới là hình ảnh về thực tế được thể hiện trong các tín hiệu ngôn ngữ và nghĩa của chúng - sự phân cắt thế giới bằng ngôn ngữ, sự sắp đặt các sự vật, hiện tượng bằng (ii) Quan niệm của cộng đồng về sự vật, hiện tượng thông qua các phương thức định danh của ngôn ngữ; (iii) Quan niệm của cộng đồng về sự vật, hiện tượng thông qua ý nghĩa của từ; (iv) Quan niệm của cộng đồng thông qua hiện tượng chuyển nghĩa từ vựng; (v) Quan niệm của cộng đồng thông qua các ngữ cố định (thành ngữ); D.V.Hung / No.06_September 2017\|p.5-7 (vi) Quan niệm của cộng đồng thông qua hiện tượng biểu trưng hoá của từ ngữ.v.v. (vi) Quan niệm của cộng đồng thông qua hiện tượng biểu trưng hoá của từ ngữ.v.v. niệm “mỗi người có 80 hồn vía, 30 hồn ở phía trước, 50 hồn ở phía sau”, tiếng Thái có tới hơn 80 từ để chỉ linh hồn, như khuân hua (hồn đầu); khuân ék (hồn óc), khuân kmom (hồn chóp), khuân phôm (hồn tóc); khuân puống nốm (hồn bầu vú); khuân hua chaư (hồn quả tim); khuân tọng nọi (hồn bụng nhỏ); khuân mốc luông (hồn bụng to)… Trong bài viết này chúng tôi bàn đến góc độ thứ nhất: Sự chia cắt thế giới của cộng đồng thể hiện qua vốn từ của ngôn ngữ Ý nghĩa của từ, trong đó có ý nghĩa biểu vật, bắt nguồn từ các sự vật, hiện tượng trong thế giới mà không phải là chính các sự vật, hiện tượng - Đó là nhận định khá phổ biến và gần như được hầu hết các nhà từ vựng học, ngữ nghĩa học chấp nhận. 2. Nội dung vấn đề Một bằng chứng quan trọng cho nhận định này là nếu ý nghĩa biểu vật của từ trùng với các sự vật, hiện tượng của thế giới thì số lượng từ ngữ trong các ngôn ngữ khác nhau phải như nhau. Nhưng trên thực tế, không phải như vậy. Số lượng các từ ngữ trong mỗi ngôn ngữ là riêng, không bằng nhau giữa các ngôn ngữ. Số lượng từ ngữ của ngôn ngữ phản ánh sự chia cắt thế giới của cộng đồng ngôn ngữ. Việc so sánh số lượng từ ngữ giữa các ngôn ngữ dẫn đến hai kết quả quan trọng. Thứ nhất, có thể thấy những sự vật, hiện tượng chỉ có ở cộng đồng này mà không có ở cộng đồng khác, chẳng hạn như nem (tên gọi một món ăn) chỉ có trong tiếng Việt. Thứ hai, điều này quan trọng hơn, là chỉ ra được cách nhìn, cách cảm nhận thế giới của mỗi cộng đồng ngôn ngữ, đặc biệt là tính khái quát và cụ thể trong nhận thức dân tộc về thế giới. Chẳng hạn, silk (loại vải tơ tằm) trong tiếng Anh có thể ứng với nhiều thứ khác nhau trong tiếng Việt: tơ, lụa, lượt, là, gấm, vóc, nhiễu, the, đoạn, lĩnh, đũi, nái, sồi, thao, vân, địa... [2, trang 404] cho thấy mức khái quát cao của từ silk trong nhận thức của người Anh về “các loại vải tơ tằm”, còn người Việt có nhận thức về những loại vải này cụ thể hơn nhiều. Tương tự, từ xanh của tiếng Việt ứng với blue, green trong tiếng Anh và 3 từ trong tiếng Nga синий, зелённый, голубой. Cũng như vậy, tiếng Việt có nhiều từ như tắm, giặt, rửa (người Việt phân biệt rất rõ các đối tượng được “làm sạch” để sử dụng tắm, giặt hay rửa) tương ứng với động từ to wash trong tiếng Anh. Hoặc người Nga phân biệt rõ tắm trong bể bơi (sông, hồ...) - купаться với tắm trong buồng tắm (принимать душ) nhưng người Việt chỉ có một từ tắm chỉ chung cho “làm sạch thân thể” (không có nét nghĩa ở đâu). 2. Nội dung vấn đề Và, việc đối chiếu này còn có thể ứng dụng trong nội bộ một ngôn ngữ đối với các phương ngữ và thổ ngữ của ngôn ngữ đó để nhận thấy “bức tranh ngôn ngữ về thế giới” của từng địa phương trong một quốc gia, một dân tộc. “Chia cắt” thế giới thành các “mẩu” nghĩa của từ làm nên sự đa dạng của tư duy và văn hoá cộng đồng, một mặt, có ảnh hưởng lớn đến sự hình thành số lượng từ của ngôn ngữ, mặt khác sự phong phú của tư duy và văn hoá còn làm nên những khu biệt tinh tế trong nghĩa từ vựng của các từ. So sánh các từ chỉ màu “xanh” (lá cây) giữa tiếng Việt với các ngôn ngữ khác như tiếng Nga - зелённый và tiếng Anh - green, dễ thấy tiếng Việt có nhiều từ hơn - xanh, xanh xanh, xanh um, xanh rì... và sự khu biệt nghĩa giữa các từ này tinh tế hơn so với nhiều ngôn ngữ khác. Điều này làm cho việc dịch nghĩa của các từ này sang các ngôn ngữ tương ứng sẽ khó khăn hơn. Thống kê, đối chiếu, so sánh số lượng từ không chỉ áp dụng đối với việc nghiên cứu các ngôn ngữ của quốc gia, dân tộc mà còn có thể sử dụng để tìm ra những đặc điểm riêng của các nhóm phương ngữ trong cộng đồng dân tộc chung. Khảo sát các từ ngữ chỉ “cá” trong phương ngữ Nghệ Tĩnh, Hoàng Trọng Canh đã thu thập được hơn 200 từ - gấp hai lần số lượng từ chỉ “cá” trong Từ điển tiếng Việt. Phân tích sự khác biệt về số lượng đó, Hoàng Trọng Canh đã nhận xét: “Sự phong phú về số lượng của lớp từ ngữ chỉ “cá” trong phương ngữ Nghệ Tĩnh không chỉ phản ánh sự phong phú về hiện thực nghề cá, nhiều loại cá được phản ánh gọi tên mà còn cho thấy đặc điểm phân cách đối tượng một cách cụ thể theo những đặc trưng lựa chọn mang tính biệt loại rõ ràng của cách cảm nhận, tri giác của người Nghệ. 2. Nội dung vấn đề Tuy nhiên, nếu chỉ dừng ở số liệu đồng đại, thì kết quả có thể chưa thật chính xác về đặc điểm nhận thức thế giới ở góc độ phân chia thế giới, nếu coi “bức tranh ngôn ngữ về thế giới” là một khái niệm hình thành trong lịch sử cộng đồng. Để chính xác hoá những nhận định về sự “chia cắt” thế giới cần kết hợp với nghiên cứu nguồn gốc các từ ngữ của cộng đồng. Phân tích nguồn gốc các tên gọi, đặc biệt chú ý đến số lượng các từ ngữ thuần Việt, so sánh với các tên gọi của tiếng Nga, Nguyễn Đức Tồn nhận định: “Những cứ liệu đã dẫn chỉ ra rằng số lượng tên gọi thuần Việt của bộ phận cơ thể người ít hơn so với các tên gọi thuần Nga. Để định danh bộ phận cơ thể, người Nga thường dùng từ ngữ trong vốn từ “của mình” hơn. Điều này cho phép giả định có lẽ xa xưa sự phạm trù hoá hiện thực khách quan ở phạm vi bộ phận cơ thể người Nga là chi tiết hơn so với người Việt. Trong “bức tranh ngôn ngữ về thế giới” ở người Việt khi đó có nhiều ô trống, hay “vết trắng” hơn so D.V.Hung/ No.06_September 2017\|p.5-7 với người Nga. Song người Việt đã nhanh chóng lấp đầy những khoảng trống ấy bằng cách vay mượn chủ yếu từ tiếng Hán...” [3, trang 228] và “nếu đối chiếu sự định danh thế giới động thực vật của người Việt và người Nga theo tham tố này (nguồn gốc tên gọi) có thể thấy rằng bức tranh ngôn ngữ về động vật của tiếng Nga có nhiều ô trống hơn so với bức tranh tương ứng trong tiếng Việt bởi vì tên gọi thuần Việt của động vật chiếm tới 93%, của thực vật tới 76,9% (505/657), trong khi đó tên gọi động vật thuần Nga chỉ chiếm 78% (155/198)” [3, trang 232, 233]. của cộng đồng ngôn ngữ thành tên gọi và qua đó xác định cách nhìn hay “bức tranh ngôn ngữ về thế giới” của mỗi dân tộc. TÀI LIỆU THAM KHẢO 1. Hoàng Trọng Canh (2011), Một kiểu định danh và biểu trưng đối với tên gọi nghề “cá” trong phương ngữ Nghệ Tĩnh, Website của Trường Đại học Vinh; 2. Nội dung vấn đề Chẳng hạn, người Nghệ không dừng lại ở tên gọi mực có ý nghĩa “chung chung” mà trong ý niệm, họ phân mực ra thành nhiều loại, nên mới có nhiều tên gọi khác nhau: mực lá, mực cơm, mực ống, mực nang, mực tuộc...”[1]. Những khác biệt về “bức tranh ngôn ngữ về thế giới” là những kết quả nghiên cứu không chỉ nhằm vào việc khám phá các đặc trưng văn hoá, tư duy của mỗi cộng đồng, sự khác biệt giữa các cộng đồng về nhận thức thế giới mà còn có ý nghĩa quan trọng trong giảng dạy ngôn ngữ cả cho người bản ngữ và người nước ngoài. 3. Kết luận Dễ dàng nhận thấy, đối chiếu đồng đại và lịch đại theo phương thức ma trận về tính [± tương ứng] từ ngữ giữa các ngôn ngữ khác nhau là hướng nghiên cứu có triển vọng lớn trong xác định mức độ “chia cắt” thế giới 2. Trần Ngọc Thêm (1998), Tìm về bản sắc văn hóa Việt Nam., Nxb TP. Hồ Chí Minh.; 2. Trần Ngọc Thêm (1998), Tìm về bản sắc văn hóa Việt Nam., Nxb TP. Hồ Chí Minh.; 3. Nguyễn Đức Tồn (2010), Đặc trưng văn hóa - dân tộc của ngôn ngữ và tư duy, Nxb Từ điển Bách khoa. 3. Nguyễn Đức Tồn (2010), Đặc trưng văn hóa - dân tộc của ngôn ngữ và tư duy, Nxb Từ điển Bách khoa.
https://openalex.org/W3195211852	https://link.springer.com/content/pdf/10.1007/s00253-021-11469-7.pdf	English	null	Identification of volatile compounds from bacteria by spectrometric methods in medicine diagnostic and other areas: current state and perspectives	Applied microbiology and biotechnology	2,021	cc-by	8,675	Key Points y • Detection of VOCs enables bacterial differentiation in various medical conditions. • Spectrometric methods may function as point-of-care diagnostics in near future. Keywords Bacteria · Volatile organic compounds · Spectrometry · Breath analyses · Diagnostics · Mass spectrometry · Ion mobility spectrometry Keywords Bacteria · Volatile organic compounds · Spectrometry · Breath analyses · Diagnostics · Mass spectrometry · Ion mobility spectrometry Applied Microbiology and Biotechnology (2021) 105:6245–6255 https://doi.org/10.1007/s00253-021-11469-7 Applied Microbiology and Biotechnology (2021) 105:6245–6255 https://doi.org/10.1007/s00253-021-11469-7 MINI-REVIEW Abstract Diagnosis of bacterial infections until today mostly relies on conventional microbiological methods. The resulting long turna- round times can lead to delayed initiation of adequate antibiotic therapy and prolonged periods of empiric antibiotic therapy (e.g., in intensive care medicine). Therewith, they contribute to the mortality of bacterial infections and the induction of multidrug resistances. The detection of species specific volatile organic compounds (VOCs) emitted by bacteria has been proposed as a possible diagnostic approach with the potential to serve as an innovative point-of-care diagnostic tool with very short turnaround times. A range of spectrometric methods are available which allow the detection and quantification of bacterial VOCs down to a range of part per trillion. This narrative review introduces the application of spectrometric analytical methods for the purpose of detecting VOCs of bacterial origin and their clinical use for diagnosing different infectious conditions over the last decade. K P i t Identification of volatile compounds from bacteria by spectrometric methods in medicine diagnostic and other areas: current state and perspectives Nils Kunze‑Szikszay1 · Maximilian Euler1 · Thorsten Perl2 Received: 8 May 2021 / Revised: 20 July 2021 / Accepted: 22 July 2021 / Published online: 20 August 2021 © The Author(s) 2021, corrected publication 2021 1 Department of Anesthesiology, University Medical Center Göttingen, Robert‑Koch‑Straße 40, 37075 Göttingen, Germany 2 Department of General, Visceral and Pediatric Surgery, University Medical Center Göttingen, Robert‑Koch‑Straße 40, 37075 Göttingen, Germany Introduction detecting volatile organic compounds (VOCs) as an option for the diagnosis of infectious diseases. Nowadays, with analytical methods allowing the detection of lowest quantities of VOCs in a headspace or exhaled breath sample, non-invasive diagnostic tools based on gas analytics seem to be within reach. In 1921, 100 years before this manuscript was put together, Harper F. Zoller and W. Mansfield Clark published a bench study on the production of volatile fatty acids by dysenteric bac- teria and the influence of different growing conditions on their production (Zoller and Clark 1921). By the use of distillation, they directly verified the types and amounts of fatty acids pro- duced by several bacterial strains and concluded that those might significantly contribute to the “unpleasant symptoms” which correlated with bacterial infections of the human intestinal tract. Most likely, neither of them was thinking about the possibility of Bacterial infections and the rise of multidrug resistances significantly contribute to morbidity and mortality world- wide. Mortality rates in septic shock, for which bacterial infections are the leading cause, do rise above 50%, even if patients are treated in a modern intensive care unit (SepNet Critical Care Trials Group 2016). Early diagnosis and patho- gen identification are crucial for the treatment of bacterial infections. The early beginning and the adequacy of the ini- tial antibiotic therapy significantly contribute to the patient’s outcome (Kumar et al. 2009). However, no biomarkers are available that reliably predict the onset of bacterial infec- tions or sepsis. Diagnosis therefore remains clinical and needs to rely on potentially life-threatening symptoms of the patients (Singer et al. 2016). Until today, culturing is the standard method for the identification of causative pathogens 2 Department of General, Visceral and Pediatric Surgery, University Medical Center Göttingen, Robert‑Koch‑Straße 40, 37075 Göttingen, Germany (0123 1 3456789) 3 Applied Microbiology and Biotechnology (2021) 105:6245–6255 6246 Table 1 List of studies discussed in this review that include clinical application of analytical methods Study Year Sample size Setting Conditions and pathogens studied Analytical method Sample matrix Sample method Respiratory infections Nasir et al 2018 60 Single-center, UK Cystic fibrosis (P. aeruginosa, S. aureus) GCxGC-TOF–MS BAL1 Direct analysis Coronel Teixeira et al 2017 106 Single-center, Paraguay Tuberculosis (M. tuberculosis) e-nose Breath Direct analysis Zetola et al 2017 71 Single-center, Botswana Tuberculosis (M. Introduction tuberculosis) e-nose Breath Pre-collected, sampling bags Van Oort et al 2017 93 Single-center, Netherlands VAP2 GC–MS Breath Pre-collected, sampling bags Gao et al 2016 60 Single-center, PR China VAP2 (A. bauma- nii) GC–MS Breath Pre-collected, des- orption tubes Neerincx et al 2016 18 Single-center, Netherlands Cystic fibrosis (S. aureus) GC–MS Breath Pre-collected, sampling bags Sahota et al 2016 21 Single-center, UK Tuberculosis (M. tuberculosis) FAIMS Breath Pre-collected, sampling bags Fowler et al 2015 46 Single-center, UK Lower airway infec- tions GC–MS Breath Pre-collected, des- orption tubes Kramer et al 2015 11 Single-center, Germany Cystic fibrosis (P. aeruginosa, S. aureus, C. albicans, A. xylososidans) GC–MS Breath SPME Schnabel et al 2015 100 Single-center, Netherlands VAP2 GC-TOF–MS Breath Pre-collected, sampling bags Filipiak et al 2014 28 Single-center, Austria VAP2 (S. aureus, C. albicans, E. coli) GC-TOF–MS Breath Pre-collected, des- orption tubes Nakhleh et al 2014 198 Multi-center, Rep. of South Africa Tuberculosis (M. tuberculosis) e-nose Breath Pre-collected, des- orption tubes Gilchrist et al 2013 20 Single-center, UK Cystic fibrosis (P. aeruginosa, S. aureus) SIFT-MS Breath Direct analysis Goeminne et al 2012 28 Single-center, Belgium Cystic fibrosis (P. aeruginosa) GC–MS Sputum SPME Gastrointestinal infections Berkhout et al 2019 843 Multi-center, Neth- erlands/Belgium Neonatal late-onset sepsis FAIMS Stool Direct headspace sampling Patel et al 2019 106 Single-center, UK CDI4 (C. difficile) GC-TOF–MS Stool Pre-collected head- space, desorption tubes Arasaradnam et al 2016 76 Single-center, UK IBD3 FAIMS Breath Pre-collected, sampling bags Bromers et al 2015 213 Two-center, Nether- lands CDI4 (C. difficile) FAIMS Stool Direct headspace sampling Arasaradnam et al 2013 62 Single-center, UK IBD3 e-nose, FAIMS Urine Direct headspace sampling Garner et al 2009 9 Single-center, Bangladesh Cholera (V. chol- era) GC–MS Stool SPME Lechner et al 2005 25 Single-center, Austria Gastritis (H. pylori) PTR-MS Breath Pre-Collected, Sampling bags Bloodstream infections Zhong et al 2019 46 Single-center, PR China BSI5 (E. coli) CDI-MS Blood SPME es discussed in this review that include clinical application of analytical methods 1 3 Applied Microbiology and Biotechnology (2021) 105:6245–6255 6247 1 BAL bronchoalveolar lavage; 2VAP ventilator-associated pneumonia; 3IBD inflammatory bowel disease; 4CDI Clostridoides difficile infection; 5BSI bloodstream infection; 6UTI urinary tract infection Table 1 (continued) Study Year Sample size Setting Conditions and pathogens studied Analytical method Sample matrix Sample method Chingin et al 2015 130 Single-center, PR China BSI5 (S. aureus, E. coli, K. pneumo- niae, A. baumanii, P. Introduction aeruginosa) APCI-MS Blood Direct headspace sampling Other infections Daulton et al 2020 19 Single-center, UK Wound infection GC-IMS Wound dressing Direct headspace sampling Lacey et al 2020 243 Single-center, UK Maternal strepto- coccal coloniza- tion (group B streptococci) GC-IMS Vaginal swabs Direct headspace sampling Kviatkovski et al 2018 26 Single-center, Israel Otitis externa (P. aeruginosa) GC–MS Pus samples Pre-collected, des- orption tubes Blankenstein et al 2015 57 Single-center, Germany Bacterial vaginosis IMS Vaginal swabs Direct headspace sampling Roine et al 2014 101 Single-center, Finland UTI6 (E. coli, S. saprophyticus, E. faecalis, Kleb- siella spp.) e-nose Urine Direct headspace sampling Chaim et al 2003 174 Single-center, Israel Bacterial vaginosis IMS Vaginal swabs Direct headspace sampling 1 BAL bronchoalveolar lavage; 2VAP ventilator-associated pneumonia; 3IBD inflammatory bowel disease; 4CDI Clostridoides difficile infection; 5BSI bloodstream infection; 6UTI urinary tract infection VAP ventilator-associated pneumonia; 3IBD inflammatory bowel disease; 4CDI Clostridoides difficile infection; TI urinary tract infection in medical microbiology. The use of these highly sensitive and very specific, but also time-consuming techniques, leads to turnaround times of up to 3 days for final results (Tabak et al. 2018). During this time, empiric broad-spectrum anti- biotic therapy is used to treat the infection (Paul et al. 2010). Without identification of the infecting organism and its sus- ceptibility, this empiric (or calculated) antibiotic therapy potentially risks inadequate treatment and induction of drug resistances and adverse effects (MacFadden et al. 2014). point, which remains a limiting factor regarding turnaround times. They need to be operated by trained personnel in specialized microbiological laboratories which, together with the high costs for acquisition and operation, leads to centralization of these systems at large hospitals or com- mercial providers (Ratiu et al. 2017). Hospitals lacking such facilities may send their microbiological specimen to such laboratories, again increasing turnaround times is increased and in some instances risking the integrity of the specimen, depending on length and circumstances of the transport. f The availability of rapid methods for the diagnosis of bac- terial infections and identification of the causative pathogen would help narrowing the empiric window and therewith contribute to a safer and more effective antibiotic therapy regimen (Battle et al. 2017; Tumbarello et al. 2010). Introduction Depending on the composition of the pathogen and resist- ance marker panel of the devices, it may, for instance, remain unclear for the user whether a detected resistance marker is associated with the detected pathogen or with a resident organism that is not part of the pathogen panel. The mecA gene is a marker for methicillin resistance and occurs in both Staphylococcus aureus and Staphylococcus epidermidis and is an example for such a possible cause of misinterpretation (Becker et al. 2006). Multiplex PCR systems incur relevant costs, and data on their ability to reduce mortality, morbid- ity, and therapy costs remain inconsistent (Warhurst et al. 2015). Their use will therefore be limited to highly equipped hospitals in economically developed countries. Depending on the composition of the pathogen and resist- ance marker panel of the devices, it may, for instance, remain unclear for the user whether a detected resistance marker is associated with the detected pathogen or with a resident organism that is not part of the pathogen panel. The mecA gene is a marker for methicillin resistance and occurs in both Staphylococcus aureus and Staphylococcus epidermidis and is an example for such a possible cause of misinterpretation (Becker et al. 2006). Multiplex PCR systems incur relevant costs, and data on their ability to reduce mortality, morbid- ity, and therapy costs remain inconsistent (Warhurst et al. 2015). Their use will therefore be limited to highly equipped hospitals in economically developed countries. The detection of VOCs of microbial origin is an alterna- tive approach with the potential to be a robust, fast, and relatively low-cost point-of-care method for pathogen differ- entiation and identification. A variety of analytical methods allows the detection and identification of VOCs (Ratiu et al. 2017). There is growing knowledge on the occurrence and composition of volatile metabolites emitted from bacteria and other microbes (Bos et al. 2013; Schulz and Dickschat 2007). With this narrative review, we aim to give a brief overview on the current state of knowledge on the use of spectrometric methods for the diagnosis of bacterial infec- tions and possible perspective of their future use. Immobility due to bulky setup and need for high effec- tive vacuum pumps and purified gas supply is a common drawback of all mass spectrometric devices. Thus, gas samples for diagnostic purposes need be transferred to the device for offline analysis. Introduction Gas bags or absorptive medi- ums like needle traps and solid-phase microextraction tools are used to transfer the sample to the mass spectrometric device. These pre-concentration techniques possibly influ- ence compound composition of gas samples by selective absorption and desorption depending on characteristics of the chosen transfer material (Slingers et al. 2021). There- fore, a favorable approach to determine VOCs is not only to measure real-time but also in a point-of-care setting with the possibility of direct sampling at the patient’s bed side (e.g., breath sampling). Introduction Inno- vative applications include microbial identification directly from positive blood cultures by the use of matrix-assisted laser desorption ionization-time-of-flight mass spectrom- etry (MALDI-TOF MS) or the Accelerate Pheno™ System, which combines fluorescence in situ hybridization (FISH) with morphokinetic cellular analysis (MCA) (Pancholi et al. 2018; Rodriguez-Sanchez et al. 2014). These proteomic (MALDI-TOF MS) and optical (FISH-MCA) methods contribute to shorter turnaround times and were shown to deliver reliable results on pathogen identification and anti- biotic resistances. Both systems are commercially available and in clinical use. However, both methods require at least a positive blood culture (or equivalent specimen) as a starting Multiplex polymerase chain reaction (multiplex PCR) is another innovative method allowing rapid pathogen identi- fication including the detection of genetic antibiotic resist- ance markers. Currently, three systems (SepsiTest [Molzym Molecular Diagnostics, Bremen, Germany], IRIDICA BAC BSI [Abbott Diagnostics, Lake Forest, USA], LightCy- cler SeptiFast [Roche, Risch-Rotkreuz, Switzerland]) are commercially available for pathogen identification from blood cultures (Stevenson et al. 2016). One cartridge sys- tem (Unyvero A50 [Curetis, Holzgerlingen, Germany]) is targeting several localized infection sides as well as blood- stream infections (Burrack-Lange et al. 2018). Of those, the Unyvero A50 is the only system that can be operated point- of-care, which leads to relevant reduction of turnaround times (Kunze et al. 2015). However, multiplex PCR devices are not part of the clinical routine in the majority of hospitals and possess some methodological challenges by themselves. 1 3 3 Applied Microbiology and Biotechnology (2021) 105:6245–6255 6248 et al. 2017). GC–MS does possess large databases for iden- tification of substances and the capability of separating and unequivocal identification of compounds. Multiple publications in the field of bacterial VOC determination with GC–MS demonstrated its applicability for this pur- pose (Liebeke et al. 2012; Tait et al. 2014). Alternatives to GC–MS are other mass spectrometric instrumentations like proton transfer reaction-mass spectrometers (PTR- MS) or selected ion flow tube-mass spectrometer (SIFT- MS). Secondary electrospray ionization mass spectrometry (SESI-MS) has been shown to be more sensitive compared to PTR-MS or SIFT-MS enabling substance detection in the ppt range (Woolfenden 2010). A strength of these devices, which are still bulky and not portable, is the pos- sibility of real-time measurements at a high sensitivity in range of ppb. However, a chemical identification of unknown VOCs is not possible. Respiratory infections Tuberculosis (TB), caused by Mycobacterium tuberculosis, is one of the leading causes of death among infectious dis- eases worldwide (Khatua et al. 2017). A reliable, simple, and cheap test for the diagnosis of TB is of outmost interest, especially for low-income countries. In 2016, Sahota et al. described the use of a FAIMS device with a non-radioactive UV ionization source for VOC pattern recognition to diag- nose TB in the UK (Sahota et al. 2016). They investigated a rather small number of 21 breath samples of TB patients compared to 19 healthy controls and found a sensitivity and specificity of 81% and 79%. Three groups used e-nose devices for the same purpose and described sensitivities and specificities of 88–94% and 90–93% for diagnosing TB (Coronel Teixeira et al. 2017; Nakhleh et al. 2014; Zetola et al. 2017). None of the four studies identified individual VOCs that were used for the prediction of TB.i The majority of pulmonary infections in cystic fibrosis (CF) is caused by Pseudomonas aeruginosa (PA) or Staphy- lococcus aureus (SA). In 2016, Neerincx et al. published a feasibility study on the identification of SA-infected CF patients in a collective of 18 patients. Using Tedlar® bags, they collected breath samples and analyzed them using GC–MS. They were able to identify nine VOCs which were useful for separating SA-infected patients. Three of these VOCs did significantly differ between the groups (1,4 pen- tadiene, acetone, undecane). Interestingly, seven of the 13 CF patients infected with SA were also infected with PA, Serratia marcescens, or Haemophilus influenzae (Neerincx et al. 2016). By measuring the concentration of hydrogen cyanide using SIFT-MS analyses of nose-exhaled breath, Gilchrist et al. successfully differentiated between chroni- cally PA-infected and non-infected CF patients (Gilchrist et al. 2013). The discrimination of PA-infected and non- infected CF patients is also possible by pattern recognition of VOCs as it was shown in a study using SPME enrichment for indirect sampling of breath for GC–MS analyses from CF patients (Kramer et al. 2015). Besides breath analysis, detection of PA-specific VOCs was also detected in sputum headspace (Goeminne et al. 2012) and over bronchoalveolar lavage fluid samples (Nasir et al. 2018). Electronic noses, often referred to as “e-noses”, repre- sent a third analytical approach by using biochemical sensor arrays for the detection of VOCs and VOC patterns (Wilson 2020). Respiratory infections E-noses are small and inexpensive and allow screen- ing for certain chemical conditions while being easy to oper- ate. However, these devices do not allow identification of individual substances. It is therefore necessary to configure a suitable sensor array for each individual analytical ques- tion. E-nose applications may be used to translate the results of more sophisticated analytical methods, like mass spec- trometric or IMS methods, into easy-to-operate and rugged bedside tools. Spectrometric methods for VOC detection Gas chromatography coupled with mass spectrometry (GC–MS) is the gold standard for VOC detection (Ratiu 1 3 1 3 Applied Microbiology and Biotechnology (2021) 105:6245–6255 6249 different applications with a focus on clinical studies pub- lished within the last 5 years (Table 1). to serve as point-of-care diagnostic tools in challenging healthcare settings. The spectrum of IMS-based analytical methods is grow- ing. Couplings of more than one detector (e.g., FAIMS- FAIMS), the use of different pre-separation methods (e.g., GC, MCC), different ionization methods, and even pre-con- centration techniques facilitate individual analytical solu- tions for specific circumstances. However, due to diverse setup of these systems, a comparison of findings is difficult. Targeted analyses are possible when known substances are investigated, and reference measurements can be provided for confirmation. The composition of volatile organic com- pounds over growing bacterial species, however, contains mostly substances that are unknown to the researcher in the first place. This untargeted analytic approach often leads to the approach of pattern recognition, where none or not all substances are identified. Using IMS techniques, the identifi- cation of (suspected) substances can be very challenging and time- and resource-consuming. The position of a substance in the topogram of a GC-IMS device is unique, but it needs to be confirmed by reference measurements of the pure substance. Once a substance is confirmed, this information can contribute to a specific database for each application, allowing identification of substances even without a refer- ence measurement. However, this information is only valid for very similar IMS applications. The diversity of analytical setups of devices makes it often hard to compare results and to deduce conclusions from results of similar but “different enough” methods. Spectrometric methods for VOC detection The metabolic pathways of bacteria have been investi- gated in detail (Schulz and Dickschat 2007). Ideally, the occurrence of a VOC should be assignable to a known metabolic pathway. However, the production of VOCs by bacteria is influenced by their growing conditions (O'Hara and Mayhew 2009). Our knowledge of bacterial metabolic pathways and therewith VOC production under various conditions in the human body is still very limited. In 2013, Bos et al. published a detailed review of VOCs produced by the six most relevant bacteria in sepsis: S. aureus, S. pneumoniae, E. faecalis, K. pneumoniae, P. aeruginosa, and E. coli (Bos et al. 2013). In contrast to mass spectrometric methods, ion mobility spectrometry (IMS) applications are portable and even available as handhelds. IMS devices are already widely used by rescue services, security authorities, and the military for the detection of hazardous substances, explosives, and chemical warfare agents (Cumeras et al. 2015). The method allows substance detection and quantification down to a range of ppt (Kunze et al. 2015). Time-of-flight-IMS (TOF-IMS), aspiration-IMS (aIMS), field asymmetric ion mobility spectrometry (FAIMS), and differential mobility spectrometers IMS (DMS) have been described for microbiological VOC detection (Costanzo et al. 2017; Ratiu et al. 2017). IMS devices can be used separately (e.g., as screening tools for a known substance), or they can be coupled with a chromatographic method, which allows analyses of more complex gas samples. GC columns or multicapillary columns (MCC) are mostly used for pre- separating complex matrices such as breath or headspace samples of complex growth media (Baumbach 2009; Speckbacher et al. 2021). IMS devices are inexpensive, robust, and portable. They therefore have the potential The analytical challenge for available devices is the detection and quantification of VOCs in a complex matrix of gaseous samples with traces down to parts per billion (ppb) or even trillion (ppt). Ideally, the detected VOCs are produced by spe- cies cultivated on defined, standardized substrate such as cul- ture media. In the clinical context, it may also be desirable to directly detect VOCs from highly diverse substrates like urine, feces, sputum, vaginal fluids, tissues, or breath (Amann et al. 2014; Eng et al. 2015; Lacey et al. 2020; Purkhart et al. 2011; Ratiu et al. 2019). Diagnostic applications for bacterial infections In 2016, Gao et al. published a study in which they differenti- ated the presence from the absence of A. baumanii in the lower respiratory tract, as well as the colonization from an actual infection using breath VOC profiles (Gao et al. 2016). volatilome. Breath analysis differentiated inflammatory bowel disease (IBD) from control and Crohn’s disease from ulcerative colitis (Arasaradnam et al. 2016). The same study groups were previously able to separate IBD patients from healthy controls by headspace analyses of urine samples using an e-nose and FAIMS, whereas both systems enabled discrimination of the groups (Arasaradnam et al. 2013). Bloodstream infections Bacterial bloodstream infections are a common cause for sepsis, a major burden for healthcare systems with high mortality, and require fast diagnosis and therapy (SepNet Critical Care Trials Group 2016). Diagnosis still depends on unspecific clinical presentations, and pathogen identifi- cation relies on culturing, which leads to relevant delays in initiating targeted antibiotic treatment (Singer et al. 2016). Gastrointestinal infections Urea breath tests using radioactive carbon isotopes are the gold standard for diagnosing Helicobacter pylori infec- tions and show specificity and sensitivity of approximately 95% (Nakayama and Graham 2004). With their PTR-TOF- S breathing gas investigations of infected and uninfected patients, Lechner et al. found significantly elevated hydrogen cyanide and hydrogen nitrate concentrations in H. pylori- infected patients (Lechner et al. 2005).l A prospective multicenter study from the Netherlands and Belgium underlined the potential of spectrometric analytical methods for individual therapy planning. By investigating the volatilome of stool samples of 127 cases of neonatal late-onset sepsis and matched controls, they were able to discriminate both groups. They used FAIMS for analyz- ing unprocessed fecal samples of neonates which provides results after very short period of time (Berkhout et al. 2019). Diagnostic applications for bacterial infections l Ventilator-associated pneumonia (VAP) is a common nosocomial infection. Diagnosis is time critical and still based on unspecific clinical criteria and pathogen detection in bronchoalveolar lavage. Breath analysis could be a fea- sible, non-invasive approach for both VAP diagnosis and pathogen identification. Schnabel et al. in 2015 used GC- TOF–MS for investigating samples of exhaled breath from 100 ventilated patients in intensive care for the occurrence of Over the last two decades, there is a growing interest in the experimental and clinical application of spectrometric methods for the detection of bacterial VOCs for diagnostic purposes. The results of the studies from a broad variety of medical fields underline the potential of the approach. Following, we aim to give a comprehensive overview of 1 Applied Microbiology and Biotechnology (2021) 105:6245–6255 6250 VAP-specific VOCs. With a set of 12 VOCs, they were able to correctly discriminate between patients with and without VAP (Schnabel et al. 2015). Other authors described changes in the volatile metabolite profile of ventilated patients with bacterial colonization of the lower respiratory tract (Fowler et al. 2015). In 2017, van Oort et al. did also discriminate VAP from control with relatively good accuracy. They analyzed 12 VOCs that changed during pneumonia and 52 VOCs that changed in colonized patients (van Oort et al. 2017). Filipiak et al. showed that the concentration of spe- cific compounds changed over the course of the infection and also correlated with illness severity (Filipiak et al. 2015). In 2016, Gao et al. published a study in which they differenti- ated the presence from the absence of A. baumanii in the lower respiratory tract, as well as the colonization from an actual infection using breath VOC profiles (Gao et al. 2016). VAP-specific VOCs. With a set of 12 VOCs, they were able to correctly discriminate between patients with and without VAP (Schnabel et al. 2015). Other authors described changes in the volatile metabolite profile of ventilated patients with bacterial colonization of the lower respiratory tract (Fowler et al. 2015). In 2017, van Oort et al. did also discriminate VAP from control with relatively good accuracy. They analyzed 12 VOCs that changed during pneumonia and 52 VOCs that changed in colonized patients (van Oort et al. 2017). Filipiak et al. showed that the concentration of spe- cific compounds changed over the course of the infection and also correlated with illness severity (Filipiak et al. 2015). 1 3 Gastrointestinal infections An alternative approach could be using VOC analyses of the headspace over inoculated blood cultures. Umber et al. used different GC couplings to investigate the headspace over experimental blood cultures, consisting of LB broth, human whole blood, and E. coli. They described six VOCs that were specific for E. coli-infected whole blood, even when compared to samples of inoculated LB broth with- out blood (Umber et al. 2013). Another group used corona discharge ionization MS to describe the increase of indole in the headspace of blood cultures incubating with E. coli (Zhong et al. 2019). Our own group recently published promising results regarding the use of GC-IMS for rapid differentiation of E. coli, P. aeruginosa, and S. aureus by analyzing the headspace of experimental blood cultures very quickly (Drees et al. 2019).i Another potential application is the investigation of fecal volatilome for the diagnosis of gastrointestinal infections. One confounding factor for such analyses is the highly inter- individual fecal microbiome. Together with a complex vola- tile background caused by fermentation processes, this leads to a challenging environment for gas analyses (Elmassry and Piechulla 2020).fi Clostridioides difficile causes antibiotic associated diar- rhea and has been found to cause a unique fecal volati- lome. Both GC-tof–MS and FAIMS discriminated between (unprocessed) infected and uninfected stool samples with high diagnostic accuracy (Bomers et al. 2015; Patel et al. 2019). GC–MS has also been successfully used to identify volatile biomarkers of Vibrio cholorae emitted from stool samples (Garner et al. 2009). Chingin et al. conducted the first clinical feasibility study using APCI-MS (atmospheric pressure chemical ionization mass spectrometry) to investigate the volatilome of blood cultures from patients with suspected or confirmed bactere- mia. Within 3–16 h, they were able to detect specific VOC patterns and successfully differentiated between five com- mon sepsis pathogens (Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa) (Chingin et al. 2015). A prospective multicenter study from the Netherlands and Belgium underlined the potential of spectrometric analytical methods for individual therapy planning. By investigating the volatilome of stool samples of 127 cases of neonatal late-onset sepsis and matched controls, they were able to discriminate both groups. They used FAIMS for analyz- ing unprocessed fecal samples of neonates which provides results after very short period of time (Berkhout et al. 2019). Other applications Urea breath tests using radioactive carbon isotopes are the gold standard for diagnosing Helicobacter pylori infec- tions and show specificity and sensitivity of approximately 95% (Nakayama and Graham 2004). With their PTR-TOF- S breathing gas investigations of infected and uninfected patients, Lechner et al. found significantly elevated hydrogen cyanide and hydrogen nitrate concentrations in H. pylori- infected patients (Lechner et al. 2005).l Urinary tract infections (UTI) are common and mostly caused by bacteria. In 2011, Storer et al. used SIFT-MS (selected ion flow tube mass spectrometry) to investigate the headspace over experimentally inoculated urine samples. They were able to distinguish between eight etiological bac- teria and fungi, namely E. coli, P. vulgaris, P. aeruginosa, S. aureus, S. epidermidis, K. pneumoniae, E. faecalis, or C. albicans (Storer et al. 2011). In their clinical study from 2014, Roine et al. demonstrated the potential of an IMS Inflammatory bowel disease diagnosis remains a clini- cal challenge, and Crohn’s disease and ulcerative colitis have shown distinct VOCs, which seem to reflect the gut 1 3 1 3 Applied Microbiology and Biotechnology (2021) 105:6245–6255 6251 based e-nose device to identify infected urine samples and to differentiate between four common causative bacteria: E. coli, S. saprophyticus, E. faecalis, and Klebsiella spp. (Roine et al. 2014). explains its occurrence in a bacterial infectious condition and therefore increases the conclusiveness of these results. During the last decade, the value of breath tests based on spectrometric methods has been investigated in a multitude of medical condi- tions ranging from several lung pathologies (e.g., infections, malignomas, COPD) up to neurological conditions, such as Alzheimer’s disease (Bach et al. 2015; Darwiche et al. 2011; Fink et al. 2014). The limitation of the pattern recognition or fingerprinting approach without identification of volatile sub- stances is the lack of causative explanation for its occurrence in the specific conditions. Most studies are monocentric, and the numbers of investigated patients are small, which increase the risk of random cohesions. In the best case, substance iden- tification would make it possible to explain the origin of an occurring VOC by referencing the underlying mechanisms of, e.g., known bacterial metabolic pathways. Bacterial vaginosis is a common infection in gynecol- ogy. IMS is able to detect malodorous biogenic amines from vaginal swabs (Chaim et al. 2003). Meanwhile, IMS devices for point-of-care testing for bacterial vaginosis are commer- cially available and show a good accuracy (Blankenstein et al. 2015). Other applications Infection with group B Streptococcus is a potentially life- threatening complication in newborn infants. By analyzing maternal vaginal swabs using a GC-IMS device, Lacey et al. recently demonstrated the ability of this method to diagnose maternal group B Streptococcus infections with high sen- sitivity and specificity. They concluded that the use of the technique as a screening tool could significantly contribute to the prevention of the condition (Lacey et al. 2020). The sampling of human breath remains challenging, as breathing air by itself provides a high degree of diversity and complexity. Factors like gender, race, age, diet, and lifestyle habits can influence the composition of VOCs in human breath. Detection of a substance depends on its chemical and physical properties and on the ability to detect it with the chosen analytical method. For instance, may the ability of an IMS to detect indole over a growing E. coli culture depend on the type of GC column chosen for pre-separation. The reviewed literature demonstrates heterogeneous analytical approaches for the same or similar questions. It also shows that our knowledge on the biochemical implication of the occurrence or non-occurrence of VOCs in certain constel- lations remains limited and should be addressed in future research.i Recently, Daulton et al. reported of the use of a GC- IMS device to distinguish between infected and uninfected wounds. Wound infections significantly complicate the treatment of surgical patients and cause a major burden on healthcare systems worldwide. Screening for specific VOCs indicating infection of wounds could therefore be an alterna- tive approach to the conventional microbiological diagnostic using wound swabs (Daulton et al. 2020). By using GC–MS, Kviatkovski et al. identified 2-ami- noacetophenone (2-AA) as a specific VOC indicating the presence of P. aeruginosa in pus samples of otitis externa patients. They assembled a device combining a whole-cell luminescent biosensor with photo-multiplier tube enabling the detection of 2-AA at very low concentrations. By using this device, they were able to diagnose otitis externa caused by P. aeruginosa with high accuracy (Kviatkovski et al. 2018). In the future, substance identification should be used to clearly link the results of basic research to the findings of clinical investigations. International and multicenter stud- ies, adequately powered, are necessary to generate high- quality evidence for the most urgent and promising fields of application. Future perspectives and challenges As it was demonstrated by Zoller and Clark a century ago, the smell of bacteria can lead to the correct diagnosis. The ability of modern spectrometric techniques makes it possible to detect smallest amounts of volatile substances and to use gas samples as a diagnostic matrix. Growing interest of the scientific community in these methods will certainly lead to more clinical applications of point-of-care diagnostics, although challenges remain. During the current SARS-CoV-2 pandemic, breath tests for the detection of viral infections have come in the focus of scientific and commercial interest (Ruszkiewicz et al. 2020). This dem- onstrates both the potential of the methodology and the chal- lenges that come with their application. Most of the investiga- tions cited in this review did not identify the actual substances that contribute to a VOC pattern or fingerprint that is consid- ered to be specific for a condition. Pattern recognition leads to valuable scientific results, and it may be justifiable to relin- quish substance identification as it would be time-consuming, expensive, and in some cases impossible. The development of urgently needed diagnostic tools may be hindered in an unjus- tifiable manner. One may however argue that identification of a substance with a clear link to a known metabolic pathway Conclusion The association of volatile organic compounds (VOCs) with bacterial presence and growth has led to a novel diagnos- tic approach, suggesting the use of spectrometric analyti- cal methods for the detection of pathogen-specific VOCs or 1 3 3 Applied Microbiology and Biotechnology (2021) 105:6245–6255 6252 VOC patterns. Basic research using complex, expensive, and space-consuming mass spectrometric methods provided knowledge on the occurrence of VOCs in association with several bacterial species. Based on these findings, less com- plex, robust, fast, and miniaturized applications, such as IMS and FAIMS, aim to transfer this knowledge into clinical, potentially point-of-care, use. Over the last two decades, a growing number of studies tested spectrometric analytical methods for diagnosing bacterial infections. 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Arch Gynecol Obstet 292(2):355-62 https://doi.org/10.1007/s00404-014-3613-x Open Access This article is licensed under a Creative Commons Attri- bution 4.0 International License, which permits use, sharing, adapta- tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. 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Funding Open Access funding enabled and organized by Projekt DEAL. p g Berkhout DJC, van Keulen BJ, Niemarkt HJ, Bessem JR, de Boode WP, Cossey V, Hoogenes N, Hulzebos CV, Klaver E, Andreessen P, van Kaam AH, Kramer BW, van Lingen RA, Schouten A, van Goudoever JB, Vijlbrief DC, van Weissenbruch MM, Wicaksono AN, Covington JA, Benninga MA, de Boer NKH, de Meij TGJ (2019) Late-onset sepsis in preterm infants can be detected pre- clinically by fecal volatile organic compound analysis: a prospec- tive, multicenter cohort study. Clin Infect Dis 68(1):70–77. https:// doi.org/10.1093/cid/ciy383 Declarations Ethics approval This article does not contain any studies with human participants or animals performed by any of the authors. Conflict of interest The authors declare no competing interests. Conflict of interest The authors declare no competing interests. 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https://openalex.org/W2148212084	https://josr-online.biomedcentral.com/track/pdf/10.1186/1749-799X-4-44	English	null	False aneurysm of the interosseous artery and anterior interosseous syndrome - an unusual complication of penetrating injury of the forearm: a case report	Journal of orthopaedic surgery and research	2,009	cc-by	3,013	Published: 24 December 2009 ; This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: Palsies involving the anterior interosseous nerve (AIN) comprise less than 1% of all upper extremity nerve palsies. Objectives: This case highlights the potential vascular and neurological hazards of minimal penetrating injury of the proximal forearm and emphasizes the phenomenon of delayed presentation of vascular injuries following seemingly obscure penetrating wounds. Case Report: We report a case of a 22-year-old male admitted for a minimal penetrating trauma of the proximal forearm that, some days later, developed an anterior interosseous syndrome. A Duplex study performed immediately after the trauma was normal. Further radiologic investigations i.e. a computer-tomographic-angiography (CTA) revealed a false aneurysm of the proximal portion of the interosseous artery (IA). Endovascular management was proposed but a spontaneous rupture dictated surgical revision with simple excision. Complete neurological recovery was documented at 4 months postoperatively. Conclusions/Summary: After every penetrating injury of the proximal forearm we propose routinely a detailed neurological and vascular status and a CTA if Duplex evaluation is negative. Open A Case report False aneurysm of the interosseous artery and anterior interosseous syndrome - an unusual complication of penetrating injury of the forearm: a case report Ramon Pini*, Stefano Lucchina, Guido Garavaglia and Cesare Fusetti Received: 17 July 2009 Accepted: 24 December 2009 Page 1 of 4 (page number not for citation purposes) Journal of Orthopaedic Surgery and Research BioMed Central Open Access Introduction p A 22 year old male sustained a penetrating injury of the forearm, after falling into a glass window during his stay in the Far East. The initial haemorrhage was treated with a simple compression. X-ray showed a small glass-like for- eign body (fig. 1). A Duplex study was apparently normal. A few days later, he developed a rapidly complete sensory deficit on the median nerve and a loss of motor function on the AIN. No specific therapy or further investigation was proposed to the patient, who, back home 4 weeks Penetrating isolated lesions of the interosseous anterior neurovascular bundle are rare. We report the case of a 22- year-old male who sustained such a lesion with formation of a false aneurysm of the proximal portion of the interos- seous artery (IA). A review of the literature showed one similar case of infective origin so that our description is the first of post-traumatic vascular compression of the anterior interosseous nerve (AIN) [1]. Page 1 of 4 (page number not for citation purposes) Page 1 of 4 (page number not for citation purposes) http://www.josr-online.com/content/4/1/44 Journal of Orthopaedic Surgery and Research 2009, 4:44 X-ray Figure 1 X-ray. The arrow shows the glass-like foreign body in the forearm. Intraoperative picture Figure 2 Intraoperative picture. The scissors shows the glass-like fragment lodged exactly into the first motor bifurcation of the anterior interosseous nerve (AIN). The main nerve is undamaged. Intraoperative picture Figure 2 Intraoperative picture. The scissors shows the glass-like fragment lodged exactly into the first motor bifurcation of the anterior interosseous nerve (AIN). The main nerve is undamaged. Intraoperative picture Figure 2 Intraoperative picture. The scissors shows the glass-like fragment lodged exactly into the first motor bifurcation of the anterior interosseous nerve (AIN). The main nerve is undamaged. X-ray Figure 1 X-ray. The arrow shows the glass-like foreign body in the forearm. is penetrating trauma secondary to gunshot wounds, stab wounds and lacerations from broken glass. However, iatrogenic traumas secondary to the widespread use of diagnostic and therapeutic intravascular techniques have also contributed to the increase in incidence [2,3]. later, consulted our unit. An established ischemic contrac- ture (Holden moderate type) was clinically suspected. Electrophysiological studies confirmed the neurological lesion, with partial denervation of the flexor pollicis lon- gus (FPL), coupled with moderate reduction of sensitive conduction in the median and ulnar nerves. We decided on surgical exploration. Introduction An extensive hematoma in the flexor's compartment was drained with extraction of the glass fragment which was lodged exactly in the first motor bifurcation of the AIN. The main trunk of the AIN was undamaged (fig. 2). The motor branch was reconstructed with a nerve graft. In the absence of evidence of a vascular lesion and active bleeding, a simple fasciotomy was per- formed before skin closure. Nerve compression from a false aneurysm is extremely rare. A review of the literature showed one similar case of infective origin and two other cases with compression of the posterior interosseous nerve [1,4,5]. All the other cases in the arm were related to compression of the brachial plexus, median and ulnar nerve [6-13]. The peculiarity of our case report is the neurologic involvement of the AIN. Angio-CT-Scan Figure 3 Angio-CT-Scan. False aneurysm of the proximal portion of the interosseous artery (IA). Two days later the arm became newly swollen and pain- ful. A computer-tomographic-angiography (CTA) (fig. 3) confirmed a false aneurysm of the IA. An endovascular embolisation was planned, but suddenly excruciating pain dictated an immediate surgical revision with aneu- rysm excision and arterial ligation. A complete neurologi- cal recovery was documented four months later. Discussion Upper extremity injuries constitute 30-50% of all periph- eral vascular injuries, more than 80% of which are from penetrating trauma. Radial and ulnar arterial injuries make up 5-30% of all peripheral vascular injuries [2]. The most common cause of upper extremity vascular injuries Angio-CT-Scan Figure 3 Angio-CT-Scan. False aneurysm of the proximal portion of the interosseous artery (IA). Page 2 of 4 (page number not for citation purposes) http://www.josr-online.com/content/4/1/44 http://www.josr-online.com/content/4/1/44 Journal of Orthopaedic Surgery and Research 2009, 4:44 Journal of Orthopaedic Surgery and Research 2009, 4:44 The AIN branches from the median nerve between the two heads of the pronator teres muscle, just distal to the ori- gins of the motor branches to the superficial forearm flexor muscles and then runs with the IA on the anterior surface of the interosseous membrane, between and deep into the FPL and flexor digitorum profundus 1+2, which it supplies [14]. to the glass fragment found in the first motor bifurcation of the AIN. We were not able to visualize the path of the glass fragment because of the hematoma and the time lapse between the accident and the exploration. Retro- spectively, the partial lesion of the IA by the glass fragment with secondary formation of a false aneurysm can explain both the hematoma and the anterior interosseous syn- drome. The glass fragment in the first motor bifurcation cannot be the sole explanation of the anterior interos- seous syndrome because the main trunk was intact. The compression of the AIN from the false aneurysm is, how- ever, instead a plausible explanation. A focused history and thorough physical examination, combined with a working knowledge of normal vascular anatomy, can help identify most vascular abnormalities of the upper extremity. Technologic improvements, such as Duplex and CTA, now allow accurate diagnosis by non- invasive methods [15,16]. Nevertheless, our case shows that in a perforating trauma of the arm a careful neurolog- ical examination must always be performed, otherwise minor neurological signs, i.e. FPL dysfunction could pass unobserved. Although conventional teaching usually holds that an electro-diagnostic study should not be done until about 3 weeks after the injury, in fact a great deal of important information can be obtained by studies carried out within the first week [17]. For the secondary revision we believe that an endovascu- lar approach would have been appropriate, to avoid a new dissection after the first microsurgical suture. Discussion Likewise, a primary endovascular approach could be considered, but only after a correct preoperative diagnosis and only in the absence of an extrinsic hematoma as the compression on the nerve would remain. However, because it is a rare condition, this kind of approach has not been described in the literature and in our case an unexpected rupture of the aneurysm during the night imposed the classical and simple intervention with resection of the aneurysm. Similar to the cases described by Illuminati and Kim the results were optimal [1,21]. For the vascular status, the first additional diagnostic modality is the Duplex: less expensive, rapidly carried out and successful in detecting significant lesions such as false aneurysms, arteriovenous fistulae, and major vessel occlu- sions [18]. In case of negative or uncertain result, a CTA or a simple arteriography should be routinely performed [1,19,20]. Nowadays, nonsurgical approaches play an important role in the treatment of peripheral false aneurysms [16,22]. Endoluminal repair of false aneurysms, large arte- riovenous fistulas, intimal flaps, and focal lacerations, is performed by using stent-graft technology. Castelli and colleagues reported a 100% immediate success rate in managing axillo-subclavian arterial injuries [23]. In the study of Onal et al, all the stent-grafts in 17 patients with iatrogenic, traumatic, or spontaneous vascular lesions, were deployed successfully [24]. However, careful patient selection must be emphasized [25]. In the study of Komorowska-Timek et al, the ultrasound-guided thrombin injection has been shown to be effective also in the treatment of peripheral pseudo-aneurysms of the radial and ulnar artery [26]. Our preoperative diagnosis was a chronic Volkmann's contracture (Holden moderate type) possibly combined with a lesion of the AIN. During wound exploration with a pneumatic tourniquet (250 mmHg) we found a muscu- lar laceration, an organized hematoma and a lesion of the first motor branch of the AIN. Our preoperative diagnosis was confirmed and the neurological deficit was attributed to compression from the hematoma. This is why the vas- cular bundle was not explored and the possibility of an arterial false aneurysm was at first not even considered. Once the circulation had been restored (pneumatic tour- niquet off) and the hematoma removed, the aneurysm had the possibility to re-expand. This explains why the lesion became clinically and radiologically evident post- operatively. Page 3 of 4 (page number not for citation purposes) Conclusions Penetrating injuries of the proximal forearm should not be underestimated because they are a potential cause of nerve and vascular injury. Technically we used a proximal anterior approach. The dissection was difficult due to the old hematoma. We do not have a clear explanation for the acute bleeding during the night but do not think that it was due to an iatrogenic lesion during the first operation. While lesions of the major neuro-vascular bundels are often evident at clinical examination, this might not be the case for less accessible structures such as the anterior or posterior interosseous neurovascular bundle. The con- sequences can be disastrous, with development of sub- acute compartment syndrome or delayed diagnosis of With regard to the pathogenesis, we assume that the false aneurysm is the result of a partial laceration of the IA due Page 3 of 4 (page number not for citation purposes) Page 3 of 4 (page number not for citation purposes) Journal of Orthopaedic Surgery and Research 2009, 4:44 http://www.josr-online.com/content/4/1/44 http://www.josr-online.com/content/4/1/44 neuro-muscular deficits, that have a less favourable func- tional prognosis. 12. O'Leary MR: Subclavian artery false aneurysm associated with brachial plexus palsy: a complication of parenteral drug addiction. Am J Emerg Med 1990, 8:129-33. J g 13. Habermann ET, Cabot WD: Median nerve compression second- ary to false aneurysm of the brachial artery. Bull Hosp Joint Dis 1974, 35:158-61. We believe that for every penetrating injury of the fore- arm, the emergency physician should perform a detailed neurological and clinical status, evaluating not only sensi- bility but also every muscle group. Vascular examination distal to the lesion is mandatory, although with deep vas- cular lesions it might initially appear to be normal. As for clinical examination, Doppler investigation distal to the lesion is often seem to be normal, initially. We therefore recommend carrying out a detailed clinical and neurolog- ical status in association with a Duplex examination of the injured region, and if there is doubt, a CTA. 14. Svizenska I, Cizmar I, Visna P: An Anatomical Study of the Ante- rior Interosseous Nerve and its Innervation of the Pronator Quadratus Muscle. J Hand Surg 2005, 30:635-7. Q J g 15. Phillips CS, Murphy MS: Vascular problems of the upper extremity: a primer for the orthopaedic surgeon. J Am Acad Orthop Surg 2002, 10:401-8. 16. Rasmussen , Todd E, et al.: Development and Implementation of Endovascular Capabilities in Wartime. Consent f Written informed consent was obtained from the patient for publication of this case report and accompanying images. 20. 20. Peng PD, Spain DA, Tataria M, Hellinger JC, Rubin GD, Brundage SI: CT angiography effectively evaluates extremity vascular trauma. Am Surg 2008, 74:103-7. g 21. Kim DH, Murovic JA, Kim YY, Kline DG: Surgical treatment and outcomes in 15 patients with anterior interosseous nerve entrapments and injuries. J Neurosurg 2006, 104:757-65. Competing interests The authors declare that they have no competing interests. 22. Buda SJ, Johanning JM: Brachial, radial, and ulnar arteries in the endovascular era: choice of intervention. Semin Vasc Surg 2005, 18:191-5. Authors' contributions 23. Castelli P, Caronno R, Piffaretti G, Tozzi M, Laganà D, Carrafiello G, et al.: Endovascular repair of traumatic injuries of the subcla- vian and axillary arteries. Injury 2005, 36:778-82. RP has made substantial contributions to conception and design, or acquisition of data. RP, SL, GG, CF have been involved in drafting the manuscript or revising it critically for important intellectual content. RP, SL, GG, CF have given final approval of the version to be published. y j y 24. Onal B, Ilgit ET, Koar S, Akkan K, Gümü T, Akpek S: Endovascular treatment of peripheral vascular lesions with stent-grafts. Diagn Interv Radiol 2005, 11:170-4. g 25. Danetz JS, Cassano AD, Stoner MC, Ivatury RR, Levy MM: Feasibil- ity of endovascular repair in penetrating axillo-subclavian injuries: a retrospective review. J Vasc Surg 2005, 41:246-54. Conclusions The Journal of Trauma: Injury, Infection, and Critical Care 2008, 64(5):1169-1176. 17. Campbell WW: Evaluation and management of peripheral nerve injury. Clin Neurophysiol 2008, 119:1951-65. j y p y 18. Schwartz M, Weaver F, Yellin A, Ralls P: The utility of color flow Doppler examination in penetrating extremity arterial trauma. Am Surg 1993, 59:375-8. 19. Busquéts AR, Acosta JA, Colón E, Alejandro KV, Rodríguez P: Heli- cal, Computed Tomographic Angiography for the Diagnosis of Traumatic Arterial Injuries of the Extremities. The Journal of Trauma: Injury, Infection, and Critical Care 2004:625-628. References j p J g 26. Komorowska-Timek E, Teruya TH, Abou-Zamzam AM, Papa D, Bal- lard JL: Treatment of radial and ulnar artery pseudoaneu- rysms using percutaneous thrombin injection. J Hand Surg 2004, 29:936-42. 1. Illuminati G, Caliò FG, Bertagni A, Vasapollo L, Vietri F, Martinelli V: Infectious aneurysm of the interosseous artery at the fore- arm. Case report. J Cardiovasc Surg 1996, 37:589-91. p J g 2. Marrero IC: Hand, Upper Extremity Vascular Injury. Emedicine, Article Last Updated: Apr 2, 2008 . p J g 2. Marrero IC: Hand, Upper Extremity Vascular Injury. 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https://openalex.org/W1819812465	https://agricultureandfoodsecurity.biomedcentral.com/track/pdf/10.1186/s40066-015-0037-1	English	null	Farmer participation in selection within segregating populations of cowpea in Volta Region, Ghana	Agriculture & food security	2,015	cc-by	4,441	RESEARCH Open Access © 2015 Egbadzor et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background: Participatory plant breeding leads to early adoption of a newly released variety as farmers would be aware of whatever advantage it might have over prior existing ones as early as possible. Cowpea farmers around Ohawu in the Volta Region of Ghana where the crop is one of the most important cash crops had no variety specifi- cally developed for their environment. This research followed farmers’ interest identified earlier for the development of varieties with preferred consumer traits. Methods: Farmers were chosen from 25 communities within the cowpea growing area by the help of agricultural extension officers. Fifty farmers including 25 males and females each, 7 scientists and 15 other persons with different backgrounds were involved in the selection exercise. The final plants selected were based on the total number of participants’ endorsement and analysis of variance for their seed size. Results: Selections were made from six F3 populations with parents of various unique traits obtained from Botswana, Ghana, Nigeria, Senegal, or United States of America. Four most liked cowpea plants from each of the six populations were selected to be advanced and tried in multi-locations towards probable release. Conclusions: Participants were very enthused with the selection exercise hoping of seeing some of their selections resulting into varieties. Farmers were interested in selecting plants with long multiple pods and early maturing. The exercise was educative for all participants. Keywords: Agricultural extension, Cowpea, Genotypes, Seed coat colour, Seed size, Varieties seed size inheritance in cowpea as well as the gene action (additive or dominance) have been made by different authors indicating the complexity of the genetic control of the trait [1]. Farmer participation in selection within segregating populations of cowpea in Volta Region, Ghana Kenneth Fafa Egbadzor1,2, Samuel Kwame Offei5, Eric Yirenkyi Danquah1, Daniel Ashie Kotey2, Dickson Korku Gamedoagbao2, Mark Dadoza3, Martin Yeboah1 and Kwadwo Ofori4 Egbadzor et al. Agric & Food Secur (2015) 4:17 DOI 10.1186/s40066-015-0037-1 © 2015 Egbadzor et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Correspondence: kegbadzor@wacci.edu.gh 1 West Africa Centre for Crop Improvement, University of Ghana, Legon, Accra, Ghana Full list of author information is available at the end of the article Background One of the important traits desired in cowpea [Vigna unguiculata (L.) Walp] by consumers is large seed size in West Africa [1–4]. Large seed size preference is not lim- ited to cowpea but known in some other crops such as rice [5]. However, much crop breeding objectives have not been directly focused on seed size compared with such traits as biotic and abiotic stress tolerance [6, 7]. This is not to say that seed size has never been studied. Conflicting reports on the number of genes controlling Seed coat pigmentation, like size, is also important for consumers of cowpea in Ghana and other parts of the world [2]. Pigmentation of seeds and other parts of the plant is due to the synthesis of anthocyanins and other flavonoids. Flavonoids represent a large class of second- ary plant metabolites within which anthocyanins are the most known [8]. These anthocyanins are water-soluble pigments and are found in all tissues in plants through- out the plant kingdom [9]. Anthocyanins are known to be important to plants in response to abiotic factors such as drought [9] as well as biotic (insect pests’ resistance) [10]. Correspondence: kegbadzor@wacci.edu.gh 1 West Africa Centre for Crop Improvement, University of Ghana, Legon, Accra, Ghana Full list of author information is available at the end of the article Correspondence: kegbadzor@wacci.edu.gh 1 West Africa Centre for Crop Improvement, University of Ghana, Legon, Accra, Ghana Full list of author information is available at the end of the article Page 2 of 7 Egbadzor et al. Agric & Food Secur (2015) 4:17 Besides the roles of anthocyanin to the plant, it controls pigmentation of the seed coat which consequently has influence on consumer preference. Fig. 1 Seeds of the cowpea genotypes used in hybridization l Contact with farmers and cowpea dealers revealed that large cream-seeded cowpea are preferred by most consumers in Ghana [1, 2, and 11]. However, the popu- lar Ghanaian cowpea varieties are inferior to imported types in these two traits. Participatory selection of large cream cowpea for varietal development would boost the production and utilization of the crop in Ghana and reduce its importation. Selection with farmers at Ohawu which is a noted cowpea production area will help not only in the timely address of gene environment interac- tion but also inculcate other traits of interest to farmers in the breeding goal. Planting at Ohawu Field for planting of the cowpea populations was at Ohawu in the Ketu North District of the Volta Region. The vegetation was cleared in the first week of June fol- lowed by ploughing. Planting was done on the 17th of June, 2013 with the spacing of 60 cm by 80 cm. The field was rain-fed (rainfall figures during the period in Table 2). Hoe weeding which was done in the 3rd and 5th week of planting was followed immediately with insec- ticide application. The insecticide used was cymethoate and it was applied at 1.5 L/ha. Harvesting was done as and when different individual plants had dried pods from the various populations from the 15th of August after the participatory selection. Background The objective of this study was to use researcher–farmer participatory approach to select cowpea lines from seven segregating populations toward variety development with focus on seed size and colour as well as agronomic traits of interest to the farmers. in the area. Some of the communities covered fell under two other Districts namely Keta Municipal and Akatsi (Fig. 2). The farmers who were drawn from 25 cowpea growing communities between Doveme, Dagbamatey, Xevi and Tadzewu were made up of 25 males and females each. Seven scientists and 15 other persons with different backgrounds were also involved in the selection exercise. Ghana has no official national regulations or guidelines for human subjects’ research. Consents of all the partici- pants were obtained towards publication of the results as a research article. Geographic location and identification of farmers Fifty cowpea farmers around Ohawu in the Ketu North District of the Volta Region were identified by the help of the Agricultural Extension Officer and his assistants Table 1 Source and trait of interest of cowpea genotypes selected for varietal development Genotype Source Distinctive trait of interest CB27 University of California riverside Large white seed UCR779 University of California riverside Large seed, aphid resistance Gh3710 CSIR-PGRRI Local adapted, early maturing Bambey21 Senegal, through UCR White, erect Tona CSIR-CRI Local variety Nhyira CSIR-CRI Local variety Gbode Farmer, Ho Farmer/consumer preferred Table 1 Source and trait of interest of cowpea genotypes selected for varietal development Genetic materials Seven cowpea genotypes from different sources were chosen for the varietal development. The genotypes are listed in Table 1. Bambey21 was crossed with Tona, Nhyira, UCR779 and Gbode in a screen house at West Africa Centre for Crop Improvement (WACCI) farm, University of Ghana in September, 2012. CB27 was also crossed with Gh3710 and UCR779. The F1s of all the crosses were advanced to F2 at Bunso, in December, 2012. In March, 2013, F2 plants were grown at Bunso and selection was made again towards farmer participatory selection at Ohawu (Fig. 1). Cowpea lines selection with farmers Selections were made from six different populations. Line selection was made from F3 population of CB27/ Gh3710. Selection from the other five F3 populations, four of which had Bambey21 as the female parent and the males as, Gbode, Nhyira, Tona and UCR779 were based on single plants. The fifth population was UCR779/CB27. Individual farmers indicated their three best lines (in the Page 3 of 7 Egbadzor et al. Agric & Food Secur (2015) 4:17 N ATTRIBUTES: TOTAL AREA (2D) = 16,687.34ha PERIMETER (3D) = 60.32km LATITUDE = 603’50.74812’’N LONGITUDE = 054’49.85784’’E Scale 1:200,000 ALTITUDE (HAE) = 52.010m 0 8.000 Kilometers Afife Doveme Avenorfeme Dagbamatey Akatsi Xevi Ohawu Netsikofe Tadzewu Fig. 2 Map of the cowpea growing areas in the Ketu North and parts of Akatsi District and Keta Municipality Afife Doveme Avenorfeme Dagbamatey Akatsi Xevi Ohawu Netsikofe Tadzewu Doveme Avenorfeme Afife Dagbamatey Ohawu Akatsi Tadzewu Netsikofe Xevi N ATTRIBUTES: TOTAL AREA (2D) = 16,687.34ha PERIMETER (3D) = 60.32km LATITUDE = 603’50.74812’’N LONGITUDE = 054’49.85784’’E Scale 1:200,000 ALTITUDE (HAE) = 52.010m 0 8.000 Kilometers Fig. 2 Map of the cowpea growing areas in the Ketu North and parts of Akatsi District and Keta Municipality ATTRIBUTES: TOTAL AREA (2D) = 16,687.34ha PERIMETER (3D) = 60.32km LATITUDE = 603’50.74812’’N LONGITUDE = 054’49.85784’’E ALTITUDE (HAE) = 52.010m Scale 1:200,000 0 Kilometers Table 2 Rainfall figures at Ohawu (Ketu North District) from June to August 2013 Total quantity of rainfall during the growth period of the cowpeas = 47.6 mm June July August Day Quantity (mm) Day Quantity (mm) Day Quantity 1st 3.6 4th 30.0 Nil 2nd 3.6 15th 3.6 5th 14.4 24th 14.0 9th 21.6 11th 9.6 case of CB27/Gh3710) or the three best individual plants (from the five other populations) 58 days after planting, by placing cards under them. Table 2 Rainfall figures at Ohawu (Ketu North District) from June to August 2013 After harvest plants with acceptable seed coat col- our were included in the next stage of selection which was based on seed size. One hundred seed weight of the selected lines were measured. Selection based on seed sizeh The number of plants selected for analysis of variance per population is shown in Table 4 in the “number of plants accepted” column. The least number of plants selected was 4 for CB27/Gh3710 whilst the most was 7 for three populations namely, Bambey21/UCR779, Bam- bey21/Gbode and UCR779/CB27. The numbers selected from Bambey21/Nhyira and Bambey21/Tona were 6 and 5, respectively. Analysis of variance for 100 seed weight was performed on the seeds of accepted plants including the parents as checks for each population and the result is shown in Table 5. The four heaviest seeds were accepted as the selected plants from each of the populations. Results Table 4 shows the number of plants selected per popu- lation and the number of them accepted based on the seed coat colour. Many of the selected plants by farmers had similar appearance and could not be developed into different varieties, so, only one was chosen among such plants. Statistical analysis Simple statistic parameters such as means and mode were used. Analysis of variance was performed on 100 seed weight. Page 4 of 7 Egbadzor et al. Agric & Food Secur (2015) 4:17 Participatory field selectionh There were three rounds of selection, each of the first two serving as curling level for the subsequent one. At the first stage, farmers and other participants in the selection indi- cated their preferred plants based on their own judgment. All plants with at least one farmer’s endorsement qualified for the next stage of the selection. The result of the selec- tion is presented in Table 3. The number of plants selected per population varied from the least of 13 for Bambey21/ Tona to the maximum of 26 for Bambey21/UCR779. Some of the plants were selected by a single participant while some were selected by many of them with the highest value of 36 for plant number 5 in the UCR779/CB27 population. Discussion At the time of selection (58 days after planting) farmers were mainly interested in yield and early maturity. They, therefore, chose plants with matured pods that were long and multiple per peduncle. Some farmers opened pods to be sure on what the seeds looked like before select- ing them, although, they were entreated not to do so. Although some of the farmers were independent and chose what they decided on, some tend to be influenced by friends leading to many of them selecting same plants. Farmers were in general enthused with the selection activities and this will contribute in developing varieties with farmers’ desired traits [12]. After harvesting, some of the plants selected were rejected because of having less desirable consumer qual- ity seed coat. Accepted seeds were mainly cream/white seed coated [2, 11]. However, few accepted plants had mottle or brown seed coat colour because of the extreme large number of farmers’ endorsement. The plants with unattractive seed coat pigmentation but with high Apart from “Plant number 6”, all of the selected ones from the population of Bambey21/UCR779 were signifi- cantly heavier than both parents. This shows effectiveness of selection for seed size in early generations of cowpea [16]. Selection based on seed coat colour Any plant selected by at least one person was har- vested at seed maturity and included in the next level of selection. Harvesting started 58 days after planting, a period within which some of the plants had dried pods. The second stage of selection was for seed coat colour. Table 3 Plants selected from various populations by participants Plant no. Percentage of participants who selected a given plant CB27/Gh3710 Bambey21/UCR779 Bambey21/Gbode Bambey21/Nhyira UCR779/CB27 Bambey21/Tona 1 7 25 3 7 1 38 2 22 4 21 32 10 24 3 15 6 17 4 24 1 4 3 6 10 3 3 3 5 3 6 1 4 50 14 6 11 1 19 35 10 1 7 29 3 21 8 28 14 8 29 4 13 7 39 44 9 15 1 7 3 1 1 10 1 1 10 18 10 21 11 1 10 14 4 3 6 12 24 13 1 13 1 14 13 3 1 13 24 1 24 14 3 1 42 19 6 15 3 6 11 7 16 3 29 17 24 1 18 7 1 19 7 31 20 14 22 21 1 22 3 23 13 24 1 25 7 26 4 Table 3 Plants selected from various populations by participants Page 5 of 7 Egbadzor et al. Agric & Food Secur (2015) 4:17 Table 4 Number of plants accepted per population based on seed coat colour from population Table 4 Number of plants accepted per population based on seed coat colour from population Population Number of field selected plants Number of plants accepted CB27/Gh3710 20 4 Bambey21/UCR779 26 7 Bambey21/Gbode 14 7 Bambey21/Nhyira 15 6 UCR779/CB27 15 7 Bambey21/Tona 13 5 endorsement by farmers may be used in further breeding programmes. A number of selected plants had similar morphology at the vegetative stage and also same seed features. It would not be possible to develop and release such plants as sep- arate varieties through single seed descent; therefore, the one with highest number of farmer’s selection was used to represent them. In so doing the use of bulking is also avoided as it is less effective than the single seed descent [3]. This led to a drastic decrease in the number of plants selected to the number accepted for seed size evaluation. Selection based on seed coat colour p All of the four plants selected from CB27/Gh3710 had average 100 seed weight significantly higher than that of the smaller seeded parent (Gh3710). The heavier par- ent, CB27 was heavier than all the selected plants except “Plant number four”. However, Plant number four was not significantly heavier than CB27. All of the plants had cream-seeded coat with three having black eye and the forth mottling. The three plants with black eyes were dif- ferent in the size of the eye as well as the seed weight. All of the selected lines had dry pods before 60 days after planting which are earlier than 65–70 days considered by [13] as early maturing. Early maturity is a very impor- tant objective in breeding cowpea and other crops as it is known to be a adaptive feature [14], especially in these times of shortening rainfall periods [15]. In this respect, the selected plants of CB27/Gh3710 are very promising. a Selected plants Conclusion Ishiyaku MF, Singh BB, Craufurd PQ. Inheritance of time to flowering in cowpea (Vigna unguiculata (L.) Walp.). Euphytica. 2005. doi:10.1007/ s10681-005-2435-0. 14. Singh BB, Ajeigbe HA, Tarawali SA, Fernandez-Rivera S, Abubakar M. Improving the production and utilization of cowpea as food and fodder Field Crops Res. 2003;84:169–77. 14. Singh BB, Ajeigbe HA, Tarawali SA, Fernandez-Rivera S, Abubakar M. Improving the production and utilization of cowpea as food and fodder. Field Crops Res. 2003;84:169–77. 15. Muchero W, Ehlers JD, Close TJ, Roberts PA. Mapping QTL for drought stress-induced premature senescence and maturity in cowpea [Vigna unguiculata (L.) Walp.]. Theor Appl Genet. 2009. doi:10.1007/ s00122-008-0944-7. References 1. Drabo I, Redden R, Smithson JB, Aggarwal VD. Inheritance of seed size in cowpea (Vigna unguiculata (L.) Walp). Euphytica. 1984;33:929–34. 1. Drabo I, Redden R, Smithson JB, Aggarwal VD. Inheritance of seed size in cowpea (Vigna unguiculata (L.) Walp). Euphytica. 1984;33:929–34. Plants of UCR779/CB27 population generally had large parts with heavy seeds. Apart from “plant number 1” all of the selected plants were transgressive segregates for large seeds. Plant number 7 of UCR779/CB27 had 100 seed weight of 29.00 g which was the largest across all populations from which selections were made. All the other plants selected across the various populations had seed size larger than at least that of the smaller parent. Although, mottling seeds are not high priced, one type was included in the selection and may be used in further improvement programme. 2. Langyintuo AS, Lowenberg-DeBoer J, Faye M, Lambert D, Ibrod G, Moussa B, Kergna A, Kushwaha S, Musa S, Ntoukam G. Cowpea supply and demand in West and Central Africa. Field Crops Res. 2003;82:215–31. 2. Langyintuo AS, Lowenberg-DeBoer J, Faye M, Lambert D, Ibrod G, Moussa B, Kergna A, Kushwaha S, Musa S, Ntoukam G. Cowpea supply and demand in West and Central Africa. Field Crops Res. 2003;82:215–31. 3. Tchiagam NJB, Youmbi E, Njintang NY, Bell JM, Maina NA. Generation means analysis of seed sucrose content in cowpea (Vigna unguiculata L. Walp.). Asian J Agric Sci. 2011;3(6):475–80. 4. Egbadzor KF, Yeboah M, Offei SK, Ofori K, Danquah EY. Farmers’ key production constraints and traits desired in cowpea in Ghana. J Agric Ex Rural Dev. 2013. doi:10.5897/jaerd.12.118. j 5. Song XJ, Huang W, Shi M, Zhu MZ, Lin HX. A QTL for rice grain width and weight encodes a previously unknown RING-type E3ubiquitin ligase. Nat Genet. 2007. doi:10.1038/ng2014. 6. Hall AE, Singh BB, Ehlers JD. Cowpea breeding. Plant Breed Rev. 1997;15:215–74. 7. Orawu M, Melis R, Laing M, Derera J. Genetic inheritance of resistance to cowpea aphid-borne mosaic virus in cowpea. Euphytica Int J Plant Breed. 2012. doi:10.1007/s10681-012-0756-3. Acknowledgements We thank the Alliance for a Green Revolution in Africa (AGRA) for the financial support for the participatory selection. We also appreciate the support given by the following: Messrs. Kudi Emmanuel, Foli Newton, Jonathan Siamey, Laryeh Samuel, Addo Ebenezer and Ms. Grace Gyamfua. if All the seeds of Bambey21/Gbode selected have cream seed coat with black eye but different shapes and sizes. Among the selected plants of Bambey21/Nhyira and Bambey21/Tona were plants with seeds similar to Bam- bey21. They, however, had different growth habits. Bambey21/Tona has some plants with seeds not having attractive pigmentation but they were included in the selection for their high yields. High yield is one of the most important traits used by farmers in selection. Seed size and numbers are directly related to yield in cowpea and other crops [19]. Discussion Effectiveness in selection in early generations for Table 5 One hundred seed weight (g) of selected plants as compared with their parents a Selected plants UCR779/CB27 Bambey21/UCR779 Bambey21/Gbode Bambey21/Tona CB27/Gh3710 Bambey21/Nhyira Genotype Weight Genotype Weight Genotype Weight Genotype Weight Genotype Weight Genotype Weight UCR779 19.20 Bambey21 16.46 Bambey21 16.46 Bambey21 16.46 CB27 20.28 Bambey21 16.46 CB27 20.28 UCR779 19.20 Gbode 17.98 Tona 16.7 Gh3710 10.54 Nhyira 13.02 Plant 1a 18.98 Plant 1a 22.78 Plant 1 16.76 Plant 1 14.84 Plant 1a 14.72 Plant 1 13.48 Plant 2 18.84 Plant 2a 21.18 Plant 2 17.88 Plant 2a 21.82 Plant 2a 15.48 Plant 2a 25.08 Plant 3 20.46 Plant 3 16.94 Plant 3a 28.28 Plant 3a 14.88 Plant 3a 18.50 Plant 3a 15.08 Plant 4a 27.54 Plant 4 18.20 Plant 4a 22.52 Plant 4a 18.84 Plant 4a 20.88 Plant 4a 17.20 Plant 5 21.66 Plant 5a 25.44 Plant 5a 20.46 Plant 5a 16.54 Plant 5 14.84 Plant 6a 22.92 Plant 6a 18.38 Plant 6 18.24 Plant 6a 17.76 Plant 7a 29.00 Plant 7 16.88 Plant 7a 18.62 Grand X 22.10 19.5 19.69 17.15 16.73 16.62 F.pr <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 SE 0.82 0.75 0.62 0.39 0.59 0.49 CV % 3.7 3.9 3.1 2.2 3.5 2.9 LSD 5 (%) 1.06 0.97 0.8 0.5 0.78 0.63 Table 5 One hundred seed weight (g) of selected plants as compared with their parents Egbadzor et al. Agric & Food Secur (2015) 4:17 Page 6 of 7 seed size in chickpea has also been reported [17]. Idea from chickpea may be applicable in cowpea as synteny has been reported between cowpea and some of its leg- ume relatives [18]. One of the plants had brown seed coat but was included in the selection because of the high number of farmers that endorsed it during the selection. The brown-seeded plant may not be released with that coat colour as a variety but rather subsequently used in hybridization. The three other plants had cream seeds with one having black eye and two with brown eyes but significantly different in seed sizes. selection exercise. They contributed to the writing of the manuscript. MD: the agricultural extension officer of the experiment area (Ohawu). He identified the farmers for the selection exercise and also supervised the cultivation of the cowpea plants. All authors read and approved the final manuscript. Competing interests The authors declare that they have no competing interests. The authors declare that they have no competing interests. Received: 1 April 2015 Accepted: 6 October 2015 Received: 1 April 2015 Accepted: 6 October 2015 Author details 1 1 West Africa Centre for Crop Improvement, University of Ghana, Legon, Accra, Ghana. 2 CSIR, Plant Genetic Resources Research Institute, Bunso, E.R. Ghana. 3 Ministry of Food and Agriculture, Ketu North District, V.R. Ghana. 4 Crop Science Department, University of Ghana, Legon, Accra, Ghana. 5 The Biotech Centre, University of Ghana, Legon, Accra, Ghana. Conclusion Crop varieties developed through farmers’ participa- tion have higher adoption rate compared with those developed by researchers alone. This was the reason for involving farmers from the cowpea growing communities around Ohawu in the Volta Region of Ghana in select- ing cowpea plants toward the development of varieties. Farmers’ participation and researchers’ knowledge were used to select 24 plants from six different bi-parental populations. These selected plants would be advanced toward varietal development focusing on seed size and colour among other traits. The expected varieties to come out of this breeding programme have a very high chance of being adopted because of the level of commit- ment shown from the onset by farmers. 8. Holton TA, Cornish E. Genetics and biochemistry of anthocyanin biosyn- thesis. Plant Cell. 1995;7:1071–83. 9. Chalker-Scott L. Environmental significance of anthocyanins in plant stress responses. Photochem Photobiol. 1999;70(1):1–9. 10. Makoi JHJR, Belane AK, Chimphango SB, Dakora FD. Seed flavonoids and anthocyanins as markers of enhanced plant defence in nodulated cowpea (Vigna unguiculata L. Walp.). Field Crops Res. 2010;118:21–7. 10. Makoi JHJR, Belane AK, Chimphango SB, Dakora FD. Seed flavonoids and anthocyanins as markers of enhanced plant defence in nodulated cowpea (Vigna unguiculata L. Walp.). Field Crops Res. 2010;118:21–7. g g 11. Quaye W, Adofo K, Buckman SE, Frempong G, Jongerden J, Ruivenkamp G. A socio-economic assessment of cowpea diversity on the Ghanaian market: implications for breeding. Int J Consum Stud. 2011;35:679–87. 11. Quaye W, Adofo K, Buckman SE, Frempong G, Jongerden J, Ruivenkamp G. A socio-economic assessment of cowpea diversity on the Ghanaian market: implications for breeding. Int J Consum Stud. 2011;35:679–87. 12. Quaye W, Frempong G, Jongerden J, Ruivenkamp G. Exploring possibili- ties to enhance food sovereignty within the cowpea production-con- sumption network in Northern Ghana. J Hum Ecol. 2009;8(2):83–92.l 12. Quaye W, Frempong G, Jongerden J, Ruivenkamp G. Exploring possibili- ties to enhance food sovereignty within the cowpea production-con- sumption network in Northern Ghana. J Hum Ecol. 2009;8(2):83–92. 12. Quaye W, Frempong G, Jongerden J, Ruivenkamp G. Exploring possibili- ties to enhance food sovereignty within the cowpea production-con- sumption network in Northern Ghana. J Hum Ecol. 2009;8(2):83–92. 13. Ishiyaku MF, Singh BB, Craufurd PQ. Inheritance of time to flowering in cowpea (Vigna unguiculata (L.) Walp.). Euphytica. 2005. doi:10.1007/ s10681-005-2435-0. 13. Ishiyaku MF, Singh BB, Craufurd PQ. Inheritance of time to flowering in cowpea (Vigna unguiculata (L.) Walp.). Euphytica. 2005. doi:10.1007/ s10681-005-2435-0. 13. Egbadzor et al. Agric & Food Secur (2015) 4:17 16. Lopes FC, Gomes RLF, Rodrigues F, Filho F. Genetic control of cowpea seed sizes. Scientia Agricola. 2003;60(2):315–8. 17. Upadhyaya HD, Sharma S, Gowda CLL. Major genes with additive effects for seed size in kabuli chickpea (Cicer arietinum L.). J Genet. 2011;90–3. 18. Lucas MR, Huynh BL, Cisse N, Drabo I, Ehlers JD, Roberts PA, Close TJ. Association studies and legume synteny reveal haplotypes determining seed size in Vigna unguiculata. Front Plant Sci. 2013. doi:10.3389/ fpls.2013.00095. 19. Monpara BA, Gaikwad SR. Combining high seed number and weight to improve seed yield potential of chickpea in India. Afr Crop Sci J. 2014;22(1):1–7. Authors’ contributions KFE: the principal investigator. SKO, EYD, KO and MY: the principal investiga- tor’s PhD Thesis research supervisors. They gave technical advice in conduct- ing the experiment. DAK and DKG: they were involved in planning of the Page 7 of 7 Page 7 of 7 Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit
https://openalex.org/W3206217819	http://pure-oai.bham.ac.uk/ws/files/154119208/Eskauriatza2021_Article_CompleteLabellingAndDomesticPr.pdf	English	null	“Complete Labelling” and Domestic Prosecutions for Crimes Against Humanity	Criminal law forum	2,021	cc-by	18,228	‘Complete labelling’ and domestic prosecutions for crimes against humanity Eskauriatza, Javier DOI: 10.1007/s10609-021-09426-0 License: Creative Commons: Attribution (CC BY) Document Version Publisher's PDF, also known as Version of record Citation for published version (Harvard): Eskauriatza, J 2021, '‘Complete labelling’ and domestic prosecutions for crimes against humanity', Criminal Law Forum, vol. 32, no. 4, pp. 473-509. https://doi.org/10.1007/s10609-021-09426-0 Link to publication on Research at Birmingham portal Download date: 24. Oct. 2024 General rights l li General rights Unless a licence is specified above, all rights (including copyright and moral rights) in this document are retained by the authors and/or the copyright holders. The express permission of the copyright holder must be obtained for any use of this material other than for purposes permitted by law. eely distribute the URL that is used to identify this publication. •Users may freely distribute the URL that is used to identify this publication. Users may freely distribute the URL that is used to identify this publication. •Users may download and/or print one copy of the publication from the University of Birmingham research portal for the purpose of private study or non-commercial research. study o o co e c a esea c •User may use extracts from the document in line with the concept of ‘fair dealing’ under the Copyright, Designs and P •Users may not further distribute the material nor use it for the purposes of commercial gain. Where a licence is displayed above, please note the terms and conditions of the licence govern your use of this document. Where a licence is displayed above, please note the terms and conditions of the licence govern your use of th When citing, please reference the published version. Dr Javier S Eskauriatza, Lecturer in Law, Birmingham Law School, University of Birmingham, Frankland 2108, Edgbaston, Birmingham, B15 2TT, UK. Contact e- mail: j.s.eskauriatza@bham.ac.uk. I am grateful to the anonymous reviewer for their comments and suggestions. Also, to the journal editors for their assistance. I would like to thank the following people for helping me to arrange my thoughts on this topic: Anthony Arnull, John Child, Robert Cryer, Cecilia Farfa´ n-Me´ ndez, Fiona de Londras, Natasa Mavronicola, Sarah M.H. Nouwen, and Mohammad Shahabud- din. Thanks also to Pablo Herna´ ndez Ramı´rez who provided valuable research assistance in the early stages. This article is dedicated to Rob. Take down policy down policy the University of Birmingham exercises care and attention in making items available there are rare occasions when an it ded in error or has been deemed to be commercially or otherwise sensitive. this is the case for this document, please contact UBIRA@lists.bham.ac.uk providing details and we will remove access ely and investigate. If you believe that this is the case for this document, please contact UBIRA@lists.bham.ac.uk providing details and we the work immediately and investigate. Download date: 24. Oct. 2024 Criminal Law Forum https://doi.org/10.1007/s10609-021-09426-0 The Author(s) 2021 JAVIER S. ESKAURIATZA 4 Joel Feinberg, Doing and Deserving: Essays in the Theory of Responsibility (Princeton, New Jersey, Princeton University Press: 1970); H.L.A. Hart, Punishment and Responsibility (Oxford, OUP: 1968) 6; Anthony Duﬀ, ‘‘Authority and Respon- sibility in International Criminal Law’’, ibid. 2 James Chalmers and Fiona Leverick, ‘‘Fair Labelling in Criminal Law’’, Modern Law Review (2008) 217–246, 236; Talita de Souza Dias, ‘‘Recharacterisation of Crimes and the Principles of Fair Labelling in International Criminal Law’’, Inter- national Criminal Law Review (2018) 788–821. 3 Anthony Duﬀ, Answering for Crime (Oxford, Hart Publishing: 2007); Anthony Duﬀ, ‘‘Authority and Responsibility in International Criminal Law’’, in Samantha Besson and John Tasoulias (Eds.) The Philosophy of International Law (Oxford, OUP: 2010) 589–604, 594. 1 I use ‘‘drug-cartel’’ to mean non-State armed groups that engage in a range of illicit activities including, but not limited to, the traﬃcking of illegal drugs. ‘‘COMPLETE LABELLING’’ AND DOMESTIC PROSECU- TIONS FOR CRIMES AGAINST HUMANITY Accepted: 13 October 2021 ABSTRACT. Fair labelling is an established principle of criminal justice that scru- tinises the way that States use language in labelling criminal defendants and their conduct. I argue that ‘‘complete labelling’’ is a related but separate principle which has not received any explicit attention from commentators. Whereas fair labelling focuses, usually, on the protection of defendant’s rights, the principle of complete labelling explains and justiﬁes whether the labels attached appropriately represent the nature and scale of the wrong done to the community. As a case study, I apply this lens in the context of regional (U.S./Mexican) criminal justice responses to crimes against humanity perpetrated by ‘‘drug-cartels’’ in the context of the Mexican Drug War. Successive administrations in Mexico and the U.S. have tended to charge cartel leaders (and/or their political supporters) with so-called ‘‘transnational crimes’’ (for example, drug-traﬃcking, money-laundering, bribery/corruption). This is despite the fact that many of the most powerful cartels have controlled territory, attacked entire towns, carried out acts of terror, and disappeared thousands of people. The principle of complete labelling is useful in normative terms because it helps in the critical exami- nation of a State’s prosecutorial practices, exposing problems that might otherwise be missed. In relation to the case study discussed, for example, a focus on complete labelling helps to expose the regional prosecutorial policy as either an unjustiﬁed exercise in selectivity or, at worst, an expression of collective denial. After considering certain counteracting reﬂexions which speak to some of the foundational anxieties of international criminal justice, the article concludes that domestic prosecutions for crimes against humanity in the context of drug-cartels may, sometimes, be justiﬁed. I INTRODUCTION Drug-cartels wreak havoc.1 Some are responsible for forced disap- pearances, mass executions, and spectacular acts of terrorism. They can exercise control over vast swathes of territory. Some capture the legitimate structures of government and exercise a sort of quasi-rule over populations. They kill thousands with impunity. So, when captured and prosecuted, should their leaders answer for crimes against humanity, as well as drug-traﬃcking, money laundering and bribery? What labels are most appropriate? What labels are fair? In criminal law, whether domestic or international, the principle of fair labelling helps to scrutinise the messages that diﬀerent criminal labels carry for a number of diﬀerent audiences – most obviously, the oﬀender, the victims and survivors, and the broader community that the criminal law purports to regulate.2 Labels should be fair, to the defendant, primarily. However, fair labelling does not stop there. Criminal trials should not be understood only as instrumental events that identify guilt, or innocence. They are also communicative pro- cesses and, therefore, labels should describe accurately the wrong- doing in question for victims, survivors, and the broader community.3 In this respect, incomplete labelling can frustrate the communicative or symbolic function of criminal trials and criminal punishment.4 In this article, I explore the concept of labelling in relation to domestic prosecutions for crimes against humanity perpetrated by leaders of powerful drug cartels. I argue that the principle of ‘‘com- plete labelling’’ helps to reveal and evaluate what may amount to an under-representation of the misconduct of individuals in the context ‘‘COMPLETE LABELLING’’ AND DOMESTIC PROSECUTIONS of prosecutorial policy. In Part 1, I begin by tracing the notion of completeness in the fair labelling scholarship of England and Wales and by explaining the relevance of this principle in domestic and international criminal law. In Part 2, I will focus on a case study that permits a practical exploration of the principle of complete labelling. This is an extreme case of criminality that has received an incomplete prosecutorial response, i.e., crimes against humanity perpetrated by the Zeta drug-cartel in North-Eastern Mexico. 5 It is also true that Mexican Armed Forces have been implicated in gross human rights violations rising to the level of crimes against humanity, however, this is not the focus of this paper. See Open Justice Initiative, Undeniable Atrocities – Con- fronting Crimes Against Humanity in Mexico, (New York, 2006). For a general introduction to the situation of violence in Mexico see Lydia Cacho, Sergio Gonza´ lez Rodrı´guez, Anabel Herna´ ndez, Diego Enrique Osorno, Emiliano Ruiz Parra, Marcela Turati, and Juan Villorio, The Sorrows of Mexico – An Indictment of their Country’s Failings by Seven Exceptional Writers, (MacLehose Press, London: 2016). 6 I do not argue that so-called transnational crimes are ‘‘less serious’’ at a con- ceptual level, although, there is a diﬀerence between some of the regulatory and ﬁnancial oﬀences prohibited in the ‘‘treaty crimes’’ regime and gross human rights abuses rising to crimes against humanity, see Douglas Guilfoyle, ‘‘Transnational Crimes’’ in Kevin Jon Heller, Fre´ de´ ric Me´ gret, Sarah MH Nouwen, Jens David Ohlin, and Darryl Robinson, (Eds.) The Oxford Handbook of International Criminal Law (Oxford, OUP: 2020) 791. 7 By ‘‘regional criminal justice’’ I simply refer to the investigation, prosecution, and conviction of cartel leaders by U.S. and Mexican criminal justice systems. 8 Andrew Ashworth, ‘‘The Elasticity of Mens Rea’’ in C. F. H. Tapper (ed.) Crime, Proof and Punishment: Essays in Memory of Sir Rupert Cross (London, Butterworth: 1981) 45. It was Glanville Williams that recommended swapping the adjective ‘‘representative’’ with ‘‘fair’’, see Glanville Williams, ‘‘Convictions and Fair Labelling’’ Cambridge Law Journal (1983) 85–95, 85. 10 For a general critique of the focus on this ‘‘subjective principle’’ see Alan Norrie, Crime, Reason, and History – A Critical Introduction to Criminal Law, 3rd Ed., (Cambridge, Cambridge University Press: 2014). I INTRODUCTION Although the cartel is responsible for the disappearance of thousands of civilians, the leaders have been charged and convicted only with money-launder- ing, bribery, drug-traﬃcking, and ﬁrearms oﬀences.5 It is undeniable that these labels represent accurately some of the wrongdoing in- volved.6 However, there is some discomfort caused by the fact that the more serious crimes, for example, attacks on entire towns, and mass forced disappearances, have remained outside the oﬃcial re- gional prosecutorial approach.7 How should we understand this prosecutorial selectivity in domestic prosecutions of international crimes? I argue that the principle of complete labelling urges the prosecutors of national criminal justice systems to consider the im- pact of their selective approach from the perspective of victims, survivors and the wider community aﬀected by the relevant conduct. This is more important in places with endemic levels of corruption and drug-related violence; where the discovery of mass graves has become depressingly common. In Part 3, however, I brieﬂy sum- marise some important counteracting reﬂexions which speak to the fundamental anxieties of international criminal justice as a project. In JAVIER S. ESKAURIATZA conclusion, I suggest that domestic prosecutions for crimes against humanity could be an important part of a multifaceted approach to tackle the root causes of widespread drug-related violence. However, the existenceofthetransnationalcriminallawregimeprovidesan‘‘easyout’’ for governments keen to avoid the stigma associated with the crimes against humanity label. After all, gross human rights abuses perpetrated against civilians on a massive scale on the national territory implicate the moral responsibility, if not the criminal liability, of the State. 9 Ashworth, ibid., 53, 54. II HISTORY OF AN IDEA(L): ‘‘REPRESENTATIVE’’, ‘‘FAIR’’, AND ‘‘COMPLETE LABELLING’’ The labels of the criminal law are linked to the moral censure ex- pressed by the operation of the system. Any general theory of crim- inal labels, therefore, ought to be founded on those principles which best explain and justify the operation of the criminal justice system as a whole. In the law of England and Wales, the principle of fair labelling was initially understood as being focused on fairness to defendants.8 Andrew Ashworth argued that the principle of ‘‘representative la- belling’’ meant that ‘‘ [t]he label applied to an oﬀence ought fairly to represent the oﬀender’s wrongdoing’’ and that ‘‘widely-felt distinc- tions’’ between oﬀences ought to be preserved.9 Otherwise, ‘‘the spirit’’ of the subjective principle of criminal liability would be vio- lated.10 The principle of fair labelling foregrounds any potential fu- ture discrimination against defendants by ensuring that the label applied to the defendant does not over-represent their wrongdoing (to victims, survivors, and the community). In response to Ashworth’s article, Glanville Williams argued that fairness to defendants was only a part of the picture, arguing that ‘‘the notion of fairness can also be made to work the other way: it may be said to be unfair to the prosecution or to the public if the conviction understates the oﬀender’s ‘‘COMPLETE LABELLING’’ AND DOMESTIC PROSECUTIONS fault [emphasis added].11 This is an implicit recognition of the importance of what I refer to here as the principle of complete labelling. Some may argue that complete labelling is really an under-appreciated dimension of the better-known principle of fair labelling. That is ﬁne. However, to avoid the same principle standing as a placeholder for a range of diﬀerent normativeconsiderations(fairness to whom?),I think it is better to appreciate that complete labelling is really a separate (but, obviously, related) principle of criminal justice. 11 Glanville Williams, ‘‘Convictions and Fair Labelling’’ Cambridge Law Journal (1983) 85–95, 85; see also Andrew P. Simester, John R. Spencer, G.R. Sullivan, and Graham J. Virgo, Simester and Sullivan’s Criminal Law – Theory and Doctrine (Hart Publishing, Oxford: 2013) 31–32. 15 Hannah Arendt, Eichmann in Jerusalem, (Penguin Books, London: 2006) 253; Robert Cryer, Darryl Robinson and Sergey Vasiliev, International Criminal Law and Procedure, 4th Ed. (Cambridge, CUP: 2019) 38–42. 11 Glanville Williams, ‘‘Convictions and Fair Labelling’’ Cambridge Law Journal (1983) 85–95, 85; see also Andrew P. Simester, John R. Spencer, G.R. Sullivan, and Graham J. Virgo, Simester and Sullivan’s Criminal Law – Theory and Doctrine (Hart Publishing, Oxford: 2013) 31–32. 12 Anthony Duﬀ, ‘‘Authority and Responsibility’’ (n. 3) 594. 13 Joel Feinberg (n. 4) 101–102. 14 Martti Koskenniemi, ‘‘Between Impunity and Show Trials’’ Max Planck Yearbook of United Nations Law (2002) 1–35, 11. 15 Hannah Arendt, Eichmann in Jerusalem, (Penguin Books, London: 2006) 253; Robert Cryer, Darryl Robinson and Sergey Vasiliev, International Criminal Law and Procedure, 4th Ed. (Cambridge, CUP: 2019) 38–42. 14 Martti Koskenniemi, ‘‘Between Impunity and Show Trials’’ Max Planck Yearbook of United Nations Law (2002) 1–35, 11. 12 Anthony Duﬀ, ‘‘Authority and Responsibility’’ (n. 3) 594. 12 Anthony Duﬀ, ‘‘Authority and Responsibility’’ (n. 3) 594. 13 Joel Feinberg (n. 4) 101–102. 18 A good review of how these labels were created and recognized at Nuremberg is provided by Ana Filipa Vrdoljak, ’Human Rights and Genocide: The Work of Lauterpacht and Lemkin in Modern International Law’, 20 European Journal of International Law (2010) 1163–1194; see also Phillip Sands, East-West Street (Lon- don, Orion Publishing Group: 2017). 17 Dias, (n. 2) 789. See also David Nersessian, ‘‘Comparative Approaches to Punishing Hate: The Intersection of Genocide and Crimes Against Humanity’’, Stanford Journal of International Law (2007) 221–264; Douglas Guilfoyle, ‘‘Re- sponsibility for Collective Atrocities: Fair Labelling and Approaches to Commission in International Criminal Law’’, Current Legal Problems (2011) 255–286; Hilmi M. Zawati, Fair Labelling and the Dilemma of Prosecuting Gender-Based Crimes at the International Criminal Tribunals (Oxford, OUP: 2014). 16 Barrie Sander, ‘‘The Expressive Turn of International Criminal Justice: A Field in Search of Meaning’’ Leiden Journal of International Law (2019) 851–872, 852. 19 Dias, (n. 2), 789. II HISTORY OF AN IDEA(L): ‘‘REPRESENTATIVE’’, ‘‘FAIR’’, AND ‘‘COMPLETE LABELLING’’ The principle of complete labelling is founded on the explanation of criminal trials as ‘‘public forums’’ where the defendant, the State, and the victims, are engaged in a communicative process; a process that involves establishing the wrongs done to the community and denouncing them.12 According to Joel Feinberg, this communicative process is an opportunity for the State to practice the ‘‘authoritative disavowal’’ or ‘‘symbolic nonacquiescence’’ of the conduct in ques- tion.13 Whether the defendant’s conduct is labelled as ‘‘murder’’ or ‘‘manslaughter’’, or ‘‘theft’’ or ‘‘robbery’’, reﬂects the outcome of a communicative process that ends with a message sent to the com- munity about the conduct in question. This process sometimes reveals an uneasy relationship between the search for the historical truth of the criminal event and the task of criminal fact-ﬁnding.14 This is, especially, true when criminal justice is adopted as a response to atrocities.15 But a failure to acknowledge the most signiﬁcant aspect of the defendant’s conduct at all sends a message that the State is indiﬀerent about that behaviour and its consequences – or even worse, the oﬃcial silence may be interpreted as connivance. Thus, complete labelling is about the eﬀectiveness and legitimacy of the public disavowal involved in a criminal justice process. A lack of accuracy in the portrayal of the wrongdoing at issue may aﬀect the legitimacy of the criminal justice response. JAVIER S. ESKAURIATZA 20 Alexander Zahar and Go ran Sluiter, International Criminal Law (Oxford, OUP: 2008) p. 274; Kai Ambos, Treatise on International Criminal Law, 2nd Edition, (Oxford, OUP: 2021). 22 Makau Mutua goes so far as to say that the London Agreement is the ‘‘birth certiﬁcate’’ of the human rights movement, see Makau Mutua, ‘‘Savages, Victims, and Saviors: The Metaphor of Human Rights’’, Harvard International Law Journal (2001) 201–245, 211; Agreement for the Prosecution and Punishment of the Major War Criminals of the European Axis, 8 August 1945, 82 UNTC 280. 21 Nersessian, supra, (n. 17) 264. The fact that Allied crimes escaped investigation and enquiry altogether is a fair point but tangential to my argument. 23 Even new developments towards the possible establishment of regional criminal courts are founded on the idea of complementarity, see Draft Statute of the Transnational Criminal Court for Latin America and the Caribbean, (‘‘COPLA Draft Statute’’), Article 1(2), Article 10, available at: https://en.coalicioncopla.org/ documentos (in Spanish). On complementarity see Sarah M.H. Nouwen, Comple mentarity in the Line of Fire (Cambridge, CUP: 2013); Phil Clark, Distant Justice (Cambridge, CUP: 2018); see also Neil Boister, ’International Tribunals for Transnational Crimes: Towards a Transnational Criminal Court?’, 23 Criminal Law Forum (2012) 295–318; Robert J. Currie and Jacob Leon, ’COPLA: A Transnational Criminal Court for Latin America and the Caribbean’, 88 Nordic Journal of Inter national Law (2019) 587–613. On regional criminal justice developments in Africa see Charles Chernor Jalloh, ’The Nature of the Crimes in the African Criminal Court’, 15 Journal of International Criminal Justice (2017) 799–826. JAVIER S. ESKAURIATZA Where the relevant prosecutorial silence amounts to conduct that may rise to crimes against humanity, the stakes, in terms of the legitimacy of the criminal justice response are far higher. As men- tioned, widespread human rights abuses implicate the responsibility of the State. Therefore, as a matter of principle, it is even more important for the State to identify accurately, and to disavow, the relevant conduct by the operation of the criminal law. There is a meaning to this public disavowal that goes beyond the immediate consequences of the criminal trial and towards the strengthening of the legitimacy of the State.16 Indeed, these considerations can be appreciated in the history of the emergence of the crimes against humanity label itself. Talita Dias has argued that fair labelling was an (implicit) concern in the early development of international crimes.17 She argues, rightly, that the terms ‘‘genocide’’ and ‘‘crimes against humanity’’ were proposed as more accurate descriptors of a special kind of oﬀending which con- cerned the international community as a whole.18 As Dias argues, ‘‘the deﬁnition of ordinary crimes and, in particular, their labels did not fully capture the international dimension, the special gravity and the degree of condemnation that were associated with acts or omis- sions that are now to be international crimes [my emphasis]’’.19 Certainly, the labels mattered. However, I am less sure that Lemkin, Lauterpacht, or the others, were overly concerned, speciﬁ- cally, with ‘‘fairness’’ in labelling; especially, in the ‘‘defendant- rights’’ dimension that concerned Ashworth, and that now appears in 19 Dias, (n. 2), 789. ‘‘COMPLETE LABELLING’’ AND DOMESTIC PROSECUTIONS modern textbooks on international criminal law.20 Instead, the cre- ation of new crimes to describe especially horriﬁc conduct might better be described as an expression of ‘‘completeness’’ in labelling – an attempt to ‘‘convey a more complete and proportionate sense of the criminal wrongdoing at issue’’.21 Furthermore, there was a sense that these criminal labels did not just describe the past, but they also shaped the future. The emergence of the European human rights movement is owed, at least, in part, to the criminal justice response to Nazi crimes.22 The labels of international criminal law are especially condem- natory, but the use of these labels is not determined exclusively by international prosecutions. JAVIER S. ESKAURIATZA Indeed, the modern system of interna- tional criminal law is established on the principle of complementarity The labels of international criminal law are especially condem- natory, but the use of these labels is not determined exclusively by international prosecutions. Indeed, the modern system of interna- tional criminal law is established on the principle of complementarity – national courts have the primary responsibility to administer the system of international criminal justice.23 This bifurcated system can lead to problems, however, from the perspective of the demands of complete labelling. In particular, as mentioned, crimes against humanity is a label that does not only condemn the behaviour of certain individuals as especially egregious but it also signals the p p p y – national courts have the primary responsibility to administer the system of international criminal justice.23 This bifurcated system can lead to problems, however, from the perspective of the demands of complete labelling. In particular, as mentioned, crimes against humanity is a label that does not only condemn the behaviour of certain individuals as especially egregious but it also signals the – national courts have the primary responsibility to administer the system of international criminal justice.23 This bifurcated system can lead to problems, however, from the perspective of the demands of complete labelling. In particular, as mentioned, crimes against humanity is a label that does not only condemn the behaviour of certain individuals as especially egregious but it also signals the 23 Even new developments towards the possible establishment of regional criminal courts are founded on the idea of complementarity, see Draft Statute of the Transnational Criminal Court for Latin America and the Caribbean, (‘‘COPLA Draft Statute’’), Article 1(2), Article 10, available at: https://en.coalicioncopla.org/ documentos (in Spanish). On complementarity see Sarah M.H. Nouwen, Comple mentarity in the Line of Fire (Cambridge, CUP: 2013); Phil Clark, Distant Justice (Cambridge, CUP: 2018); see also Neil Boister, ’International Tribunals for Transnational Crimes: Towards a Transnational Criminal Court?’, 23 Criminal Law Forum (2012) 295–318; Robert J. Currie and Jacob Leon, ’COPLA: A Transnational Criminal Court for Latin America and the Caribbean’, 88 Nordic Journal of Inter national Law (2019) 587–613. On regional criminal justice developments in Africa see Charles Chernor Jalloh, ’The Nature of the Crimes in the African Criminal Court’, 15 Journal of International Criminal Justice (2017) 799–826. JAVIER S. 24 For example, in the context of western policies of migration control, see Ioannis Kalpouzos and Itamar Mann, ‘‘Banal Crimes against Humanity: The Case of Asy- lum Seekers in Greece’’, Melbourne Journal of International Law (2015) 1; see also Ioannis Kalpouzos, ‘‘International Criminal Law and the Violence against Mi- grants’’, 21 German Law Journal (2020) 179. 28 For a sanguine view of the expansion of U.S. extraterritorial criminal juris- diction over transnational crimes, see Michael Fabiarz, ‘‘Extraterritorial Criminal Jurisdiction’’ Michigan Law Review (2016) 507–557. 27 Darryl Robinson, ‘‘Mexico: The War on Drugs and the Boundaries of Crimes Against Humanity’’, 26 May 2015, EJIL:Talk!, available online at: https://www. ejiltalk.org/mexico-the-war-on-drugs-and-the-boundaries-of-crimes-against-human ity/ (last accessed 11 November 2020). 26 More nuanced rationales in favour of the regional courts also make reference to the dismantling of criminal networks, COPLA Draft Statute, (n. 23) Article 5. 25 Cryer et al., (n. 15) p. 73. 25 Cryer et al., (n. 15) p. 73. JAVIER S. ESKAURIATZA ESKAURIATZA 24 partial breakdown of liberal democratic systems of governance.24 Some of the complexity and sensitivity of international criminal justice stems from the fact that criminal proceedings are often putting the State itself on trial (directly, or indirectly).25 These tensions be- tween individual and collective wrongdoing at the centre of the international criminal justice project raise countervailing considera- tions that will be discussed in Part 3. For now, suﬃce it to say that it is unsurprising that domestic prosecutions for crimes against humanity are still rare and that is, of course, one reason for the emergence of international courts.26 The principle of complete labelling has a general application in liberal criminal justice systems. In order to demonstrate its theoretical and practical application, this article is focused on the regional prosecutorial response to crimes perpetrated by drug-cartels as a result of the Mexican Drug War. Despite very high levels of violence and human suﬀering, this is the type of situation that is usually considered outside of the legal and moral geography of international criminal law.27 Therefore, it is a very usefulcase studyforthe evaluationof thecontent, impact, andoperation of the principle of complete labelling. In the context of the ‘‘war on drugs’’, successive Mexican and U.S. governments have been unwilling to attach accurate, complete labels to those implicated in atrocity crimes – international criminal law, as a part of a domestic, or regional, com- municative system of denunciation against mass violence and human suﬀering lays dormant in these jurisdictions.28 Instead, the regional re- sponse is to investigate and prosecute leaders of powerful and violent organised crime groups for so-called ‘‘transnational crimes’’ (usually, drug-traﬃcking, bribery, and money-laundering). These labels ﬁt, in ‘‘COMPLETE LABELLING’’ AND DOMESTIC PROSECUTIONS part. Some ofthe wrongdoing is accurately identiﬁedandcommunicated to the communities in question. However, thinking about completeness in criminal labels puts us in mind of the thousands of victims and sur- vivors of Zeta cartel attacks – in this sense, the labels appear to under- represent, at least, some of the wrongdoing. p g g Consider the convictions of the leaders of the Zeta cartel, which forms the basis of the case study in this article. Between 2009–2012, the Zeta cartel was one of the most powerful organisations in the country and it controlled vast swathes of territory (often in collusion with local gov- ernment). 29 ‘‘Un juez dicta formal prisio´ n a Miguel A´ ngel Trevin˜ o Morales’’, el ‘‘Z-40’’, Expansio´n, 25/07/2013 (available online: https://expansion.mx/nacional/2013/07/25/ un-juez-dicta-formal-prision-a-miguel-angel-trevino-morales-el-z-40). 30 U.S. Department of State, Narcotics Rewards Program Targets, available on- line: https://www.state.gov/narcotics-rewards-program-target-information-brought- to-justice/miguel-angel-trevino-morales-captured/ (last accessed 13/03/2021). 31 Rube´ n Mosso, ‘‘Dan 18 an˜ os de ca´ rcel a lı´der de Los Zetas, responsible de incendio en Casino Royale’’, Milenio, 21/07/2019, available online at https://www. milenio.com/policia/Zetas-18-anos-lider-responsable-incendio-casino-royale-mon terrey (last accessed 24/03/2021). 29 ‘‘Un juez dicta formal prisio´ n a Miguel A´ ngel Trevin˜ o Morales’’, el ‘‘Z-40’’, Expansio´n, 25/07/2013 (available online: https://expansion.mx/nacional/2013/07/25/ un-juez-dicta-formal-prision-a-miguel-angel-trevino-morales-el-z-40). 30 U.S. Department of State, Narcotics Rewards Program Targets, available on- line: https://www.state.gov/narcotics-rewards-program-target-information-brought- to-justice/miguel-angel-trevino-morales-captured/ (last accessed 13/03/2021). 31 Rube´ n Mosso, ‘‘Dan 18 an˜ os de ca´ rcel a lı´der de Los Zetas, responsible de incendio en Casino Royale’’, Milenio, 21/07/2019, available online at https://www. milenio.com/policia/Zetas-18-anos-lider-responsable-incendio-casino-royale-mon terrey (last accessed 24/03/2021). 32 ‘‘En Texas, condenan a cadena perpetua a ex lı´der de Los Zetas’’, Aristegui Noticias, https://aristeguinoticias.com/0811/Mexico/en-texas-condenan-a-cadena- perpetua-a-ex-lider-de-los-Zetas/ (last accessed 24/03/2021). 33 U.S. Department of Justice, ‘‘Former Mexican governor sent to U.S. prison for money-laundering’’, Press Release, 23/06/2021, available online: https://www.justice. gov/usao-sdtx/pr/former-mexican-governor-sent-us-prison-money-laundering (last accessed 20/07/2021). 32 ‘‘En Texas, condenan a cadena perpetua a ex lı´der de Los Zetas’’, Aristegui Noticias, https://aristeguinoticias.com/0811/Mexico/en-texas-condenan-a-cadena- perpetua-a-ex-lider-de-los-Zetas/ (last accessed 24/03/2021). 34 Allegations that Mexico’s ‘‘crackdown’’ on the cartels during the Caldero´ n administration (2006–2012) was actually only a crackdown on the enemies of the Sinaloa Cartel have been raised by Mexican investigative journalists, see generally Anabel Herna´ ndez, Los Sen˜ores del Narco, (Penguin, Random House: 2010), available in English as Narcoland – The Mexican Drug Lords and their Godfathers (Verso, London: 2014); see also Ryan Deveraux, ‘‘Prosecution of Top Mexican Security Oﬃcial Exposes The Fac¸ ade of the Drug War’’, The Intercept, 26/01/2020, available at: https://theintercept.com/2020/01/26/mexico-drug-war-el-chapo-garcia- luna-trial/. 35 Indeed, in some instances, this a legal obligation that emerges from the principle of sovereignty. JAVIER S. ESKAURIATZA It did not have a political objective other than to control, expand, and defend, lucrative, and illegal, income streams. The leaders were eventually caught, prosecuted, and convicted. Miguel A´ ngel Tre- vin˜ o (alias ‘‘Z-40’’) was charged with the possession of an illegal ﬁrearm and money-laundering type oﬀences.29 He still faces charges in a U.S. indictment, but these extend only to violations of drug-traﬃcking char- ges under Title 21 USC Sections 959, 960 and 963.30 Omar Trevin˜ o, who replacedMiguelasleaderofthecartel,wassentencedto18yearsinprison for money-laundering and ﬁrearms charges.31 Another leader of the Zetas (their nephew) Juan Francisco Trevin˜ o, was sentenced to 20 years after being convicted of seven charges of drug-traﬃcking.32 High-rank- ing political leaders, such as the governors of Coahuila at the material time, have also been investigated and prosecuted. JorgeTorres Lo´ pez, an interim governor during some of theworst violence, was extraditedto the U.S. on 29th October 2019, and he pleaded guilty to money-laundering and bribery charges, attracting a 36-month sentence.33 29 ‘‘Un juez dicta formal prisio´ n a Miguel A´ ngel Trevin˜ o Morales’’, el ‘‘Z-40’’, Expansio´n, 25/07/2013 (available online: https://expansion.mx/nacional/2013/07/25/ un-juez-dicta-formal-prision-a-miguel-angel-trevino-morales-el-z-40). 30 U.S. Department of State, Narcotics Rewards Program Targets, available on- line: https://www.state.gov/narcotics-rewards-program-target-information-brought- to-justice/miguel-angel-trevino-morales-captured/ (last accessed 13/03/2021). 30 U.S. Department of State, Narcotics Rewards Program Targets, available on- line: https://www.state.gov/narcotics-rewards-program-target-information-brought- to-justice/miguel-angel-trevino-morales-captured/ (last accessed 13/03/2021). 31 Rube´ n Mosso, ‘‘Dan 18 an˜ os de ca´ rcel a lı´der de Los Zetas, responsible de incendio en Casino Royale’’, Milenio, 21/07/2019, available online at https://www. milenio.com/policia/Zetas-18-anos-lider-responsable-incendio-casino-royale-mon terrey (last accessed 24/03/2021). 32 ‘‘En Texas, condenan a cadena perpetua a ex lı´der de Los Zetas’’, Aristegui Noticias, https://aristeguinoticias.com/0811/Mexico/en-texas-condenan-a-cadena- perpetua-a-ex-lider-de-los-Zetas/ (last accessed 24/03/2021). 33 U.S. Department of Justice, ‘‘Former Mexican governor sent to U.S. prison for money-laundering’’, Press Release, 23/06/2021, available online: https://www.justice. gov/usao-sdtx/pr/former-mexican-governor-sent-us-prison-money-laundering (last accessed 20/07/2021). 33 U.S. Department of Justice, ‘‘Former Mexican governor sent to U.S. prison for money-laundering’’, Press Release, 23/06/2021, available online: https://www.justice. gov/usao-sdtx/pr/former-mexican-governor-sent-us-prison-money-laundering (last accessed 20/07/2021). JAVIER S. ESKAURIATZA JAVIER S. ESKAURIATZA In all these cases, no attempt has been made to investigate and prosecute the responsibility of these individuals for crimes against humanity in relation to gross human rights abuses perpetrated by the Zeta cartel upon the citizens of Coahuila (and other regions). Other leaders of transnational organised crime in Mexico have faced investigations and prosecutions but crimes against humanity are absent from the prosecution and conviction of Joaquı´n Guzman, aka ‘‘El Chapo’’ (in the U.S.), and in the ongoing investigation and prosecution of the ex-Minister for Public Security, Genaro Garcı´a Luna.34 The prosecutorial policy, on both sides of the border, seems to be focused on avoiding those labels that best explain, at least, some of the responsibility in question. The labels used (money-laundering, possession of ﬁrearms, and drug-traﬃcking) may only represent one dimension of the wrongdoing done to the communities by the leaders of organised crime. True, as a matter of fair labelling, it is not, necessarily, unfair to them – a drug cartel can be built around drug- traﬃcking and the laundering of the proceeds. However, it does not tell the complete story. The ‘‘turn to transnational crimes’’ tends to de-emphasize that part of the story which is founded on the realities of State failure and the breakdown of governance systems. It tends to emphasise that part of the story which tells of a State struggling against the emergence of criminal gangs and lays the blame for the violence on matters beyond State control. The criminal labels, and the regional prosecutorial response, at times appear incomplete. Given the nature and scale of some of the violence, the most obvious avenue towards a more complete labelling of the wrongdoing involved is to investigate and prosecute cartel leaders for the com- mission of crimes against humanity when the evidence so demands.35 This label captures more fully the scale of some of the wrongdoing as a systematic abuse of human rights perpetrated by a non-State armed involved is to investigate and prosecute cartel leaders for the com- mission of crimes against humanity when the evidence so demands.35 This label captures more fully the scale of some of the wrongdoing as a systematic abuse of human rights perpetrated by a non-State armed group. 36 For a view that this ‘‘direct criminalization theory’’ is an exercise in naturalism, see Kevin Jon Heller, ‘‘What is an International Crime? (A Revisionist History) Harvard International Law Journal (2017) 353–420; on the concept of transnational criminal law see Neil Boister, ‘‘Transnational Criminal Law’’, European Journal of International Law (2003) 953–976; see also Douglas Guilfoyle, ‘‘Transnational Crimes’’ (n. 6). 38 See comments submitted by Mexico to the International Law Commission in relation to its work on a Draft Convention on Crimes Against Humanity, available online at: https://legal.un.org/ilc/sessions/69/pdfs/spanish/cah_Me´ xico.pdf at §2 (in Spanish). 37 This article focuses on Mexico, as the territorial State where most of the worst violence occurs, and as a State party to the Rome Statute. 40 Mahmoud Cherif Bassiouni, Crimes against Humanity in International Criminal Law (Martinus NijhoﬀPublishers, Dordrecht: 1992) and Crimes against Humanity: Historical Evolution and Contemporary Application (Cambridge, CUP: 2011); Darryl Robinson, Deﬁning ‘‘Crimes Against Humanity’’ at the Rome Conference’, 93 AJIL (1999) 43–57; William Schabas, The International Criminal Court – A Commentary on the Rome Statute (Oxford, OUP: 2010) p. 137; Charles Jalloh, ‘‘What Makes a Crime Against Humanity a Crime Against Humanity?’’, 28 Am. U. Int’ L. Rev. (2013) 381– 441; Leila Sadat, ‘‘Crimes Against Humanity in the Modern Age’’, AJIL (2013) 334– 377; Roger O’Keefe, International Criminal Law (Oxford, OUP: 2015) p. 137; Cryer et al, supra, (n. 15) p. 227; Kai Ambos, Rome Statute of the International Criminal Court - Article-by-Article Commentary, 4th Edition, (2022), Article 7. JAVIER S. ESKAURIATZA After all, the principle of complete labelling is being discussed here as a general principle of international criminal law. JAVIER S. ESKAURIATZA It also carries important signals to local, national, and inter- This label captures more fully the scale of some of the wrongdoing as a systematic abuse of human rights perpetrated by a non-State armed group. It also carries important signals to local, national, and inter- ‘‘COMPLETE LABELLING’’ AND DOMESTIC PROSECUTIONS national communities about the scale of the problem of the Mexican Drug War (and the related global ‘‘war on drugs’’). Indeed, it is this message that is occluded by the current approach which focuses on transnational regulatory and ﬁnancial oﬀences. A focus on the principle of complete labelling may reveal this prosecutorial practice as an exercise in selectivity – a way of ensuring that the war on drugs is not tainted by the morally-loaded labels of international criminal law. In this sense, the principle of complete labelling also contributes to an understanding of the way that States (through their domestic prosecutions) weaponize the fuzzy boundary between so-called ‘‘treaty crimes’’ (drug-traﬃcking, money-laundering) and those so- called ‘‘core crimes’’ which are directly criminalised under interna- tional law (crimes against humanity).36 In actual fact, crimes against humanity, perpetrated by State and non-State actors, have been a direct consequence of the region’s war on drugs. To be clear, not all drug-related violence (in Mexico, or elsewhere) necessarily rises to the level of crimes against humanity. The question explored in this article, however, is founded on the view that the worse of it does cross the threshold into international crimes, and that ignoring this fact as a matter of prosecutorial policy is unjusti- ﬁed. To demonstrate, this article turns to a doctrinal analysis of the law on crimes against humanity. After all, there cannot be an incomplete labelling where the criminal label is unavailable to pros- ecutors as a matter of legal doctrine.37 Mexico signed the Rome Statute on 7 September 2000 and ratiﬁcation followed on 28th October 2005. Curiously, though, the deﬁnition of crimes against humanity in Mexico’s domestic law does not reﬂect that which is found in the Rome Statute.38 There are no contextual elements in the Mexican law. This removes an important legal obstacle to domestic JAVIER S. ESKAURIATZA prosecutions.39 However, the following analysis applies the more stringent customary international law rules on point, indicating where the Rome Statute law may have diverged from these. 41 Article 3 ICTRS; Article 7(1) ICCS; Prosecutor v. Tadic´, ICTY, Trial Chamber, 07/05/1997, §644; Prosecutor v. Tadic´, ICTY Appeals Chamber, 15/07/1999, §248. 39 I am grateful to Sarah Nouwen for helping me to see that point more clearly. III THE LAW ON CRIMES AGAINST HUMANITY AND THE MEXICAN SITUATION OF VIOLENCE (COAHUILA, 2009–2012) The historical development of the law on crimes against humanity has been very disorderly and there are still some doubts about its scope.40 As a matter of customary international law, crimes against humanity can be identiﬁed by the commission of a prohibited act (i.e. murder, torture, rape) in a speciﬁc context, namely, a ‘‘widespread or systematic attack directed against any civilian population’’.41 In addition to the contextual requirements, any individual’s culpability also turns on whether, the prohibited act forms part of the attack on the civilian population and whether the individual had ‘‘knowledge’’ of the attack on civilians. The central purpose of the following analysis is to discover whether the Zetas crimes in North-Eastern Mexico rise to the level of crimes against humanity so that this label was available to prosecutors in doctrinal terms. Whether the relevant individuals were or were not guilty can only be determined in a court of law. The focus, then, is on the contextual elements. It is the contextual threshold (referred to by the Trial Chamber in Tadic´ as ‘‘conditions of applicability’’) that ‘‘elevates crimes that might otherwise fall ‘‘COMPLETE LABELLING’’ AND DOMESTIC PROSECUTIONS exclusively under national jurisdiction to crimes of concern to the international community as a whole’’.42 Previous contextual require- ments, such as the ‘‘war nexus’’ and the need for a discriminatory intent are no longer required to ‘‘cross the threshold’’ into crimes against humanity.43 This is important insofar as there may be legitimate doubts about whether or not the Mexican situation displays the conditions of one (or more) non-international armed conﬂicts.44 The most important element to consider, for present purposes, is the so-called ‘‘policy element’’, so this will be dealt with ﬁrst. The ﬁrst controversy is about whether this is even required as a matter of custom. 48 Gue´ nae l Mettraux, ‘‘Crimes against Humanity in the Jurisprudence of the International Criminal Tribunals for the Former Yugoslavia and for Rwanda’’ Harvard International Law Journal (2002) 237–316, 270 and The Deﬁnition of Crimes Against Humanity and the Question of a ‘‘Policy Element’’’, in Leila Sadat, Forging a Convention on Crimes against Humanity, (Cambridge, CUP: 2011) pp. 142– 176. 45 Robinson, supra, (n. 40) 43–57. 46 Article 7(2)(a), ICCS. 43 Save for the need for a discriminatory intent in relation to persecution as crimes against humanity. 42 Cryer et al, supra, (n. 15) p. 227; Tadic´, ibid, Trial Chamber Judgment, §625. Therefore, unlike domestic crimes, international crimes raise the possibility that other States will assert prescriptive (universal) jurisdiction over the relevant conduct. 44 Alejandro Rodiles, Law and Violence in the Global South: The Legal Framing of Mexico’s ‘‘NARCO WAR’’’, Journal of Conﬂict and Security Law (2018) 269–281. 47 Cryer et al, supra, (n. 15), p. 227. III THE LAW ON CRIMES AGAINST HUMANITY AND THE MEXICAN SITUATION OF VIOLENCE (COAHUILA, 2009–2012) At the 1998 Rome Conference, disagreements about whether the ‘‘widespread or systematic’’ test should be conjunctive or dis- junctive led to the adoption of Article 7(2)(a) Rome Statute.45 According to this provision the attack [on a civilian population] must be ‘‘pursuant to or in furtherance of a State or organizational policy to commit such attack [emphasis added]’’.46 The inclusion of this element has been very controversial, but it depends on what the policy ele- ment means.47 If the policy element is required as a matter of cus- tomary law, the key question is whether drug-cartels, such as the Zetas cartel, qualify as the kinds of organisations that can perpetrate crimes against humanity. 3.1 ‘‘State or Organisational Policy’’: Do the Zetas Qualify? 3.1 ‘‘State or Organisational Policy’’: Do the Zetas Qualify? Some commentators have argued that the policy element forms no part of the customary law deﬁnition.48 For example, writing in 2002, Some commentators have argued that the policy element forms no part of the customary law deﬁnition.48 For example, writing in 2002, 42 Cryer et al, supra, (n. 15) p. 227; Tadic´, ibid, Trial Chamber Judgment, §625. Therefore, unlike domestic crimes, international crimes raise the possibility that other States will assert prescriptive (universal) jurisdiction over the relevant conduct. JAVIER S. ESKAURIATZA Mettraux argued that ‘‘this requirement appears to be contradicted by almost all relevant writing on the subject and by the overwhelming state practice’’.49 A review of the international legislation prior to the Rome Statute reveals that no previous instrument mentions a ‘‘State or organisational policy’’ element.50 Therefore, it is worth placing the ‘‘re-emergence’’ of the policy element, as a matter of customary international law, in its historical context. As Leila Sadat and others have explained, at the Rome Confer- ence, the relatively late addition of the policy element into the deﬁ- nition of ‘‘an attack’’ was ‘‘designed to break a deadlock’’ between States (not as a proposal to change the customary law).51 It was included to ensure that random, isolated, unconnected crimes would be excluded from the jurisdiction of the Court.52 After the Rome Conference, the International Criminal Tribunal for the Former Yugoslavia (ICTY) dispensed with any policy requirement in the Kunarac case.53 Subsequent case-law at the International Criminal Tribunal for Rwanda (ICTR) followed suit. The Special Court for Sierra Leone (SCSL), whose Statute was drafted after the Rome Conference, also rejected the need for any attack on civilians to be linked to any State or organisational policy. Thus, Mettraux and others have a point – there is a strong argument to the eﬀect that customary law does not require the attack to be pursuant to a ‘‘State or organisational policy’’ as required by Article 7(2)(a) Rome Statute. 49 Mettraux, ‘‘Crimes against Humanity in the Jurisprudence of the International Criminal Tribunals for the Former Yugoslavia and for Rwanda’’, ibid., p. 270. 55 Schabas, supra, (n. 40) p. 137; UN Doc. A/CONF.183/C.1/SR.36, para. 13 and UN Doc. A/CONF.183/C.1/SR.34, para. 15. 53 Prosecutor v. Kunarac et al, Case IT-96-23 & IT-96-23/1-A, Appeals Chamber, Judgment, 12/06/2002, §98. 50 See Article 6(c) Nuremberg Charter, Article 5 ICTYS, Article 3 ICTRS. 51 Sadat, supra, (n. 40), 353. 50 See Article 6(c) Nuremberg Charter, Article 5 ICTYS, Article 3 ICTRS. 51 d ( ) 52 ibid., 354. 54 William Schabas, ‘‘State Policy as an Element of International Crimes’’ 98 Journal of International Criminology (2008) 953–982, 960. 52 ibid., 354. 53 Prosecutor v. Kunarac et al, Case IT-96-23 & IT-96-23/1-A, Appeals Chamber, Judgment, 12/06/2002, §98. 54 William Schabas, ‘‘State Policy as an Element of International Crimes’’ 98 Journal of International Criminology (2008) 953–982, 960. 55 Schabas, supra, (n. 40) p. 137; UN Doc. A/CONF.183/C.1/SR.36, para. 13 and UN Doc. A/CONF.183/C.1/SR.34, para. 15. 49 Mettraux, ‘‘Crimes against Humanity in the Jurisprudence of the International Criminal Tribunals for the Former Yugoslavia and for Rwanda’’, ibid., p. 270. 50 See Article 6(c) Nuremberg Charter, Article 5 ICTYS, Article 3 ICTRS. 51 Sadat, supra, (n. 40), 353. 52 ibid., 354. 53 Prosecutor v. Kunarac et al, Case IT-96-23 & IT-96-23/1-A, Appeals Chamber, Judgment, 12/06/2002, §98. 54 William Schabas, ‘‘State Policy as an Element of International Crimes’’ 98 Journal of International Criminology (2008) 953–982, 960. 55 Schabas, supra, (n. 40) p. 137; UN Doc. A/CONF.183/C.1/SR.36, para. 13 and UN Doc. A/CONF.183/C.1/SR.34, para. 15. 62 Sergio Aguayo and Jacobo Daya´ n, El Yugo Zeta – Norte de Coahuila, 2010– 2011 (Working Paper: Violence and Peace Seminar, Colegio de Me´ xico: 2017) p. 7. I will use my own translations from the original Spanish. 56 Go ran Sluiter, ‘‘Chapeau Elements’’ of Crimes Against Humanity in the Jurisprudence of the UN Ad-Hoc Tribunals’’, in Sadat (ed.), supra, (n. 48). 102–41. 57 It would, therefore, be preferable for Mexico to amend its legislation accord- ingly. 58 58 Cryer et al, supra, (n. 15) p. 235. 63 ibid., 7. 60 Tadic´, supra, (n. 41), Trial Chamber Judgment, §653. 63 ibid., 7. 57 It would, therefore, be preferable for Mexico to amend its legislation accord- ingly. 59 ibid., 236. 60 Tadic´, supra, (n. 41), Trial Chamber Judgment, §653. 61 ibid 56 Go ran Sluiter, ‘‘Chapeau Elements’’ of Crimes Against Humanity in the Jurisprudence of the UN Ad-Hoc Tribunals’’, in Sadat (ed.), supra, (n. 48). 102–41. if the CNDH’s [National Human Rights Commission] estimates are used as the baseline, there may have been over 18,000 migrants abducted in Mexico in 2009; this means that these crimes could bring in around 50 million dollars a year for the organized crime groups that engage in smuggling migrants, human traﬃcking, and drug traﬃcking.68 3.1 ‘‘State or Organisational Policy’’: Do the Zetas Qualify? On the other hand, some commentators have argued that the policy requirement was rejected erroneously by the ad-hoc tri- bunals.54 It is also signiﬁcant, as Schabas has noted, that at the Rome Conference, only representatives from the Democratic Republic of the Congo and Jamaica objected to the (re-)insertion of a policy requirement in the deﬁnition of the crime.55 The Rome Statute deﬁ- nition is also the only deﬁnition of crimes against humanity to be ‘‘COMPLETE LABELLING’’ AND DOMESTIC PROSECUTIONS reached after extensive multilateral negotiations. This gives the Rome deﬁnition a special weight in terms of the ‘‘legitimacy in the devel- opment of international law’’.56 Thus, in my view, it is no longer appropriate to simply disregard the policy requirement out of hand when it comes to the identiﬁcation of customary international law on point.57 Instead, it is better to focus on what the policy requirement might mean. One sensible approach out of the dilemma has been found by accepting the requirement as a matter of customary law but ‘‘inter- preting down’’ the evidentiary threshold.58 The policy requirement when accepted by national jurisdictions (and the ICC) must be interpreted in a way that is consistent with the previous authorities, ‘‘as a modest threshold that simply excludes random action’’.59 This means that it is not necessary for the policy to be formally adopted or declared.60 Furthermore, its existence as a matter of fact can be satisﬁed by inference from the way the acts occurred.61 In this respect, the Zeta cartel atrocities in Coahuila and environs easily satisfy the standard required of an organisational policy. As recounted by Aguayo and Daya´ n, during Zeta control over the re- gion, there was a prison in Piedras Negras (just the other side of Eagle Pass, Texas) that was run by the Zeta cartel.62 This is not necessarily rare in Mexico where the National Human Rights Commission has found that 71 out of 154 prisons which they visited could be classiﬁed as either ‘‘self-governed’’ or ‘‘co-governed’’.63 In the case of Piedras Negras, however, it was used as a regional headquarters by the cartel for a variety of activities. Aguayo and Daya´ n explain: This enclave was key for the Zeta organisation because a) it was a safehouse for Zeta leaders that wanted to hide from federal law enforcement, b) they used 64 ibid. 67 Inter-American Commission on Human Rights, Human Rights of Migrants and Other Persons in the Context of Human Mobility in Mexico, OEA/Ser.L/V/O, Doc. 48/13, 30/12/2013, §114ﬀ. 66 Comisio´ n Nacional de Derechos Humanos, Informe especial sobre los casos de secuestro contra migantes, 15/06/2009, at 10. The report is available online at: https:// www.cndh.org.mx/sites/all/doc/Informes/Especiales/2009_migra.pdf (last accessed 11/11/2020). 65 WOLA Statement, ‘‘A Decade After San Fernando Massacre, Migrants Still Face Violence, Impunity for Abuses in Mexico’’, 20/08/2020, available online: https://www.wola.org/2020/08/justice-massacre-san-fernando-Mexico-migrants/ (last accessed 23/03/2021). 64 ibid. 65 WOLA Statement, ‘‘A Decade After San Fernando Massacre, Migrants Still Face Violence, Impunity for Abuses in Mexico’’, 20/08/2020, available online: https://www.wola.org/2020/08/justice-massacre-san-fernando-Mexico-migrants/ (last accessed 23/03/2021). 66 Comisio´ n Nacional de Derechos Humanos, Informe especial sobre los casos de secuestro contra migantes, 15/06/2009, at 10. The report is available online at: https:// www.cndh.org.mx/sites/all/doc/Informes/Especiales/2009_migra.pdf (last accessed 11/11/2020). 67 Inter-American Commission on Human Rights, Human Rights of Migrants and Other Persons in the Context of Human Mobility in Mexico, OEA/Ser.L/V/O, Doc. 48/13, 30/12/2013, §114ﬀ. 68 ibid, attributed to Father Pedro Pantoja, General Advisor to domestic civil society organisations. 68 ibid, attributed to Father Pedro Pantoja, General Advisor to domestic civil society organisations. JAVIER S. ESKAURIATZA it to generate income selling drugs, snacks, charging the prisoners rents for the use of the cells, and renting rooms for conjugal visits, c) it oﬀered them a safe and discrete location to install secret compartments in the vehicles that would transport narcotics to the United States, d) it served as a base to recruit sicarios [hired assassins], and e) it was a place to temporarily conﬁne those who had been kidnapped and to torture, execute, and disappear bodies.64 The systematic nature with which the cartel disappeared their victims has even given rise to a new verb in the Mexican Spanish: los zac- ahuileaban (to make them zacahuil, a kind of stew). The Zetas en- gaged in signiﬁcant and repeated attacks against migrants and refugees heading to the Texas border. In many cases, kidnapping them, for the purposes of extortion and, in some cases, conducting mass executions (e.g., as in San Fernando).65 According to the Mexican National Commission for Human Rights, 9,758 migrants disappeared across a six-month timespan in 2009.66 In 2013, the Inter- American Commission on Human Rights published a report citing the involvement of criminal organizations such as the Zetas (but also the Gulf Cartel) in the organized kidnapping and disappearance of (mostly) Central American migrants.67 The report continues: 65 WOLA Statement, ‘‘A Decade After San Fernando Massacre, Migrants Still Face Violence, Impunity for Abuses in Mexico’’, 20/08/2020, available online: https://www.wola.org/2020/08/justice-massacre-san-fernando-Mexico-migrants/ (last accessed 23/03/2021). 66 Comisio´ n Nacional de Derechos Humanos, Informe especial sobre los casos de secuestro contra migantes, 15/06/2009, at 10. The report is available online at: https:// www.cndh.org.mx/sites/all/doc/Informes/Especiales/2009_migra.pdf (last accessed 11/11/2020). 67 Inter-American Commission on Human Rights, Human Rights of Migrants and Other Persons in the Context of Human Mobility in Mexico, OEA/Ser.L/V/O, Doc. 48/13, 30/12/2013, §114ﬀ. 68 ibid, attributed to Father Pedro Pantoja, General Advisor to domestic civil society organisations. ‘‘COMPLETE LABELLING’’ AND DOMESTIC PROSECUTIONS The UN Committee on the Protection of the Rights of Migrant Workers went further and mentioned explicitly the collusion between criminal gangs and state oﬃcials in its 2011 report, The Committee is deeply concerned by the alarming number of cases of kid- napping and extortion of undocumented migrant workers coming up from the southern border and by the acts of torture and cruel, inhuman and degrading treatment, disappearances and killings of these migrants, primarily at the hands of national and international organized crime groups. 71 Pre-Trial Chamber I, Prosecutor v Katanga, ICC-01/04-01/07, 30/09/2008, §396; Pre-Trial Chamber II, Decision Pursuant to Article 61(7)(a) and (b) of the Rome Statute on the Charges of the Prosecutor against Jean-Pierre Bemba Gombo, ICC-01/ 05-01/08-424, 15/06/2009, §81; Prosecutor v Blasˇkic´, Case No. IT-95-14-T, ICTY, Judgment, 03/03/2000, §204. 70 Fre´ de´ ric Me´ gret, ‘‘Is the ICC Focusing Too Much on Non-State Actors?’’ in Margaret de Guzman and Diane M. Amann, Arcs of Global Justice: Essays in Honour of William A. Schabas, (Oxford, OUP: 2018) pp. 173–201, at 198. 69 UN, Committee on the Protection of the Rights of All Migrant Workers and Members of their Families, Concluding Observations to Mexico, 03/05/2011, §29. 72 Romaine Le Cour Grandmaison, ‘‘Order, Sovereignty, and Violence in Mex- ico’’, Mexico Violence Resource Project, 07/12/2020, available online at: https:// www.mexicoviolence.org/post/order-sovereignty-and-violence-in-mexico (last ac cessed 20/07/2021); Ioan Grillo, ‘‘How the Sinaloa Cartel Bested the Mexican Army’’, Time, 18/10/2019, https://time.com/5705358/sinaloa-cartel-Mexico-culiacan/ …the [U.S.] Drug Enforcement Administration scored an unexpected coup. An agent persuaded a high-level Zetas operative to hand over the trackable cellphone identiﬁcation numbers for two of the cartel’s most wanted kingpins, Miguel A´ ngel Trevin˜ o and his brother Omar. Then the DEA took a gamble. It shared the intelligence with a Mexican federal police unit that has long had JAVIER S. ESKAURIATZA The Committee is also concerned by allegations that public authorities participate in these human rights violations, or that they are carried out with the complicity, consent and/or collusion of federal, state and municipal authorities [emphasis added].69 It is sometimes argued that the attacking organisation must be ‘‘State-like’’ before they qualify as capable of committing crimes against humanity. But this interpretation can be challenged as pure Eurocentrism. International criminal law should not simply reﬂect the European history of the legal frameworks. Today, it is simply not clear what kind of ‘‘State’’ is being imagined when these interpretive arguments are made. As Me´ gret has argued, in many places ‘‘the state is simply not what it was traditionally imagined to be by international (criminal) lawyers, and its ability to inﬂict harm may pale in com- parison to that of armed groups [sic]’’.70 Indeed, the ICC has acknowledged the same in Katanga, when it ruled that ‘‘…a policy may be made by groups of persons who govern a speciﬁc territory or by any organization with the capability to commit a widespread or systematic attack…’’.71 Mexico is a vast territory (by European standards). In Mexico, the State’s presence in several of the more violent regions, including in North-Eastern Mexico, is a matter of ongoing (sometimes, violent) negotiation with multiple armed ac- It is sometimes argued that the attacking organisation must be ‘‘State-like’’ before they qualify as capable of committing crimes against humanity. But this interpretation can be challenged as pure Eurocentrism. International criminal law should not simply reﬂect the European history of the legal frameworks. Today, it is simply not clear what kind of ‘‘State’’ is being imagined when these interpretive arguments are made. As Me´ gret has argued, in many places ‘‘the state is simply not what it was traditionally imagined to be by international (criminal) lawyers, and its ability to inﬂict harm may pale in com- parison to that of armed groups [sic]’’.70 Indeed, the ICC has acknowledged the same in Katanga, when it ruled that ‘‘…a policy may be made by groups of persons who govern a speciﬁc territory or by any organization with the capability to commit a widespread or systematic attack…’’.71 Mexico is a vast territory (by European standards). In Mexico, the State’s presence in several of the more violent regions, including in North-Eastern Mexico, is a matter of ongoing (sometimes, violent) negotiation with multiple armed ac- JAVIER S. 73 Situation in the Republic of Kenya, Decision Pursuant to Article 15 of the Rome Statute on the Authorisation of an Investigation in the Situation in the Republic of Kenya, ICC-01/19, 31/03/2010, §95. 74 ibid §93 JAVIER S. ESKAURIATZA ESKAURIATZA tors.72 Rather than imagining the archetypal State and comparing diﬀerent organisations to that image, other indicators, such as control over territory, control of the movement of persons, informal taxes/ extortion, or the mere capacity to attack civilians must be brought to the fore in the interpretation of the kinds of organisations which qualify as part of the customary law deﬁnition.73 In this respect, the interpretation of ‘‘organisational’’ provided by the majority in the Kenya decision should be preferred. The major- ity’s (non-exhaustive) factor-based approach identiﬁed the following as most relevant: (i) a responsible command or established hierarchy, (ii) capacity to carry out the widespread or systematic attack against a civilian population, (iii) control over part of the territory of a State, (iv) having as a primary purpose criminal activities against the civilian population, (v) the articulation of an intention (explicit or implicit) to attack a civilian population, or (vi) being part of a larger group which fulﬁls some or all of the above-criteria.74 The Zetas organisation qualiﬁes as a relevant organisation on these criteria. It was created out of Mexican ex-special forces per- sonnel and, at the relevant time, had a clearly deﬁned hierarchical leadership. The Zetas were heavily armed, and they had a capacity to carry out widespread or systematic attacks against civilians. Fur- thermore, the organisation exercised control over the territory in question. This is clear when we consider the ‘‘revenge attacks’’ on the northern towns of Coahuila. As Ginger Thompson explains: ‘‘COMPLETE LABELLING’’ AND DOMESTIC PROSECUTIONS problems with leaks — even though its members had been trained and vetted by the DEA. Almost immediately, the Trevin˜ os learned they’d been betrayed.75 In response to the betrayal, the Zeta leadership unleashed a wave of attacks on several towns of Northern Coahuila. Although the details are still unclear (owing to a lack of a full investigation) Piedras Ne- gras and Allende were particularly aﬀected with thousands of emer- gency calls reporting ﬁres breaking out all over the towns.76 In particular, the facts of what happened in Allende are illustrative of what happens when an area is handed over to an organisation like the Zetas drug cartel.77 As explained by the investigative director, Jose´ Juan Morales, Coahuila State Prosecutor’s Oﬃce: We have testimony from people who say they participated in the crime. They described some 50 trucks arriving in Allende, carrying people connected to the cartel. 75 Ginger Thompson, ‘‘How the U.S. Triggered a Massacre in Mexico’’, ProPu- blica, 12/06/2017. For background to Thompson’s research see, Alice Driver, ‘‘How One Reporter Uncovered the US Role in Mexico Massacre’’, Global Investigative Journalism Network, 18/06/2018, available online at: https://gijn.org/2018/06/18/ how-one-reporter-uncovered-the-us-role-in-a-Mexico-massacre/ (last accessed 13/03/ 2021); see also Sarah Chayes, Thieves of State – Why Corruption Threatens Global Security (New York, Norton: 2016), p. 185. 76 Aguayo and Daya´ n report that there were approximately 1,450 emergency calls from Piedras Negras and Allende, supra, (n. 62) p. 62. 77 Ginger Thompson, supra, (n. 75). 75 Ginger Thompson, ‘‘How the U.S. Triggered a Massacre in Mexico’’, ProPu- blica, 12/06/2017. For background to Thompson’s research see, Alice Driver, ‘‘How One Reporter Uncovered the US Role in Mexico Massacre’’, Global Investigative Journalism Network, 18/06/2018, available online at: https://gijn.org/2018/06/18/ how-one-reporter-uncovered-the-us-role-in-a-Mexico-massacre/ (last accessed 13/03/ 2021); see also Sarah Chayes, Thieves of State – Why Corruption Threatens Global Security (New York, Norton: 2016), p. 185. 76 Aguayo and Daya´ n report that there were approximately 1,450 emergency calls from Piedras Negras and Allende, supra, (n. 62) p. 62. 77 Ginger Thompson, supra, (n. 75). 78 ibid. 79 ibid. JAVIER S. ESKAURIATZA They broke into houses, they looted them and burned them. Afterward, they kidnapped the people who lived in those houses and took them to a ranch just outside of Allende. First, they killed them. They put them inside a storage shed ﬁlled with hay. They doused them with fuel and lit them on ﬁre, feeding the ﬂames for hours and hours.78 In relation to the oﬃcial response, Allende’s deputy mayor has said that ‘‘there was a two-sided government, the oﬃcial one and the criminal one that was in charge. We knew that the police were con- trolled by criminals’’.79 To summarise, it may well be that a policy element is now required as a matter of customary international law. However, the threshold is low, and it would be easily satisﬁed in respect of Zeta cartel crimes. Therefore, the most important con- textual element for the purposes of applying the crimes against humanity label to some of Mexico’s drug-related violence is satisﬁed. In relation to the oﬃcial response, Allende’s deputy mayor has said that ‘‘there was a two-sided government, the oﬃcial one and the criminal one that was in charge. We knew that the police were con- trolled by criminals’’.79 To summarise, it may well be that a policy element is now required as a matter of customary international law. However, the threshold is low, and it would be easily satisﬁed in respect of Zeta cartel crimes. Therefore, the most important con- textual element for the purposes of applying the crimes against humanity label to some of Mexico’s drug-related violence is satisﬁed. JAVIER S. ESKAURIATZA 80 Prosecutor v. Kayishema, ICTR-95-1-T, Judgment, 21/05/1999, §122. 81 Prosecutor v Limaj (IT-03-66-T) Judgment, 30/11/2005, para. 182. 82 Tadic´, supra, (n. 41), Trial Chamber Judgment, §649; Prosecutor v. Kunarac et al, Case IT-96-23-T & IT96-23/1-T, Trial Chamber, Judgment, 22/02/2001, §415. 83 Prosecutor v. Akayesu, Case No. ICTR-96-4-T, 2 September 1998, §581. 84 Although, some of this was paid labour (i.e., working in the ‘‘kitchens’’ drew a salary of $300 per night). Those inmates in maximum security/special isolation were required to dispose of the remains around the prison grounds, and there is no indication this was paid. 84 Although, some of this was paid labour (i.e., working in the ‘‘kitchens’’ drew a salary of $300 per night). Those inmates in maximum security/special isolation were required to dispose of the remains around the prison grounds, and there is no indication this was paid. 87 Tadic´, supra, (n. 41), Appeals Chamber Judgment, §248. 85 ibid. 86 ICTR Statute, Art. 3; Tadic´, supra, (n. 41), Trial Chamber Judgment, §656; Akayesu, supra (n. 83), §579. 85 ibid. 86 ICTR Statute, Art. 3; Tadic´, supra, (n. 41), Trial Chamber Judgment, §656; Akayesu, supra (n. 83), §579. 87 Tadic´, supra, (n. 41), Appeals Chamber Judgment, §248. 88 Tadic´, (n. 41), Trial Chamber Judgment, §206. 89 Akayesu, supra (n. 83) §580. 90 Katanga et al., supra, (n. 71) paras. 394–398; Bemba, supra, (n. 71), para. 83; Kenya Decision, supra (n. 73), §95. 91 Kunarac et al, supra, (n. 82), §428. 88 Tadic´, (n. 41), Trial Chamber Judgment, §206. 90 Katanga et al., supra, (n. 71) paras. 394–398; Bemba, supra, (n. 71), para. 83; Kenya Decision, supra (n. 73), §95. ‘‘COMPLETE LABELLING’’ AND DOMESTIC PROSECUTIONS ‘‘COMPLETE LABELLING’’ AND DOMESTIC PROSECUTIONS down, out of fear of the threats we faced. There were too many armed men. We were afraid for our lives. We couldn’t ﬁght bullets with water.85 89 Akayesu, supra (n. 83) §580. 91 Kunarac et al, supra, (n. 82), §428. 3.2 The Requirement of a ‘‘Widespread or Systematic Attack’’ For the label of crimes against humanity to apply, there must also be an ‘‘attack’’ on a civilian population. The attack is ‘‘the event’’ during which one of the prohibited crimes must take place. However, it need not be a military attack.80 An ‘‘attack’’ is a ‘‘course of conduct involving the commission of acts of violence’’.81 Any individual need only be implicated in a single act (i.e., kill one person, or help to imprison them, or deport them) but this prohibited act must form part of the broader attack on civilians.82 The Zeta revenge attacks on the northern towns of Coahuila were military-style attacks, as such. Therefore, this aspect of the deﬁnition is easily satisﬁed. It bears mentioning that, as a matter of customary international law, the mistreatment of a civilian population also qualiﬁes as ‘‘an attack’’. In Akayesu, the Trial Chamber held that ‘‘exerting pressure on the population to act in a particular manner, may come under the purview of an attack, if orchestrated on a massive scale or in a systematic manner [emphasis added]’’.83 As reported by Aguayo and Daya´ n, the Zeta cartel charged non-cartel inmates for use of their cells in Piedras Negras prison. They also ‘‘forced’’ in- mates to carry out a number of cartel-related activities, including modifying vehicles and participating in the burning of the victims’ bodies.84 In relation to the attack on Allende, the population was directed to ignore the attacks on the town. Public service personnel, summoned by the burning of the bodies in nearby ranches, have explained that upon their arrival, they met the Zetas gunmen who told them to withdraw: They said there were going to be numerous incidents. We were going to get numerous emergency calls about gunshots, ﬁres and things like that. They told us we were not authorized to respond. In my capacity as ﬁre chief, what I did was to advise my boss, who in this case was the mayor. I told him that we were facing an impossible situation and that the only thing we could do was to stand Kenya Decision, supra (n. 73), §95. 89 Akayesu, supra (n. 83) §580. 85 ibid. down, out of fear of the threats we faced. There were too many armed men. We were afraid for our lives. We couldn’t ﬁght bullets with water.85 down, out of fear of the threats we faced. There were too many armed men. We were afraid for our lives. We couldn’t ﬁght bullets with water.85 There is clear evidence that the Zetas cartel perpetrated attacks on numerous towns in the territory controlled by the organisation. But in addition, the law requires that the attack must be either widespread or systematic.86 As the Appeals Chamber put it in Tadic´, ‘‘the acts of the accused must comprise part of a pattern of widespread or sys- tematic crimes directed against a civilian population [emphasis ad- ded]’’.87 In Tadic´, the Trial Chamber thought that the standard for ‘‘widespread’’ was satisﬁed by evidence of the ‘‘large-scale nature of the attack and the number of victims’’.88 The ICTR may have em- braced a slightly higher threshold in Akayesu where the Trial Chamber deﬁned ‘‘widespread’’ as ‘‘massive, frequent, large scale action, carried out collectively with considerable seriousness and di- rected against a multiplicity of victims’’.89 The ICC jurisprudence has not deviated materially from this customary law understanding of ‘‘widespread’’.90 It is not possible to be absolutely clear about how many victims are needed for there to be a ‘‘multiplicity’’ of victims (beyond the exclusion of isolated acts of violence).91 However, the revenge at- tacks, which, at least, killed hundreds, must be appreciated in the context of repeated crimes against civilians by the Zetas across the North-Eastern territory during the three-year period, including the kidnapping and disappearance of thousands of migrants, and the systematic torture and disappearances at Piedras Negras prison. The precise scope of application of the legal terms cannot be separated from the factual circumstances in question. Thus, a reﬂexive ap- 91 Kunarac et al, supra, (n. 82), §428. JAVIER S. ESKAURIATZA proach might be wise.92 As Jalloh has argued, ‘‘the focus of the analysis should […] hone in on the wrongful conduct that has caused international social harm or alarm [sic]’’, rather than, exclusively, on the meaning of particular words.93 This is helpful. The alarm in question is that thousands of persons in North-Eastern Mexico dis- appeared and mass graves are being discovered with alarming fre- quency. Many of these are victims of Zeta attacks (though owing to lack of investigations by the State it is impossible to say, at this stage, how many).94 An excessively narrow theory of the situation may consider that the Zetas attack(s) are not ‘‘widespread’’. 92 Suggesting reﬂexivity in the context of general principles of liability in inter- national criminal law see Robert Cryer, ‘‘Imputation and Complicity in Common Law States – A (Partial) View from England and Wales’’, Journal of International Criminal Justice (2014) 267–282, 281: ‘‘…the point is to develop, reﬂexively, princi- ples and rules from which there may be (again reﬂexive) deduction in the future [emphasis added]’’. 94 For context, there are now 90,000 disappeared in Mexico, Sandra Weiss, ‘‘Me´ xico: Ma´ s de 90,000 desaparecidos y los crı´menes continu´ an’’ DW, 3 August 2021 available online at: https://www.dw.com/es/m%C3%A9xico-m%C3%A1s-de- 90000-desaparecidos-y-los-cr%C3%ADmenes-contin%C3%BAan/a-59031428. 93 Charles C. Jalloh, ‘‘What Makes a Crime Against Humanity a Crime Against Humanity?’’, 28 Am. U. Int’ L. Rev. (2013) 381–441, 435. 96 Kunarac et al, supra, (n. 53), §94. 95 Akayesu, supra (n. 81) §580. 97 Katanga et al., supra, (n. 71), paras. 397–398. 98 ibid. 99 Article 5, ICTYS; Article 3, ICTRS; Article 2, SCSLS; Article 5, ECCCS. 100 Kunarac et al, supra, (n. 82), §423. 101 Leila Sadat, ‘‘Putting Peacetime First: Crimes against Humanity and the Civilian Population Requirement’’, Emory International Law Review (2017) 197–270; Kai Ambos, The ECCS’s Contribution to Substantive ICL – The Notion of ‘‘Civilian Population’’ in the Context of Crimes Against Humanity’, JICJ (2020) 689–700, 690. 102 A b b d 691 98 ibid. 102 Ambos, ibid., 691. down, out of fear of the threats we faced. There were too many armed men. We were afraid for our lives. We couldn’t ﬁght bullets with water.85 Even so, they may be considered ‘‘systematic’’. The jurisprudence of the ad-hoc tribunals has equated this criterion with evidence of planning, organisation, and the existence of a regular pattern of conduct. For example, in Akayesu, the Trial Chamber deﬁned ‘‘systematic’’ as ‘‘thoroughly organised and following a regular pattern on the basis of a common policy involving substantial public or private resources [emphasis added]’’.95 In Kunarac, the Appeals Chamber agreed with the Trial Chamber’s view that ‘‘patterns of crimes – that is the non-accidental repetition of similar criminal conduct on a regular basis – are a common expression of such systematic occurrence’’.96 Again, the Rome Statute jurisprudence has followed a similar line. In Katanga, a Pre-Trial Chamber stated that ‘‘systematic’’ meant the Prosecutor needed evidence of ‘‘an organised plan in furtherance of a common policy, which follows from a regular pattern and results in a con- tinuous commission of acts’’.97 Alternatively, the Pre-Trial Chamber ‘‘COMPLETE LABELLING’’ AND DOMESTIC PROSECUTIONS required evidence of ‘‘non-accidental repetition of similar criminal conduct on a regular basis’’.98 The revenge attacks were certainly systematic. The Zetas arrived in town with a purpose and set to the destruction of property and the rounding up of persons associated that they believed were associated with the DEA informant. Violent revenge is by deﬁnition non-acci- dental. Further, the repeated assaults on migrants can also be de- scribed as one systematic attack – repeated kidnappings and disappearances are not random events because an income-stream, linked to the repetition of human rights abuses, by deﬁnition cannot be random or isolated. Finally, it is not possible to run a prison as a regional headquarters, and site of extermination, without also satis- fying the systematic standard. 3.3 ‘‘Directed Against Any Civilian Population’’ 3.3 ‘‘Directed Against Any Civilian Population’’ The crimes against humanity label requires that it is a ‘‘civilian population’’ that is protected from widespread or systematic attack.99 This, again, suggests that evidence of a certain amount of scale is required in terms of the victims of the attack. It also serves to exclude isolated acts against, even multiple, individuals. But it is clear that customary law adopts a wide deﬁnition of a ‘‘civilian population’’. This is evidenced by the use of the word ‘‘any’’ which means that the distinguishing characteristics of the civilian population are immate- rial (i.e. their race, their ethnicity, their nationality).100 Problemati- cally, the deﬁnition of a ‘‘civilian’’ population tends to suggest a normative coupling with international humanitarian law.101 But as Ambos has argued, the two bodies of law are supported by diﬀerent rationales – namely, the prohibition of crimes against humanity does not arise from the ‘‘principle of distinction’’ and so automatic transfers from one body of law to the other should be avoided.102 In peacetime situations, such as (ostensibly) North-Eastern Mexico be- tween 2009–2012, the core of crimes against humanity should be The crimes against humanity label requires that it is a ‘‘civilian population’’ that is protected from widespread or systematic attack.99 JAVIER S. ESKAURIATZA understood as the protection of everyone from organised and sys- tematic attacks, regardless of their status, and regardless of the kind of organisation that is carrying out the widespread or systematic attack. This interpretation is most faithful to the rationale of crimes against humanity as protecting the human rights of individuals from organised attack by groups in power; groups that control the territory in which they happen to live, or need to pass through, as a matter of simple chance. As has been explained, the Zetas organisation was in control of vast swathes of Coahuila and colluded with local gov- ernment to carry out their activities, in Piedras Negras prison, and across the territory, attacking towns and kidnapping and disap- pearing migrants. Yet, another issue that might arise is the question of how many civilians must be targeted before the ‘‘population’’ criterion is satis- ﬁed. 103 Tadic´, supra, (n. 41), Trial Chamber Judgment, §644. 104 Kunarac et al, supra, (n. 53), §90. 3.3 ‘‘Directed Against Any Civilian Population’’ The Trial Chamber in Tadic´ stated that the reference to ‘‘pop- ulation’’ meant that the crimes must have been ‘‘crimes of a collective nature’’ and this, therefore, excluded ‘‘single or isolated acts’’ which might be ordinary crimes or war crimes.103 This does not mean, however, that the entire population living in the sphere of the attack must have been subjected to the attack.104 During the material time, some parts of North-Eastern Mexico may even have been relatively peaceful. Also, in the context of the revenge attacks, the Zetas se- lected certain families and individuals and spared others. In the prison, there was an aspect of selection in the context of the killings, and in the selection of migrants to kidnap, extort, and disappear. According to witness testimony, for example, the victims in the prison were often drug dealers that were in competition with the Zetas (so- called ‘‘grasshoppers’’), or people who owed the cartel money, and the family members of these persons. The so-called ‘‘kitchens’’ were also used to disappear people who were taken from the towns of Allende, Morelos, Nava, Villa Unio´ n and Zaragoza during the re- venge attacks of March 2011. Yet, an aspect of selection of victims is not determinative. The victims and survivors were not randomly selected individuals – the killing and burning of civilians was a part of how the Zetas (and other cartels) governed in Coahuila. The fact that other cartels operate on a similar basis explains why there are approximately 90,000+ oﬃcial ‘‘COMPLETE LABELLING’’ AND DOMESTIC PROSECUTIONS missing persons in Mexico.105 It does not matter that the civilians were only temporarily on the territory in question (i.e., migrants); nor would it matter that the victims were police or local law enforce- ment.106 Even the presence of so-called ‘‘self-defence groups’’ (autodefensas) would not deprive the population attacked of its civilian character. The law states that it is suﬃcient if an organisation attacks a part of the population in the area subjected to attack. 105 Jacobo Daya´ n, ‘‘Se supero´ la cifra de 80 mil desaparecidos’’, Aristegui Noticias, 13/01/2021; Luis Astorga, ‘‘The Limits of anti-drug policy in Mexico’’, (UNESCO: 2001) pp. 427–431. 106 This approach has been followed at the ICC, see Prosecutor v Jean Pierre Bemba Gombo Case No. ICC-01/05-01/08, Judgment pursuant to Article 74 of the Statute, 21/03/2016, §153. 108 ibid., §154; Draft Articles on Prevention and Punishment of Crimes Against Humanity, International Law Commission, A/74/10 at 13. 106 This approach has been followed at the ICC, see Prosecutor v Jean Pierre Bemba Gombo Case No. ICC-01/05-01/08, Judgment pursuant to Article 74 of the Statute, 21/03/2016, §153. 107 ibid. 108 ibid., §154; Draft Articles on Prevention and Punishment of Crimes Against Humanity, International Law Commission, A/74/10 at 13. 109 ILC Draft Articles, ibid, at 36. 109 ILC Draft Articles, ibid, at 36. 3.3 ‘‘Directed Against Any Civilian Population’’ The Appeals Chamber in Kunarac determined that it was: suﬃcient to show that enough individuals were targeted in the course of the attack, or that they were targeted in such a way as to satisfy the Chamber that the attack was in fact directed against a civilian population, rather than against a limited and randomly selected number of individuals.107 This approach has been endorsed by recent ICC jurisprudence.108 It has also been followed in the 2019 Draft Convention on Crimes against Humanity prepared by the International Law Commission which states that population simply means that the attack must be directed against ‘‘multiple victims’’.109 None of the Zetas victims were ‘‘randomly selected’’ – for example, in the context of the revenge attacks, they were aiming at those individuals and families that they decided were linked to the informant. It is true that innocent persons were harmed as a consequence of the application of the policy, seemingly, at random – however, the policy was not to attack random people. Neither is the policy to kidnap, extort, and disappear mi- grants, random, for the purposes of the law. Lastly, there is a question about what is meant by ‘‘directed against’’ (or the notion of an attack being ‘‘on’’ civilians). At the ICTY, the ‘‘war nexus’’ in the Statute meant that it was necessary to exclude civilians that were ‘‘collateral damage’’. The Appeals Chamber in Kunarac explained that ‘‘directed against’’ signals that the civilian population is the ‘‘primary object of the attack’’, thus JAVIER S. ESKAURIATZA excluding incidental harm to civilians.110 It found evidence for ‘‘di- rection’’ from a range of factors including: the means and methods used to attack the victims; their status and number; and the nature of the crimes committed during the attack.111 Recent jurisprudence at the ICC has reﬂected the approach of the ad-hoc tribunals. 3.3 ‘‘Directed Against Any Civilian Population’’ In Bemba, the Trial Chamber determined that it was necessary to prove that ‘‘the civilian population was the primary, as opposed to incidental, target of the attack…’’.112 This ﬁnding was evidenced by the ‘‘means and methods used in the course of the attack, the status of the vic- tims, their number, the discriminatory nature of the attack, the nature of the crimes committed in its course, [and] the form of resistance to the assailants at the time of the attack,[…] ’’113 In relation to the activities of the Zetas in Coahuila, the ‘‘kitchens’’ of Piedras Negras, the extermination of migrants in San Fernando, and elsewhere, this is not really in doubt. 114 Three ‘‘communications’’ pursuant to Article 15 Rome Statute have been sent to the Oﬃce of the Prosecutor (OTP): Mexico: Report on the Alleged Commission of Crimes Against Humanity in Baja California between 2006–2012 (2014) available online at: https://www.ﬁdh.org/IMG/pdf/mexique642ang2014web.pdf (this petition was rejected); Mexico Coahuila: Ongoing Crimes Against Humanity (2017) available online at: https://www.ﬁdh.org/IMG/pdf/angmexico_coahuila_ongoing_crimes_ 110 Kunarac et al, supra, (n. 53), §91–92. 111 ibid., §91. 112 Bemba Gombo, supra, (n. 71), §674. 113 ibid., §153. 114 Three ‘‘communications’’ pursuant to Article 15 Rome Statute have been sent to the Oﬃce of the Prosecutor (OTP): Mexico: Report on the Alleged Commission of Crimes Against Humanity in Baja California between 2006–2012 (2014) available online at: https://www.ﬁdh.org/IMG/pdf/mexique642ang2014web.pdf (this petition was rejected); Mexico Coahuila: Ongoing Crimes Against Humanity (2017) available online at: https://www.ﬁdh.org/IMG/pdf/angmexico_coahuila_ongoing_crimes_ 113 ibid., §153. 112 Bemba Gombo, supra, (n. 71), §674. 117 Beverley Goldberg, ‘‘Ayotzinapa: 1,826 days later, and still no justice’’, Open Democracy, 30 September 2019, available online: https://www.opendemocracy.net/ en/democraciaabierta/masacre-de-ayotzinapa-5-a%C3%B1os-sin-resolver-en/ (last accessed 21 September 2021). For a reconstruction of the events that occurred on the 26th September 2014 (based on: i) eyewitness accounts, ii) video-footage, iii) testi mony of the surviving student Fernando Marı´n, iv) information from Tlachinollan IV COMPLETE LABELLING AND COUNTERVAILING CONSIDERATIONS Given the nature and scale of the Zeta crimes in Coahuila, and sur- roundings, I have argued in Part 2 that the most complete and appropriate label for these crimes is crimes against humanity. Simi- larly, national and international civil society actors have expressed inconformity with the current approach by turning to the language of international criminal law to describe the situation of violence and the responsibility of those involved (including the State). National and international human rights organisations have produced a number of Rome Statute Article 15 communications in recent years alleging that crimes against humanity have been committed by State and non-State actors on Mexican territory in Baja California, Coa- huila, and Chihuahua.114 In Coahuila, speciﬁcally, they have de- ‘‘COMPLETE LABELLING’’ AND DOMESTIC PROSECUTIONS scribed and evaluated the violence in terms of crimes against humanity as a more appropriate label that tells a more complete story about what happened when the Zeta cartel governed large swathes of Coahuila.115 However, there are important counterarguments to consider and this section looks at some of these. The worry is that the prosecution and conviction of drug-cartel leaders for crimes against humanity is, in a diﬀerent way, also failing to adhere to the principle of complete labelling. In the ﬁrst place, to avoid selectivity claims, domestic prosecutions for international crimes would seem to require a more serious investigation of the Mexican Armed Forces and their conduct during the Mexican Drug War. Successive governments have been unwilling to hold the military to account.116 Thus, any beneﬁts of the criminal trial as a more complete communicative process would appear to be available only to those who happen to be victims and survivors of cartel violence.117 This is, clearly, not justiﬁable, as a matter of principle. Footnote 114 continued against_humanity_ﬁdh-ﬁnal_a_revisar-1.pdf; Article 15 Communication Under the Rome Statute to the International Criminal Court Regarding the alleged commission of crimes against humanity in Chihuahua, Me´xico between 2008 and 2010 (2018) https:// www.ﬁdh.org/IMG/pdf/mexique715anglais-1_ﬁnal.pdf (all the reports last accessed on 11 November 2020). Of these communications, only the ﬁrst has received a response from the OTP (rejected). against_humanity_ﬁdh-ﬁnal_a_revisar-1.pdf; Article 15 Communication Under the Rome Statute to the International Criminal Court Regarding the alleged commission of crimes against humanity in Chihuahua, Me´xico between 2008 and 2010 (2018) https:// www.ﬁdh.org/IMG/pdf/mexique715anglais-1_ﬁnal.pdf (all the reports last accessed on 11 November 2020). against_humanity_ﬁdh-ﬁnal_a_revisar-1.pdf; Article 15 Communication Under the Rome Statute to the International Criminal Court Regarding the alleged commission of crimes against humanity in Chihuahua, Me´xico between 2008 and 2010 (2018) https:// www.ﬁdh.org/IMG/pdf/mexique715anglais-1_ﬁnal.pdf (all the reports last accessed on 11 November 2020). Of these communications, only the ﬁrst has received a response from the OTP (rejected). Footnote 117 continued Human Rights Centre) see Anabel Herna´ ndez, ‘‘The Hours of Extermination – The Disappearance of the Forty-Three Students in Iguala, Guerrero’’, in Lydia Cacho, et al (n. 5) at 102–120. 120 Decision on the Prosecutor’s request for authorization of an investigation pursuant to Article 15(3) of the Statute, Situation in the Republic of the Philippines, Pre-Trial Chamber I, 15 September 2021, ICC-01-21, available online from the Court’s website. 119 Naomi Roht-Arriaza and Santiago Martı´nez, Grand Corruption and the International Criminal Court in the ‘‘Venezuela Situation’’’, Journal of International Criminal Justice (2019) 1057–1082, 1062. 118 Sarah Chayes, Thieves of State – Why Corruption Threatens Global Security (New York, Norton: 2016), 185. IV COMPLETE LABELLING AND COUNTERVAILING CONSIDERATIONS Of these communications, only the ﬁrst has received a response from the OTP (rejected). 115 Aguayo and Daya´ n, supra, (n. 62); Coahuila: Ongoing Crimes Against Humanity (2017) available online at:https://www.ﬁdh.org/IMG/pdf/angMexico_coa huila_ongoing_crimes_against_humanity_ﬁdh-ﬁnal_a_revisar-1.pdf. 115 Aguayo and Daya´ n, supra, (n. 62); Coahuila: Ongoing Crimes Against Humanity (2017) available online at:https://www.ﬁdh.org/IMG/pdf/angMexico_coa huila_ongoing_crimes_against_humanity_ﬁdh-ﬁnal_a_revisar-1.pdf. 116 Article 15 Communication Under the Rome Statute to the International Criminal Court Regarding the alleged commission of crimes against humanity in Chihuahua, Me´xico between 2008 and 2010 (2018) https://www.ﬁdh.org/IMG/pdf/mex ique715anglais-1_ﬁnal.pdf (all the reports last accessed on 11 November 2020). On selectivity see Robert Cryer, Prosecuting International Crimes: Selectivity and the International Criminal Law Regime (Cambridge, CUP: 2005); J. Eskauriatza, The ‘‘Cienfuegos Aﬀair’’ and the Proposal for a New Regional Transnational Criminal Court in Latin America (COPLA)’, OpinioJuris, 16 July 2021, available online at: http://opiniojuris.org/2021/07/16/the-cienfuegos-aﬀair-and-the-proposal-for-a-new- regional-transnational-criminal-court-in-latin-america-copla/ (last accessed 11 Au gust 2021). 117 Beverley Goldberg, ‘‘Ayotzinapa: 1,826 days later, and still no justice’’, Open Democracy, 30 September 2019, available online: https://www.opendemocracy.net/ en/democraciaabierta/masacre-de-ayotzinapa-5-a%C3%B1os-sin-resolver-en/ (last accessed 21 September 2021). For a reconstruction of the events that occurred on the 26th September 2014 (based on: i) eyewitness accounts, ii) video-footage, iii) testi mony of the surviving student Fernando Marı´n, iv) information from Tlachinollan Footnote 117 continued Human Rights Centre) see Anabel Herna´ ndez, ‘‘The Hours of Extermination – The Disappearance of the Forty-Three Students in Iguala, Guerrero’’, in Lydia Cacho, et al (n. 5) at 102–120. 118 Sarah Chayes, Thieves of State – Why Corruption Threatens Global Security (New York, Norton: 2016), 185. 119 Naomi Roht-Arriaza and Santiago Martı´nez, Grand Corruption and the International Criminal Court in the ‘‘Venezuela Situation’’’, Journal of International Criminal Justice (2019) 1057–1082, 1062. 120 Decision on the Prosecutor’s request for authorization of an investigation pursuant to Article 15(3) of the Statute, Situation in the Republic of the Philippines, Pre-Trial Chamber I, 15 September 2021, ICC-01-21, available online from the Court’s website. JAVIER S. ESKAURIATZA Secondly, questions may be raised about the expressive or sym- bolic power of domestic criminal trials in Mexico tout court. For example, it may be asked how prosecuting cartel leaders for crimes against humanity would adequately express and denounce the col- lusion between the cartels and State oﬃcials that has led to the overall situation of widespread violence and bloodshed. As Chayes has ar- gued, ‘‘ [i]n…Mexico […] among other acutely corrupt countries, public oﬃcials have entered into destabilizing alliances, even sym- biosis, with transnational criminal superpowers: drug and weapons syndicates whose activities span continents’’.118 In this context, what good is it to insist upon the communicative beneﬁts of domestic prosecutions? Criminal trials are likely to be counter-productive or weaponised when domestic criminal justice systems are compromised. Of course, it may be countered that this is the very reason for the emergence of international criminal courts. Indeed, some commen- tators have argued that the modern law of crimes against humanity means that the ICC should begin to consider Mexico-type situations more seriously. As Arriaza and Martı´nez have argued, Increasingly, situations involving crimes against humanity are likely to involve a murky mix of actions to control territory and resources for personal, orga- nizational or political gain, combined with ever-more sophisticated interna- tional networks to fund these actions and hide the proceeds […] For the ICC, and international justice more generally, continued relevance will increasingly require grappling with this underlying reality.119 The recent opening of an investigation in the Republic of the Philippines has, at least, put oﬃcial violence linked to the ‘‘war on drugs’’ on the international criminal justice agenda.120 Yet, whether The recent opening of an investigation in the Republic of the Philippines has, at least, put oﬃcial violence linked to the ‘‘war on drugs’’ on the international criminal justice agenda.120 Yet, whether ‘‘COMPLETE LABELLING’’ AND DOMESTIC PROSECUTIONS or not the ongoing ‘‘pre-preliminary’’ examination of Mexico leads anywhere is uncertain. Thirdly, some may argue that to apply the supposedly universal standards of Anglo-American criminal law theory to a radically diﬀerent context is at best wrongheaded, or at worst, an exercise in a kind of ‘‘academic imperialism’’. 121 Martti Koskenniemi, The Gentle Civilizer of Nations: The Rise and Fall of International Law 1870–1960 (Cambridge: Cambridge University Press: 2001), 489. 122 Anthony Anghie, Imperialism, Sovereignty and International Law, (New York, CUP: 2004). 123 Dias, (n. 2) 790, referring to the International Convention on Civil and Political Rights (‘‘ICCPR’’), Article 14(3)(a). 124 Mexico acceded to the ICCPR on 23 March 1981. 124 Mexico acceded to the ICCPR on 23 March 1981. 123 Dias, (n. 2) 790, referring to the International Convention on Civil and Political Rights (‘‘ICCPR’’), Article 14(3)(a). 130 See Propuesta Ciudadana Para La Construccio´n de Una Polı´tica Sobre Verdad, Justicia y Reparacio´n a las Vı´ctimas de la Violencia y de las Violaciones a Derechos Humanos (2019), (‘‘Citizens Proposal’’); see also https://jtMexico.org/; On the turn to transformative justice in transitional justice literature see, Kieran McEvoy, ‘‘Beyond Legalism: Towards a Thicker Understanding of Transitional Justice’’, Journal of 129 Mark Drumbl, Atrocity, Punishment and International Law (Cambridge, CUP: 2009). 127 Immi Tallgren, ’The Sensibility and Sense of International Criminal Law’ 13 European Journal of International Law (2002) 561–595; Carsten Stahn, A Critical Introduction to International Criminal Law (Cambridge, CUP: 2019), 117–158; Guilfoyle, (n. 17) at 257, citing Gerry Simpson, ‘‘Men and Abstract Entities: Indi- vidual Responsibility and Collective Guilt in International Criminal Law’’ in Andre´ Nollkaemper and Herman van der Wilt, System Criminality in International Law (Cambridge, CUP: 2009); see also Janine Natalya Clark, ’The Limits of Retributive Justice: Findings of an Empirical Study in Bosnia and Hercegovina’ 7 Journal of International Criminal Justice (2009) 463–487. 128 Koskenniemi, (n. 13) 14. 125 John Gardner, ‘‘Legal Positivism - 5 1/2 Myths’’, 46 American Journal of Jurisprudence 1 (2001) 199, 200. 126 For a comprehensive list see Dias, (n. 2) at fn. 23, 793. JAVIER S. ESKAURIATZA Fair labelling and complete label- ling may be relevant to questions of ‘‘murder’’ and ‘‘manslaughter’’ at the ‘‘Old Bailey’’, but this theory of criminal law is simply not transferable to a prosecutorial policy that involves transnational organised crime and the ‘‘war on drugs’’ in Mexico. To be sure, Feinberg, Duﬀ, and others were simply not thinking about the communicative power of criminal trials in this kind of context. This is a serious charge. As Martii Koskenniemi has argued, when actors refer to the ‘‘special character’’ of certain norms this ‘‘enables them to transgress the preferences of single individuals, clans or nations’’.121 Because reason (in contrast to State will or State consensus) is pre- sented as universal, these commands are presented as enjoying uni- versal validity. Of course, this approach to the identiﬁcation of the law has a historical resonance in the ‘‘civilization’’ of the peoples of North and South America.122 At the same time, the principle of fair labelling is reﬂected in core texts of international human rights law, and it may be said to form part of customary international law.123 My argument is that complete labelling is an under-appreciated dimension of fair labelling which has a distinctive normative pull. In my view, it is better to distinguish complete labelling as a separate principle. It may not have been re- ferred to explicitly in the traditional sources of international law, but I argue that its validity as a general principle can be inferred from the general acceptance of fair labelling. Mexico has accepted the principle of fair labelling insofar as it acceded to the International Convention on Civil and Political Rights.124 This provides the necessary consent to the principle of complete labelling. As Gardner explains, JAVIER S. ESKAURIATZA JAVIER S. ESKAURIATZA …a norm is valid as a norm of that system in virtue of the fact that at some relevant time and place some relevant agent or agents announced it, practiced it, invoked it, enforced it, endorsed it, or otherwise engaged with it.125 Mexico is also a part of the Rome Statute system, and the ICC has referred to the principle of fair labelling in its jurisprudence.126 p p g j p Fourthly, and from a slightly diﬀerent critical perspective, there is the question of whether the individualisation of guilt sits comfortably with the collective nature of widespread violence in Mexico. It may be an excessively radical liberalism that wants to hold a few individuals to account for what is, in essence, the breakdown of State governance in several places in Mexico.127 The ﬂipside of individualisation could also function as a sort of collective amnesty for those elements of the State that have contributed to the breakdown in public security and mass violence in Mexico.128 Therefore, perhaps, a more collective public reckoning is required.129 The principle of complete labelling may require a synergistic approach – one that attempts to fuse individual and collective accountability. In an eﬀort to acknowledge the seriousness of the situation, na- tional and international civil society organisations have argued that the current state of aﬀairs is so beyond the pale, that some form of transitional justice mechanism must be established to aid the failing Mexican criminal justice system.130 The ‘‘Citizen’s Proposal’’ is one ‘‘COMPLETE LABELLING’’ AND DOMESTIC PROSECUTIONS attempt to delineate a transitional justice roadmap that would go some way towards a more complete labelling of the criminality that arises from the horrors of the war on drugs. Among other things, it provides for an international mechanism for the investigation of atrocities in Mexico (Mecanismo Internacional Contra la Impunidad en Mexico, known as ‘‘MICIM’’) and it also sets out plans for a parallel truth commission and reparations programme. 133 See Christopher Paul, Colin P. Clarke and Chad C. Serena, Mexico is Not Colombia, (Rand Corporation: 2014) xvii. Even so, the authors identify the following cases as the most analogous to Mexico’s situation: Colombia, Peru, the Balkans (especially Bosnia and Croatia), West Africa (especially Sierra Leone and Liberia) and the Caucasus (Georgia and Chechnya). Other cases that made the shortlist are Angola, Myanmar, Tajikistan and Afghanistan. Footnote 130 continued Law and Society (2007) 411–440; Dustin Sharp, ‘‘Interrogating the Peripheries: The Preoccupations of Fourth Generation Transitional Justice’’, 26 Harvard Human Rights Journal (2013) 149–178; Paul Gready and Simon Robins (eds.) From Tran- sitional to Transformative Justice (Cambridge, CUP: 2019). 134 I do not assert that the ‘‘war on drugs’’ began in 2006. 131 Part 5, ‘‘Final Agreement for the End of Conﬂict and the Construction of a Stable and Long Lasting Peace’’, (hereafter the Peace Agreement) signed on 24 November 2016 and ratiﬁed by Congress 1 December 2016. The full text of the peace agreement is available at: http://www.altocomisionadoparalapaz.gov.co/procesos-y- conversaciones/Documentos%20compartidos/24-11-2016NuevoAcuerdoFinal.pdf. Footnote 130 continued Law and Society (2007) 411–440; Dustin Sharp, ‘‘Interrogating the Peripheries: The Preoccupations of Fourth Generation Transitional Justice’’, 26 Harvard Human Rights Journal (2013) 149–178; Paul Gready and Simon Robins (eds.) From Tran- sitional to Transformative Justice (Cambridge, CUP: 2019). 131 Part 5, ‘‘Final Agreement for the End of Conﬂict and the Construction of a Stable and Long Lasting Peace’’, (hereafter the Peace Agreement) signed on 24 November 2016 and ratiﬁed by Congress 1 December 2016. The full text of the peace agreement is available at: http://www.altocomisionadoparalapaz.gov.co/procesos-y- conversaciones/Documentos%20compartidos/24-11-2016NuevoAcuerdoFinal.pdf. 132 ibid. 133 See Christopher Paul, Colin P. Clarke and Chad C. Serena, Mexico is Not Colombia, (Rand Corporation: 2014) xvii. Even so, the authors identify the following cases as the most analogous to Mexico’s situation: Colombia, Peru, the Balkans (especially Bosnia and Croatia), West Africa (especially Sierra Leone and Liberia) and the Caucasus (Georgia and Chechnya). Other cases that made the shortlist are Angola, Myanmar, Tajikistan and Afghanistan. 134 I do not assert that the ‘‘war on drugs’’ began in 2006. 135 See for example, Article 15 Communication Under the Rome Statute to the International Criminal Court Regarding the alleged commission of crimes against humanity in Chihuahua, Me´xico between 2008 and 2010 (2018) https://www.ﬁdh.org/ IMG/pdf/mexique715anglais-1_ﬁnal.pdf (last accessed 11 October 2020). See also the Report of the Special Rapporteur on torture and other cruel, inhuman or degrading treatment or punishment, Juan E. Me´ ndez, 29 December 2014, A/HRC/ 28/68/Add.3, available online at: http://antitorture.org/mexico-2014/ (last accessed 11 November 2020) and United Nations Convention against Torture and Other Cruel, Inhuman and Degrading Treatment or Punishment, Concluding Observations on the seventh periodic report submitted of Mexico, CAT/C/MEX/CO/7, 24th July 2019, at §8, available online at: https://tbinternet.ohchr.org/_layouts/15/treatybody external/Download.aspx?symbolno=CAT/C/MEX/CO/7&Lang=En (last accessed 11 November 2020). JAVIER S. ESKAURIATZA As ever, comparisons and lessons might be learned from other ‘‘models’’, such as, the ‘‘Integral System for Truth, Justice, Reparation and Non- Repetition’’ in Colombia.131 The Colombian system includes a number of separate but ‘‘integrated’’ mechanisms including i) a Truth, Reconciliation and Non-Repetition Commission, ii) a Special Search Unit for Conﬂict-Related Disappeared Persons, iii) the Spe- cial Jurisdiction for Peace and iv) a range of speciﬁc measures on reparations.132 But Mexico is not Colombia – in particular, the most powerful cartels (Cartel de Jalisco Nueva Generacio´ n, Cartel de Sinaloa) are not in the same position as the FARC-EP in 2016.133 The homicide rate across Mexico remains at an all-time high. A cursory look at the oﬃcial government ﬁgures tells the story of approximately 350,000 drug-related deaths since the war on drugs was declared by ex-Pres- ident Felipe Caldero´ n in 2006.134 Torture continues to be widely practiced by State oﬃcials throughout the criminal justice process, including during so-called precautionary detention without charge JAVIER S. ESKAURIATZA (arraigo).135 The femicide rate has increased by 145% since 2015.136 Even the Covid-19 pandemic failed to make a dent in the stories of violence and bloodshed.137 As is well-known, the general ‘‘impunity rate’’ across Mexico is over 90% and access to justice is, in practical terms, non-existent.138 Despite the situation, however, the current Mexican administra- tion (led by President Andre´ s Manuel Lo´ pez Obrador, hereafter, ‘‘AMLO’’) has not been amenable to calls for the implementation of a Mexican transitional justice programme. AMLO came to power in late 2018 with the promise to put an end to what he referred to as the ‘‘war on the cartels’’ – under the now much-ridiculed slogan: ‘‘hugs, not bullets’’.139 At ﬁrst, during the campaign trail, it appeared that 135 See for example, Article 15 Communication Under the Rome Statute to the International Criminal Court Regarding the alleged commission of crimes against humanity in Chihuahua, Me´xico between 2008 and 2010 (2018) https://www.ﬁdh.org/ IMG/pdf/mexique715anglais-1_ﬁnal.pdf (last accessed 11 October 2020). See also the Report of the Special Rapporteur on torture and other cruel, inhuman or degrading treatment or punishment, Juan E. JAVIER S. ESKAURIATZA Me´ ndez, 29 December 2014, A/HRC/ 28/68/Add.3, available online at: http://antitorture.org/mexico-2014/ (last accessed 11 November 2020) and United Nations Convention against Torture and Other Cruel, Inhuman and Degrading Treatment or Punishment, Concluding Observations on the seventh periodic report submitted of Mexico, CAT/C/MEX/CO/7, 24th July 2019, at §8, available online at: https://tbinternet.ohchr.org/_layouts/15/treatybody external/Download.aspx?symbolno=CAT/C/MEX/CO/7&Lang=En (last accessed 11 November 2020). 136 Linnea Sandin, ‘‘Femicides in Mexico: Impunity and Protests’’, Centre for Strategic & International Studies, 19th March 2020, available online at: https://www. csis.org/analysis/femicides-mexico-impunity-and-protests (last accessed 11 Novem ber 2020). 137 Throughout 2018, 2019, and 2020, the murder rate has remained steady at 29 homicides per 100,000 https://politica.expansion.mx/mexico/2021/07/27/homicidios- en-mexico-2020-inegi (last accessed 22 September 2021). It amounts to about 36,000 oﬃcial murders a year. 137 Throughout 2018, 2019, and 2020, the murder rate has remained steady at 29 homicides per 100,000 https://politica.expansion.mx/mexico/2021/07/27/homicidios- en-mexico-2020-inegi (last accessed 22 September 2021). It amounts to about 36,000 oﬃcial murders a year. 138 ‘‘El taman˜ o de la impunidad en Me´ xico’’, Impunidad Cero, available online: https://www.impunidadcero.org/impunidad-en-mexico/#/ (last accessed 13 October 2021); Alejandro Anaya-Mun˜ oz, James Cavallaro, and Patricia Cruz-Marı´n, La im punidad active en Me´xico: Co´mo entender y enfrentar las violaciones masivas a los derechos humanos, ITESO-Universidad Jesuita de Guadalajara and University Net work for Human Rights (ITESO, Guadalajara: 2021). 138 ‘‘El taman˜ o de la impunidad en Me´ xico’’, Impunidad Cero, available online: https://www.impunidadcero.org/impunidad-en-mexico/#/ (last accessed 13 October 2021); Alejandro Anaya-Mun˜ oz, James Cavallaro, and Patricia Cruz-Marı´n, La im punidad active en Me´xico: Co´mo entender y enfrentar las violaciones masivas a los derechos humanos, ITESO-Universidad Jesuita de Guadalajara and University Net work for Human Rights (ITESO, Guadalajara: 2021). 139 The slogan rhymes in Spanish: abrazos no balazos. See National Development Plan and National Security and Peace Plan, Spanish language versions available online: National Development Plan https://www.gob.mx/cenace/acciones-y-pro gramas/plan-nacional-de-desarrollo-2019-2024-195029 and National Peace and Security Plan: https://lopezobrador.org.mx/wp-content/uploads/2018/11/PLAN- DE-PAZ-Y-SEGURIDAD_ANEXO.pdf (each last accessed 25/03/2021). 139 The slogan rhymes in Spanish: abrazos no balazos. See National Development Plan and National Security and Peace Plan, Spanish language versions available online: National Development Plan https://www.gob.mx/cenace/acciones-y-pro gramas/plan-nacional-de-desarrollo-2019-2024-195029 and National Peace and Security Plan: https://lopezobrador.org.mx/wp-content/uploads/2018/11/PLAN- DE-PAZ-Y-SEGURIDAD_ANEXO.pdf (each last accessed 25/03/2021). ‘‘COMPLETE LABELLING’’ AND DOMESTIC PROSECUTIONS this meant that a transitional justice approach was within the realms of the possible. 142 ‘‘Mexican President authorizes 4 more years of military policing’’, CBS News, 21/05/2020, https://www.cbsnews.com/news/mexico-military-authorized-president- lopez-obrador-4-more-years-domestic-policing-cartel-crime/#:~:text=Mexican% 20president%20authorizes%204%20more%20years%20of%20military%20poli cing&text=Mexico%20City%20%E2%80%94%20Mexico’s%20president%20pub lished,more%20years%2C%20to%20March%202024. JAVIER S. ESKAURIATZA During one of his daily televised morning press conferences, AMLO announced that: [t]he main function of the government is to guarantee public security, and it is not the strategy of the security forces to detain drug kingpins. What we are seeking is security, that we may diminish the number of daily homicides.’140 When asked directly whether his administration would end the ‘‘war on the cartels’’, he responded: ‘‘There is no war, oﬃcially, there is no more war. We want peace, and we are going to get peace’’.141 However, AMLO has backtracked. Mexican armed forces (together with the newly created National Guard) will continue to carry out crucial public security tasks through to the end of his administration 2024.142 As such, the war on drugs has continued, along with the concomitant human loss, and the lack of any serious attempt to ac- knowledge what is happening. 141 Morning Press Conference, 30/01/2019, transcript available online: https:// www.gob.mx/presidencia/prensa/conferencia-de-prensa-del-presidente-andres-man uel-lopez-obrador-30-de-enero-de-2019 (last accessed 11/11/2020). V CONCLUSION To quote the Mexican writer, Juan Rulfo: ‘‘No decimos lo que pensamos. Hace ya tiempo que se nos acabaron las ganas de ha- 140 AMLO speech on 30/01/2019, as reported by Hannia Novell, ‘‘AMLO: el ﬁn de la guerra contra el narco y su amnistı´a’’, Eje Central, 07/02/2019, available online: http://www.ejecentral.com.mx/bitacora-de-guerra-amlo-el-ﬁn-de-la-guerra-contra- el-narco-y-su-amnistia/. See also, Arturo de Dios Palma, ‘‘AMLO analiza amnistı´a a lı´deres del narco para garantizar la paz, El Universal, 02/12/2017, available online: https://www.eluniversal.com.mx/elecciones-2018/amlo-plantea-analizar-amnistia-li deres-del-narco-para-garantizar-la-paz and David Agren, ‘‘Fury as Mexico presi dential candidate pitches amnesty for drug cartel kingpins’’, The Guardian, 04/12/ 2017, available online: https://www.theguardian.com/world/2017/dec/04/mexican- presidential-candidate-pitches-amnesty-deal-for-drug-cartel-kingpins (all last ac cessed 06/09/2019). The translations from the original Spanish are my own. 140 AMLO speech on 30/01/2019, as reported by Hannia Novell, ‘‘AMLO: el ﬁn de la guerra contra el narco y su amnistı´a’’, Eje Central, 07/02/2019, available online: http://www.ejecentral.com.mx/bitacora-de-guerra-amlo-el-ﬁn-de-la-guerra-contra- el-narco-y-su-amnistia/. See also, Arturo de Dios Palma, ‘‘AMLO analiza amnistı´a a lı´deres del narco para garantizar la paz, El Universal, 02/12/2017, available online: https://www.eluniversal.com.mx/elecciones-2018/amlo-plantea-analizar-amnistia-li deres-del-narco-para-garantizar-la-paz and David Agren, ‘‘Fury as Mexico presi dential candidate pitches amnesty for drug cartel kingpins’’, The Guardian, 04/12/ 2017, available online: https://www.theguardian.com/world/2017/dec/04/mexican- presidential-candidate-pitches-amnesty-deal-for-drug-cartel-kingpins (all last ac cessed 06/09/2019). The translations from the original Spanish are my own. 141 Morning Press Conference, 30/01/2019, transcript available online: https:// www.gob.mx/presidencia/prensa/conferencia-de-prensa-del-presidente-andres-man uel-lopez-obrador-30-de-enero-de-2019 (last accessed 11/11/2020). JAVIER S. ESKAURIATZA blar’’.143 It may be countered that the crimes against humanity label ‘‘covers only a small part of the crime committed’’ in Mexico as a whole.144 As such, the argument goes, the label is not really necessary, it is inaccurate, or perhaps, it unfairly places the con- sequences of a more collective social breakdown on the shoulders of a few individuals. This may be true in some cases. Even so, Wirken and Bosdriesz are mistaken in thinking that the elevation of some of the worst violence to the level of crimes against humanity would be ‘‘artiﬁcial’’.145 This under-appreciates the value in conceptualizing the worst atrocities in a particular situation as international crimes and the jurisgenerative power of labels in helping to support and implement the most eﬀective policy solutions. At the same time, it may underestimate the damage done by current policy approaches which underplay the le- vels of violence by ensuring that those individuals who are most responsible are either i) not investigated or ii) convicted of transna- tional crimes, such as money-laundering and bribery. 144 Sander Wirken and Hanna Bosdriesz, ‘‘Privatization and Increasing Com- plexity of Mass Violence in Mexico and Central America: Exploring Appropriate International Responses’’ in Harmen Van der Wilt and Christophe Paulussen (eds), Legal Responses to Transnational and International Crimes – Towards an Integrative Approach (Edward Elgar Publishing, Cheltenham: 2017) 245–251. 143 Juan Rulfo, ‘‘Nos han dado la tierra’’, in Pedro Pa´ramo y el Llano en Llamas (Planeta, Barcelona: 2006) 137. One possible English translation would be: ‘‘We do not say what we think. It has been a long time since we felt the desire to say anything.’’ 146 The ﬁrst being the War of Independence from Spain (1810–1821), the second refers to the Benito Jua´ rez Reform Law Period (1858–1861) and, thirdly, the Revolutionary uprising, (1910–1917). V CONCLUSION In terms of criminal justice as communication of moral wrongs, the acknowl- edgment that international crimes are taking place in, and being produced by the ‘‘war on drugs’’, could, at least, help to ‘‘move the needle’’ in States like Mexico, closer towards more signiﬁcant dis- cussions about what should be done with civil society actors and external partners. The Mexican President has been engaged in pro- ducing a supposed ‘‘4th Transformation’’ of Mexico.146 But this is mere rhetoric, or jargon, unless his government (and those that fol- low) are willing to accept the nature and scale of the public security problems which show no signs of going away. One strategy could be to pursue domestic prosecutions for crimes against humanity, when the evidence demands it, in order that Mexico can more eﬀectively ‘‘COMPLETE LABELLING’’ AND DOMESTIC PROSECUTIONS disavow the relevant conduct, uphold victims’ rights, and create space and support for more meaningful policy responses that tackle the root causes of the Mexican Drug War. Further, from a moral per- spective, a failure to condemn international crimes may not be unrelated to a certain connivance in their commission. The incomplete labelling of defendants has tended to portray the Mexican Drug War problem in a half-light, or even worse, as narco- entertainment. In October 2015, to the profound embarrassment of Mexican authorities, Kate del Castillo (an LA-based Mexican TV and movie star) and Sean Penn (a Hollywood A-lister) met with Joaquı´n ‘‘El Chapo’’ Guzma´ n in Mexico to carry out an interview for Rolling Stone magazine and to discuss plans for a biopic – all the time while El Chapo was a leader of one of the most violent criminal groups in the world.147 The glamourisation of narco culture is part of the problem.148 This is not to say that labelling cartel leaders for crimes against humanity will eliminate, as if by magic, the perpetration of wide- spread human rights abuses in the context of the ‘‘war on drugs’’. However, at least, in terms of the ongoing ﬁght against ‘‘grand cor- ruption’’, indictments that equate the criminality of the most pow- erful drug-cartels with that of other violent non-State armed groups (e.g., Islamic State/Al-Qaeda) may at least illuminate the impacts of kleptocracy, and help to generate the political will towards imple- menting, inter alia, the Citizens Proposal. 150 Gerry Simpson, ‘‘International Criminal Law – The Next Hundred Years’’, in Heller et al, (n. 5) 841–849, 847. 147 Whether this meeting led to the capture of El Chapo, as the Mexican authorities have claimed, is disputed, see Benjamin Lee and Scott Bixsby, Sean Penn on El Chapo interview: ‘‘I have a terrible regret’’’, 15 Jan 2016, The Guardian, available online at: https://www.theguardian.com/ﬁlm/2016/jan/15/sean-penn-on-el- chapo-interview-i-have-a-terrible-regret (last accessed on 11 November 2020). 147 Whether this meeting led to the capture of El Chapo, as the Mexican authorities have claimed, is disputed, see Benjamin Lee and Scott Bixsby, Sean Penn on El Chapo interview: ‘‘I have a terrible regret’’’, 15 Jan 2016, The Guardian, available online at: https://www.theguardian.com/ﬁlm/2016/jan/15/sean-penn-on-el- chapo-interview-i-have-a-terrible-regret (last accessed on 11 November 2020). 148 Howard Campbell and Tobin Hansen, ‘‘Getting out of the Game: Desistance from Drug-Traﬃcking’’, International Journal of Drug Policy, (2012) 481–487, 486. 149 Ruti Teitel, Transitional Justice (Oxford, OUP: 2000) 27–67. 150 Gerry Simpson, ‘‘International Criminal Law – The Next Hundred Years’’, in Heller et al, (n. 5) 841–849, 847. 148 Howard Campbell and Tobin Hansen, ‘‘Getting out of the Game: Desistance from Drug-Traﬃcking’’, International Journal of Drug Policy, (2012) 481–487, 486. 149 Ruti Teitel, Transitional Justice (Oxford, OUP: 2000) 27–67. V CONCLUSION This article is founded on the view that international criminal law should be understood as a positive dimension of a broader system of transitional justice – part of the toolkit.149 This conclusion may be criticised as an exercise in what Gerry Simpson has called ‘‘incre- mentalism’’.150 It may also be seen as idealist, as if the critiques, in fact, run deeper and have more problematic implications for the international criminal justice project. However, again, to quote JAVIER S. ESKAURIATZA Simpson, the great strength of the criminal justice project, interna- tional and domestic, is that most people know that ‘‘bad individuals should be jailed in the name of justice and […] it would be uncon- scionable to remain passive in the face of this’’.151 As part of this project, I have tried to describe and evaluate the general principle of complete labelling which responds to the requirement that criminal prosecutions represent appropriately the wrongs done by individuals to the broader community. It is related to the principle of fair la- belling, but it is less focused on the defendant’s fair trial rights. In fact, it may pull in a diﬀerent direction, one that is more focused on the victims of crimes and on the future of a broken society. In some instances, this may cause tension where fairness to the community may be unfair to defendants who are identiﬁed unreasonably as the bearers of what should be a more collective guilt. At the same time, the individualisation of guilt allows for criminal processes to pursue the aims of retribution and deterrence more eﬀectively.152 Finally, as a general principle, complete labelling may inform non-prosecutorial approaches to accountability, i.e., as a foundational norm of transi- tional justice programmes. Ultimately, if the project of (international) criminal justice is to have any legitimacy, it must be to play a con- structive role in helping societies to tell more complete and accurate stories about mass suﬀering in the hope that stronger communities will emerge. Hope, in this sense, is about commitment. Sometimes, that commitment must be expressed via domestic prosecutions of international crimes. 152 Robert Cryer, ‘‘The Role of International Criminal Prosecutions in Increasing Compliance with International Humanitarian Law in Contemporary African Con- ﬂicts’’ in Heike Krieger, Inducing Compliance with International Humanitarian Law: Lessons from the African Great Lakes Region (Cambridge, CUP: 2015) 188–216; Jakob von Holderstein Holstermann, A ‘‘Slice of Cheese’’ – a Deterrence-Based Argument for the International Criminal Court’, 11 Human Rights Review 289–315. 151 ibid., 848. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional aﬃliations. OPEN ACCESS This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, dis- tribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if ‘‘COMPLETE LABELLING’’ AND DOMESTIC PROSECUTIONS changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons. org/licenses/by/4.0/. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional aﬃliations.
https://openalex.org/W2162680114	https://opendermatologyjournal.com/VOLUME/2/PAGE/77/PDF/	English	null	Hairy Concerns: Use of Instructional/Informational Sheets for Assistance with Disorders of Hair	The Open dermatology journal	2,008	cc-by	5,635	Address correspondence to this author at the University of Toledo College of Medicine, 5600 Monroe Street, Suite 106B, Sylvania, OH 43560, USA; Tel: 419-885-3403; Fax: 419-885-3401; E-mail: cgbakb@aol.com TRICHORRHEXIS NODOSA Unlike most physical characteristics, hair can be altered and manipulated to the dictates of culture and fashion. The chemical reactions from coloring, perming, and straightening hair affect the normal structure of the hair shaft. Many hair cosmetics are used without any visual effects on the integrity of hair, they nonetheless cause microscopic weathering ren- dering the hair structure weaker and more prone to breakage. When hair breakage occurs with visible hair loss, the term that is utilized is ‘trichorrhexis nodosa [2]’. Hair is a major part of one's self-identity, and central to one's feeling of personal attractiveness. Physical appearance symbolizes one's perceptions, thoughts, feelings, and behav- iors. People use this physical attribute to express one's indi- viduality. Hair styling is a major part of one's daily grooming ritual. Quoting a psychologist, "Preparing one's hair is prepa- ration to face one's social world. The expression 'bad hair day' is testimony to the psychological importance of hair. Hair loss can turn every day into a bad hair day [1]". The trichorrhexis nodosa sheet (informational sheet 1) is very informative as patients with this entity often question the validity of this disease entity. Hair styling and hair cos- metic habits are hard to change, but after reading through this synopsis, my patients are more receptive to considering altering their hair style and using fewer chemicals. Treating a patient with hair loss and/or hair issues is not easy because of psychosocial overtones and patients' distress and body image concerns. These patients often require con- siderable support. They need education on their condition as well as a realistic appraisal of possible treatments, length of time before resolution, and alternatives in terms of hair re- placement. After discussing the 'hairy' situation, patients who remain greatly distressed and/or are totally unrealistic with expectations of magical cures, one might ask them to go to the internet or book store and research their stated hair dis- ease as well as the topic of 'body image.' In short, for some individuals, the loss of hair represents a body image impair- ment which can undermine self-esteem and quality of life. The Open Dermatology Journal, 2008, 2, 77-82 The Open Dermatology Journal, 2008, 2, 77-82 77 77 Open Access Craig G. Burkhart,1 and Craig N. Burkhart2 1University of Toledo College of Medicine, USA; Ohio University College of Osteopathic Medicine, 5600 Monroe Street, Suite 106B, Sylvania, OH 43560, USA 2Department of Dermatology, Northwestern University, Chicago, Illinois, USA 2Department of Dermatology, Northwestern University, Chicago, Illinois, USA Abstract: Hair serves as a social sign of gender, age, status, values, and group membership. Hair is a major part of one's self-identity, and central to one's feeling of personal attractiveness. Treating a patient with hair loss and/or hair issues is not easy because of psychosocial overtones and patients' distress and body image concerns. In this paper, several common hair diseases will be discussed in consort with sample patient instructional sheets. They include trichorrhexis nodosa, te- logen effluvium, pattern alopecia, alopecia areata, excess facial hair in females, and male pattern baldness. This paper therefore presents a template from which individual physicians can assess whether any portion of the material is worth be- ing incorporated into their individual practices. In this paper, several common hair diseases will be dis- cussed in consort with sample patient instructional sheets. This paper therefore presents a template from which individ- ual physicians can assess whether any portion of the material is worth being incorporated into their individual practices. One option is to allow patients time to read the informational sheet after completing the history and physical, and there- fore, before outlining the treatment program. Scalp hair has been endowed with greater social and psy- chological significance than biological importance. Medi- cally, it merely provides some shielding from ultraviolet rays and some cushioning of the cranium from head gear. Histori- cally and culturally, hair serves as a social sign of gender, age, status, values, and group membership. As stated previ- ously, "from monks to skinheads, prisoners of war to warri- ors, bigwig European aristocrats to mop top Beatles, and hippies to head-shaven celebrity athletes, hair makes a statement whether chosen or imposed [1]". Hairy Concerns: Use of Instructional/Informational Sheets for Assistance with Disorders of Hair Craig G. Burkhart*,1 and Craig N. Burkhart2 EXCESS FACIAL HAIR IN A WOMAN Alopecia areata (informational sheet 4) is a non-scarring hair loss condition which can affect any hair-bearing surface. The only predictable thing about the progress of the condi- tion is that it is unpredicatable. Several episodes of hair loss and hair regrowth are not unusual in this condition. It is re- ported that as many as 7% of individuals with alopecia areata develop permanent hair loss in patches or in significant por- tions of their scalp. Alopecia areata is due to a hair cycle dysfunction, but the pathogenesis of the condition is still unknown, with non-specific immune factors appearing to be contributory. While hair loss is devastating to women, an equally trou- bling problem is unwanted hair growth. Our society has deemed body hair on women is undesirable, and women have developed a habit of shaving such unwanted hairs as on the underarms and legs. Indeed, the sight of a woman with natural hair development that is unshaven is considered gro- tesque. Thus, this is another example of multimillion dollar industries motivating women to try to alter their bodies to match some ideal seen in fashion magazines. Thus shaving joins dieting, exercise, cosmetics, hair dying, and cosmetic surgeries as the method to achieve beauty in our society. These body-altering behaviors certainly attest to the fact that a woman’s body is not acceptable the way it naturally is [8]. At present, there is no cure that works every time. Be- cause of the chronic nature of the disease, any mode of treatment needs to be used for prolonged periods of time. The prognosis tends to be worse when the condition begins in early childhood and when extensive areas of the scalp as involved. Thus, patients devoid of scalp hair (alopecia to- talis) and/or total body hair (alopecia universalis) normally have a poor prognosis. Millions of women experience what could be considered heavy hair growth on the face. Some ethnic background and genetic parentage normally have some facial hair, which in our generation, is still considered undesirable. Additionally, abnormal hair growth can also accompany altered androgen metabolism, increase androgen production, decreased andro- gen binding in the circulation, and/or exogenous androgens. Having tried innumerable treatments through the years, I presently start with topical immunomodulators and a kerato- lytic agent [4]. The treatment can be performed by patient at home and response rates have been very encouraging. EXCESS FACIAL HAIR IN A WOMAN In- deed, patients are asked to call for reappointments in 2 to 3 months if no growth is experienced. An overview of available methods for hair removal is discussed on the instructional sheet for excess facial hair (informational sheet 5). There is hope that safer, more selec- tive, and more economical lasers will be developed for hair removal. For non-responders, I consider intralesional corticoster- oid injections for localized patchy alopecia areata. Normally a concentration of 2.5 mg/mL is used and a total of 3mL of triamcinolone acetonide would be the most utilized in any one visit. A 30 gauge needle is helpful for the intradermal injections. In areas of total hair loss, a series of blebs are raised with approximately 0.1 mL per site, 1 cm apart. Such PATTERN ALOPECIA IN WOMEN Topical immunotherapy has been recommended for ex- tensive alopecia areata that fails with conventional therapy [7]. The three contact sensitizers that have been used are dinitrochlorobenzene (DNCB), squaric acid dibutyl ester (SADBE), and diphenylcyclopropenone (DPCP). DNCB has shown mutagenic effects and is rarely used presently. The contact sensitizer is applied weekly trying to put a concentra- tion of the sensitizer to maintain a low-grade tolerable erythema, scaling, and pruritus on the treated side for 36 hours after application. Side effects of such treatment in- cluded eczema, possible extention of the dermatitis to other body parts, itching, edema, lymphadenopathy, and pigmen- tary alterations. Pattern alopecia is the result of genetics, aging, and hor- monal changes that combine to cause changes in the hair follicle, namely, miniaturization of the terminal hair into vellus hair. The end result is generalized thinning of hair over the crown and top areas of the head. The patient hand- out (informational sheet 3) divides the condition into pattern alopecia secondary to low estrogen levels and pattern alope- cia secondary to an increase in (what is often referred to as) male hormones (specifically testosterone and 5- dihydroxyepiandrosterone). The sheet further outlines the options of treatment for patients experiencing low estrogen. The Open Dermatology Journal, 2008, Volume 2 78 Burkhart and Burkhart a treatment would be repeated every 6 weeks, if proving suc- cessful. Nutritional factors are mentioned on the informational sheet for this condition. Rather than elaborate studies, our minimal knowledge on the subject comes from studies of protein-energy malnutrition, starvation, and eating disorders [3]. Otherwise healthy individuals may not find great benefit in dietary supplementation. However, there is medical evi- dence that major deficiencies in zinc and biotin can cause hair loss, so it doesn’t hurt to offer patients these inexpensive agents that may be somewhat helpful. In the very least, it allows more time for the body to return to normal hair cy- cling and reduce the amount of hair shedding. To a great extent, time heals telogen effluvium, especially if the stresses that caused the hair shedding are eliminated. The other options which I consider in recalcitrant cases includes sulfasalazine [5, 6]. This is an anti-inflammatory drug composed of sulfonamide and a salicylate which has shown to be of benefit for rheumatoid arthritis, arthritis, in- flammatory bowel disease, psoriasis, and dermatitis herpeti- formis. It has numerous immunological effects including immunosuppression and immunomodulation. It leads to in- hibition inflammatory cell chemotaxis, cytokine production, antibody proliferation, and IL-2 release. Other alternatives for treatment include topical glucocorticoids, anthralin (Dithranol, Drithocreme), and topical minoxidil. TELOGEN EFFLUVIUM When presenting telogen effluvium (informational sheet 2) to patients, one has to familiarize patients with the con- cept that our hair grows in cycles. At any given time, a small percentage of our hair follicles are in the stage in which hair is shed in preparation for new hair that follows. One loses hair every day. Evidence for shedding is noted in one’s hair brush as part of one’s normal grooming routine. With telo- gen effluvium, there is a shift in the growth cycle in which an abnormal number of follicles have moved into the telo- gen, or resting, phase of the growth cycle. 1874-3722/08 2008 Bentham Open 2008 Bentham Open The Open Dermatology Journal, 2008, Volume 2 Hairy Concerns: Use of Instructional/Informational Sheets Hairy Concerns: Use of Instructional/Informational Sheets The Open Dermatology Journal, 2008, Volume 2 79 There has been significant advance in the medical treat- ment of androgenetic alopecia with two very good treatment options. Finasteride is a type II 5-alpha-reductase inhibitor, and thereby blocks the major enzyme responsible for the condition. The drug has been shown to slow the progression of hair loss and leads to some regrowth of hair recently lost. Although studied most extensively in males under the age of 50, it also has benefit for those in the sixth and seventh dec- ade of their life as well. Additionally, the side effect of re- ducing prostatic size is more welcomed with these latter age group. I prefer giving the flier provided by the manufacturer for finasteride, rather than an informational sheet, because it contains a monetary rebate and adequate information on the drug. only semi-permanent hair dyes to relaxed hair as it has no ammonia and less peroxide than permanent color. Moreo- ver, it is standard to wait at least two to four weeks before applying any hair color to recently relaxed hair. The most common misuse of relaxers is over- processing. Reapplications should be touch-ups to only the new hair growth, and the frequency between treatments should be at least six, and preferably eight or more weeks. In other words, applying hair straightener to already re- laxed hair will greatly increase the risk of breakage. Another possible error with hair straighteners is that all chemical relaxer has to be completely removed with warm water. Of assistance, some shampoos contain “color ac- tion” agents, which indicate by the color of the lather, if all the relaxer chemical has been effectively removed. Most importantly, a neutralizing formula, or stabilizer, is added next to the hair to lower the pH. If the hair is not neutral- ized and its pH not restored to normal levels, the chemical will continue to further weaken the hair shaft. Until this neutralizing step is completed, the hair is extremely sensi- tive and so all pulling, tugging, and excessive combing of the hair should be avoided until this maneuver is com- pleted. The other product for male pattern alopecia is minoxidil. The method of action of minoxidil is not as direct as finas- teride. Nevertheless, clinical results are satisfactory with minoxidil. General Information on Trichorrhexis Nodosum Trichorrhexis nodosum is a distinctive type of hair loss resulting from physical and/or chemical trauma to the hair shaft. It clinically presents with patchy or diffuse hair loss. The hair readily fractures and snaps off on mild pulling or combing, and the hair is not able to achieve its normal length. The base of the hair still is present, and will proceed to grow out if the offending physical and chemical agents are discontinued, or greatly diminished. One must realize that the condition does not represent a failure of her hair to grow; but rather, an actual destruction of the hair follicle from the chemicals. It is hard to give up straight hair which you have grown comfortable with. Not that it helps, but you have been trying to make the hair dif- ferent from what nature intended. By the way, the sodium hydroxide in relaxers is the same ingredient in drain clean- ers, so it isn't unexpected that it causes hair breakage. The most practical therapy is avoidance of physical and chemi- cal trauma to the hair. However, since many no longer yearn for the afro look, compromises may have to be con- sidered. Some patients might merely have to use the prod- ucts less frequently, or for less time duration. Many may be able to tolerate relaxers if they adhere to all of the safety precautions outlined above. The most common cause of trichorrhexis nodosum is hair straighteners which are also referred to as hair relaxers. There are two types of hair straighteners, namely lye relax- ers containing sodium hydroxide as the active ingredient, and “no-lye" products in which calcium hydroxide and guanidine carbonate are mixed to form guanidine hydrox- ide. Both types of relaxers can cause trichorrhexis nodo- sum, although "no-lye" products tend to have less incidence of scalp irritation. With both straightening products, one needs to follow common safety rules in terms of leaving the products in for no longer than the prescribed time, carefully washing out with neutralizing shampoo, and following up with regular conditioning. Conditioners are very important as they re- store some of the natural oils and proteins removed by these chemicals. Blow drying and curling further damage the hair after relaxers and should be avoided. Also, color- ing the hair after using a relaxer also increases the risk of hair damage. The FDA actually recommends the use of Hairy Concerns: Use of Instructional/Informational Sheets There is some evidence with monkeys and with several human case reports, that maximum benefit is achieved when both finasteride orally and minoxidil topi- cally are administered to balding individuals; but given monetary constraints, if one is choosing one product, I would vote for finasteride. Presently the preferred surgical approach to male pattern baldness is hair micrografts. With this method, tiny follicular units of one to four hairs are inserted in closely spaced fash- ion over the entirety of the bald or thinning areas. In our practices, such a procedure would be referred to a different physician. Of note, the use of hot styling tools including blow dry- ers, hot combs, and curling irons should be avoided, if pos- sible, to hair that is relaxed. If one chooses to use the home relaxer box kits, it would be helpful to have someone else on hand to assist in the complete rinse and removal of the chemical. ADDITIONAL INFORMATION Many stylists recommend applying a layer of petroleum jelly on the scalp before applying the relaxer to create a protective barrier between the chemical and the skin. Addi- tionally, before using a relaxer, avoid scratching, brushing, and combing as this makes the scalp more susceptible to chemical irritation. MALE PATTERN BALDNESS Male pattern baldness is a genetically predisposed condi- tion mediated by androgen metabolism. It is not a disease in the conventional sense, but in our culture, hair loss can threaten one’s sense of personal and social acceptance and self-esteem. Information on Telogen Effluvium (Note: some physicians may feel com- fortable expanding possible treatments to include propecia and higher dosages of spironolactone, but I feel both these alternatives are risky.) The cause of the body to direct more hair to go into the resting state than normal has to do with energy conserva- tion—the body typically has a certain amount of energy it uses for making the brain, muscles, heart, and hair work and grow. If the body senses some stress, it will redirect the energy away from hair growth and direct this energy to more important functions (like fighting infection, or mak- ing the heart work faster). Such stresses that cause telogen effluvium can be emotional (i.e. loss of a loved one, di- vorce), or physical (i.e. childbirth, surgery, extreme dieting, drug reaction, severe infection). If there is no obvious source of stress in a severe case of telogen effluvium, I will often suggest a blood test looking for any clues as to what might be causing the body to redistribute its energy away from hair growth. If there is a serious problem detected by the blood test, I will call you within 24 hours of receiving these results. The normal technique to assess hair cycling abnormali- ties is the simple pull test. Hair is pulled with gentle trac- tion to assess the amount of shedding and to examine the proximal ends of the hairs. Note: These suggested treatments may retard further hair loss fairly well, but one should expect only minimal regrowth of lost hair. In some cases of advanced hair loss, one may have to consider wigs and/or hair transplantation. This condition is self-limited, meaning that with time and elimination of the body’s stresses, the hair will regrow. Telogen effluvian can occur at any age. The treatment that I suggest is taking zinc and biotin (which are over-the- counter minerals and vitamins sold at drug stores and at health food stores). These products may be in certain vita- min products. I would prefer if you took at least 8mg of zinc/day and 30mcg/day of biotin. With these products and removal of body stresses, the body should readjust with new hair growth in two to four months. Pattern alopecia secondary to an increase in male hor- mones (specifically testosterone and 5-dihydroxyepiandros- terone) is determined by laboratory tests. These tests are important to obtain if under the age of 45. Information on Telogen Effluvium Hair on the scalp typically grows for three to six years before going into a resting state for three months. This hair cycling is the reason why one notices a few hairs coming out with brushing and washing throughout one’s life. Normally, 90% of scalp hairs are in the growing state. Average hair loss per day is twenty-five hairs because of this cycling process. However, up to 100 hairs lost per day is still within normal limits. The Open Dermatology Journal, 2008, Volume 2 80 Burkhart and Burkhart With telogen effluvium, more hairs are in the resting state rather than the growing state. Therefore, one notices more hair shedding than normal, and less total hair on the scalp. Various degrees of hair loss can occur with telogen effluvium. become more visible. The condition can be caused by (1) too little estrogen, or (2) too much testosterone (often re- ferred to as male hormones, even thought women have small amounts normally). Pattern alopecia secondary to lower estrogen levels of- ten occurs with menopause when the ovaries stop produc- ing estrogen, or after female surgery, such as a tubal liga- tion (in which the blood supply to the ovaries is compro- mised (or greatly reduced), causing a diminishment of es- trogen levels and basically early menopause). If you fit into this category, we have 5 options for treatment. The best option for growing hair is to (1) take prescription oral sup- plemental estrogen, but only if your primary care physician feels you are a candidate, as estrogen does have side ef- fects. The alternative option is (2) topical, over-the-counter Rogaine for Women. A third option would be (3) natural estrogens sold over-the-counter at drug stores health food stores (if your primary care physician agrees). Another choice would be (4) topical ketoconazole cream or sham- poo which recently has been shown to somewhat suppress androgen receptors. (5) Low dose spironolactone 25mg daily also has shown merit when used in consort with Ro- gaine for Women. Information About Pattern Alopecia in Women To all women, hair loss is devastating. There are many concerns and fears associated with alopecia (a general term meaning hair loss). Additionally, there are many agents that claim to grow hair, but almost all of these products are more hype than science. Of note, even medicine and re- search hasn’t solved this type of hair loss. Information on Telogen Effluvium (Note: if you are over 45 and without an increase in facial hair, these tests offer little help in our evaluation.) These tests allow an assessment of your ovarian and adrenal secretions of these two hormones. These tests are performed preferentially during one’s period. (If you are no longer having periods, then you can take the test at any time.) (Inasmuch as estro- gen values fluctuate so much, most physicians feel that estrogen levels do not aid in the assessment of pattern alopecia.) Note: Make sure that the lab also sends the re- sults also to your primary care physician. If these blood tests reveal an abnormality, I will call you…..however, your primary care physician would determine what course to take in that scenario. If your tests are normal, then you would follow the discussion listed above in the paragraph discussing pattern alopecia due to low estrogen. Society makes a person with hair loss feel uncomfort- able. Remember that this condition does not lead to bald- ness, it is not a medical disaster, and the hair will regrow (eventually). You have to lose 80% of your scalp hairs for someone to notice definite baldness when passing you in the mall. So God has given us a lot of hair so that mild hair loss will not be readily noticed in our society. This protein is impairing hair from growing normally. The natural history of alopecia areata has been well studied. The prognosis for the majority of people with alopecia areata is excellent. Most patients have a few shiny bald patches that persist for 6 months to two years, and then enjoy complete and permanent regrowth of the lost hair. It is only in the minority of people with alopecia areata in which the hairs do not return. In such patients the hair loss may be long standing and may continue to spread over much of the scalp. Your primary care physician or gynecologist are better able to assess if there might be a serious medical condition causing the hirsutism. For example, I do not do pelvic ex- ams. Some of the diseases to consider are polycystic ovary syndrome, idiopathic hirsutism, Cushing’s disease, and hormone-secreting tumors of the ovary or adrenal. Your primary care physician or gynecologist would also be the one to decide on whether any oral drugs are indicated for your hirsutism such as oral contraceptives, antiandrogens (such as spironolactone, finasteride, cyproterone acetate, metformin). There is no treatment that is 100% effective for alopecia areata. Recent articles suggest new topical immunomodula- tors (namely, elidil or protopic) may be very effective. These drugs are not readily absorbed through scalp skin and so a topical agent improving its skin penetration is helpful. We are presently using a topical retinoid (such as tazorac, retin-A, or differin) for application at night, with elidel or protopic applied twice daily to the areas of hair loss. It often takes two months to see improvement with this therapy. Other treatment options are tried in two months if necessary. The decision to treat excess facial hair is a matter of personal choice. Our culture often determines how much hair is cosmetically acceptable and how important it is to treat. The psychological impact of hirsutism can range from annoying to severely disabling. In terms of topical measures for your excess facial hair, no topical treatment is perfect, as you probably already know. First, some people lighten (bleach) their hair so that the course hair is less noticeable. Shaving is actually the most common way of controlling facial hair, with over 50% of females choosing this method. Shaving does not change hair diameter, but stubbles can look large to some individu- als. The hairs that regrow in the affected areas are occasion- ally white, and sometimes quite fine. This protein is impairing hair from growing normally. The tendency is for the hair to regrow in this condition within several months. However, an occasional patient may have recurrences. Un- fortunately, there is that rare patient with this condition who gets worse, experiences entire loss of scalp hair, and does not respond to any therapy. Plucking and waxing can be used to remove hair, but can cause pigmentary changes and scarring. In rare instances, the condition has been associated with other diseases. Citing from a textbook by Moschella, “It should be emphasized that, from a practical standpoint, patients with alopecia areata are generally in good health and ordinarily do not require special study. Extensive labo- ratory work-ups fail to reveal any unusual findings associ- ated with alopecia areata.” Thus, unless there are other signs of medical problems, I do not usually pursue investi- gational blood studies with this condition. Chemical depilatories are available but can cause skin irritation. Electrolysis damages individual hair follicles with an electric current so that they do not grow back. This treat- ment can leave pigmentary changes in some individuals and can be expensive. One would have to seek a qualified electrologist for this procedure. Laser hair removal has reports of success, but it is ex- pensive and often requires return treatments, as hair re- moval is not usually permanent by this method. You would have to seek a physician who does this procedure on your own (as I do not make any specific recommendations for this cosmetic procedure). If considering lasers, you might be smart to read on the internet as to which laser would work best for your skin type (i.e. ruby, alexandrite, and/or neodymium-yttrium) and then find a physician who has that particular laser. For more information, go to the web site of the National Alopecia Areata Foundation: (www.naaf.org). What You Should Know About Excess Facial Hair Hirsutism is defined as excessive growth of course, dark hair in areas where women normally only grow fine hair, such as above the lip, on the chin and sideburns. It can be hereditary or it could be secondary to an excess of any of the hormones called androgens (male hormones). All women do make some of these hormones, but when they are at levels seen in men, then that can be a problem. I will offer you a screening blood test to check the levels of these hormones. If possible, get the blood test during your pe- riod. Also, please make sure that the blood drawers know to also send the results to your other doctors. I will only call you if your tests are abnormal. Vaniqa is a skin cream approved by the FDA for treat- ment of unwanted facial hair. It does not remove hair per- manently, but seems to slow its growth. It appears to offer limited benefit for patients in my opinion. Information on Alopecia Areata Alopecia areata is a common hair loss disorder account- ing for 2% of new outpatient visits to dermatologists. In- deed, Humphrey Bogart and J. D. Rockefeller both had this condition. Patients present with sharply outlined, shiny bald patches on the scalp. These bald spots appear suddenly without any preceding event. The hairs in these areas are basically “on strike.” Everything is in place to produce hair, but the hair-making machine is no longer working. It is believed that the body’s white blood cells have made a protein (or antibody) that has attached to the hair roots. My job is to see if the hair loss is due to some medical problem (of the ovaries or adrenal gland) as well as direct- ing you to reasonable treatments available for your condi- tion. Pattern alopecia is a common hereditary condition in which there is central diffuse hair thinning. Women with pattern alopecia usually first notice a gradual thinning of their hair mostly on the top of their heads and their scalps The Open Dermatology Journal, 2008, Volume 2 Hairy Concerns: Use of Instructional/Informational Sheets 81 This protein is impairing hair from growing normally. [1] Cash TF. The psychology of hair loss and its implications for pa- tient care. Clin Dermatol 2001; 19: 161-66. p This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.5/), which permits unrestrictive use, distribution, and reproduction in any medium, provided the original work is properly cited. Burkhart and Burkhart Burkhart and Burkhart 82 The Open Dermatology Journal, 2008, Volume 2 Burkhart and Burkhart [2] Burkhart CG, Burkhart CN. Trichorrhexis nodosa revisited. Skin- Med 2007; 6: 57-8. [3] Rushton DH. Nutritional factors and hair loss. Clin Exp Dermatol 2002; 27: 396-404. [4] Burkhart CN, Burkhart CG. Use of topical immunomodulators with agents to increase absorption. J Drugs Dermatol 2002; 1: 122-123. [5] Ellis CN, Brown MR, Voorhees JJ. Sulfasalazine for alopecia areata. J Am Acad Dermatol 2002; 46: 541-4. [6] Bakar O, Gurbuz O. Is there a role for sulfasalazine in the treatment of alopecia areata? J Am Acad Dermatol 2007; 57: 703-6. [7] Madani S, Shapiro J. Alopecia areata update. J Am Acad Dermatol 2000; 42: 549-66. [8] Tiggemann M, Lewis C. Attitudes toward women’s’ body hair: relationship with disgust sensitivity. Psychol Women Quar 2004; 28: 381-7. [2] Burkhart CG, Burkhart CN. Trichorrhexis nodosa revisited. Skin- Med 2007; 6: 57-8. [3] Rushton DH. Nutritional factors and hair loss. Clin Exp Dermatol 2002; 27: 396-404. [4] Burkhart CN, Burkhart CG. Use of topical immunomodulators with agents to increase absorption. J Drugs Dermatol 2002; 1: 122-123. [5] Ellis CN, Brown MR, Voorhees JJ. Sulfasalazine for alopecia areata. J Am Acad Dermatol 2002; 46: 541-4. [6] Bakar O, Gurbuz O. Is there a role for sulfasalazine in the treatment of alopecia areata? J Am Acad Dermatol 2007; 57: 703-6. [7] Madani S, Shapiro J. Alopecia areata update. J Am Acad Dermatol 2000; 42: 549-66. [8] Tiggemann M, Lewis C. Attitudes toward women’s’ body hair: relationship with disgust sensitivity. Psychol Women Quar 2004; 28: 381-7. [2] Burkhart CG, Burkhart CN. Trichorrhexis nodosa revisited. Skin- Med 2007; 6: 57-8. [3] Rushton DH. Nutritional factors and hair loss. Clin Exp Dermatol 2002; 27: 396-404. [4] Burkhart CN, Burkhart CG. Use of topical immunomodulators with agents to increase absorption. J Drugs Dermatol 2002; 1: 122-123. [5] Ellis CN, Brown MR, Voorhees JJ. Sulfasalazine for alopecia areata. J Am Acad Dermatol 2002; 46: 541-4. [6] Bakar O, Gurbuz O. Is there a role for sulfasalazine in the treatment of alopecia areata? J Am Acad Dermatol 2007; 57: 703-6. [7] Madani S, Shapiro J. Alopecia areata update. J Am Acad Dermatol 2000; 42: 549-66. [8] Tiggemann M, Lewis C. Attitudes toward women’s’ body hair: relationship with disgust sensitivity. Psychol Women Quar 2004; 28: 381-7. [2] Burkhart CG, Burkhart CN. Trichorrhexis nodosa revisited. Skin- Med 2007; 6: 57-8. © Burkhart and Burkhart; Licensee Bentham Open. s is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecomm mits unrestrictive use, distribution, and reproduction in any medium, provided the original work is properly cited. Received: May 20, 2008 Burkhart and Burkhart [3] Rushton DH. Nutritional factors and hair loss. Clin Exp Dermatol 2002; 27: 396-404. [4] Burkhart CN, Burkhart CG. Use of topical immunomodulators with agents to increase absorption. J Drugs Dermatol 2002; 1: 122-123. [5] Ellis CN, Brown MR, Voorhees JJ. Sulfasalazine for alopecia areata. J Am Acad Dermatol 2002; 46: 541-4. [6] Bakar O, Gurbuz O. Is there a role for sulfasalazine in the treatment of alopecia areata? J Am Acad Dermatol 2007; 57: 703-6. [2] Burkhart CG, Burkhart CN. Trichorrhexis nodosa revisited. Skin- Med 2007; 6: 57-8. [7] Madani S, Shapiro J. Alopecia areata update. J Am Acad Dermatol 2000; 42: 549-66. [8] Tiggemann M, Lewis C. Attitudes toward women’s’ body hair: relationship with disgust sensitivity. Psychol Women Quar 2004; 28: 381-7. g p g [5] Ellis CN, Brown MR, Voorhees JJ. Sulfasalazine for alopecia areata. J Am Acad Dermatol 2002; 46: 541-4. Accepted: June 5, 2008 Revised: June 5, 2008 Received: May 20, 2008
https://openalex.org/W2767138860	https://europepmc.org/articles/pmc5665418?pdf=render	English	null	Phenome-wide association study using research participants’ self-reported data provides insight into the Th17 and IL-17 pathway	PloS one	2,017	cc-by	8,490	Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. RESEARCH ARTICLE * meg.g.ehm@gsk.com OPEN ACCESS Citation: Ehm MG, Aponte JL, Chiano MN, Yerges- Armstrong LM, Johnson T, Barker JN, et al. (2017) Phenome-wide association study using research participants’ self-reported data provides insight into the Th17 and IL-17 pathway. PLoS ONE 12(11): e0186405. https://doi.org/10.1371/journal. pone.0186405 Phenome-wide association study using research participants’ self-reported data provides insight into the Th17 and IL-17 pathway Margaret G. Ehm1, Jennifer L. Aponte2, Mathias N. Chiano3, Laura M. Yerges- Armstrong1, Toby Johnson3, Jonathan N. Barker4, Suzanne F. Cook5, Akanksha Gupta6, David A. Hinds7, Li Li2, Matthew R. Nelson1, Michael A. Simpson4, Chao Tian7, Linda C. McCarthy3, Deepak K. Rajpal1, Dawn M. Waterworth1 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Target Sciences, GlaxoSmithKline, Collegeville, PA, United States of America, 2 Genomic Medicine, PAREXEL International, Durham, NC, United States of America, 3 Target Sciences, GlaxoSmithKline, Stevenage, United Kingdom, 4 Division of Genetics and Molecular Medicine, Faculty of Life Sciences & Medicine, Kings College London, London, United Kingdom, 5 Suzanne F Cook Epidemiology Associates LLC, Chapel Hill, NC, United States of America, 6 Dermatology, GlaxoSmithKline, Research Triangle Park, NC, United States of America, 7 Statistical Genetics, 23andMe, Inc., Mountain View, CA, United States of America meg.g.ehm@gsk.com Abstract A phenome-wide association study of variants in genes in the Th17 and IL-17 pathway was performed using self-reported phenotypes and genetic data from 521,000 research partici- pants of 23andMe. Results replicated known associations with similar effect sizes for auto- immune traits illustrating self-reported traits can be a surrogate for clinically assessed conditions. Novel associations controlling for a false discovery rate of 5% included the asso- ciation of the variant encoding p.Ile684Ser in TYK2 with increased risk of tonsillectomy, strep throat occurrences and teen acne, the variant encoding p.Arg381Gln in IL23R with a decrease in dandruff frequency, the variant encoding p.Asp10Asn in TRAF3IP2 with risk of male-pattern balding, and the RORC regulatory variant (rs4845604) with protection from allergies. This approach enabled rapid assessment of association with a wide variety of traits and investigation of traits with limited reported associations to overlay meaningful phenotypic context on the range of conditions being considered for drugs targeting this pathway. Editor: Marie-Pierre Dube´, Universite de Montreal, CANADA Editor: Marie-Pierre Dube´, Universite de Montreal, CANADA Received: February 4, 2017 Accepted: September 29, 2017 Published: November 1, 2017 Copyright: © 2017 Ehm et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: © 2017 Ehm et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Phenome-wide association study of Th17 IL-17 pathway genes increase both disease activity and adverse events including infection when investigated in patients with active Crohn’s disease [CD] [2, 3]. A study to identify novel associations for genes in this pathway has the potential to suggest new indications and highlight possible safety concerns for existing and emerging drugs [4]. This is particularly useful in the drug develop- ment context since genetically supported targets are demonstrated to have increased success rates in clinical development [5]. authors [MGE, MNC, LMY-A, TJ, AG, MRN, LCM, DKR, DMW], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section. Competing interests: Margaret G. Ehm, Mathias N. Chiano, Laura M. Yerges-Armstrong, Toby Johnson, Akanksha Gupta, Matthew R. Nelson, Linda C. McCarthy, Deepak K. Rajpal, and Dawn M. Waterworth are employed by GlaxoSmithKline and may be stockholders of GlaxoSmithKline. Jennifer L. Aponte, Li Li and Suzanne F. Cook were employed by GlaxoSmithKline during the term of the research project and may own GlaxoSmithKline stock. Jennifer L. Aponte is an employee of PAREXEL International. David A. Hinds and Chao Tian are employed by 23andMe and own stock or stock options in 23andMe. This does not alter our adherence to PLOS ONE policies on sharing data and materials. In the last 10 years, the genetic contribution to common disease has been investigated by performing increasingly large genome wide association studies (GWAS), whereby polymor- phisms along all chromosomes are tested for association with a trait of interest. GWAS results have provided a growing catalog of the genetic loci that play a role in disease pathogenesis. Identifying causal variants is difficult and usually more work is required to identify the mecha- nism by which a variant influences a clinical trait [6]. An alternate approach which can provide insight into possible biological disease mechanisms is a phenome-wide association study (Phe- WAS), whereby a particular variant is studied for association with a wide variety of traits to profile the phenotypic effects of a gene variant. This approach is relevant to drug discovery when a variant is known to have a functional effect and the available traits include conditions which have not been previously studied, since there is focus on anticipating the effects of mod- ulating the gene products as drug targets. Denny et al., [7] applied the PheWAS paradigm using electronic medical records (EMRs) to study the relationships between genetic variants and multiple traits in 13,835 participants. In the present study, we used the approach with a much larger database (521,000) of self-reported health histories from research participants of the genetics company, 23andMe, to investigate the relationship between variants in genes from the Th17 and IL-17 pathway to provide insight into the functional consequences of mod- ulating the proteins encoded by these genes. We selected seven variants from seven genes known to be in the Th17 and IL-17 pathway, for PheWAS analysis. We included variants with established GWAS associations and/or those predicted to have functional consequences. We evaluated associations across the selected gene variants to look for common patterns of association. Selected variants were also compared with previous association reports for autoimmune diseases, to evaluate how genetic analysis of these self-reported phenotypes compare to more traditional case-control study results. Results for two of the variants (encoding p.Arg381Gln in IL23R and p.Ile684Ser in TYK2) which were included in a PheWAS of 1,358 EMR-derived traits evaluated in 13,835 European-ancestry individuals from five sites of the Electronic Medical Records and Genomics (eMERGE) net- work (7) were compared with the results from this study. Lastly, PheWAS results from each variant were more comprehensively assessed, evaluating each gene from a drug target perspec- tive. While this study focused on only 7 genes within the pathway, the results provide insight into the use of PheWAS in drug discovery research. Introduction Funding: MGE, MNC, LMY-A, TJ, AG, MRN, LCM, DKR, DMW are employees of GlaxoSmithKline. JA is an employee of PAREXEL International. SFC is an employee of Suzanne F Cook Epidemiology Associates LLC. DAH and CT are employees of 23andMe. GlaxoSmithKline funded the study. The funder provided support in the form of salaries for T helper (Th) 17 cells produce the cytokine interleukin 17 (IL-17) and play a role in multiple autoimmune and infectious diseases. There are promising drugs, including several anti IL-17 or anti IL-17 receptor (IL-17R) antibodies, targeting the Th17 and IL-17 pathway for dermatol- ogy and autoimmune diseases, some of which have been recently approved and others that are in development [1]. The anti IL-17 antibodies were efficacious for psoriasis but seemed to PLOS ONE \| https://doi.org/10.1371/journal.pone.0186405 November 1, 2017 1 / 14 Association results Association results for all seven variants with all 1254 traits are shown in Fig 1, S3 Table lists all results significant at 5% FDR and S4 Table lists all results. For four variants, the IL23R mis- sense variant encoding p.Arg381Gln (rs11209026; c.1142G>A; MAF = 0.07), the RORC regu- latory variant (rs4845604; c.70+225C>T; MAF = 0.14), the TRAF3IP2 missense variant encoding p.Asp10Asn (rs33980500; c.28G>A; MAF = 0.08), and the TYK2 missense variant encoding p.Ile684Ser (rs12720356; c.2051T>G; MAF = 0.09), we observed a large number of highly significant associations (six or more per variant at 5% FDR), including traits with both previously known and novel associations. Fig 2 shows a heat map of association statistics, for all traits associated with at least one variant at 5% FDR. A cluster of autoimmune traits (ulcera- tive colitis, psoriasis, inflammatory bowel disease [IBD]—Crohn’s disease or ulcerative colitis) have associations for variants in IL23R and TRAF3IP2 genes. Beyond the associations with autoimmune traits, the variants in IL23R, RORC, TRAF3IP2 and TYK2 seem to have variant specific, rather than pathway-related, association patterns. For these four variants, and also for the IL17RB nonsense variant encoding p.Gln484Stop (rs1043261; c.1450C>T; MAF = 0.08), we identified, at 5% FDR, a number of previously unreported associations, albeit some at bor- derline significance levels, which we discuss further below. We did not observe any significant associations for the IL17A promoter variant (rs2275913; c.-197G>A; MAF = 0.35) or the IL17F missense variant encoding p.His161Arg (rs763780; c482A>G; MAF = 0.05). These neg- ative results are consistent with the relatively weaker a priori evidence for either large func- tional effects or phenotypic associations for these two variants, but are also consistent with a possible functional effect but lower power for the IL17F variant encoding p.His161Arg due to the lower MAF. Fig 2 shows that, for some traits, the IL17F variant encoding p.Asp10Asn may have comparable effect sizes to other variants evaluated here (shown by size of the circles), although at lower statistical significance levels (shown by color intensity). These results pro- vide evidence that the IL17A variant we selected is unlikely to have a large effect on the human traits studied (upper bounds shown by outer circle size in Fig 2), likely due to insufficient func- tional impact. Overall, these results suggest that genes involved in the Th17 and IL-17 pathway have variable and heterogeneous mechanistic influences on a range of human disease phenotypes. Variant selection and association analyses We evaluated variants in 57 genes (Wikipathways www.wikipathways.org) from the Th17 and IL-17 pathway, denoted IL-17 signaling pathway in homo sapiens (S1 Table) and identified seven variants either known or predicted to be functional, or associated with multiple common conditions with minor allele frequencies (MAF) greater than 0.5%. S2 Table lists the seven var- iants along with associations previously reported for each variant [5]. Genotype data for these seven variants were available from genome wide array genotyping data and any missing values were imputed. Each variant was tested for association with 1254 health-related traits, derived from self-reported phenotype data. A total of 521,000 study participants with greater than 97% PLOS ONE \| https://doi.org/10.1371/journal.pone.0186405 November 1, 2017 2 / 14 Phenome-wide association study of Th17 IL-17 pathway genes European ancestry (determined through analysis of local ancestry) with phenotype data for the questions of interest were included. Analyses were adjusted for age, gender, and the top five ancestry principal components to account for residual population structure. The false discov- ery rate (FDR) was controlled at 5% over analyses for all 7 variants [8]. We report FDR q-val- ues throughout the paper, except when compared to previously reported p-values. Power was estimated across a variety of traits (with widely differing sample sizes) for the MAFs observed for all variants to assist with interpretation of results. European ancestry (determined through analysis of local ancestry) with phenotype data for the questions of interest were included. Analyses were adjusted for age, gender, and the top five ancestry principal components to account for residual population structure. The false discov- ery rate (FDR) was controlled at 5% over analyses for all 7 variants [8]. We report FDR q-val- ues throughout the paper, except when compared to previously reported p-values. Power was estimated across a variety of traits (with widely differing sample sizes) for the MAFs observed for all variants to assist with interpretation of results. PLOS ONE \| https://doi.org/10.1371/journal.pone.0186405 November 1, 2017 Table). These differing estimates could be attributed to factors such as differences in sample size, phenotype definitions, patient demographics or to the winner’s curse [9]. Table). These differing estimates could be attributed to factors such as differences in sample size, phenotype definitions, patient demographics or to the winner’s curse [9]. https://doi.org/10.1371/journal.pone.0186405.g001 https://doi.org/10.1371/journal.pone.0186405.g001 Power analyses For PheWAS studies, power will be highly variable due to the varying sample sizes across phe- notypes, precision of the phenotype and allele frequency of the variant. To support interpreta- tion, we performed a comprehensive power analysis of traits, for the given allele frequencies. S7 Table summarizes results from an analysis of power across a variety of traits including the minimum odds ratio (OR) or effect size (β), in response standard deviations per allele, for binary or continuous traits, respectively, for each variant where there is 80% power to identify an additive effect assuming the given allele frequency, sample size and α = 0.0002, the p-value threshold which empirically corresponds to the FDR cut-off of 5%. No adjustment to account for precision of the phenotype was made. For the variants studied, there is 80% power to iden- tify small effects (OR>1.15; β>0.06) for 26–71% of the traits across the MAF range, moderate effects (OR>1.5; β>0.11) for 87–95% of the traits and large effects (OR>2; β>0.15) for nearly 90% of the traits (S8 Table). Review of the power results by trait category highlights that there is 80% power to detect moderate (OR>1.5; β>0.11) or large effects (OR>2; β>0.15) for more than 75% of the traits within all trait categories except drug side effects, drug efficacy and Par- kinson’s disease related traits. However, there is 80% power to detect small effects (OR>1.15; Fig 2. Heat map of selected PheWAS associations. Associations are shown for each variant with each trait, for the set of all traits that had association with any -variant at 5% FDR. For each association, black circles are drawn with area proportional to the effect size point estimate (inner circle) and 95% upper confidence limit (outer circle), scaled relative to the maximum within each trait. The inner circle is colored according to the effect direction (blue to red) and significance (color scale according to association Z score, with corresponding q value cut-offs shown in the legend). Traits and [variants] are ordered using hierarchical clustering on Euclidean distances between columns [and rows] of the association Z score matrix. https://doi.org/10.1371/journal.pone.0186405.g002 Fig 2. Heat map of selected PheWAS associations. Associations are shown for each variant with each trait, for the set of all traits that had association with any -variant at 5% FDR. Comparison to GWAS results We investigated similarities and differences of the current PheWAS results, with association results available in the literature that were based on clinician assessment or validated diagnos- tic instruments. Fig 3 and S5 Table compare the association statistics for Crohn’s disease, IBD, psoriasis, psoriatic arthritis and eczema. Of fourteen previously reported associations summarized in Fig 3, nine were previously associated with genome-wide significance levels (p5.0×10−8). For all fourteen of the previously reported associations, the odds ratios for this dataset were in the same direction as the previously reported associations although we observed significantly different (p<0.05) effect estimates for 7 of the 14 associations (S6 PLOS ONE \| https://doi.org/10.1371/journal.pone.0186405 November 1, 2017 3 / 14 Phenome-wide association study of Th17 IL-17 pathway genes 71/journal.pone.0186405 November 1, 2017 4 / 14 PLOS ONE \| https://doi.org/10.1371/journal.pone.0186405 November 1, 2017 4 / 14 PLOS ONE \| https://doi.org/10.1371/journal.pone.0186405 November 1, 2017 4 / 14 Phenome-wide association study of Th17 IL-17 pathway genes Fig 1. PheWAS results for variants in seven genes. False Discovery Rate q-values for 1254 traits are plotted for each variant evaluated. Closed circles depict association of the minor allele with increased risk of the conditions and open circles with protection from the conditions. Horizontal dashed lines indicate the q = 0.05 FDR threshold. Phenome-wide association study of Th17 IL-17 pathway genes Fig 3. Association and literature results. Summary of association statistics for study results and previously reported associations for Crohn’s disease, IBD, psoriasis, psoriatic arthritis and eczema. https://doi org/10 1371/journal pone 0186405 g003 Fig 3. Association and literature results. Summary of association statistics for study results and previously reported associations for Crohn’s disease, IBD, psoriasis, psoriatic arthritis and eczema. Fig 3. Association and literature results. Summary of association statistics for study results and previously reported associations for Crohn’s dis psoriasis, psoriatic arthritis and eczema. https://doi.org/10.1371/journal.pone.0186405.g003 https://doi.org/10.1371/journal.pone.0186405.g003 β>0.06) for 65% of the traits only within the trait categories of cognition, sleep, infection, life- style, sensation, longevity and personality. Power analyses For each association, black circles are drawn with area proportional to the effect size point estimate (inner circle) and 95% upper confidence limit (outer circle), scaled relative to the maximum within each trait. The inner circle is colored according to the effect direction (blue to red) and significance (color scale according to association Z score, with corresponding q value cut-offs shown in the legend). Traits and [variants] are ordered using hierarchical clustering on Euclidean distances between columns [and rows] of the association Z score matrix. Fig 2. Heat map of selected PheWAS associations. Associations are shown for each variant with each trait, for the set of all traits that had association with any -variant at 5% FDR. For each association, black circles are drawn with area proportional to the effect size point estimate (inner circle) and 95% upper confidence limit (outer circle), scaled relative to the maximum within each trait. The inner circle is colored according to the effect direction (blue to red) and significance (color scale according to association Z score, with corresponding q value cut-offs shown in the legend). Traits and [variants] are ordered using hierarchical clustering on Euclidean distances between columns [and rows] of the association Z score matrix. PLOS ONE \| https://doi.org/10.1371/journal.pone.0186405 November 1, 2017 5 / 14 Interpretation of results by variant The IL23R gene is involved in Th17 regulation and regulates IL-17 production [10]. The IL17-induced Th17 effector function is impaired in the presence of rs11209026 encoding p. Arg381Gln. This minor allele encoding the glycine residue has also been previously associated with twofold protection against psoriasis and threefold protection against Crohn’s disease [10]. Consistent with previous reports, in this study, the minor allele was associated with protection from Crohn’s disease (OR = 0.53; q = 6.63x10-28), IBD (OR = 0.67; q = 8.0x10-31), psoriasis (OR = 0.82; q = 7.64x10-20), and a composite of 25 auto-immune conditions (OR = 0.88; q = 9.37x10-20). The minor allele was also associated with traits likely driven by these disease associations: in participants reported as taking anti-TNFα medications (OR = 0.72; q = 3.92x10-8) and in patients reported as taking prednisone or other steroid medications (OR = 0.95; q = 0.026). We also observed a novel and highly significant association with a PLOS ONE \| https://doi.org/10.1371/journal.pone.0186405 November 1, 2017 6 / 14 Phenome-wide association study of Th17 IL-17 pathway genes decrease in dandruff frequency (β = -0.08; q = 6.61x10-7). This is unlikely to be driven by other trait associations since dandruff frequency was not significantly correlated with the other asso- ciated traits (S1 Fig). The variant was included in a previously reported PheWAS analysis which failed to identify any statistically significant associations using a Bonferroni threshold of p<0.05/1358 = 3.7x10-5 [7]. The RORγT transcript of the RORC gene is an important transcription factor in Th17 dif- ferentiation. The rs4845604 variant is hypothesized to have a regulatory effect on expression and may be associated with reduced levels of the transcript. This variant was the only common variant in RORC with a previously reported genetic association–with IBD [11]. The q-value for association of the rs4845604 with IBD was q = 0.18 which failed to meet the q<0.05 threshold. We observed an association with the minor allele (A) of this variant with decreased hair curl (β = -0.09; q = 9.92x10-31). The variant is 261kb from rs17646946 (MAF = 0.24; r2 = 0.02 and D’ = 0.20 with rs4845604) within the TCHH gene, which has been associated with hair curl in multiple studies [12, 13]. The Eriksson et al paper [12] studied participants that were included in this PheWAS. Interpretation of results by variant The association of rs4845604 with hair curl is likely to be novel, but further conditional analyses would be required to determine if this signal and previously report associ- ations implicating TCHH may tag the same underlying genetic effect. We observed associa- tions of the minor allele with protection from multiple allergies including dog (OR = 0.92; q = 0.019) and any animal (OR = 0.95; q = 0.042), and with subjects receiving medications for asthma or other lung conditions (OR = 0.95; q = 0.008). Additionally, there were associations with q-values just above the 0.05 threshold with protection from mold allergy (OR = 0.95; q = 0.062), any food allergy (OR = 0.94; q = 0.069) and grasses allergy (OR = 0.96; q = 0.072). The minor allele was not associated with protection from asthma (OR = 0.97; q = 0.22). The associated traits showed high levels of correlation between different allergy traits and between allergy and the asthma related traits (S1 Fig). TRAF3IP2 encodes ACT1, a signaling adaptor involved in the regulation of adaptive immu- nity. The variant, rs33980500, encoding p.Asp10Asn has been shown to prevent ACT1 from interacting with TRAF6, suggesting that this variant may modulate downstream signals of dif- ferent crucial immunoreceptors through altered TRAF interactions [14]. The variant had been previously reported as associated with IBD [11, 15], psoriasis [16], and psoriatic arthritis [14]. Only psoriasis (OR = 1.09; q = 0.0019) and IBD (OR = 1.11; q = 0.042) were found to have a significant association in this dataset compared to previously reported odds ratios (OR = 1.53; 95% CI [1.41,1.67]) for psoriasis [16] and (OR = 1.15; 95% CI [1.09,1.22]) for IBD [15]. Despite previous reports of an association with psoriatic arthritis (OR = 1.95; p = 1.13x10-20) [14], and 80% power to detect an OR of 1.29 in this dataset, the association with psoriatic arthritis is non-significant. Significant associations were observed for protection from frequent nose bleeds (OR = 0.87; q = 0.0063) and severe acne (OR = 0.93; q = 0.022), for green eye color as measured on a blue versus green scale (β = -0.04; q = 0.026), for risk of male pattern balding before age 40 (OR = 1.17; q = 0.021) and male pattern balding (analyzed using survival analysis —Hazard Ratio = 1.09; q = 0.042). PLOS ONE \| https://doi.org/10.1371/journal.pone.0186405 November 1, 2017 Interpretation of results by variant Further evaluation of the association of this variant and acne vulgaris in 1893 cases and 5132 controls (imputation r2 = 0.99 with p = 0.51) did not rep- licate the observed results [17, 18]. A look-up in an association analysis of male pattern balding in CoLaus including 578 cases and 547 controls demonstrated that this variant was associated with male pattern balding (OR = 1.34, p = 0.02; 95% CI of [1.05, 1.72]) with imputation r2 of 0.91 [19]. Review of a published GWAS using 23andMe database for blue versus green eye color (31,037 cases and 30,741 controls) identified association with the G allele of rs13200059 (OR = 1.26; 95% CI [1.18, 1.35]; p = 2.7x10-11). The credible set for the region includes the TRAF3IP2 gene. The effector gene in this locus is more likely to be SLC16A10 which is an aro- matic amino acid transporter where the drosophila orthologue of this gene, KAR, is known to PLOS ONE \| https://doi.org/10.1371/journal.pone.0186405 November 1, 2017 7 / 14 Phenome-wide association study of Th17 IL-17 pathway genes be involved in visual pigmentation (33). Review of Pearson correlation coefficients between associated traits (S1 Fig) highlighted correlation within the related male pattern baldness traits and within the related eye color traits but not between the trait groupings suggesting that these associations may represent true pleiotropy. TYK2 is a member of the Janus kinase (JAK) family of proteins that mediates signaling downstream of several cytokine receptors and intercellular adhesion molecule genes [20]. This gene contains a number of common coding variants including the missense variants rs34536443, encoding p.Pro1104Ala, and rs12720356 encoding p.Ile684Ser. We selected the rs12720356 variant for study as it was the only missense variant previously predicted to be del- eterious with MAF greater than 5% with previous disease association reports: Crohn’s disease [11] and psoriasis [21]. A recent analysis of the functional impact of variants in the TYK2 gene established that minor allele homozygosity at the rs34536443 SNP drives a near-complete loss of TYK2 function which impairs type I IFN, IL-12 and IL-23 signaling whereas rs12720356 does not have similar biological effects [22]. Interpretation of results by variant Analysis of publicly available eQTL data suggests that the rs1270356 or the rs5030390 variant that is in linkage disequilibrium with it, may have an effect on ICAM1 expression in stimulated CD14+ monocytes suggesting the ICAM1 gene which may explain why rs34536443 confers protection from multiple diseases while more het- erogeneous effects were noted for rs1270356 [22]. We observed significant associations between this variant and increased weight (β = 0.88; q = 4.10x10-5) and height (β = 0.06; q = 0.0011) as well as associations with risk of tonsillectomy (OR = 1.06; q = 0.0061), IBD (OR = 1.12; q = 0.0061), more severe acne as a teenager (β = 0.03; q = 0.034), strep throat occurrences (β = 0.04; q = 0.038), and BMI (β = 0.09; q = 0.039). None of these associations were replicated in a PheWAS of 13,835 European-ancestry individuals which included the variant, although the case counts for tonsillitis, IBD and acne related phenotypes (S9 Table) were all less than 262 [7]. The associations with height and BMI were not replicated with N = 119,206 partici- pants (p = 0.21) [23] and N = 112,363 (p = 0.17) [24], respectively. Review of both the BMI and height studies failed to identify any variants within 1 Mb of the p.Ile684Ser variant that could explain the association of this variant with height and BMI that we observed. The association with IBD was previously reported (OR = 1.16; p-value = 4.3x10-16) [11]. In addition to the sig- nificant association with risk of more severe acne as a teenager, we observed an association with risk of severe acne with a q-value just exceeding the 0.05 threshold (OR = 1.07; q = 0.056). This variant was evaluated for association with risk of acne vulgaris in 1893 cases and 5132 controls and was found to be modestly associated (OR = 1.135; 95% CI [1.0048, 1.283]; p = 0.045) [17, 18]. An association with protection from psoriasis has been reported in the lit- erature (OR = 0.71; p = 4.00x10-11) [21] but not found in this dataset (OR = 0.95; q = 0.20) despite 80% power to identify a protective association with an OR less than 0.92 (S7 Table). A previous, smaller PheWAS study of rs12720356 in 29,377 European participants for 502 com- mon and 30 clinical traits related to TYK2 biology did not identify any significant associations [20]. Discussion Our PheWAS study of variants in the Th17 and IL-17 pathway rediscovered known and iden- tified novel significant associations which provide insight into conditions that may be affected by modulation of the protein encoded by the gene. We reviewed results for the four variants that appear to have an effect on human clinical traits, to assess possible interpretations of the PheWAS findings [25]. Considering the results for the variant encoding p.Arg381Gln in IL23R gene, we noted that Il-23R and Th17 cytokines are down-regulated after successful therapies and there are monoclonal antibodies directed against IL-12/23p40, IL-17A and IL-17RA 8 / 14 PLOS ONE \| https://doi.org/10.1371/journal.pone.0186405 November 1, 2017 Phenome-wide association study of Th17 IL-17 pathway genes which are effective in psoriasis [1, 26]. In addition to psoriasis, therapies directed against IL23 have been tested in other diseases including psoriatic arthritis, multiple sclerosis and Crohn’s diseases [1]. The association with decrease in dandruff frequency is likely to be due to true plei- otropy since Di Meglio et al [10] showed that the individuals homozygous for the Arg381Gln allele in Il23R gene were strikingly unresponsive to IL-23, with minimal or no IL-17A and IL- 17F production suggesting a shared biological mechanism, and we did not observe correlation between the associated traits of dandruff frequency, Crohn’s disease and psoriasis. The PheWAS results for RORC regulatory variant highlighted associations for the minor allele with protection from allergies, a phenotype that has not been comprehensively studied using a whole genome scan approach. Multiple companies have programs targeting RORC including Vitae Pharmaceuticals with a phase II program in psoriasis, and Japan Tobacco with a phase I program for autoimmune and allergic asthma (Pharmaprojects: https://citeline.com/ products/pharmaprojects/). If rs4845604 is shown to be associated with reduced levels of the RORγT transcript, then a RORC inhibitor may be an effective treatment for diseases for which this variant has a protective effect, such as asthma. The PheWAS results for the variant encoding p.Ile684Ser in TYK2 (which we noted was working through the ICAM1 gene) highlighted a known risk association with IBD and a pro- tective association for psoriasis. While there are no marketed drugs targeting ICAM1, efalizu- mab is an integrin alpha-L/beta-2 inhibitor which was approved for the treatment of psoriasis but then withdrawn due to a potential risk to patients of developing progressive multifocal leu- koencephalopathy (FDA. Efalizumab withdrawal. 2009 [updated 11 Apr 2011] http://www.fda. gov/drugs/drugsafety/postmarketdrugsafetyinformationforpatientsandproviders/ucm143347. htm). PLOS ONE \| https://doi.org/10.1371/journal.pone.0186405 November 1, 2017 Discussion The integrins and their receptors potentially represent a class of targets of interest for inflammatory and immune diseases. While our study is not a comprehensive evaluation of the Th17 pathway, the results high- lighted that the promoter variant, rs2275913, in IL17A and variant encoded by p.Gln484STOP in IL17RB are unlikely to have a functional impact on the human traits studied since they are either not associated or associated with a small number with good power while the variant encoding p.Arg381Gln in IL23R, regulatory variant, rs4845604, in RORC, variant encoding p. Asp10Asn in TRAF3IP2 and variant encoding p.Ile684Ser in TYK2 do show evidence that they have an impact on human traits with multiple trait associations. The TRAF3IP2 and TYK2 genes impact downstream or more distal signaling and we observed both protective and risk associations with variants in these genes. In contrast, the IL23R and RORC genes have a more direct impact on IL-17 levels and we observed only protective trait associations for these genes. The strategy provides a more holistic view of the impact of gene variation across the pathway that is not as apparent when genes are evaluated one at a time. Analysis of these data provided insight into the use of self-reported data. These data included traits that have not been previously studied using a genome scan approach such as dandruff frequency, as well as infection traits like strep throat occurrences. The results obtained using the self-reported data are similar to association results reported by cohort stud- ies (which focus on clinically assessed patients). One example is illustrated by the results for the variant encoding p.Asn10Asp in TRAF3IP2. While associations with psoriasis and IBD were reported, the results failed to identify a statistically significant association with psoriatic arthritis–reported as p = 1.13x10-20 with OR = 1.95 [14]. Psoriatic arthritis is relatively uncom- mon (prevalence is estimated to vary from 0.3% to 1%). In this dataset we observed a higher than expected prevalence of reported cases in the 23andMe community (1.3%) which may be due to customer misreport, use of inconsistent diagnostic criteria compared to past GWAS cohorts (which used the CASPAR and Moll and Wright criteria [14]), and/or differences in disease spectrum in the 23andMe cohort compared to other clinically ascertained cohorts. Discussion PLOS ONE \| https://doi.org/10.1371/journal.pone.0186405 November 1, 2017 9 / 14 Phenome-wide association study of Th17 IL-17 pathway genes In summary, we were able to confirm numerous reported associations and extend our find- ings of variants in the TH17 pathway. Specifically, we characterized a regulatory variant in RORC gene associated with multiple allergy traits and asthma related traits which have not been reported previously. We identified that the Ile684Ser variant within the TYK2 gene, which may act through the ICAM1 gene, is associated with many traits including psoriasis, Crohn’s disease, and infections, and may suggest integrins and their receptors as potential drug targets. Addi- tional associations, beyond those reported in the literature, were observed for the variants with previous association reports suggesting this information provides valuable information about the effect of the variant on human traits. This PheWAS study provided a comprehensive profile of human traits associated with each variant evaluated, including skin, infection and allergy traits, which haven’t been comprehensively studied thus far. This PheWAS does have limita- tions. Important variants or genes may not have been included in this study which was limited to 7 variants in 7 different genes. The analyses were well powered for moderate and large effect sizes greater than 1.5 for nearly 80% of the traits for all variants reported in European partici- pants but samples sizes were too small to provide a meaningful analysis for other ethnicities and traits. The use of PheWAS in the drug discovery context provides an approach for identifying variants most likely to have an observable effect on gene function and context for the direction of effects suggesting whether a gene activator or inhibitor is needed. Furthermore, clinical trials may want to select or eliminate patients carrying variants with an effect on gene function for drug target genes depending on size and direction of the effect. Interpretation of PheWAS study findings can be complicated by nearby genes, as illustrated by the association of RORC and hair curl, and variable power for each trait assessed. We hope that the emergence of EMR-linked biobanks with genetic data will facilitate more routine use of the PheWAS screen providing insights into drug development strategies for targeting these genes and pathways. PLOS ONE \| https://doi.org/10.1371/journal.pone.0186405 November 1, 2017 Methods and materials All research participants included in the analyses provided informed consent and answered surveys online according to 23andMe’s human subject’s protocol, which was reviewed and approved by Ethical & Independent Review Services, a private institutional review board (http://www.eandireview.com). Informed consent is neither written nor oral—it is electronic. 23andMe customers consent to participate in research online and consent is captured electronically. Ethical & Independent Review Services approved this form of informed consent and waived the requirement to obtain signed consent under 45 CFR 46.117(c). Participants have access to all consent documents through their accounts and may change their consent selection at any time. Samples were genotyped on one of four genotyping platforms. The genotyping platforms, quality control procedures, ancestry determination and imputation protocols have been previ- ously described [27]. Restriction to greater than 97% European ancestry excluded about 25% of otherwise eligible research participants. Minor allele frequencies (MAF) for the variants included in this study for each genotyping platform are listed in S10 Table. We tested the association with 1254 well-curated phenotypes, which are distributed among different phenotypic categories (e.g., cognitive, autoimmune, psychiatrics, etc). An overview of the trait data collection process has been previously described [12]. Specific definitions for all traits associated with variants with q<0.05 FDR are summarized in S1 Appendix. For case con- trol comparisons, we computed association test results by logistic regression. For quantitative traits, association tests were performed by linear regression. For survival traits, we computed association test results by fitting Cox proportional hazards regression. All regression analyses were performed using R version 3.2.2. We typically assumed additive allelic effects and PLOS ONE \| https://doi.org/10.1371/journal.pone.0186405 November 1, 2017 10 / 14 Phenome-wide association study of Th17 IL-17 pathway genes included covariates for age (as determined by participant date of birth), gender, and the top five principal components to account for residual population structure. The association test P value we report was computed using a likelihood ratio test. Power calculations were performed for binary and continuous traits estimating the mini- mum OR or effect size (β) in response standard deviations per allele to identify associations using logistic or linear regression, respectively, with an additive model assuming the given sample size, 80% power, minor allele frequencies corresponding to the variants selected (0.05– 0.35) and β = 0.0002 which empirically corresponds to the FDR cut-off of 5% in these results. Methods and materials For each allele frequency, the proportion of traits (of 1250 binary or continuous traits) with power to identify small OR and effect sizes (OR>1.15; β>0.06), moderate OR and effect sizes (OR>1.5; β>0.11) and large OR and effect sizes (OR>2; β>0.15) is calculated. The OR ranges were determined based on the 25th, 50th, and 75th quantiles of the ORs and hazard ratios from the GWAS catalog with p-values 5x10-8 for non-pharmacogenetic and non-anthropometric traits [28]. The effect size ranges were selected so that the distribution of effect sizes within the small, moderate and large effect size range was similar to that for the odd ratios. Correlation between traits involved in associations with FDR less than 5% was summarized using the Pear- son correlation coefficient. Welch’s t-test is used to test for consistency of OR estimates using OR estimates, confidence intervals and sample sizes. Supporting information S1 Fig. Pearson correlation coefficients for associated traits. Pearson correlation coefficients for traits associated with traits at 5% FDR. (TIF) S1 Fig. Pearson correlation coefficients for associated traits. Pearson correlation coefficients for traits associated with traits at 5% FDR. (TIF) S1 Table. Th17 and IL-17 pathway genes. List of genes evaluated as associated with Th17 and IL-17 pathway (Wikipathways https://www.wikipathways.org and pathway knowledge) (XLSX) S1 Table. Th17 and IL-17 pathway genes. List of genes evaluated as associated with Th17 and IL-17 pathway (Wikipathways https://www.wikipathways.org and pathway knowledge) (XLSX) S2 Table. Previously reported genetic associations. Variants evaluated and traits reported as associated by combining data from multiple sources including the National Human Genome Research Institute (NHGRI) GWAS Catalog, the tables and supplementary materials of manu- scripts archived in the NHGRI GWAS Catalog, and the database of Genotypes and Phenotypes (dbGaP), among others [5]. Acknowledgments We thank Kijoung Song, Justin Rubio, Soumitra Ghosh, Sue Zelt, and Andrea Itano from Glaxo SmithKline, Heiko Runz from Merck and Elizabeth Noblin and Bethann Hromatka from 23andMe for helpful discussions. We thank the research participants of 23andMe who answered surveys and made this work possible. We thank Kijoung Song, Justin Rubio, Soumitra Ghosh, Sue Zelt, and Andrea Itano from Glaxo SmithKline, Heiko Runz from Merck and Elizabeth Noblin and Bethann Hromatka from 23andMe for helpful discussions. We thank the research participants of 23andMe who answered surveys and made this work possible. (XLSX) Comparison of effect sizes for immune traits. Summary of p-values for Welch’s t- test which was used to test for consistency of OR estimates. Ratio of odd ratios and 95% CI is for effect estimates from this study verses previously reported associations. (XLSX) literature with references. (XLSX) S6 Table. Comparison of effect sizes for immune traits. Summary of p-values for Welch’s t- test which was used to test for consistency of OR estimates. Ratio of odd ratios and 95% CI is for effect estimates from this study verses previously reported associations. (XLSX) S7 Table. Power table for each variant and phenotype. Maximum OR and effect size where there is 80% power to identify an effect assuming an additive model, β = 0.0002, and given number of cases and controls or N and given allele frequency. (XLSX) S8 Table. Power summary for each variant. Summary of the proportion of traits with 80% power to detect small, moderate and large effects for each variant. (XLSX) S9 Table. Number of cases for eMERGE PheWAS study. The number of cases for pheno- types of interest in a PheWAS [7]. (XLSX) S10 Table. MAF stratified by array. Minor allele frequencies for all variants for each genotyp- ing array. (XLSX) S1 Appendix. Phenotype definitions. Survey names, survey questions, and logic used to define cases and controls for traits associated with variants with q<0.05 FDR. (PDF) S7 Table. Power table for each variant and phenotype. Maximum OR and effect size where there is 80% power to identify an effect assuming an additive model, β = 0.0002, and given number of cases and controls or N and given allele frequency. (XLSX) S8 Table. Power summary for each variant. Summary of the proportion of traits with 80% power to detect small, moderate and large effects for each variant. (XLSX) S9 Table. Number of cases for eMERGE PheWAS study. The number of cases for pheno- types of interest in a PheWAS [7]. (XLSX) S10 Table. MAF stratified by array. Minor allele frequencies for all variants for each genotyp- ing array. (XLSX) S1 Appendix. Phenotype definitions. Survey names, survey questions, and logic used to define cases and controls for traits associated with variants with q<0.05 FDR. (PDF) S1 Appendix. Phenotype definitions. Survey names, survey questions, and logic used to define cases and controls for traits associated with variants with q<0.05 FDR. (PDF) (XLSX) S3 Table. Study association statistics. Association statistics (trait, description, gene, rs num- ber, p-value, experiment wide q-value, number of cases, number of controls, OR and 95% confidence interval, number of participants, effect size and 95% confidence interval) from 23andMe analysis for traits with FDR significance level of 5%. We report OR for binary traits and effect sizes for quantitative traits including some traits that analyzed ordinal numbers. (XLSX) S3 Table. Study association statistics. Association statistics (trait, description, gene, rs num- ber, p-value, experiment wide q-value, number of cases, number of controls, OR and 95% confidence interval, number of participants, effect size and 95% confidence interval) from 23andMe analysis for traits with FDR significance level of 5%. We report OR for binary traits and effect sizes for quantitative traits including some traits that analyzed ordinal numbers. (XLSX) S4 Table. Study association statistics. Association statistics (trait, description, gene, rs num- ber, p-value, experiment wide q-value, number of cases, number of controls, OR and 95% confidence interval, number of participants, effect size and 95% confidence interval) from 23andMe analysis for traits and levels of significance. We report OR for binary traits and effect sizes for quantitative traits including some traits that analyzed ordinal numbers. (XLSX) S4 Table. Study association statistics. Association statistics (trait, description, gene, rs num- ber, p-value, experiment wide q-value, number of cases, number of controls, OR and 95% confidence interval, number of participants, effect size and 95% confidence interval) from 23andMe analysis for traits and levels of significance. We report OR for binary traits and effect sizes for quantitative traits including some traits that analyzed ordinal numbers. (XLSX) S5 Table. Study and literature association results. Summary of association statistics (p-val- ues, OR, 95% confidence intervals and sample sizes) for results from this study and from the S5 Table. Study and literature association results. Summary of association statistics (p-val- ues, OR, 95% confidence intervals and sample sizes) for results from this study and from the PLOS ONE \| https://doi.org/10.1371/journal.pone.0186405 November 1, 2017 11 / 14 Phenome-wide association study of Th17 IL-17 pathway genes literature with references. (XLSX) S6 Table. Comparison of effect sizes for immune traits. Summary of p-values for Welch’s t- test which was used to test for consistency of OR estimates. Ratio of odd ratios and 95% CI is for effect estimates from this study verses previously reported associations. (XLSX) literature with references. (XLSX) S6 Table. References 1. Bartlett HS, Million RP. Targeting the IL-17-T(H)17 pathway. Nature Reviews Drug Discovery. 2014; 14 (1):11–2. 2. 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Hinds, Li Li, Chao Tian. Investigation: Jennifer L. Aponte, Mathias N. Chiano, Jonathan N. Barker, Akanksha Gupta, Michael A. Simpson. Methodology: Laura M. Yerges-Armstrong, Toby Johnson, David A. Hinds, Li Li, Chao Tian. Methodology: Laura M. Yerges-Armstrong, Toby Johnson, David A. Hinds, Li Li, Chao Tian. Project administration: Margaret G Ehm Jennifer L Aponte Project administration: Margaret G. Ehm, Jennifer L. Aponte. Resources: Margaret G. Ehm, Matthew R. Nelson. Software: David A. Hinds, Chao Tian. Software: David A. Hinds, Chao Tian. Supervision: Margaret G. Ehm. Validation: Jonathan N. Barker, Michael A. Simpson. 12 / 14 PLOS ONE \| https://doi.org/10.1371/journal.pone.0186405 November 1, 2017 PLOS ONE \| https://doi.org/10.1371/journal.pone.0186405 November 1, 2017 Phenome-wide association study of Th17 IL-17 pathway genes Visualization: Mathias N. Chiano, Laura M. Yerges-Armstrong, Li Li. Writing – original draft: Margaret G. Ehm, Linda C. McCarthy, Dawn M. Waterworth. 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https://openalex.org/W4224302630	https://bmcplantbiol.biomedcentral.com/track/pdf/10.1186/s12870-022-03570-4	English	null	Tea plant roots respond to aluminum-induced mineral nutrient imbalances by transcriptional regulation of multiple cation and anion transporters	BMC plant biology	2,022	cc-by	14,910	Tea plant roots respond to aluminum‑induced mineral nutrient imbalances by transcriptional regulation of multiple cation and anion transporters Jing Hao1†, Anqi Peng1†, Yingying Li1†, Hao Zuo1, Ping Li1, Jinsong Wang1, Keke Yu1, Chun Liu2, Shancen Zhao2, Xiaochun Wan1, Jon K. Pittman3 and Jian Zhao1* Abstract Background: Tea is one of the most popular non-alcoholic beverages in the world for its flavors and numerous health benefits. The tea tree (Camellia sinensis L.) is a well-known aluminum (Al) hyperaccumulator. However, it is not fully understood how tea plants have adapted to tolerate high concentrations of Al, which causes an imbalance of mineral nutrition in the roots. Results: Here, we combined ionomic and transcriptomic profiling alongside biochemical characterization, to probe the changes of metal nutrients and Al responsive genes in tea roots grown under increasing concentrations of Al. It was found that a low level of Al (~ 0.4 mM) maintains proper nutrient balance, whereas a higher Al concentra- tion (2.5 mM) compromised tea plants by altering micro- and macro-nutrient accumulation into roots, including a decrease in calcium (Ca), manganese (Mn), and magnesium (Mg) and an increase in iron (Fe), which corresponded with oxidative stress, cellular damage, and retarded root growth. Transcriptome analysis revealed more than 1000 transporter genes that were significantly changed in expression upon Al exposure compared to control (no Al) treat- ments. These included transporters related to Ca and Fe uptake and translocation, while genes required for N, P, and S nutrition in roots did not significantly alter. Transporters related to organic acid secretion, together with other putative Al-tolerance genes also significantly changed in response to Al. Two of these transporters, CsALMT1 and CsALS8, were functionally tested by yeast heterologous expression and confirmed to provide Al tolerance. Conclusion: This study shows that tea plant roots respond to high Al-induced mineral nutrient imbalances by transcriptional regulation of both cation and anion transporters, and therefore provides new insights into Al tolerance mechanism of tea plants. The altered transporter gene expression profiles partly explain the imbalanced metal ion accumulation that occurred in the Al-stressed roots, while increases to organic acid and Al tolerance gene expression partly explains the ability of tea plants to be able to grow in high Al containing soils. The improved transcriptomic understanding of Al exposure gained here has highlighted potential gene targets for breeding or genetic engineering approaches to develop safer tea products. Correspondence: jzhao2@qq.com; jianzhao@ahau.edu.cn †Jing Hao, Anqi Peng and Yingying Li contributed equally to this work. 1 State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, 130 West Changjiang Road, Hefei 230036, China Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Hao et al. BMC Plant Biology (2022) 22:203 https://doi.org/10.1186/s12870-022-03570-4 Background Tea is one of the most popular non-alcoholic beverages, consumed by over two-thirds of the world’s population due to its health-beneficial, refreshing and pleasant stim- ulant effects. Because tea made from the fresh buds and leaves of tea plants (Camellia sinensis L.) contains various nutritional trace elements, drinking tea provides intake of many essential macro- and micro-nutrients for humans [1–4]. Meanwhile, environmental exposure of tea plants to metal pollutants may inhibit growth and therefore the yield of tea, and may cause the accumulation of signifi- cant amounts of non-essential and potentially toxic met- als in tea leaves, which is a public health concern for tea products [2, 3]. For example, trace elements including, aluminum (Al), arsenic (As), cadmium (Cd), copper (Cu), fluoride (F), manganese (Mn), chromium (Cr), and nickel (Ni) have been reported in different tea infusions, and in some cases metal concentrations exceed the maximum permissible limits [3]. Tea plants are perennial evergreen woody trees or shrubs, which are widely grown in subtropical mountain- ous regions of southern China, the Assam area in India, and other parts of the world [5]. Many tropical and sub- tropical plant species such as tea plants grow in acidic soils that are prevalent in these regions. Metals includ- ing Al are more bioavailable in acidic soils leading to Al3+ toxicity, which is one of the major factors limiting crop production on acid soils [6]. Elevated Al (as Al3+) causes rhizotoxicity symptoms on plants: root elongation is severely inhibited, which could result from the binding of Al to multiple cellular targets and subsequent disrup- tion of the functions of the cell wall, cell membrane, as well as various proteins and enzymes, DNA replication, and other cellular processes [6–10]. Most often, plants grown on acidic soils also suffer from other alterations to essential mineral nutrition that is influenced indirectly by high Al and H+ availability. Furthermore, the avail- ability of Al may alter the uptake characteristics of other non-essential but toxic metals such as Cd. Although many metals such as iron (Fe), Mn, Cu, and zinc (Zn) are essential trace micronutrients, excessive accumulation of these metals can be toxic, while deficiencies of micronu- trients and macronutrients, such as nitrogen (N), phos- phorus (P), sulfur (S), calcium (Ca), magnesium (Mg), and potassium (K) are deleterious to plants [6, 7]. Keywords: Metal transporter, Transcriptome, Ionome, Ion imbalance, Aluminum tolerance Keywords: Metal transporter, Transcriptome, Ionome, Ion imbalance, Aluminum tolerance grow normally on most acidic soils containing higher concentrations of Al, without any obvious rhizotoxicity symptoms [4, 12, 13]. So far, at least two broad resistance mechanisms involving external or internal detoxifica- tion have been proposed to explain most plant adaptive responses to Al toxicity [6]. For external exclusion mech- anisms, many plants secrete compounds including organic acids from their root tips to chelate Al and avoid Al toxicity at the cell wall or to prevent Al entering into growing root tip cells, thus providing a certain degree of Al resistance [14–16]. Organic acids are also used for internal tolerance mechanisms, whereby Al-organic acid complexes accumulate in the root symplasm and then facilitate the partitioning of Al into older shoot tissues for storage in a non-toxic form. In addition, tea plants are Al hyperaccumulators that can tolerate these acidic, high Al conditions due to their ability to accumulate high amounts of Al, such that Al concentrations in older leaves are typically 100-fold higher (e.g., ~ 30,000 mg kg− 1) than in leaves of two of the most Al-tolerant monocot plants, rice and common buckwheat, and which in turn are approximately 2–7 times more Al-tolerant than wheat, sorghum, or maize [10, 12, 13]. Tea plants are able to hyperaccumulate high concentrations of Al in part due to the ability to maintain and even enhance root growth in the presence of Al, to allow mobile forms of Al to be translocated into aerial tissues, and having enhanced leaf anti-oxidant processes [9, 10, 12]. However, the accu- mulation of Al, F, and other metals in tea plant leaves [1, 2] introduces food safety issues and health concerns for drinking tea or consumption of tea products. While some of the physiological and biochemical details of Al tolerance and accumulation mechanisms by tea plants are known, there are still gaps in knowledge particularly with regard to the identification of genetic details of these processes. Therefore research to investigate the uptake, transport, and distribution mechanisms of Al, as well as other Al affected metals is of interest in order to poten- tially breed low Al-accumulating and reduced metal con- taining tea plant varieties for safer drinks. Understanding the plant’s Al tolerance mechanisms is also of interest to identify new molecular tools to engineer Al-tolerance to other major crops and to expand their cultivation on acidic soils. Keywords: Metal transporter, Transcriptome, Ionome, Ion imbalance, Aluminum tolerance The aim of this study was to explore the Al accumu- l ti ll Al d t ifi ti h i f t © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Hao et al. BMC Plant Biology (2022) 22:203 Page 2 of 20 Effects of Al exposure on tea seedling root growth and Al accumulation Macronutrients such as K, Ca and Mg, and some transi- tion metal micronutrients such as Fe, Cu, Mn, and Zn are essential mineral nutrients for normal plant growth and development, but they also can be toxic to plants when present in excess [18]. To understand how high Al and acidic conditions effects mineral nutrition in tea plant roots, metal elements were measured in the roots from seedlings grown in 0, 0.4, and 2.5 mM Al over a 10 day period. Overall, we observed significantly reduced Ca, Mg and Mn root content in the highest Al treatments, and fluctuations in the root concentrations of Fe, Zn, and Cu (Fig. 2). Ca and Mg content showed no significant change over time in the absence of Al (0 mM treatment), but there was a decrease over time in Ca content in the 0.4 and 2.5 mM Al treatments (Fig. 1B). Likewise, Mg concentration of roots steadily decreased over time, par- ticularly in the 2.5 mM Al treatment where Mg content dropped quickly by 12 h after treatment and continued to decrease to ~ 400 mg kg− 1 by day-10 of the treatment period. Mn content also decreased in response to Al. Fe contents in roots fluctuated in larger ranges between 140 to over 300 mg kg− 1 with both 0.4 and 2.5 mM Al treat- ments inducing rapid increases in Fe content after 6 h. The exposure of Al gave a much smaller and more vari- able response to Cu and Zn contents. For example, some increases in Zn content were observed in the 0.4 and 2.5 mM Al treatments but only a few time points were significantly higher than in the 0 mM Al treatment roots (Fig. 2). To gain further understanding of the Al-tolerance mech- anisms of tea plants, we treated tea seedlings with differ- ent concentrations of Al (0, 0.4, and 2.5 mM, pH 4.3) in modified Shigeki Konishi nutrient solution (SK-solution). Standard SK-solution contains 0.4 mM Al2(SO4)3 since previous studies have demonstrated that Al at this con- centration effectively promotes tea seedling root growth and development in contrast to an absence of Al [12, 17]. In the weekly replaced SK-solutions with consistent pH 4.3, we determined Al3+ activity at 0.4 mM Al equated to approximately 200 μM Al3+ [9, 12]. Background These indirect effects of Al and H+ toxicity provide further plant stress, by evoking subsequent oxidative stresses and triggering cell death [8, 11]. While most major crops dis- play significant sensitivity to acidic soils, tea plants can The aim of this study was to explore the Al accumu- lation as well as Al detoxification mechanisms of tea plants, and the consequential mineral nutritional changes induced by Al exposure, by integrating transcriptomic and ionomic profiling, and biochemical analyses on Hao et al. BMC Plant Biology (2022) 22:203 Hao et al. BMC Plant Biology (2022) 22:203 Page 3 of 20 high of approximately 1200 mg kg− 1 after 10 days of incu- bation (Fig. 1B). Root Al accumulation was further exam- ined using the fluorescent Al-sensitive morin dye. Root tip Al accumulation was clearly observed after 3 h expo- sure within the stele of the root, with a low level of stain- ing for the 0.4 mM Al treatment but very intense staining for the 5 mM Al treatment (Fig. 1C). At earlier time points (1 h) only the 5 mM Al treatment showed strong staining, while some extreme tip staining was consist- ently observed for the 2.5 mM Al treated roots. No stain- ing was observed for any treatments after just 30 min. seedlings grown hydroponically under various Al stress conditions in acidic solutions. We observed imbalanced accumulation of various metal ions in roots, and altered expression of many metal ions and organic acid trans- porter genes, as well as genes encoding oxidative stress- related enzymes and other putative Al tolerance genes. Since these membrane transporters play essential roles in metal ion uptake, translocation, distribution, and homeo- stasis, their expression changes are implicated in the tea plant’s adaptive responses to Al and H+ stresses. Effects of Al exposure on tea seedling root growth and Al accumulation When tea seed- lings were grown hydroponically in SK-solution without Al or containing higher (> 0.4 mM) Al concentrations, the roots displayed clear differences: roots in 0 mM Al- solution were shorter and grew less strongly than those from the 0.4 mM-solution, which showed the healthiest growth (Fig. 1A). But roots from the 2.5 mM Al-solution showed substantially worse root growth (Fig. 1A). We also observed that roots grown in SK-solution containing 5 to 10 mM Al displayed significantly worse growth per- formance: roots became brown and root growth was sev- erally inhibited (Fig. 1A; data not shown), consistent with other reports [12, 17]. Quantification of root elongation found that seedlings grown in 2.5 mM Al were not signifi- cantly changed until 10 days of treatment, and then after a further 5 days of growth there was 16% root inhibition compared to the seedlings grown in 0.4 mM Al.h The root stress symptoms observed were coincident with significantly increased Al accumulation over time as external Al concentration was increased (Fig. 1B). Al content in roots of seedlings treated with no added Al showed no significant change over time and fluctuated slightly at a baseline concentration of approximately 300 mg kg− 1. However, the Al content rapidly increased to 600–800 mg kg− 1, even after just 6 h of cultivation in 0.4 mM Al-SK solution, but then with very little change over the rest of the 10 day incubation period. In 2.5 mM Al-SK solution, Al content in roots also rapidly increased to 700–800 mg kg− 1 after 6 h, and eventually reached a Cd, like Al, is a non-essential and very toxic metal which is able to easily accumulate into plant roots. It was also examined whether Al exposure provides any protec- tion against Cd accumulation by exposing plants to the three Al treatments (0 mM, 0.4 mM and 2.5 mM) and then measuring Cd content. In tea seedlings that were exposed to 0 mM Al treatment, an initial rapid increase in Cd content after 6 h was followed by a decline in root Cd content after 12 h, with a significant drop after 10 days, possibly due to a transfer of Cd into shoot tissues Hao et al. BMC Plant Biology (2022) 22:203 Page 4 of 20 Fig. 1 Effects of Al stress on root growth of hydroponically grown tea plants. Effects of Al exposure on tea seedling root growth and Al accumulation a Two-year old tea plant seedlings of the same genetic background were cultivated hydroponically in SK-medium containing 0.4 mM Al2(SO4)3. The seedlings were treated 0 mM Al or 5 mM Al for the effects of growth. b Contents of Ca and Mg in the roots of tea plant seedlings under 0, 0.4, and 2.5 mM Al stress for different times measured by ICP-MS. c Imaging of Al accumulation in root tips of tea plant seedlings. The Al-specific fluorescence dye morin was used for imaging. The representative photos shown are from three independent experiments. Data are presented as means ± SD from three independent experiments and statistical significance is based on Student’s t-test (P < 0.05; ** P < 0.001) compared with controls Fig. 1 Effects of Al stress on root growth of hydroponically grown tea plants. a Two-year old tea plant seedlings of the same genetic background were cultivated hydroponically in SK-medium containing 0.4 mM Al2(SO4)3. The seedlings were treated 0 mM Al or 5 mM Al for the effects of growth. b Contents of Ca and Mg in the roots of tea plant seedlings under 0, 0.4, and 2.5 mM Al stress for different times measured by ICP-MS. c Imaging of Al accumulation in root tips of tea plant seedlings. The Al-specific fluorescence dye morin was used for imaging. The representative photos shown are from three independent experiments. Data are presented as means ± SD from three independent experiments and statistical significance is based on Student’s t test (P<0 05; * P<0 001) compared with controls Fig. 1 Effects of Al stress on root growth of hydroponically grown tea plants. a Two-year old tea plant seedlings of the same genetic background were cultivated hydroponically in SK-medium containing 0.4 mM Al2(SO4)3. The seedlings were treated 0 mM Al or 5 mM Al for the effects of growth. b Contents of Ca and Mg in the roots of tea plant seedlings under 0, 0.4, and 2.5 mM Al stress for different times measured by ICP-MS. c Imaging of Al accumulation in root tips of tea plant seedlings. The Al-specific fluorescence dye morin was used for imaging. The representative photos shown are from three independent experiments. Data are presented as means ± SD from three independent experiments and statistical significance is based on Student’s t-test (P < 0.05; * P < 0.001) compared with controls and/or efflux out of the roots (Fig. Effects of Al exposure on tea seedling root growth and Al accumulation 2). In response to Al treatment, the initial Cd accumulation into the root was mostly inhibited but more Cd was retained in the roots by 10 d, particularly in the 2.5 mM Al treatment. transcript contigs were assembled and 86% were able to be matched to the C. sinensis var. sinensis genome. Compared with the non-Al (0 mM) control treatment, the 2.5 mM Al treatment resulted in 2617 up-regulated and 1544 down-regulated genes at 12 h, 1165 up-regu- lated and 1304 down-regulated genes at 24 h, and 1611 up-regulated and 1704 down-regulated genes at 48 h. However, compared with the 0.4 mM Al treatment, the 2.5 mM Al treatment resulted in 1766 up-regulated and Global gene expression changes in roots upon Al exposure To understand the molecular mechanisms taking place in the Al-stressed roots in more detail, an RNA-Seq transcriptome analysis was performed. About 45,737 Hao et al. BMC Plant Biology (2022) 22:203 Page 5 of 20 Fig. 2 Effects of Al stress on the metal content in roots of hydroponically grown tea plants. Metal element accumulation (Mg, Fe, Zn, Mn, Cu and Cd) in roots of two-year-old tea seedlings grown hydroponically in SK medium containing 0.4 mM Al, and exposed to a series of Al concentrations (0, 0.4, 2.5 mM Al) for different times, then measured by ICP-MS. Data are presented as means ± SD from three independent experiments and statistical significance is based on Student’s t-test (P < 0.05; * P < 0.001) compared with controls Fig. 2 Effects of Al stress on the metal content in roots of hydroponically grown tea plants. Metal element accumulation (Mg, Fe, Zn, Mn, Cu and Cd) in roots of two-year-old tea seedlings grown hydroponically in SK medium containing 0.4 mM Al, and exposed to a series of Al concentrations (0, 0.4, 2.5 mM Al) for different times, then measured by ICP-MS. Data are presented as means ± SD from three independent experiments and statistical significance is based on Student’s t-test (P < 0.05; * P < 0.001) compared with controls of each of these identified genes were determined (Table S1-S9, Fig. S1–24). 1768 down-regulated genes at 12 h, 1859 up-regulated and 1329 down-regulated genes at 24 h, and 1719 up- regulated and 1484 down-regulated genes at 48 h. Genes assigned to four categories, signal transduction, environmental adaptation, immune system and sensory system, were over-represented in Al-stressed roots. Effects of Al exposure on tea seedling root growth and Al accumulation In contrast, some of the transcripts encod- ing proteins responsible for moving Ca2+ into internal organelles, including a putative MCU gene TEA000129 (CsMCU2) and two ECAs TEA015853 (CsECA9) and TEA011305.1 (CsECA11), showed up-regulation by 2 to 8-fold over the 3 time points examined in response to excess (2.5 mM) Al (Fig. 3A). The ACA genes TEA023534 (CsACA6) and TEA026012 (CsACA11) were down-reg- ulated in response to both Al treatments in compari- son to no Al. There was also evidence of Al-responsive reduced transcript abundance for TEA008626 (CsACA3). However, CAX genes including TEA007963 (CsCAX1) and TEA000101 (CsCAX2) did not show any significant expression change in response to Al (Fig. 3A, Table S1, Fig. S1-S7). HMAs belong to the P-type ATPase superfamily, which is further classified into several sub-families, accord- ing to their transport specificity [28]. The P1B HMA sub-family can either mediate the transport of divalent Zn2+/Co2+/Cd2+/Pb2+ (including AtHMA1 to 4) or monovalent Cu+/Ag+ (including AtHMA5 to 8) [18, 29]. Most tea HMAs showed just moderate responses to Al exposure. Those most closely related to AtHMA1 to 4 and therefore putative Zn2+ and Cd2+ efflux transport- ers, including TEA019557 (CsHMA1) and TEA018983 (CsHMA2), were moderately increased (but less than 2-fold) in response to 2.5 mM Al (Fig. 3B, Table S4, Fig. S16). Likewise, those more closely related to AtHMA5 to 8 and therefore putative Cu+ efflux transporters, includ- ing TEA018281 (CsHMA3), TEA000616 (CsHMA7), TEA000615 (CsHMA9) were also moderately up-reg- ulated in response to 2.5 mM Al treatment (Fig. 3B), and data was validated by qRT-PCR for representa- tive gene CsHMA9 (Fig. 3C). The Cu influx transporter (COPT) family are high affinity plasma membrane Cu transporters [30]. Three of the tea COPT genes were highly expressed in the roots: TEA022572 (CsCOPT1), TEA030188 (CsCOPT2) and TEA024734 (CsCOPT4). These all show induced expression of approximately 1.4-fold following by 48 h following 0.4 mM Al exposure (Fig. 3B, Table S3, Fig. S16-S17). The CCX gene family has been proposed to play a role in the transport of various ions, with some A. thaliana members such as AtCCX3 and AtCCX4 implicated in K+ and possibly Na+ transport, while AtCCX1 and AtCCX2 are likely to function as Ca2+ transporters [22, 23]. Effects of Al exposure on tea seedling root growth and Al accumulation One of the tea plant CCX genes TEA032754 (CsCCX3), was markedly down-regulated by Al treatment, while TEA021659 (CsCCX6) was highly up-regulated by between 2 to 8-fold in response to 0.4 mM and 2.5 mM Al treatment (Fig. 3A, Table S1, Fig. S1-S7). Mg plays a positive role in plant resistance to Al stress [24, 25]. Two classes of Mg transporters have been well studied in plants, the Mg2+/H+ exchang- ers (MHX) and the Mg2+ transporters (MGT). AtMHX is a tonoplast protein mediating transport of Mg2+ into the vacuole in a proton-dependent manner [26, 27]. A. thaliana MGTs including AtMGT1 and AtMGT10 are plasma membrane high affinity Mg2+ transporters that Effects of Al exposure on tea seedling root growth and Al accumulation These included a large number of putative membrane proteins and transporters, including essential and non- essential metal transporters, and organic acid trans- porters, as well as genes involved in oxidative stress response. Phylogenetic analyses, in comparison with Arabidopsis thaliana genes, were performed to group tea genes into specified gene families (Table S1-S9, Fig. S1–24). In addition, the plant tissue expression patterns Al stress‑induced changes in ca and mg transporter genes Two major essential nutrients Mg and Ca, were sig- nificantly reduced in tea plant roots in response to high concentration of Al. Therefore, it was relevant to further examine the gene candidates known to be involved in plant Ca2+ and Mg2+ transport and homeostasis. In addi- tion to the RNA-Seq data, validation of selected genes was performed by RT-PCR. The cellular homeostasis of Ca2+ in plant cells is enabled by membrane transporters at the plasma membrane and at endomembranes, includ- ing various Ca2+-permeable ion channels for Ca2+ influx Hao et al. BMC Plant Biology (2022) 22:203 Page 6 of 20 into the cytosol [19]. and active Ca2+ efflux transport- ers, which can include the endoplasmic reticulum-type Ca2+-ATPase (ECA) and autoinhibited Ca2+-ATPase (ACA), the Ca2+/H+ exchanger (CAX), and the mito- chondrial Ca2+ uniporter (MCU) proteins [20]. Ca2+ influx is mediated by several families of selective and non-selective ion permeable channels, including cyclic nucleotide-gated channels (CNGCs), ionotropic gluta- mate-like receptors (GLR), two-pore channel 1 (TPC1), annexins, and mechanosensitive-like (MSL) ion channels, although it is important to highlight that many of these channels can also mediate the movement of other ions in addition to Ca2+ (Table S1, Fig. S1-S7) [21]. The RNA-Seq data as shown in the heatmaps indicated that many of the Ca2+ permeable channel genes were down-regulated in response to 0.4 or 2.5 mM Al, including the GLR genes TEA014867 (CsGLR1) and TEA006325 (CsGLR15), and the MSL genes TEA001140 (CsMSL1) and TEA019176 (CsMSL8), while TEA024970 (CsGLR13) was up-reg- ulated in Al treated roots at 12 h and 24 h (Table S1, Fig. S1-S7). In contrast, some of the transcripts encod- ing proteins responsible for moving Ca2+ into internal organelles, including a putative MCU gene TEA000129 (CsMCU2) and two ECAs TEA015853 (CsECA9) and TEA011305.1 (CsECA11), showed up-regulation by 2 to 8-fold over the 3 time points examined in response to excess (2.5 mM) Al (Fig. 3A). Effects of Al exposure on tea seedling root growth and Al accumulation The ACA genes TEA023534 (CsACA6) and TEA026012 (CsACA11) were down-reg- ulated in response to both Al treatments in compari- son to no Al. There was also evidence of Al-responsive reduced transcript abundance for TEA008626 (CsACA3). However, CAX genes including TEA007963 (CsCAX1) and TEA000101 (CsCAX2) did not show any significant expression change in response to Al (Fig. 3A, Table S1, Fig. S1-S7). mediate Mg uptake into roots and also have low affin- ity to other cations including Fe2+, Mn2+ and Cu2+, and yet are also highly sensitive to Al stress [24, 26]. Some MGTs even can transport Al [27]. From the 14 putative MGT genes in tea, TEA005607 (CsMGT5), TEA011435 (CsMGT8), TEA015654 (CsMGT11), and TEA015667 (CsMGT12) were up-regulated by approximately 2 to 3.2- fold in response to 2.5 mM Al stress, while TEA030537 (CsMGT13) was up-regulated (between 2.6 and 9-fold) following both Al treatments (Fig. 3B). In contrast, TEA010637 (CsMGT4), TEA011435 (CsMGT8) and TEA011455 (CsMGT9) showed down-regulation in response to 0.4 mM Al, with CsMGT8 and CsMGT9 giv- ing markedly higher transcript abundance in the 2.5 mM treatments compared to the 0.4 mM treatments, and the representative gene CsMGT4 was verified with qRT- PCR. (Fig. 3B,C, Table S2, Fig. S8). Only the MHX gene TEA032978 (CsMHX3) was slightly up-regulated in response to 2.5 mM Al (Fig. 3B). into the cytosol [19]. and active Ca2+ efflux transport- ers, which can include the endoplasmic reticulum-type Ca2+-ATPase (ECA) and autoinhibited Ca2+-ATPase (ACA), the Ca2+/H+ exchanger (CAX), and the mito- chondrial Ca2+ uniporter (MCU) proteins [20]. Ca2+ influx is mediated by several families of selective and non-selective ion permeable channels, including cyclic nucleotide-gated channels (CNGCs), ionotropic gluta- mate-like receptors (GLR), two-pore channel 1 (TPC1), annexins, and mechanosensitive-like (MSL) ion channels, although it is important to highlight that many of these channels can also mediate the movement of other ions in addition to Ca2+ (Table S1, Fig. S1-S7) [21]. The RNA-Seq data as shown in the heatmaps indicated that many of the Ca2+ permeable channel genes were down-regulated in response to 0.4 or 2.5 mM Al, including the GLR genes TEA014867 (CsGLR1) and TEA006325 (CsGLR15), and the MSL genes TEA001140 (CsMSL1) and TEA019176 (CsMSL8), while TEA024970 (CsGLR13) was up-reg- ulated in Al treated roots at 12 h and 24 h (Table S1, Fig. S1-S7). Altered regulation of Fe transport and homeostasis genes in tea plant root by Al stressi Fe concentrations were significantly increased by Al stress in tea plant roots. This correlated with observed alteration in the expression of Fe metabolism and trans- port related genes. For most dicots, the ferric reductase oxidase (FRO) reduces Fe3+ to Fe2+, which can then Hao et al. BMC Plant Biology (2022) 22:203 Page 7 of 20 Fig. 3 Effects of Al stress on the expression of transporter genes involved in root transport of Ca, Mg, and Cu and other divalent cations. a Heatmap analysis of expression patterns of genes encoding various transporters in roots in response to 0.4 mM and 2.5 mM Al stresses at different time points. The transporters include Type IIB Ca2+-ATPases (CsACA), Type IIA ER-Ca2+-ATPases (CsECA), Ionotropic glutamate receptor (CsGLR), Ca2+/ H+ antiporter (CsCAX), Cyclic nucleotide-gated channels (CsCNGCs), Mechanosensitive ion channel like (CsMSL), Annexins (CsAnn), Mitochondria calcium uniporter (CsMCU). b Heatmap analysis of expression patterns of genes encoding tonoplast Mg/H transporter (CsMHX), Magnesium transporter (CsMGT), Heavy metal H-ATPase (CsHMA), and Copper transporter (CsCOPT). c qRT-PCR verification of expression patterns of several key transporter genes in roots in response to 0.4 mM and 2.5 mM Al. The heatmaps were made with transcriptome data normalized as Log10(1 + log (1 + FPKM)). MeV software was used to depict transcript levels. qRT-PCR data are presented as means ± SD from three independent experiments and statistical significance is based on Student’s t-test (P < 0.05; * P < 0.001) compared with controls Fig. 3 Effects of Al stress on the expression of transporter genes involved in root transport of Ca, Mg, and Cu and other divalent cations. a Heatmap analysis of expression patterns of genes encoding various transporters in roots in response to 0.4 mM and 2.5 mM Al stresses at different time points. The transporters include Type IIB Ca2+-ATPases (CsACA), Type IIA ER-Ca2+-ATPases (CsECA), Ionotropic glutamate receptor (CsGLR), Ca2+/ H+ antiporter (CsCAX), Cyclic nucleotide-gated channels (CsCNGCs), Mechanosensitive ion channel like (CsMSL), Annexins (CsAnn), Mitochondria calcium uniporter (CsMCU). b Heatmap analysis of expression patterns of genes encoding tonoplast Mg/H transporter (CsMHX), Magnesium transporter (CsMGT), Heavy metal H-ATPase (CsHMA), and Copper transporter (CsCOPT). c qRT-PCR verification of expression patterns of several key transporter genes in roots in response to 0.4 mM and 2.5 mM Al. The heatmaps were made with transcriptome data normalized as Log10(1 + log (1 + FPKM)). MeV software was used to depict transcript levels. Altered regulation of Fe transport and homeostasis genes in tea plant root by Al stressi qRT-PCR data are presented as means ± SD from three independent experiments and statistical significance is based on Student’s t-test (P < 0.05; * P < 0.001) compared with controls be transported into the root epidermis by the divalent metal transporter IRT1, which is a member of the ZRT/ IRT-like protein (ZIP) family of transporter. Vacuolar iron transporter (VIT) proteins at the tonoplast can in turn store labile Fe2+ into vacuolar compartments, such as performed by A. thaliana AtVIT1, while some mem- bers of the natural resistance-associated macrophage protein (NRAMP) family can mediate Fe release from the vacuole [31, 32]. Of the seven putative FRO genes, TEA033298 (CsFRO6) expression increased over 2-fold Hao et al. BMC Plant Biology (2022) 22:203 Hao et al. BMC Plant Biology (2022) 22:203 Page 8 of 20 Fig. S9-S12). In comparison to the control condition, TEA021515 (CsVIT3) was down-regulated by 2-fold and 4-fold at 12 h and 24 h, respectively after 2.5 mM Al treat- ment, while the transcript that is most closely related to AtVIT1, TEA015983 (CsVIT6), was increased by nearly 3-fold at 12 h under 2.5 mM Al treatment in comparison in response to both 0.4 and 2.5 mM Al treatments (Fig. 4A). Expression of a putative tea CsIRT1 gene (TEA023203) was undetectable in control or 0.4 mM Al treated roots, but low-level expression was observed following 2.5 mM Al treatment (Fig. 4A). Six VIT genes were found within the tea root transcriptome (Table S3, Fig. 4 Effects of Al stress on transporter genes involved in the transport of Fe, Zn, Cu, and other metals. a Expression patterns for genes encoding transporters or enzymes involved in transport of Fe or Fe-organic acid complex across various membranes, including Ferric reductase oxidase (CsFRO), Fe-binding proteins ferritin (CsFER), vacuolar iron transporters (CsVITs), nicotianamine synthase (CsNAS), yellow-stripe like (CsYSL) transporters, and IRON REGULATED/Ferroportin (CsIREG/Ferroportin). b Expression patterns for other genes encoding multi-function transporters, such as NRAMP transporters, ZRT/IRT-like proteins (CsZIPs), plasma membrane ATPases (CsPMAs), oligopeptide transporter (CsOPTs). c qRT-PCR verification of expression patterns of key genes involved in metal ion transport in roots in response to 0.4 mM and 2.5 mM Al. The heatmaps were made with transcriptome data normalized as Log10(1 + log (1 + FPKM)). MeV software was used to depict transcript levels. Altered regulation of Fe transport and homeostasis genes in tea plant root by Al stressi qRT-PCR data are presented as means ± SD from three independent experiments and statistical significance is based on Student’s t-test (P < 0.05; * P < 0.001) compared with controls Fig. 4 Effects of Al stress on transporter genes involved in the transport of Fe, Zn, Cu, and other metals. a Expression patterns for genes encoding transporters or enzymes involved in transport of Fe or Fe-organic acid complex across various membranes, including Ferric reductase oxidase (CsFRO), Fe-binding proteins ferritin (CsFER), vacuolar iron transporters (CsVITs), nicotianamine synthase (CsNAS), yellow-stripe like (CsYSL) transporters, and IRON REGULATED/Ferroportin (CsIREG/Ferroportin). b Expression patterns for other genes encoding multi-function transporters, such as NRAMP transporters, ZRT/IRT-like proteins (CsZIPs), plasma membrane ATPases (CsPMAs), oligopeptide transporter (CsOPTs). c qRT-PCR verification of expression patterns of key genes involved in metal ion transport in roots in response to 0.4 mM and 2.5 mM Al. The heatmaps were made with transcriptome data normalized as Log10(1 + log (1 + FPKM)). MeV software was used to depict transcript levels. qRT-PCR data are presented as means ± SD from three independent experiments and statistical significance is based on Student’s t-test (P < 0.05; * P < 0.001) compared with controls Fig. 4 Effects of Al stress on transporter genes involved in the transport of Fe, Zn, Cu, and other metals. a Expression patterns for genes encoding transporters or enzymes involved in transport of Fe or Fe-organic acid complex across various membranes, including Ferric reductase oxidase (CsFRO), Fe-binding proteins ferritin (CsFER), vacuolar iron transporters (CsVITs), nicotianamine synthase (CsNAS), yellow-stripe like (CsYSL) transporters, and IRON REGULATED/Ferroportin (CsIREG/Ferroportin). b Expression patterns for other genes encoding multi-function transporters, such as NRAMP transporters, ZRT/IRT-like proteins (CsZIPs), plasma membrane ATPases (CsPMAs), oligopeptide transporter (CsOPTs). c qRT-PCR verification of expression patterns of key genes involved in metal ion transport in roots in response to 0.4 mM and 2.5 mM Al. The heatmaps were made with transcriptome data normalized as Log10(1 + log (1 + FPKM)). MeV software was used to depict transcript levels. qRT-PCR data are presented as means ± SD from three independent experiments and statistical significance is based on Student’s t-test (P < 0.05; * P < 0.001) compared with controls Hao et al. BMC Plant Biology (2022) 22:203 Page 9 of 20 to the 0.4 mM Al treatment (Fig. 4A). Altered regulation of Fe transport and homeostasis genes in tea plant root by Al stressi Based on the phylogenetic analysis, these three genes are most closely related to OsNRAMP4 from rice, which is a root plasma membrane transporter that can provide Al3+ accumulation into root tip cells but does not appear to transport other cations including Fe2+, Mn2+ or Cd2+ [42]. These tea NRAMPs are also closely grouped with AtNRAMP1, which is a plasma membrane Mn2+ and Fe2+ transporter [18, 29]. Altered regulation of Fe transport and homeostasis genes in tea plant root by Al stressi NRAMPs are broad substrate transporters including AtNRAMP3 and AtN- RAMP4, which are associated with Fe2+ and Mn2+ trans- port [32, 33]. TEA011223 (CsNRAMP2) is most closely related to these genes and was slightly down-regulated in response to 0.4 mM Al (Fig. 4A, Table S3, Fig. S12). Other transport proteins that are associated with Fe homeosta- sis are members of the oligopeptide transporter (OPT) family, which include the Yellow Stripe-like (YSL) pro- teins that can mediate the transport of Fe chelate com- plexes such as Fe bound to phytosiderophores (PS) or nicotianamine (NA) [34–36]. The IRON REGULATED (IREG) / ferroportin (FPN) proteins are efflux transport- ers, such as AtFPN2, a vacuolar membrane protein that is expressed during Fe deficiency [37], while soybean and buckwheat GmIREG3 and FeIREG3 are tonoplast trans- porters that contribute to Al resistance in plants [38]. OPT genes, such as TEA015243 (CsOPT3), TEA016756 (CsOPT6) and TEA021263 (CsOPT7) were down-reg- ulated in roots under 0.4 mM and 2.5 mM Al treatment (Fig. 3A, Table S3, Fig. S9-S12). Most of the YSL genes examined did not show a clear response to Al exposure, with the exception of TEA025648 (CsYSL6) that was induced by 2.5 mM Al, while TEA019706 (CsYSL1) and TEA018013 (CsYSL3) were down-regulated by 2.5 mM Al treatment compared with the control (Fig. 4B). The IREG TEA008315 (CsIREG2) was up-regulated by 2.5 mM Al stress at 48 h after treatment (Fig. 4B). included slightly reduced Cu and Mn content and slightly increased Zn and Cd content, which corresponded with changes in expression of various metal transport- ers. Many of the transporter classes described above, including non-selective cation channels, ZIPs, NRAMPs, and OPTs, are all broad substrate transporters that can mediate accumulation and cellular distribution of these and other metals [18, 40, 41]. Of the various ZIP genes identified, many showed transient fluctuation in expres- sion in response to Al stress. TEA032109 (CsZIP5) and TEA028928 (CsZIP8) were up-regulated by 2.5 mM Al treatment by over 2-fold while TEA028921 (CsZIP7) was up-regulated in 0.4 mM Al treatment at 48 h compared with the control (Fig. 4B). Tea NRAMP transporter genes TEA017264 (CsNRAMP1), TEA002435 (CsNRAMP3) and TEA009385 (CsNRAMP6), which are all specifically expressed in root tissue (Table S3, Fig. S12-S13), were all were up-regulated substantially in response to Al. Antioxidant and oxidative stress under Al3+/H+ rhizotoxicityi NA is synthesized in higher plants by NA synthase (NAS). NA complexes with Fe2+ and Fe3+, and other metal ions, such as Cu2+, Ni2+, Co2+, Zn2+, and Mn2+ to facilitate transport into cells via YSL transporters and the long-distance translocation of mineral nutrients from roots to shoots or vice versa. Among the two NAS genes, TEA020761 (CsNAS1) was up-regulated in response to 2.5 mM Al at 24 h (Fig. 4B), suggesting the potential for enhanced Fe-NA complexes for translocation within the tea plant. The Fe-binding protein ferritin (FER) also plays an important role in plant Fe homeostasis [39]. Altered transcript abundance was seen for most of the CsFER genes. TEA016450 (CsFER1), TEA002014 (CsFER3) and TEA026599 (CsFER4) were up-regulated follow- ing 0.4 mM Al treatment, while TEA016447 (CsFER2) showed increased abundance in 2.5 mM Al (Fig. 4B, Table S3, Fig. S15). Some of these significantly up-regulated genes as mentioned above were selected for being veri- fied with qRT-PCR (Fig. 4C). One of the most significant changes in most plant roots under Al stress is oxidative damage caused by Al tox- icity and the Al-induced accumulation of metal ions such as Fe2+ [11]. We therefore used the 2,7-DCFDA reporter to monitor for increasing reactive oxygen spe- cies (ROS) in roots. While fluorescence was observed in all roots including the control treatment without Al after the 1 h time point, fluorescence at 30 min was only seen in response to Al exposure and fluorescent intensi- ties increased in response to increasing Al concentrations (Fig. 5A). It was also observed that the epidermal cells and root elongation zones showed more fluorescence than other parts of the roots. We also determined H2O2 generation and localization using DAB staining (Fig. 5B), which showed that root tips were the regions that pro- duced more H2O2, which accumulated over the time of Al exposure and in response to increasing concentra- tion of Al. Glutaredoxin (GRX) and thioredoxin (TRX) proteins play important roles in glutathione metabolism, which is important for heavy metal-induced oxidative stress tolerance [43]. Among 14 GRX genes tested, a few showed increased expression levels under Al exposure, including TEA005028 (CsGRX1), TEA012816 (CsGRX8), TEA015447 (CsGRX20), and TEA006700 (CsGRX28), Changes in expression of metal transporter genes for trace metalsi Finally, there were a large number of putative NIP genes includ- ing TEA008904 (CsNIP32), TEA009357 (CsNIP33), TEA015178 (CsNIP35), TEA016839 (CsNIP37), TEA019674 (CsNIP38) and TEA009696 (CsNIP40) that were decreased in transcript abundance following both 0.4 mM and 2.5 mM Al treatment compared to the no Al control (Fig. 6A, Table S6, Fig. S21). Boron (B) is an essential plant micronutrient that can be transported via boric acid channels AtNIP5;1, AtNIP6;1 and AtNIP7;1, and by a boric acid/borate exporter AtBOR1 in A. thali- ana [46, 48, 49]. B decreases root Al uptake and translo- cation to the shoot in pea plants, and thus alleviates Al toxicity [50]. We observed a slight increased expression of B transporter genes, TEA013947 (CsBOR1/CsNIP54), TEA001638 (CsBOR2/CsNIP55), and TEA000759 (CsBOR3/CsNIP56) in 2.5 mM Al treated roots. Two tea plant homolog genes for AtNIP5;1 and AtNIP6;1 are TEA019654 (CsNIP5) and TEA030821 (CsNIP4), which were transiently up-regulated by 0.4 mM and 2.5 mM Al stress (Fig. 6B, Table S6, Fig. S21). Several highly up- regulated CsNIP genes by Al treatment were selected and verified with qRT-PCR (Fig. 6C). while others including TEA002408 (CsGRX11) and TEA026750 (CsGRX18) were decreased by Al (Fig. 5C). Many TRX genes were mostly up-regulated by Al stress, suggesting significant changes in cellular redox status (Table S5, Fig. S18-S20). while others including TEA002408 (CsGRX11) and TEA026750 (CsGRX18) were decreased by Al (Fig. 5C). Many TRX genes were mostly up-regulated by Al stress, suggesting significant changes in cellular redox status (Table S5, Fig. S18-S20). Several genes encoding ROS scavenging enzymes were examined. These included superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and per- oxidase (POD) (Fig. 5C-D), as well as other protective enzymes, such as glutathione S-transferases (GSTs) that are also important for the synthesis of metal binding pep- tides such as metallothionein (Fig. 5). Interestingly, sev- eral major GST genes were clearly down-regulated by 0.4 mM Al, but also significantly up-regulated by 2.5 mM Al, such as the tau-type GST genes, TEA014619.1, TEA015778.1, TEA025564.1, TEA025571.1 (Fig. 5). But other classes of GST genes were down-regulated by 2.5 mM Al stress, indicating strong toxicity of Al (Table S5, Fig. S18-S20). Several genes for SOD and CAT iso- forms as well as APX and POD were up-regulated in roots treated with 2.5 mM Al as compared with the con- trol, suggesting that these genes were responsive to Al- induced oxidative stresses. Fig. 5 Oxidative stress in tea plant roots upon Al stress. a, b 2,7-DCFDA staining of ROS (a) and DAB staining of H2O2 (b) in tea seedling roots in response to 0, 0.4, 2.5, and 5 mM Al2(SO4)3. The root tips were stained with 2,7-DCFDA or 3,3-diaminobezidin (DAB) in 10 mM Tris-HCl (pH 7.6) and then imaged using a fluorescence microscope. c Heatmap analysis of expression of glutaredoxin (CsGRX) genes in tea seedling roots in response to Al stress. d Analysis of expression patterns of superoxide dismutase (CsSOD) and catalase (CsCAT) genes in tea seedling roots in response to Al stress. The data are the ratio of transcriptome data at different treatment times. e Analysis of expression patterns of ascorbate peroxidase (CsAPX) and peroxidase (CsPOD) genes in tea seedling roots in response to Al stress. Data are presented as averages of calculated FPKM readings and representative fluorescence microscopy images are shown. All results were from three independent experiments (See figure on next page.) Changes in expression of metal transporter genes for trace metalsi All these data support the conclusion that in Al accumulating tea plants, heavy metal imbalance occurred and caused massive ROS gen- eration and oxidative stress. Changes in expression of metal transporter genes for trace metalsi Some subtle but significant changes in the content of Cu, Mn and Zn, as well as the non-essential toxin Cd were observed in tea roots in response to Al exposure. These Hao et al. BMC Plant Biology (2022) 22:203 Page 10 of 20 Page 10 of 20 TEA024296 (CsNIP14) were up-regulated in response to Al as compared with the control (Fig. 6A, Table S6, Fig. S21). Some NIPs can also facilitate the transfer of silicon, which can benefit plant growth and help allevi- ate different abiotic stresses including metal toxicities [45–47]. The homologous NIP genes in tea plants are TEA004662 (CsNIP2) and TEA013255 (CsNIP3) that were all slightly down-regulated initially (12 h) after Al treatment, but then up-regulated at 48 h, particu- larly for CsNIP3 with nearly a 6-fold increase. Finally, there were a large number of putative NIP genes includ- ing TEA008904 (CsNIP32), TEA009357 (CsNIP33), TEA015178 (CsNIP35), TEA016839 (CsNIP37), TEA019674 (CsNIP38) and TEA009696 (CsNIP40) that were decreased in transcript abundance following both 0.4 mM and 2.5 mM Al treatment compared to the no Al control (Fig. 6A, Table S6, Fig. S21). Boron (B) is an essential plant micronutrient that can be transported via boric acid channels AtNIP5;1, AtNIP6;1 and AtNIP7;1, and by a boric acid/borate exporter AtBOR1 in A. thali- ana [46, 48, 49]. B decreases root Al uptake and translo- cation to the shoot in pea plants, and thus alleviates Al toxicity [50]. We observed a slight increased expression of B transporter genes, TEA013947 (CsBOR1/CsNIP54), TEA001638 (CsBOR2/CsNIP55), and TEA000759 (CsBOR3/CsNIP56) in 2.5 mM Al treated roots. Two tea plant homolog genes for AtNIP5;1 and AtNIP6;1 are TEA019654 (CsNIP5) and TEA030821 (CsNIP4), which were transiently up-regulated by 0.4 mM and 2.5 mM Al stress (Fig. 6B, Table S6, Fig. S21). Several highly up- regulated CsNIP genes by Al treatment were selected and verified with qRT-PCR (Fig. 6C). TEA024296 (CsNIP14) were up-regulated in response to Al as compared with the control (Fig. 6A, Table S6, Fig. S21). Some NIPs can also facilitate the transfer of silicon, which can benefit plant growth and help allevi- ate different abiotic stresses including metal toxicities [45–47]. The homologous NIP genes in tea plants are TEA004662 (CsNIP2) and TEA013255 (CsNIP3) that were all slightly down-regulated initially (12 h) after Al treatment, but then up-regulated at 48 h, particu- larly for CsNIP3 with nearly a 6-fold increase. Aquaporin transporter genes in tea roots induced by Al stressh The aquaporin nodulin 26-like intrinsic protein (NIP) type transporter can allow influx of water and other sub- strates such as metals into plant cells. For example, one route for Al entry across the plant plasma membrane in A. thaliana has been proposed to be via AtNIP1;2, which can be specifically induced by Al stress and can transport an Al-malate complex, but not Al3+ ions, into the sym- plasm, followed by further translocation from the root to the shoot, after subsequent Al xylem loading [44]. The formation of the Al-malate complex requires a functional root malate exudation system mediated by the Al-acti- vated malate transporter ALMT1 [16, 44]. Involvement of NIP-like aquaporin transporters in Al tolerance have also been reported in other plants [44]. NIP1-like aqua- porin transporter genes including TEA012255 (CsNIP8), TEA027942 (CsNIP10), TEA019952 (CsNIP11), and i q g Multidrug and toxin extrusion (MATE) transporters can mediate citrate secretion in root and vascular cells for metal ion transport and detoxification of Al3+ extracellu- larly [14–16]. MATE transporters include the A. thali- ana ferric reductase defective 3 (FRD3), which plays an important role in Fe homeostasis by transporting citrate into vascular tissues and facilitating Fe or Al transport through the xylem [14, 15]. We also observed that FRD3- like MATE transporter genes were significantly up-regu- lated by Al-treatment, including TEA027978 (CsMATE1) and TEA013673 (CsMATE2), which were induced by 2.5 mM Al but not by 0.4 mM Al, while TEA008152 (CsMATE5) and TEA027978 (CsMATE8) were increased Hao et al. BMC Plant Biology (2022) 22:203 Hao et al. BMC Plant Biology Page 11 of 20 Fig. 5 (See legend on previous page.) Fig. 5 (See legend on previous page.) Page 12 of 20 Hao et al. BMC Plant Biology (2022) 22:203 expressed in the roots and up-regulated in response to 2.5 mM Al (Fig. 7, Table S7–8, Fig. S23–24). expressed in the roots and up-regulated in response to 2.5 mM Al (Fig. 7, Table S7–8, Fig. S23–24). in both 0.4 mM and 2.5 mM Al stress (Fig. 6C). CsMATE5 is highly expressed in tea old leaves, but also was induced by 2.5 mM Al stress, indicating that it may be involved in Al translocation to the old leaves (Fig. 6C, Fig. S24). TEA017874 (CsMATE6) was the only gene markedly down-regulated by Al exposure (Fig. 6C, Table S7–8, Fig. S23–24). A representative one, CsMATE1, were con- firmed and verified with qRT-PCR (Fig. 6C). Aquaporin transporter genes in tea roots induced by Al stressh g g Next, CsALMT1 and CsALS8 were heterologously expressed in yeast cells to examine their effects on the resistance of yeast cells to 10 mM and 15 mM AlCl3 in the growth media at pH 4.5 (Fig. 7F). The growth of the wild type YPH449 yeast strain was inhibited by 10 mM Al when grown on both solid growth medium and liq- uid medium, while the growth of YPH449 cells trans- formed with the empty vector pDEST52 was similarly inhibited by 10 mM Al. However, the CsALS8-express- ing yeast cells grew much faster than the control yeast cells expressing empty vector pDEST52, and was able to mediate growth on both 10 mM and 15 mM Al. Also, the CsALMT1-expressing yeast cells grew less well than CsALS8-expressing yeast cells, but faster than the control cells, indicating increased resistance to Al stress in solid growth medium and liquid medium (Fig. 7F). We also analyzed Al contents in the growth media of yeast cells expressing CsALS8, CsALMT1, and pDEST52. Al content remaining in the growth media after 2 days of cultiva- tion was higher for the CsALS8-expressing yeast cultures than others, and after 3 days of cultivation, the Al content in the media was lowest for the CsALMT1-expressing yeast cultures (Fig. 7H). This was consistent with CsALS8 expression in yeast cells conferring resistance to Al stress, potentially by restricting Al entry into the cells, such as through Al efflux. Furthermore, CsALMT1 expression also conferred more resistance to Al than the pDEST52 control cells, potentially by enhancing Al-organic acid conjugate formation in the apoplastic space and reduc- ing the availability of free Al3+ ions, which are otherwise toxic to yeast cells. (See figure on next page.) Fig. 6 Effects of Al stress on NIP like aquaporin and other transporter genes. a, b Heatmap analysis of expression of genes encoding nodulin 26-like intrinsic protein (CsNIP) (a) and multidrug and toxin extrusion protein (CsMATE) and urate transporter (CsUPS/UAT) (b) in tea seedlings in response to Al. c qRT-PCR verification of gene expression for CsNIP genes in tea seedling roots in response to Al stress. The heatmaps were made with transcriptome data normalized as Log10(1 + log (1 + FPKM)). MeV software was used to depict transcript levels. qRT-PCR data are presented as means ± SD from three independent experiments and statistical significance is based on Student’s t-test (P < 0.05; * P < 0.001) compared with controls ABC and ALMT transporters were involved in transport of Al and other metal cations ATP-binding cassette (ABC) family transporters repre- sent one of the largest protein families in living organ- isms of most life domains [18]. Several ABC-transporters have been reported to directly be involved in transport Al/Al-organic acid (Al-AO) complexes, including into the vacuole and confer plant Al tolerance, such as A. thaliana AtALS1 and AtALS3, rice OsSTAR1 and OsS- TAR2 [51–54]. OsSTAR1 homologous genes, including TEA027830 (CsALS9) and TEA023178 (CsALS10) were up-regulated in response to 2.5 mM Al treatment by up to 1.4-fold (for CsALS10) and up to 3-fold (for CsALS9) (Fig. 7, Table S7–8, Fig. S22). The AtALS3 homologous gene TEA002530 (CsALS7) was up-regulated in 2.5 mM Al treatment at 12 h by 2.8-fold, but down-regulated in response to 0.4 mM, while another AtALS3 homolog TEA025228 (CsALS8) was also up-regulated by 1.6-fold in 2.5 mM Al treatment at 48 h. The AtALS1 homolo- gous gene TEA031570 (CsALS1) showed moderate up- regulation in response to Al (Fig. 7, Table S9, Fig. S22). The previously mentioned rice NRAMP transporter, NRAMP ALUMINUM TRANSPORTER 1 (OsNRAT1) can directly take up Al3+ from the extracellular space, and work together with the tonoplast ABC transporter ALS1 to detoxify Al3+ by sequestration into the vacuole [42]. Its homologous genes in tea plants, TEA017264 (CsNRAMP1) and TEA002435 (CsNRAMP3), were up- regulated by more than 2–3-fold by 2.5 mM Al stress (Fig. 4A). Discussion It has long been known that tea plants can accumulate and tolerate higher concentrations of Al compared to many other plant species [12, 17, 55], and that this char- acteristic is determined in part by large scale transcrip- tional changes in response to Al exposure, which include genes involved in Al transport and sequestration, biosyn- thesis and secretion of organic acids, and modification to the cell wall [12, 13]. It is also well known that metal responses are interactive and typically affect other metals, such that changes in the availability and plant accumula- tion of one metal, such as Al, typically alters the behavior Aluminum-induced malate transporters (ALMTs) are involved in organic acid release into the apoplast, par- ticularly for malate to provide Al tolerance by chelation of excess Al3+ [6, 7, 15, 16]. The tea genome contains 16 putative ALMT genes, including homologs of HvALMT, TaALMT and AtALMT1; TEA013305 (CsALMT1) and TEA013312 (CsALMT2), which were both highly Hao et al. BMC Plant Biology (2022) 22:203 Page 13 of 20 Fig. 6 (See legend on previous page.) Fig. 6 (See legend on previous page.) Fig. 6 (See legend on previous page.) Hao et al. BMC Plant Biology (2022) 22:203 Page 14 of 20 Page 14 of 20 using yeast heterologous expression to be able to pro- vide strong Al tolerance (Fig. 7). Members of this fam- ily include AtALS1 and OsALS1 found on the vacuolar membrane of root tip cells that may transport Al-organic acid complexes into the vacuole to provide detoxification [51–54]. Furthermore, AtALS3 or OsSTAR2 localized at the plasma membrane of root phloem cells may upload Al-organic acid complexes into the vascular tissues for translocation of Al to above-ground tissues, such as old leaves, that are insensitive to Al stress [51, 53]. The cor- relation analyses on the expression changes of nearly 1000 transporter genes with altered metal contents in tea plant roots under various Al conditions showed that many of these transporter genes and metal changed are tightly correlated, either negatively or positively (Table. S10). Also, expression changes of a AtBOR1-like CsNIP5 was highly correlated to Al accumulation, indicating the possible involvement of this transporter in Al tolerance (Table. S10). of other metals and mineral nutrients. For example, in maize plants, Al exposure reduced the accumulation of Ca and Mg, while Al supply to tea leaves alters the uptake and transport of Fe and B [55]. Discussion While other studies have examined broad-scale biochemical and transcriptional responses to Al exposure in tea plants, this study aimed to specifically examine the ionomic and transcriptional responses in relation to metal transport and mineral nutrient homeostasis mechanisms in tea roots exposed to Al and found that there were indeed widespread changes in many processes related to many different metals and mineral nutrients. As expected, increased Al exposure led to increased Al content in roots, potentially facilitated by plasma mem- brane channels, such as the aquaporins including NIP1;2 [44]. NIP1;2 like genes from tea, as well as other NIP iso- forms, were transcriptionally increased in response to Al. AtNIP1;2 was characterized to transport Al-malate complex from cell wall into the symplasm, through which Al-malate can be further translocated from the root to the shoot, after subsequent Al xylem loading [15]. Most plants share common mechanisms to tolerate Al expo- sure and the increased Al content in cells. These include the exudation of organic acids such as malate and citrate from the roots to chelate and detoxify excess Al3+ in the apoplastic space and the sequestration of cellular Al into the vacuole [15, 16, 56]. While intracellular transport of citrate or malate may provide external or internal detoxi- fication of Al in roots and leaves, ALS1 on the tonoplast, together with other transporters such as Nrat1, remove Al3+ from the cell wall and sequester it in the root-cell vacuole [15, 54]. Key organic acid transporters involved in Al tolerance include the putative ALMT type malate transporter [16], while other organic acid transporters involved in Al tolerance include the MATE type trans- porters [17]. We showed that a putative malate exporter CsALMT1 in tea plant roots could confer a moderate tol- erance to Al when over-expressed in yeast cells (Fig. 7). Some half-size ABC transporters, such as ALS1 and ALS3 in Arabidopsis [51, 52], and their tea homologs, including CsALS5, 6, 7 and 8, were all induced by Al in roots. Particularly, CsALS8 was further demonstrated by The Al-citrate complex is a widely detected organic acid complex in many plants [15, 16]. The MATE gene FRD3 has been demonstrated to be involved in the trans- location of citrate in the xylem to control Fe movement, but when overexpressed, FRD3 can provide Al tolerance [6, 7, 15]. The expression of two orthologs of FRD3 were highly induced by the Al treatment. Fig. 7 Functional identification of CsALS8 and CsALMT1 from tea plants. a, b Phylogenetic analysis of aluminum sensitive transporter (CsALS) (a) and aluminum-inducible malate transporter (CsALMT) (b) gene families in tea genome. c Heatmap analysis of expression of CsALS7 and CsALS8 homolog genes in tea seedling roots in response to Al stress. d qRT-PCR verification of CsALMT1 and CsALMT15 gene expression in tea roots in response to Al stress. e qRT-PCR verification of CsALS8 and CsALMT1 gene expression in tea roots in response to Al stress. f Growth test on the functions of CsALS8 and CsALMT1 in resistance to Al stress in yeast cells. g Growth curve of yeast strain YPH499 expressing empty vector pYESDEST52, CsALS8, or CsALMT1 in response to Al stress. h Al content in the medium of yeast strain YPH499 expressing empty vector pYESDEST52, CsALS8, or CsALMT1 in response to Al stress for 2 and 3 days. The heatmap was made with transcriptome data normalized as Log10(1 + log (1 + FPKM)). MeV software was used to depict transcript levels. qRT-PCR and Al content data are presented as means ± SD from three independent experiments and statistical significance is based on Student’s t-test (P < 0.05; * P < 0.001) compared with controls (See figure on next page.) Discussion Al stress often causes an increase in cytosolic Ca2+ spiking, which plays a role in activating defense responses against Al toxicity [7, 21]. A correlation was observed between the rapid Al- induced increase in cytosolic Ca2+ and root growth inhi- bition in wheat, leading to the hypothesis that disruption of Ca2+ homeostasis may be the primary cause of Al3+ toxicity [21]. However, the increased intracellular concentration of free Fe is also cytotoxic since Fe is highly reactive via the Fenton reaction to generate ROS [31, 33]. Thus, plants tightly control Fe homeostasis by multiple strategies, depending on the types of plants, to react to Fe deficiency as well as Fe overload. So the transcriptional increase of the ROS scavenging mechanisms that were observed, components to sequester Fe into intracellular stores such as the ortholog of the vacuolar transporter VIT1 were increased [32]. However, the increased intracellular concentration of free Fe is also cytotoxic since Fe is highly reactive via the Fenton reaction to generate ROS [31, 33]. Thus, plants tightly control Fe homeostasis by multiple strategies, depending on the types of plants, to react to Fe deficiency as well as Fe overload. So the transcriptional increase of the ROS scavenging mechanisms that were observed, components to sequester Fe into intracellular stores such as the ortholog of the vacuolar transporter VIT1 were increased [32]. Conclusions Tea plants actively take up Al under acidic conditions, and transport Al from roots to above ground tissues, therefore it was important to understand the processes of Al accumulation and other metals in root tissues. Al stress significantly reduced root Mg, Ca, and Mn uptake, but increased Fe uptake. Al stress modified metal cat- ion transporter gene profiles as compared with con- trols. These altered transporter gene expression patterns may explain why imbalanced metal ion accumulation occurred in the Al-stressed roots. Over-accumulation of Al, and other toxic metals, such as Cd in tea plants could cause public concerns over the safety of consuming tea products [1, 2]. The improved understanding gained from this study of the genetic responses to Al exposure, par- ticularly in relation to metal transport and homeostatic processes, has highlighted potential gene targets for breeding or genetic engineering approaches to develop safer tea products. The Al-induced inhibition of root Mg content might also have been due to the repression of Mg transporter genes, such as members of the MGT family. The change in profile of tea MGT genes was variable as some were enhanced by Al exposure and others were reduced. Al-Mg interaction via this class of transporter has previ- ously been observed. Overexpression of MGT1 enhanced Al tolerance [24, 25]. Mg2+ in the cytoplasm directly regulates H+-ATPase activity, which maintain the mem- brane potential and pH gradient across the plasma mem- brane [24, 25]. Al stress inhibits H+-ATPase activity, but Mg addition can restore activity, thus alleviating Al toxic- ity [62]. In addition, it is hypothesized that enhanced Mg nutrition under Al stress may prevent Al-induced pertur- bation of cytosolic Ca2+ signaling [24]. Discussion This supports the speculation that in the presence of Al, the amount of cit- rate release to the xylem is increased in order to enhance Al-citrate translocation, requiring the induction of genes such as MATE transporters involved in citrate release. Once inside the root cells, Al can induce the peroxida- tion of membrane lipids and the production of ROS, which can lead to further cellular damage. The increased expression of antioxidant genes including members of the GST, GRX, SOD, and CAT families, indicate addi- tional roles in mitigating Al toxicity [8, 11]. Our data clearly showed that high Al stresses caused significant losses of macronutrients, such as Mg and Ca, which are essentially required for plant growth, photo- synthesis, and cell wall structure, function, and stability. Because of important cell wall structural and physiologi- cal roles of these two elements in plants, maintenance of Hao et al. BMC Plant Biology (2022) 22:203 Page 15 of 20 Fig. 7 (See legend on previous page.) Fig. 7 (See legend on previous page.) Fig. 7 (See legend on previous page.) Hao et al. BMC Plant Biology (2022) 22:203 Hao et al. BMC Plant Biology (2022) 22:203 Hao et al. BMC Plant Biology (2022) 22:203 Page 16 of 20 Page 16 of 20 Page 16 of 20 high levels of Mg and Ca levels in roots and shoots are essential for normal plant growth and development [24, 25]. Deficiencies of these two major nutrients cause vari- ous symptoms, which could connect to the major causes for reduced root cell elongation and root growth by high Al and low pH. Changes in expression of candidate Ca transporter genes may in part explain the reduced Ca content. For example, members of the GLR and MSL families of Ca2+ influx channels that play a role in cellular Ca accumulation showed reduced expression. Likewise, some putative organellar Ca2+ transporters such as ACA Ca2+ pump isoforms that are important in providing Ca content into intracellular stores were also reduced in response to Al. However, loss of these Ca transport path- ways is unlikely to significantly decrease total root Ca content [57]. In fact, transcriptional modification of these Ca2+ transporters is more likely to play a key role in alter- ing the generation of cellular Ca2+ signals, which may in turn mediate adaptive responses [20]. Microscopic observations After treatment of tea plant seedlings with 0, 0.4, 2.5 and 5 mM Al in SK medium for various times, roots from dif- ferent plants were fixed with ethanol and stained with DAPI for 15 min for cell death examination. ROS gen- eration in roots was observed by using 2,7-DCFDA as a reporter. Seedlings were treated with Al for different time and then were put into a fresh PBS buffer; 5 μg ml− 1 of 2,7-DCFDA were added to stain the ROS [8]. Roots were imaged under a fluorescence microscope (Nikon Eclipse 800, Japan) with excitation at 488 nm and emission at 540 nm to detect green fluorescence intensity. Roots were stained with 1 mg ml− 1 DAB solution (pH 4.0) for 8 h for examination of the presence of H2O2 in root cells [59]. Tissue- or cell-specific accumulation of Al in tea seedling roots were determined using Morin staining according to the method described previously [8]. Briefly, 10 μM morin in DMSO solution was added to the root tips on the glass plate for staining for 15 min. Excitation and emission wavelengths of 420 nm and 515 nm, respectively, were used for imaging the Al–morin complex in the roots [8]. A range of 55.1–66.6 million clean reads was obtained for each repeat sample, and in total there were 767.4 mil- lion clean reads generated for all samples. Both a de novo assembly strategy (the Trinity method) with the regu- lar parameters [58], and assembly against the tea plant reference genome [58] was used. All-predicted coding sequences were verified with protein databases including NCBI, Swiss-Prot, KEGG and COG. 45,737 transcript contigs were also assembled by BLAST against tea plant genome sequences. About 86% of the transcript con- tigs were mapped to the tea plant genome sequence of C. sinensis var. sinensis [58]. The differential expression genes (DEGs) were identified and modified by a rigor- ous algorithm. A false discovery rate (FDR) ≤ 0.001 and an absolute value of log2Ratio ≥ 1 were chosen as the thresholds to determine the significance of differentially expressed genes. For a given unigene, the corresponding FPKM value for transcripts were determined. Hierarchi- cal clustering was carried out using Cluster 3. Differen- tially expressed genes were extracted for GO functional enrichment analysis and KEGG pathway enrichment analysis. Plant materials and growth conditionsh The collection of tea plant material in this study had com- plied with relevant institutional, national, and interna- tional guidelines and legislation. Seedlings of the tea plant (Camellia sinensis L.O. Kuntze) cultivar “Shuchazao” were used for all experiments. This cultivar was obtained from the Guohe tea garden (Anhui Agricultural Univer- sity), Hefei, Anhui province, China. About 1.5-year old tea plant (Shuchazao) seedlings were used for Al treat- ment experiments in hydroponic solutions. Preliminary Al treatments were performed to ascertain the optimum Al concentration and the toxic exposure concentration for tea plants, by using a concentration range of 0, 0.4, 2.5, and 5 mM Al delivered to the plants using a hydropon- ics system. The Al concentration at 0.4 mM provided the best performance of root growth, but for analysis of Al responses, the no Al (0 mM Al) treatment was used as a control concentration. The hydroponics SK-solution (pH was set to 4.3 as the initial value) contained (NH4)2SO4 (1.07 mM), Ca (NO3)2·4H2O (0.357 mM), KH2PO4 (0.1 mM), K2SO4 (0.413 mM), CaCl2·2H2O (0.392 mM), Enhanced Fe uptake was observed in the tea plant roots under Al stress, likely to result from altered expression of Fe transporter genes, although this observation contrasts with previous studies that showed Al stimulated reduc- tion in Fe uptake and transport [55]. Most dicots, includ- ing tea plants, use a FRO isoform to reduce Fe3+ to Fe2+, which can then be transported into the root epidermis by the divalent metal transporter IRT1, both of which were increased. Other components that can enhance the accumulation and transfer of Fe such as NAS, FER and YSL were also enhanced [31, 33]. Fe is essential for plant growth, respiration, and synthesis of Fe-S clusters. Page 17 of 20 Hao et al. BMC Plant Biology (2022) 22:203 Hao et al. BMC Plant Biology (2022) 22:203 Page 17 of 20 FDR. The assembled contigs had a length distribution ranging from 201 to 25,284 bp, with an average length of 1184 bp with a high read coverage for the contigs. MgSO4·7H2O (1.029 mM), C10H12FeN2NaO8 (6.27 μM), H3BO3 (9.25 μM), MnSO4·7H2O (18.2 μM), ZnSO4·7H2O (1.53 μM), CuSO4·5H2O (0.39 μM), Al2(SO4)3·18H2O (0.4 mM) and Na2MoO4·2H2O (0.52 μM), with modifi- cations to the Al concentration as needed. The solution was aerated by using an air pump and refreshed with fresh medium once every 5 days. RNA‑Seq, DEG analysis, and qRT‑PCR validation RNA Seq, DEG analysis, and qRT PCR validation After tea seedlings were exposed to 0, 0.4, or 2.5 mM Al for up to 48 h in SK solution (about 50 seedlings for each concentration of Al), roots of at least 3 seedlings were harvested and then frozen in liquid nitrogen before total RNA was extracted using a General Plant RNA Extraction Kit (BioTeke, China). The extracted RNA was digested with DNase I (TAKARA) to remove contaminated genomic DNA. mRNAs were purified from the total RNA using Dynabeads Oligo (dT) (Life Technologies). The derived mRNAs were reverse transcribed into first-strand cDNAs with random hexamer and then the second-strand cDNAs were synthesized according to the manufacturer’s instructions. The double-stranded cDNAs were purified and ligated to adaptors for Illumina paired-end sequenc- ing. The cDNA library was sequenced using the Illumina HiSeq2500 system by BGI (Shengzhen, China). Plant materials and growth conditionsh Tea plant seedlings were grown under a natural photosynthetic photon flux density of 1300 μmol m− 2 s− 1 with a diurnal cycle of 18 h day/6 h night, Temperature (24 °C /18 °C, day/night) and humidity (70–80%) were controlled in the cabinet. To validate the RNA-Seq data for the expression of key genes involved in organic acid and metal ion transport, qRT-PCR was performed with RNA samples prepared from Al treatment experiments. The single-stranded cDNAs used for real-time PCR analysis were synthe- sized from the RNAs using a Prime-Script™ 1st Strand cDNA Synthesis Kit (TaKaRa, Dalian, China) based on three biological replicates and three technical replica- tions. In addition, detailed information of the validated transcripts, including unigene IDs and the primer pairs designed for qPCR analysis in this study, is presented in Supplemental Table S11. An IQ5 real-time PCR detection system (Bio-Rad) was utilized as previously described [15, 23]. CsACTIN was used as an internal reference gene, and relative expression was calculated relative to the optimal Al concentration of 0.4 mM Al using the 2ΔCt method. A total of 11 transcripts were originally validated by qPCR and then compared with transcriptome data, that we found the transcriptome data were highly reliable. Funding This work was supported by the National Key Research and Development Program of China (2018YFD1000601), the Key Research and Development (R&D) Program of Anhui Province (18030701155), and the Funding from Anhui Agricultural University, and Funding from the State Key Laboratory of Tea Plant Biology and Utilization. Determination of metal accumulation by ICP‑MS y Accumulation of Al, Ca, Cd, Cu, Fe, Mg, Mn, and Zn in tea roots, and of Al in yeast cells or in yeast growth medium upon Al treatment were determined by using ICP-MS (inductively coupled argon plasma mass spec- trometer) [60]. The roots or yeast cells were washed with 5 mM CaCl2 solution for 10 min before further rinsed in ddH2O. Al in the yeast growth medium was measured, according to the method described previously [8, 60]. Availability of data and materials The datasets generated and/or analyzed during the current study are available in the supporting information. The original RNA-seq data have been deposited in the NCBI Sequence Read Archive, accession number: PRJNA- (https://www. ncbi.nlm.nih.gov/sra/PRJNA-). References Phylogenetic analysis was carried out by using the MEGA4.0 program (http://www.megasoftware.net/). The neighbor-joining method was used to construct the phy- logenetic tree with 1000 bootstrap trials by MEGA4.0. 1. Fung KF, Zhang ZQ, JWC W, Wong MH. Aluminium and fluoride con- centrations of three tea varieties growing at lantau island, Hong Kong. Environ Geochem Health. 2003;25:219–32. 1. Fung KF, Zhang ZQ, JWC W, Wong MH. Aluminium and fluoride con- centrations of three tea varieties growing at lantau island, Hong Kong. Environ Geochem Health. 2003;25:219–32. 2. Wong MH, Fung KF, Carr HP. Aluminium and fluoride contents of tea with emphasis on brick tea and their health implications. Toxicol Lett. 2003;137:111. 2. Wong MH, Fung KF, Carr HP. Aluminium and fluoride contents of tea with emphasis on brick tea and their health implications. Toxicol Lett. 2003;137:111. Author details 1 State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, 130 West Changjiang Road, Hefei 230036, China. 2 BGI Institute of Applied Agriculture, BGI–Shenzhen, Shenzhen 518083, China. 3 Department of Earth and Environmental Sciences, Faculty of Science and Engineering, The University of Manchester, M13 9PT, Manchester, UK. Received: 19 January 2022 Accepted: 28 March 2022 Received: 19 January 2022 Accepted: 28 March 2022 Declarations Ethics approval and consent to participate Not applicable. Authors’ contributions J.Z., designed and supervised the research; J.H., A.P., Y.L., H.Z., P.L., J.W., K.Y.,, C.L., performed experiments; A. P, J.H., Y.L., H.Z., P.L., J.W., K.Y., C. L, and S. Z, analyzed data; J. Z., J. K. P, and X.W. wrote the article. All authors read and approved the final manuscript. For the functional characterization of CsALS8 and CsALMT1, the wild-type yeast strain YPH499 (MATa ura3–52 lys2-801_amber ade2–101_ochre trp1-Δ63 his3-Δ200 leu2-Δ1) was used for transformation with pYESDEST52-CsALS8, or pYESDEST52-CsALMT1 con- structs, with empty vector pYESDEST52 as a control, by using the PEG/lithium acetate method [60]. Transfor- mants were selected on YNB lacking Uracil and func- tional expression and induction of CsALS8 and CsALMT1 were performed as described previously [60]. YPH499 cells expressing empty vector pYESDEST52, CsALS8, or CsALMT1 were grown in media containing galactose or glucose, supplemented with different concentrations of Al, then were examined for their growth response to Al stress for 2 and 3 days. Growth rates were assayed in 24-well plates and Al contents in yeast cells were deter- mined under different conditions. Competing interests h h d l h The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. 4. Gao HJ, Zhao Q, Zhang XC, Wan XC, Mao JD. Localization of fluoride and aluminum in subcellular fractions of tea leaves and roots. J Agric Food Chem. 2014;62:2313–9. 3. Karak T, Bhagat RM. Trace elements in tea leaves, made tea and tea infu- sion: a review. Food Res Int. 2010;43:2234–52.l Microscopic observations The enrichment analysis was tested using a hypergeometric test at a significance cutoff of ~ 0.1% Transporter gene cloning and functional characterization The open reading frames (ORFs) for CsALS8 and CsALMT1 genes were obtained by PCR amplification using gene-specific primer pairs based on RNA-Seq sequence information. Total RNA was extracted from roots, 5 μg of which were used to synthesize the first-strand cDNA using the first-strand synthesis system Superscript III (Invitro- gen, USA). The cDNA library was used as a template for Hao et al. BMC Plant Biology (2022) 22:203 Hao et al. BMC Plant Biology (2022) 22:203 Page 18 of 20 Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s12870-022-03570-4. Additional file 1. Additional file 2. gene amplification and performed using short primers with the Ex-Taq DNA polymerase (TAKARA): 5 min at 94 °C, followed by 30 s at 94 °C, 30 s at 58, 1.30 min at 72 °C, and then 10 min at 72 °C, for 30 cycles. The PCR products were purified and cloned into the Gateway entry vector pDONR221 by using BP Clonase (Invitrogen, USA). The resulting pDONR221 constructs harboring target genes were confirmed by sequencing and recombined by using Gateway LR Clonase (Invitrogen, USA) into the destina- tion vector pYEST-DEST52 for yeast strains expression. These final constructs containing CsALS8 and CsALMT1 genes were also confirmed by sequencing. The Gen- bank accession numbers for these genes are: CsALMT1 (OK562691) and CsALS8 (OK562692). Supplementary Information Acknowledgements We are grateful for the funding support. Statistical analysis Thomine S, Lelièvre F, Debarbieux E, Schroeder JI, Barbier-Brygoo H. AtNRAMP3, a multispecific vacuolar metal transporter involved in plant responses to iron deficiency. Plant J. 2003;34:685–95. 16. Liu JP, Magalhaes JV, Shaff J, Kochian LV. Aluminum-activated citrate and malate transporters from the MATE and ALMT families function independently to confer Arabidopsis aluminum tolerance. Plant J. 2009;57:389–99. 42. Xia JX, Yamaji N, Kasai T, Ma JF. 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https://openalex.org/W2946042374	https://link.springer.com/content/pdf/10.1007/s10846-019-01022-0.pdf	English	null	Using Pre-Computed Knowledge for Goal Allocation in Multi-Agent Planning	Journal of intelligent & robotic systems	2,019	cc-by	19,979	Abstract Many real-world robotic scenarios require performing task planning to decide courses of actions to be executed by (possibly heterogeneous) robots. A classical centralized planning approach has to find a solution inside a search space that contains every possible combination of robots and goals. This leads to inefficient solutions that do not scale well. Multi-Agent Planning (MAP) provides a new way to solve this kind of tasks efficiently. Previous works on MAP have proposed to factorize the problem to decrease the planning effort i.e. dividing the goals among the agents (robots). However, these techniques do not scale when the number of agents and goals grow. Also, in most real world scenarios with big maps, goals might not be reached by every robot so it has a computational cost associated. In this paper we propose a combination of robotics and planning techniques to alleviate and boost the computation of the goal assignment process. We use Actuation Maps (AMs). Given a map, AMs can determine the regions each agent can actuate on. Thus, specific information can be extracted to know which goals can be tackled by each agent, as well as cheaply estimating the cost of using each agent to achieve every goal. Experiments show that when information extracted from AMs is provided to a multi-agent planning algorithm, the goal assignment is significantly faster, speeding-up the planning process considerably. Experiments also show that this approach greatly outperforms classical centralized planning. Keywords Multi agent planning · Actuation maps · Goal allocation · Robotics · Distributed planning · Path planning Using Pre-Computed Knowledge for Goal Allocation in Multi-Agent Planning Nerea Luis1 · Tiago Pereira2,3,4 · Susana Fern´andez1 · Ant´onio Moreira2,4 · Daniel Borrajo Received: 24 June 2018 / Accepted: 11 April 2019 © The Author(s) 2019 / Published online: 15 2019 May Received: 24 June 2018 / Accepted: 11 April 2019 © The Author(s) 2019 / Published online: 15 2019 May Nerea Luis nluis@inf.uc3m.es 1 Universidad Carlos III de Madrid, Avda. Universidad 30, Madrid, Spain 2 Faculty of Engineering, University of Porto, Porto, Portugal 3 Carnegie Mellon University, Pittsburgh, PA, USA 4 INESC-TEC, Porto, Portugal https://doi.org/10.1007/s10846-019-01022-0 Journal of Intelligent & Robotic Systems (2020) 98:165–190 https://doi.org/10.1007/s10846-019-01022-0 Journal of Intelligent & Robotic Systems (2020) 98:165–190 1 Introduction Here we consider actuation as a robot performing an operation that results in some change to the environment. However, the actuation capabilities could be modeled not only as the operations where a robot changes its surrounding, but also as any perception-like operation. The planning problem to solve would be equivalent if the overall goal is, for instance, to clean all reachable space or to mea- sure the temperature everywhere. Our framework allows to solve planning problems where sensing operations need to be executed at specific waypoint locations, such as mapping the Wi-Fi signal strength in buildings, taking measurements of temperature and humidity on a set of pre-defined loca- tions such as a computer cluster and server sites, inspecting some regions or even executing some surveillance tasks. For vacuum cleaning robots, the actuation is performed through the robot’s actuator that cleans the floor. Cleaning a specific waypoint location can be seen as the robot accomplishing a specific cleaning operation, which is an actuation goal. Thus the coverage problem would represent a set of robots moving through a map and accomplishing multiple actua- tion goals. To accomplish those goals, the robots would have to execute multiple cleaning operations, i.e. actuating on all available waypoints on the environment with the objec- tive of cleaning all reachable regions of the environment. In the cleaning case, the actuation capabilities of each robot depend on the specific actuator each robot uses. In general, for each robot and domain a different actuation model can be considered. For example, the actuation radius can either be smaller than the robot’s footprint, equal to the robot’s footprint, or extend further than the robot’s footprint. An example of the last case is a mobile manipulator, where an arm can be extended and actuate on regions beyond the robot’s range in terms of its shape and footprint. In this paper, for simplicity we only focus on two cases. First, cir- cular robots where the actuation range is smaller or equal to the robot footprint’s radius, and second, any-shape robots whose actuation range is the same as its footprint. We expect the planner to output a plan that accomplishes all the feasible actuation goals, by moving the robots to all almost impossible for a planner to solve this problem in a reasonable amount of time by just assigning all goals to all agents (following a centralized planning approach). 1 Introduction For example, the actuation radius can either be smaller than the robot’s footprint, equal to the robot’s footprint, or extend further than the robot’s footprint. An example of the last case is a mobile manipulator, where an arm can be extended and actuate on regions beyond the robot’s range in terms of its shape and footprint. In this paper, for simplicity we only focus on two cases. First, cir- cular robots where the actuation range is smaller or equal to the robot footprint’s radius, and second, any-shape robots whose actuation range is the same as its footprint. Therefore, we contribute a methodology that uses Actu- ation Maps (AMs) to extract path-planning related informa- tion. That information is used to later boost the performance of a multi-agent planner. In concrete, we use these maps as a preprocessing step to speed-up the goal assignment phase. AMs are built only once before the planning pro- cess, one per robot, at a very low cost in comparison to the impact on time savings observed later in goal assignment. The AMs, from the robot-dependent reachability maps [27], not only determine the feasibility of each pair robot-goal but also allow us to efficiently compute an estimated cost of achieving that goal. As a result, the planner receives the estimated cost information as input, and saves time by sim- plifying the goal allocation computation, directly assigning goals to robots. In our previous work, we showed that combining a pre- processing operation with MAP could bring huge savings on planning time [29]. More specifically, we used AMs to esti- mate the cost of each robot when actuating each goal way- point. Then, we introduced those estimations into the goal assignment phase to distribute the goals among the robots and plan individual paths that together solved the coverage problem. In this paper, we extend that approach with various contributions: – Introduction of collision avoidance, with a replanning phase solving the conflicts between the individual paths of each robot. – Generalization of robot shape models, from circular only to any-shape, updating both the AMs and the information extraction to generate the PDDL problem. We expect the planner to output a plan that accomplishes all the feasible actuation goals, by moving the robots to all the reachable locations from where they can actuate the goal waypoints. 1 Introduction time. Vacuum cleaning robots can be potential candidates for this problem. We assume that we have a team of het- erogeneous robots with different sizes. While the smallest robot can reach more areas, a bigger robot cleans a wider area while traveling a smaller distance. Nevertheless, other similar problems can also be solved with our contributed technique e.g. heterogeneous robots executing surveillance tasks, cooperative mapping of the environment or search and rescue tasks. As long as there exist (1) some naviga- tion graph where we can extract information to help the planner; and (2) agents with similar or different capabilities, it will be a potential domain to solve with our approach. We have encoded our problem as a Multi-Agent Planning (MAP) task. Automated planning is the field of Artificial Intelligence which deals with the computation of plans. A plan is a sequence of actions that, if executed in order from the initial state, reaches another state where all the feasible goals are achieved. Real-world robotic scenarios, in which a set of robots need to solve a certain amount of tasks, usually require the com- bination of path-planning and motion-planning techniques. An example of this type of scenarios is the coverage prob- lem, which consists of distributing the space among the set of robots, so that each one explores a certain region of the environment. The coverage problem planning task is to find a route for each robot so that all the feasible space is covered by the robots’ actuators, while minimizing the execution Nerea Luis nluis@inf.uc3m.es 1 Universidad Carlos III de Madrid, Avda. Universidad 30, Madrid, Spain 2 Faculty of Engineering, University of Porto, Porto, Portugal 3 Carnegie Mellon University, Pittsburgh, PA, USA 4 INESC-TEC, Porto, Portugal The problem is modeled with the standard PDDL lan- guage [14]. For that purpose, we use a discrete represen- tation of the map, e.g., a 2D grid of waypoints, which is 8-connected regarding robot motion. This means robots can 166 J Intell Robot Syst (2020) 98:165–190 move horizontally, vertically and diagonally in the grid of waypoints. Robots can move from one waypoint to another as long as they are grid neighbors and do not collide with obstacles. Moreover, robots can actuate other waypoints if their distance to the robot’s current position is less than their actuation radius. 1 Introduction On the other hand, some Multi-Agent planners invoke a goal-allocation phase before starting to plan to decrease the effort of computing individual plans [4, 24]. During goal allocation, a relaxed plan is computed per goal and robot to either return an estimated cost or to identify unfeasibility. This process would be repeated multiple times, concretely \|Agents\| × \|Goals\| resulting in a huge loss of computation time, especially when exploring all of the search space to identify unfeasible goals. move horizontally, vertically and diagonally in the grid of waypoints. Robots can move from one waypoint to another as long as they are grid neighbors and do not collide with obstacles. Moreover, robots can actuate other waypoints if their distance to the robot’s current position is less than their actuation radius. Here we consider actuation as a robot performing an operation that results in some change to the environment. However, the actuation capabilities could be modeled not only as the operations where a robot changes its surrounding, but also as any perception-like operation. The planning problem to solve would be equivalent if the overall goal is, for instance, to clean all reachable space or to mea- sure the temperature everywhere. Our framework allows to solve planning problems where sensing operations need to be executed at specific waypoint locations, such as mapping the Wi-Fi signal strength in buildings, taking measurements of temperature and humidity on a set of pre-defined loca- tions such as a computer cluster and server sites, inspecting some regions or even executing some surveillance tasks. For vacuum cleaning robots, the actuation is performed through the robot’s actuator that cleans the floor. Cleaning a specific waypoint location can be seen as the robot accomplishing a specific cleaning operation, which is an actuation goal. Thus the coverage problem would represent a set of robots moving through a map and accomplishing multiple actua- tion goals. To accomplish those goals, the robots would have to execute multiple cleaning operations, i.e. actuating on all available waypoints on the environment with the objec- tive of cleaning all reachable regions of the environment. In the cleaning case, the actuation capabilities of each robot depend on the specific actuator each robot uses. In general, for each robot and domain a different actuation model can be considered. 1 Introduction Given the robot heterogeneity, some goals might only be feasible for a subset of the robots. – Evaluation of four different configurations of our approach against two centralized planners and three multi-agent planners. – Description of the overall planning architecture, now organized into four modules, and detailed explanation of the preprocessing step. From a MAP point of view, the multi-robot problem we propose forces us to deal with two issues regarding the performance of the planning process: (1) the size of the search space grows with the number of waypoints and goals; and (2) some goals are not feasible for some robots. On one hand, real-world scenarios are big enough to make From a MAP point of view, the multi-robot problem we propose forces us to deal with two issues regarding the performance of the planning process: (1) the size of the search space grows with the number of waypoints and goals; and (2) some goals are not feasible for some robots. On one hand, real-world scenarios are big enough to make – Description of the type of planning instances in the coverage problem where our approach has the greatest impact. 167 J Intell Robot Syst (2020) 98:165–190 – Description of the Goal Allocation process and the deletion of unfeasible goals. – Description of the Goal Allocation process and the deletion of unfeasible goals. For the coverage problem, we compute the estimated cost as the distance from the robots to each of the waypoints. Again, on alternative problems, the estimated cost could be related to the distance to a required object, the dangerousness or reliability of a path, or the features of a robot, such has maximum velocity or the existence of manipulation capabilities. For the coverage problem, we compute the estimated cost as the distance from the robots to each of the waypoints. Again, on alternative problems, the estimated cost could be related to the distance to a required object, the dangerousness or reliability of a path, or the features of a robot, such has maximum velocity or the existence of manipulation capabilities. – Description of the MAP algorithm - including a new version that detects and solves interactions among agents. This paper is structured as follows: in Section 2 we describe the kind of problems for which our approach has been designed, and we include some planning formaliza- tions. 1 Introduction Then, on Section 3 we describe the coverage problem we want to solve. On Section 4, we present our con- tributed planning approach with pre-computed knowledge. Section 5 explains in detail the preprocessing step, which is our main contribution. After that, Section 6 contains the description of the MAP algorithm. On Section 7 we briefly extend our approach to MAP problems that involve inter- actions between robots. Then, on Section 8, we extend the formalization of circular robots to any-shape robots. On Section 9 we show illustrative experimental results of our algorithm on different scenarios. Finally, we discuss the related work and we present our conclusions and directions for future work. In order to transform this kind of problems into PDDL we have to model (1) a domain; (2) a problem; and compute (3) a set of estimated costs. The domain and problem are a lifted representation in predicate logic of the planning task. However, most of the planners always perform a grounding transformation from the domain and problem to generate the planning task. The aim of solving a planning task is to find a plan that reaches the goals specified on the task while minimizing some cost. Definition 1 (Planning Task (Single Agent)) A single-agent classical planning task [12] is a tuple = ⟨F, A, I, G⟩, where F is a set of propositions, A is a set of instantiated actions, I ⊆F is an initial state, and G⊆F is a set of goals. Definition 1 (Planning Task (Single Agent)) A single-agent classical planning task [12] is a tuple = ⟨F, A, I, G⟩, where F is a set of propositions, A is a set of instantiated actions, I ⊆F is an initial state, and G⊆F is a set of goals. Each action a ∈ A is described by (1) a set of preconditions (pre(a)) that represent literals that must be true in a state to execute the action; (2) and a set of effects (eff(a)), which are literals that are expected to be added (add(a) effects) or removed (del(a) effects) from the state after the execution of the action. The definition of each action might also include a cost function, C(a) (the default cost is one). The solution of a planning task is a plan, which is a sequence of actions π = (a1, . . . 1 Introduction , an) that, if executed in order from the initial state, reaches a state where all the goals in G are satisfied. 2 General Problem Formulation Our approach can be easily applied to any robotic problem that involves at least the following elements: – a map of the environment; – a set of potential goals, such as cleaning actuation goals, to be executed by a set of agents over the environment; – a way to model that scenario into a PDDL domain and problem. As we are working with multiple agents, we consider a MAP formalization where a set of m agents, = {φ1, . . . , φm}, has to solve the given task. The map can be modeled in different ways. In this work, we start with an image representing the world’s floor plan. From that image, we extract a navigation graph over a grid of waypoints, which is used to generate the PDDL problem. However, from the floor plan we can also extract additional information related to the environment, which is accomplished through the process of building the AMs. Definition 2 Multi-Agent Planning task. The MAP task is formed by a set of planning subtasks, one for each agent, M = {1, . . . , m} where M refers to the MAP task. Each planning subtask i includes only the facts, actions, goals and initial state related to the agent φi. The set of potential goals can vary depending on the problem to solve. In this paper we are focusing on the coverage problem and as a result it is enough for the robots to move through the environment. On alternative problems, the goals could be: looking for objects, opening doors or achieving some clients’ orders through the environment. The potential of our approach relies on the ability to extract information from the map related to the goals to be achieved. The aim is to transform that information into a set of estimation costs that can speed up the planning process. Last, we define the estimated cost per agent and goal. The set of potential goals can vary depending on the problem to solve. In this paper we are focusing on the coverage problem and as a result it is enough for the robots to move through the environment. On alternative problems, the goals could be: looking for objects, opening doors or achieving some clients’ orders through the environment. Last, we define the estimated cost per agent and goal. Definition 3 Estimated cost per agent and goal (EC). EC = {(g, φi, c) \| g ∈G, φi ∈, c = C(g, φi)} such that the C function represents the cost for an agent φi to reach the goal position g from the agent’s initial state. If a goal cannot be reached by an agent, c will be ∞. The potential of our approach relies on the ability to extract information from the map related to the goals to be achieved. The aim is to transform that information into a set of estimation costs that can speed up the planning process. Usually, these estimated costs are computed to divide the goals among the agents before the planning process starts. In J Intell Robot Syst (2020) 98:165–190 168 Listing 1 Action Navigate in PDDL Listing 1 Action Navigate in PDDL planning, c is obtained with a heuristic function. However, in our approach, the C function is given by the AMs. Even though MAP is domain-independent, our function to compute the estimated cost is domain-dependent and should be set up differently on each domain, though it would be very similar to the one we propose here for most of the related robotic domains. heterogeneity, some connections might be traversable by some robots and not by others; – Actuated (waypoint): indicates which way- points were already actuated; this predicate is used to specify goals; – Actuated (waypoint): indicates which way- points were already actuated; this predicate is used to specify goals; – Actuable (robot, waypoint, waypoint): shows which waypoints can be actuated by a robot when located on a different waypoint location. – Actuable (robot, waypoint, waypoint): shows which waypoints can be actuated by a robot when located on a different waypoint location. For the coverage problem, robots have to actuate every waypoint in G (as long as the goal is feasible). The waypoints, when connected, generate a navigation graph for a certain robot. Last, we define the estimated cost per agent and goal. The three actions that are defined in the domain are called navigate (Listing 1), actuate-on (Listing 2) and actuate-other (Listing 3). The action actuate-on is used to actuate the current position of the robot. The third action is employed to mark a waypoint as actuated if the waypoint is identified as actuable from the robot’s current location i.e. the waypoint is located inside the robot’s actuation radius on the real environment. Actions navigate and actuate-/on/other can be executed by an agent when it is placed on a waypoint. Both actuate-on and actuate-other have as effect the predicate actuated. 3 The Coverage Problem Description For the coverage problem, the feasible actuation goals are waypoints that are reachable to some agent (in terms of actuation reachability). In alternative problems, the actuation goal of the coverage problem can be replaced by other types of goals. 3 The Coverage Problem Description In this Section we consider heterogeneous teams of robots that actuate in a 2D environment, where the world is represented by a 2D image that can be downsampled to a 2D grid of waypoints. The Actuation Map gives information about the actuation capabilities of each robot, as a function of robot size and initial position [27]. At first, we assume that robots are circular and thus the only robot feature is its size, with 2D grid positions being rotation-invariant. Other shapes can also be trivially considered in our approach by extending the PDDL domain file to take into consideration robot orientation as well. This aspect is further explained in Section 8. As it was previously said, we modeled the domain and problem using PDDL. The domain has two types of objects: robots, which act as agents; and waypoints, which represent positions in the discretized world. In order to generate a PDDL problem, the waypoints’ grid resolution is defined in advance using a discretization step. After that, a navigation graph and a set of actuable waypoints are defined for each robot, taking into account their physical characteristics. All this information is generated on the preprocessing step, further explained in Section 5. The planning task of the coverage problem consists on finding a sequence of navigation and actuation actions for each robot so that all actuation goals are covered by any of the robots’ actuators, while minimizing the execution time. An actuation goal refers to the atomic action of marking a waypoint as “actuated”. Going back to the vacuum cleaning example, the actuation goal represents the objective of cleaning a certain waypoint position. For the coverage problem, the feasible actuation goals are waypoints that are reachable to some agent (in terms of actuation reachability). In alternative problems, the actuation goal of the coverage problem can be replaced by other types of goals. The planning task of the coverage problem consists on finding a sequence of navigation and actuation actions for each robot so that all actuation goals are covered by any of the robots’ actuators, while minimizing the execution time. An actuation goal refers to the atomic action of marking a waypoint as “actuated”. Going back to the vacuum cleaning example, the actuation goal represents the objective of cleaning a certain waypoint position. Listing 2 Action Actuate-on in PDDL and MAP. We introduce here the base work needed to later explain the preprocessing step and the MAP algorithm. 1. Actuation Maps module: it is in charge of generating the AMs for each given robot. It also extracts the map fea- tures that can potentially alleviate the planning process (e.g. estimation costs), and it generates the planning problem in PDDL. It is explained on Section 5.2. 4 Architecture As it was previously said, this work combines Actuation Maps (AMs) with Multi-Agent Planning (MAP). The contributed architecture can be seen in Fig. 1. It has been divided into four modules and receives as input the map of the environment, the general knowledge related to the task Therefore, the set G is a list of waypoints to actuate on (positions that need to be covered). The PDDL domain we created has four predicates: Listing 2 Action Actuate-on in PDDL – At (robot, waypoint):definestherobotposition; – Connected (robot, waypoint, waypoint): establishes the navigation connectivity between way- points, specified for each robot, and given the robot Listing 2 Action Actuate-on in PDDL 169 J Intell Robot Syst (2020) 98:165–190 Listing 3 Action Actuate-other in PDDL Listing 3 Action Actuate-other in PDDL to solve and the features of the set of robots. The aim of each module is described below. and MAP. We introduce here the base work needed to later explain the preprocessing step and the MAP algorithm. Fig. 1 Complete architecture that combines actuation maps and multi-agent planning 4.1 Actuation Maps Our system receives as input the Environment map which represents a 2D environment (e.g. building floor plan) and m Robot models with the agents’ features. There is a third input provided by the user that refers to the General knowl- edge of the environment (i.e. tasks to solve). These three inputs represent the input information described in Fig. 1. 2. Multi-Agent Planning Task Generation module: once the outputs from the prior module and the domain are received as input, the goal assignment process is launched. This module is in charge of dividing the goals among the agents following some goal-strategy. Then, a set of domain and problem is generated for each agent, which is known as factorization. It is explained on Section 5.3. We briefly summarize here the process of building the AMs [27]. We assume there is an occupancy grid map, i.e., a gray-scale image representing the environment. In this image each pixel has a value with the probability of the corresponding world position being occupied by an obstacle. This occupancy grid map is first transformed into a binary image of free and obstacle pixels, using a fixed threshold. An example of the resulting black and white image is shown in Fig. 2a. 3. Multi-Agent Planning Algorithm module: the individ- ual planning process and the merging phase are run on this module. It is explained on Section 6. 4. Conflicts solver module: if any interactions need to be solved, this module employs a plan-reuse-planner to fix them. It is explained on Section 7. We define G as the set with all pixel positions from the input binary image. The input image is a visual representa- tion of M, the set with the obstacle pixel positions, where white represents free space and black represents obstacles. The following subsections explain the essential informa- tion regarding both components of our contribution: AMs Fig. 1 Complete architecture that combines actuation maps and multi-agent planning Multi-Agent Planning Task Generation Actuation Maps (Feature Extraction) If any conﬂicts arise Multi-Agent Planning Algorithm Conflicts solver (Plan-Reuse Planner) Environment Map Features Robot1 Features Robot2 Features Robotm ... Input information 2 1 3 4 General knowledge Multi-Agent Planning Task Generation Actuation Maps (Feature Extraction) If any conﬂicts arise Multi-Agent Planning Algorithm Conflicts solver (Plan-Reuse Planner) Environment Map Features Robot1 Features Robot2 Features Robotm ... Input information 2 1 3 4 General knowledge Fig. 4.1 Actuation Maps For circular vacuum cleaning robots, corners of the environment are non-actuable regions they cannot clean due to the robot’s circular shape. 4.1 Actuation Maps The free configuration space, Cf ree, is then defined as: Cf ree i = {p ∈G \| p /∈M ⊕Ri} (2) (2) Ai(r0 i ) = Li(r0 i ) ⊕Ti (4) Ai(r0 i ) = Li(r0 i ) ⊕Ti (4) where G is the grid set with all the pixel positions. The free configuration space represents the feasible positions for the robot center, but does not give any information about the regions that can be actuated by the robot. While the actuation space, Ai(r0 i ), is the set containing all the waypoints that can be actuated by a robot, its representation as an image, as shown in Fig. 3, is the Actuation Map. We also use the term Actuation Map to refer to the overall technique to determine actuation capabilities of robots. In the figures below we show the actuation map, obtained after applying the partial morphological closing operation to the original map. The actuation map can be used as a visual representation to show what the robot can actuate from any point reachable from its initial position, and we use them to visually represent in figures the sets corresponding to each robot’s actuation space. In order to determine which regions of the environment are actuable, the partial morphological closing opera- tion is used. Morphological closing is a dilation operation followed by a morphological erosion. Because dilation and erosion are dual operations, the morphological closing of obstacles (erosion applied to the image with inflated obsta- cles) is equivalent to the dilation of free configuration space. However, the morphological closing cannot be used to determine the actuation capabilities, because it does not consider which points are reachable from the initial robot position, and different initial positions do change the overall reachability in terms of actuation. The partial morphological closing was introduced in order to consider the initial robot position when determining the actuation capabilities. In order to use the partial morphological closing, the algorithm needs to find the navigable regions first. The set of navigable regions from a starting robot point r0 i is always a subset of Cf ree i . As an example, we can consider again the vacuum cleaning robot case. The configuration space represents the possible center positions for the robot; the actuation space A represents the regions the robot can clean; and the non-actuable regions are positions that the robot cannot clean. 4.2 Multi-Agent Planning Li(r0 i ) = {p ∈G \| p connected to r0 i ∧p ∈Cf ree i } (3) (3) 4.1 Actuation Maps 1 Complete architecture that combines actuation maps and multi-agent planning Multi-Agent Planning Task Generation 2 Actuation Maps (Feature Extraction) 4 Multi-Agent Planning Algorithm 3 If any conﬂicts arise Conflicts solver (Plan-Reuse Planner) 170 J Intell Robot Syst (2020) 98:165–190 Fig. 2 Simulated map and two heterogeneous robots with different sizes in (a); colored regions represent the navigable space, Li(r0 i ), for 2 robots with different sizes, depending on size and initial position of robots Fig. 2 Simulated map and two heterogeneous robots with different sizes in (a); colored regions represent the navigable space, Li(r0 i ), for 2 robots with different sizes, depending on size and initial position of robots Fig. 2 Simulated map and two heterogeneous robots with different sizes in (a); colored regions represent the navigable space, Li(r0 i ), for 2 robots with different sizes, depending on size and initial position of robots In Fig. 2 we show a simulated map with 2 robots with different sizes, and the respective navigable space. We define the structuring element as an image that repre- sents the robot shape. Assuming first the circular robot case, we represent the robot with Ri, the set with pixel positions from a circle with radius equal to the robot size. The mor- phological operation dilation on the obstacle set M by Ri is: In Fig. 2 we show a simulated map with 2 robots with different sizes, and the respective navigable space. Finally, by applying the the second dilation operation of the morphological closing to the navigable set (subset of Cf ree) instead of applying it to the free configuration space, we obtain the partial morphological closing operation. The actuation space is thus the dilation of the navigable space. The structuring element for this second operation is the one that models the actuation capabilities, T , which dilates the navigable space according with the actuation model. If instead the structuring element R is used again, that would be equivalent to assuming an actuating ability completely coincident with the entire robot footprint. M ⊕Ri = r∈Ri Mr (1) (1) where Mr is the translation of M by vector r. The visual output of applying this dilation operation to a map of obstacles is the inflation of obstacles by the robot size. Fig. 2 Simulated map and two heterogeneous robots with different sizes in (a); colored regions represent the navigable space, Li(r0 i ), for 2 robots with different sizes, depending on size and initial position of robots 5.1 Discretization For the planning problem, it is possible to consider each individual pixel as a waypoint. However, that approach results in a high density of points that would make the planning problem excessively complex. Moreover, there is some redundancy in having points that are too close to each other, as their difference is not significant in terms of the environment size and localization accuracy. Therefore, we reduced the set of locations from all pixels to a smaller set of locations. We considered again waypoints distributed into a grid, but now the grid-size is greater than one pixel. The reduced grid of waypoints is obtained using a downsampling rate sr, such as the number of waypoints in each direction in the new grid is sr times less than the number of waypoints in the original grid of pixels. In Multi-Agent Planning two main approaches have been commonly used: centralized and distributed. The centralized approach involves a master agent which knows everything about the agents and the environment. The master agent sees the rest of agents as resources and is also responsible for coordinating and solving the interactions that might arise during the planning process. On the distributed approach each agent builds its own plan synchronously with the rest of the agents. Depending on the amount of communication allowed among the agents, they will need to share their information during the planning process or they have to later merge their plans and solve the conflicts that might have arisen. Our MAP algorithm follows the latter, using plan merging [13, 25] to build the solution plan after the individual planning process. Thus, no cost of communication is involved and there is no implementation inside the algorithm regarding communication. After downsampling, we can find the connectivity between points to construct the navigation graph of each robot, shown in Fig. 5a. It is also possible to find which waypoints can be actuated from other waypoints using the distance between them, as shown in Fig. 5b, by considering the maximum actuation radius. The problem of such discretization is the change in the actuation space topology. Adjusting the position of waypoints could allow a better representation of the topology of the environment, but the multi-robot nature of the problem compromises that solution. 5.1 Discretization In order to deal with multiple robots with different configuration spaces, for each agent, we independently adjust the waypoint position -temporarily- in a hidden manner invisible to the other agents. When discretizing each robot’s configuration space, we might consider a waypoint as belonging to the free configuration space even if it is strictly outside it, as we assume an error margin to compensate for the discretization error. Nevertheless, we still maintain the original waypoint position in further steps, such as determining actuation Li(r0 i ) = {p ∈G \| p connected to r0 i ∧p ∈Cf ree i } (3) Usually, MAP tasks have to deal with some coordination issues among agents or the shar- ing of resources. From the perspective of MAP, planning domains exhibit a coupling level that ranges from loosely- coupled to tightly-coupled, depending on the degree of interaction between agents plans [7]. Agents on loosely- coupled domains barely interact with each other. Tightly- coupled domains have a considerable level of interac- tion among agents. This implies solving interactions while planning (agents’ communication) or afterwards (conflict solving). Our domain is loosely-coupled, as agents barely interact with each other. However, there might be colli- sions that are not detected by the planner. A different approach involving collision-detection is further explained on Section 7. Li(r0 i ) = {p ∈G \| p connected to r0 i ∧p ∈Cf ree i } (3) When multiple agents are involved (e.g. robots, workers, drivers) we talk about Multi-Agent Planning (MAP) [34]. MAP computes a plan for/by a set of agents that jointly The navigable set Li(r0 i ) is the set of points that are connected to the initial position r0 i through a path of adjacent cells in the free configuration space. J Intell Robot Syst (2020) 98:165–190 171 Fig. 3 Colored regions in (b) and (c) represent actuation spaces for respective robots, i.e. the points in the environment that each robot can actuate, depending on their size and initial position shown in (a); the actuation capability in this example is completely coincident with the entire robot footprint, i.e., the actuation range is equal to the robot radius l l i bl U ll MAP k h d l i f l h d i f f ibl i d generation of goals, the detection of unfeasible regions and the computation of estimated costs. Section 5.3 describes the process of generating the MAP task and how the task is factorized (divided) in subtasks. Fig. 3 Colored regions in (b) and (c) represent actuation spaces for respective robots, i.e. the points in the environment that each robot can actuate, depending on their size and initial position shown in (a); the actuation capability in this example is completely coincident with the entire robot footprint, i.e., the actuation range is equal to the robot radius solve a planning problem. Usually, MAP tasks have to deal with some coordination issues among agents or the shar- ing of resources. From the perspective of MAP, planning domains exhibit a coupling level that ranges from loosely- coupled to tightly-coupled, depending on the degree of interaction between agents plans [7]. Agents on loosely- coupled domains barely interact with each other. Tightly- coupled domains have a considerable level of interac- tion among agents. This implies solving interactions while planning (agents’ communication) or afterwards (conflict solving). Our domain is loosely-coupled, as agents barely interact with each other. However, there might be colli- sions that are not detected by the planner. A different approach involving collision-detection is further explained on Section 7. generation of goals, the detection of unfeasible regions and the computation of estimated costs. Section 5.3 describes the process of generating the MAP task and how the task is factorized (divided) in subtasks. solve a planning problem. 5 Preprocessing Input information Environment Map Features Robot1 Features Robot2 Features Robotm ... Input information General knowledge PDDL Domain ation purposes graph of each ght be moved to the closest point in the configuration space, if the adjustment is under a given margin δ. As stated previously, the adjustment is always temporary to the construction of Fig. 4 Preprocessing stage before the planning process starts. First, inputs are processed in order to generate the AM for each agent. Then, a discretization is applied to generate all the required information for planning such as the navigation graph for the PDDL problem, and the list of estimated costs. Once this information has been generated, GA starts and the MAP problem is divided into subproblems with specific goals assigned to each individual problem PDDL Problem Generation Goal Assignment Actuation Transformation Robot1 Actuation Transformation Robot2 Actuation Transformation Robotm ... #robots #waypoints Grid of waypoints List of goals Discretization and Merging of Maps Estimated Cost per Goal-Agent List of feasible and unfeasible goals per agent Factorization Domain1 Problem1 Domain2 Problem2 Domainm Problemm ... Environment Map Features Robot1 Features Robot2 Features Robotm ... nput information Environment Map Features Robot1 Features Robot2 Features Robotm ... Input information General knowledge PDDL Domain on purposes aph of each ht be moved to the closest point in the configuration space, if the adjustment is under a given margin δ. As stated previously, the adjustment is always temporary to the construction of atures bot1 Features Robot1 nt General knowledge Envir M Environment Map Actuation Transformation Robot2 Actuation Transformation Robotm Discretization and Merging of Maps PDDL Domain Goal Assignment feasibility of that waypoint, and for visualization purposes as well. When determining the navigation graph of each robot, an unreachable waypoint position might be moved to the closest point in the configuration space, if the adjustment is under a given margin δ. As stated previously, the adjustment is always temporary to the construction of of the free configuration space by allowing points in the navigation graph that were originally unfeasible for the robot. In b, the actuation map of the same robot, and the respective actuation graph represented with yellow lines Fig. 5 In a we have an example of the free configuration space, with the discretization waypoints shown as green dots. The blue lines repre- sent the connectivity between waypoints in the navigation graph of the robot. 5 Preprocessing The contributed preprocessing step is shown in Fig. 4. This is the point where both techniques, AMs and MAP, are com- bined and complement each other. Section 5.1 describes the discretization process, downsampling the waypoint density from the original pixel grid. Section 5.2 describes the J Intell Robot Syst (2020) 98:165–190 172 Fig. 4 Preprocessing stage before the planning process starts. First, inputs are processed in order to generate the AM for each agent. Then, a discretization is applied to generate all the required information for planning such as the navigation graph for the PDDL problem, and the list of estimated costs. Once this information has been generated, GA starts and the MAP problem is divided into subproblems with specific goals assigned to each individual problem PDDL Problem Generation Goal Assignment Actuation Transformation Robot1 Actuation Transformation Robot2 Actuation Transformation Robotm ... #robots #waypoints Grid of waypoints List of goals Discretization and Merging of Maps Estimated Cost per Goal-Agent List of feasible and unfeasible goals per agent Factorization Domain1 Problem1 Domain2 Problem2 Domainm Problemm ... Environment Map Features Robot1 Features Robot2 Features Robotm ... Input information Environment Map Features Robot1 Features Robot2 Features Robotm ... Input information General knowledge PDDL Domain feasibility of that waypoint, and for visualization purposes as well. When determining the navigation graph of each robot, an unreachable waypoint position might be moved to the closest point in the configuration space, if the adjustment is under a given margin δ. As stated previously, the adjustment is always temporary to the construction of Fig. 5 In a we have an example of the free configuration space, with the discretization waypoints shown as green dots. The blue lines repre- sent the connectivity between waypoints in the navigation graph of the robot. Using parameters δ and α it is possible to maintain the topology of the free configuration space by allowing points in the navigation graph that were originally unfeasible for the robot. In b, the actuation map of the same robot, and the respective actuation graph represented with yellow lines PDDL Problem Generation Goal Assignment Actuation Transformation Robot1 Actuation Transformation Robot2 Actuation Transformation Robotm ... #robots #waypoints Grid of waypoints List of goals Discretization and Merging of Maps Estimated Cost per Goal-Agent List of feasible and unfeasible goals per agent Factorization Domain1 Problem1 Domain2 Problem2 Domainm Problemm ... Environment Map Features Robot1 Features Robot2 Features Robotm ... 5.2 Extracting Cost Information from Actuation Maps Moreover, when determining the connectivity of way- points for the navigation graph, only the eight grid neigh- bors are considered. A∗is then used to determine the real distance between waypoints (e.g., around obstacles), and connectivity is only considered if the real distance is at most a factor of α = 1.2 the straight line distance between them. The downsampling rate sr was set manually, depending on the resolution one wants for the grid of waypoints. If the original pixel resolution would be used, the resulting grid of waypoints G′ would contain all pixels and it would be equivalent to G. Otherwise the set G′ represents the grid waypoint positions after downsampling. Finally, all waypoints that belong to the robot actuation map should be connected to some waypoint of its navigable graph. If that is not the case after the previous steps, we connect the isolated waypoints to the closest navigable vertex in line of sight, even if their distance is greater than the maximum actuation distance, again to compensate for the discretization error. Therefore, while the planner may return an actuate action to cover waypoint A from the navigable waypoint B in the discretized world, a real robot would have to move closer from the waypoint B to waypoint A in order to actuate the latter. Using the concept of AMs it is possible to very easily find UG, the list of unfeasible goals per agent: UG = {g ∈G′, φi ∈ \| g ̸∈Ai(r0 i )} (5) (5) The positions in the actuation space Ai(r0 i ) are feasible actuation goals for agent φi. The information from the UG list can speed-up goal assignment by avoiding computation related to unfeasible goals, but it does not provide any information about the cost for each robot to accomplish a feasible actuation goal. For that purpose, we present the following extension. We build the navigable space Li in an iterative procedure, from the starting position r0 i . In the first iteration we have L0 i (r0 i ) ←{r0 i }, and then the following rule applies: The grid density is chosen manually in order to adjust the level of discretization. As for the α and δ parameters, they were tuned empirically such as the free space topology is still maintained even while using lower density discretization of the environment. 5.2 Extracting Cost Information from Actuation Maps By trial and error, we found empirically that α = 1.2 works for all the tested scenarios. As for the δ parameter, we set it to always start with a value of 3 pixels, then build the discretized model and verify if it is valid, i.e., if all the waypoints belonging to the actuation map become feasible for the respective robot in terms of the discretized representation. If not, we increment the parameter until a topologically consistent representation is found (number of feasible goals equals number of waypoints inside actuation map). Even though this fine-tuning methodology seems sensitive to the robot heterogeneity, the truth is that the final δ value depends on the size of the bigger robot, because the correct discretization of the configuration space is more sensitive to the δ parameter for bigger robots. Through experimentation, we found out that if a certain value of the δ parameter works well for the biggest robot, it always produces the correct discretization for smaller robots. Moreover, we also observed that δ = 4 pixels worked well for all the different and very diverse maps we tested in our experiments with circular robots, only failing for the any-shape experiments where the configuration space discretization is more sensitive to the possible robot orientation. For the any-shape robot experiments, we found that δ = 6 pixels was enough to obtain good a discretization for all the environments tested. The consistency of the δ parameter over different environment maps shows that these Lj i (r0 i ) = {p ∈G \| ∃q ∈Lj−1 i (r0 i ) : p neighbor of q ∧p ∈Cf ree i ∧p ̸∈La i (r0 i ) ∀a < j} (6) (6) When using this recursive rule to build the navigable space, we guarantee that any point in the set Lj i (r0 i ) is exactly at distance j from the initial position r0 i . Because we want to build the complete actuation space, the iterative computation of Eq. 6 stops when all pixels have been expanded at least once. 5 Preprocessing Using parameters δ and α it is possible to maintain the topology of the free configuration space by allowing points in the navigation graph that were originally unfeasible for the robot. In b, the actuation map of the same robot, and the respective actuation graph represented with yellow lines of the free configuration space by allowing points in the navigation graph that were originally unfeasible for the robot. In b, the actuation map of the same robot, and the respective actuation graph represented with yellow lines Fig. 5 In a we have an example of the free configuration space, with the discretization waypoints shown as green dots. The blue lines repre- sent the connectivity between waypoints in the navigation graph of the robot. Using parameters δ and α it is possible to maintain the topology J Intell Robot Syst (2020) 98:165–190 173 the connectivity graph of each robot. After the navigation connectivity is tested, the waypoint position resets to its default grid position for the next steps, such as determing the actuation feasibility, and the navigation and actuation graphs of other robots. parameters can be map-independent to a certain extent, with most of the work being easily automated. parameters can be map-independent to a certain extent, with most of the work being easily automated. 5.2 Extracting Cost Information from Actuation Maps Furthermore, if we build the actuation space sets with the intermediate navigable sets Lj i (r0 i ), Aj i (r0 i ) = Lj i (r0 i ) ⊕Ti (7) (7) then the intermediate actuation set Aj i (r0 i ) represents the points that can be actuated by the robot from positions whose distance to r0 i is j. The actuation space defined in the previous Section can also be alternatively defined as Ai(r0 i ) = {p ∈G \| ∃a : p ∈Aa i (r0 i )} (8) (8) The actuation cost is defined for g ∈Ai(r0 i ): ACi(r0 i , g) = min{j \| g ∈Aj i (r0 i )} + 1 (9) (9) The actuation cost ACi(r0 i , g) represents, for each g ∈ Ai(r0 i ), the minimum number of actions needed for the robot to actuate the grid waypoint g if starting from the initial position r0 i , measured in the pixel-based grid G. In 174 J Intell Robot Syst (2020) 98:165–190 possible, assigning more goals than k to each agent. The LB assignment strategy is used when minimizing the maximum number of actions per agent (makespan). As a second option, we also took the Best-Cost (BC) strategy also defined before in [2], which simply assigns each goal to the agent that can achieve it with the least cost. The BC strategy is used when minimizing the total number of actions over all robots (plan length). Eq. 9, the minimum j∗represents the minimum distance (i.e., minimum number of navigate actions) needed to travel from r0 i to some point from where g can be actuated. The added one in Eq. 9 accounts for the one actuate action needed to actuate g, after the j∗navigate actions needed to reach a place from where the robot can actuate g. Eq. 9, the minimum j∗represents the minimum distance (i.e., minimum number of navigate actions) needed to travel from r0 i to some point from where g can be actuated. The added one in Eq. 9 accounts for the one actuate action needed to actuate g, after the j∗navigate actions needed to reach a place from where the robot can actuate g. Thus, the cost function C presented previously in Section 2 is defined in Eq. 10, where sr is the downsampling rate. 5.3 Multi-Agent Planning Task Generation Once the discretization of maps has been performed, we have all the information needed to generate the MAP task M, which is formed by a domain (received as input) and a problem (generated through the discretization). The inputs to the Goal Assignment (GA) phase are (1) the PDDL domain; (2) the PDDL problem; (3) the list of estimated costs where c is computed as the number of steps for an agent to reach the goal position g from its initial position; and (4) the list of unfeasible goals UG = {g ∈G, φi ∈ \| C(g, φi) = ∞}. The cost of navigating between two neighbor grid waypoints is 1 unit. As long as EC is provided, UG is not used inside the MAP algorithm. The case when EC is not provided is later explained in this Section. The first step is to allocate the feasible goals to the agents. This step uses the information of estimated costs received from AMs. Goal assignment phase (GA) returns as output (1) a subset of ′ agents, ′ = {φ′ 1, . . . , φ′ n}, that will be the only ones who will plan to solve the problem; and (2) a new MAP task M′ = {1, . . . , n}. As a result, a specific PDDL domain and problem will be generated for each φ′ i agent which only includes the goals each agent has to achieve. As it previously mentioned, if a goal is unfeasible for all the ′ agents, the MAP algorithm will discard it from the new MAP task M′, so that the process of planning will not fail. The output of this process is a pair (domain, problem) for each agent as in Fig. 4 after the Factorization step and in Eq. 12. In Multi-Agent systems, in order to perform task allocation [8, 18] some strategy has to be determined or implemented, as the aim it is to divide the MAP task in subtasks to alleviate the planning process afterwards. In addition, a goal-assignment strategy (GAS) needs to be chosen to define the way goals are assigned to agents by the system. In our approach we took the Load-Balance (LB) strategy previously defined in [2], that first calculates k = ⌈\|G\| \|\|⌉, which represents the average number of goals per agent. 5.2 Extracting Cost Information from Actuation Maps The division by sr transforms the estimated cost of actions measured in the pixel-based grid G, ACi(r0 i , g), to the respective cost value in the downsampled grid of waypoints G′. The ceil function rounds up the result of the division to the smallest integral value that is not less than ACi(r0 i , g)/sr. The cost function C is domain-dependent and works for the coverage problem. If a different problem is given as input, the cost function should be redefined. (p g ) As in [2], only when the information about estimated cost per pair robot-goal is not available for some reason, our MAP algorithm would perform GA computing a relaxed plan using the FF heuristic [19]. This is not a contribution of the paper itself, as it was already in [24]. However, in that work, when a goal was unfeasible for every agent, it was assigned to all of them. In our approach, when the goal has been identified as unfeasible by all agents, the relaxed plan is not computed for that pair robot-goal and the goal is not included into the M task. This behavior is not common in classical deterministic Automated Planning, as planners expect that the problem does not contain any unfeasible goal. As our approach separates goal allocation from planning, we can easily deal with unfeasible goals. This small contribution gives us more flexibility when working for real environments, as it is better to obtain a plan that solves 95% of the goals than just failing during planning. To plan using soft-goals [23] or working on oversubscription planning [17, 31] would have been other ways to deal with unfeasibility, but they are out of the scope of this work. In summary, there are two contributions to the GA process: (1) the detection and deletion of unfeasible goals is a contribution that helps not only on skipping the computation of those relaxed plans but also avoids the planning process to fail; and (2) to use information from AMs, as the algorithm receives and processes the estimated costs from the AMs to skip the computation of the relaxed plans. C(g, φi) = ceil ACi(r0 i , g)/sr (10) (10) Finally, the Estimated Cost per Goal-Agent list EC is defined in Eq. 11. EC ={⟨g, φi, c⟩\| g ∈G′∧φi ∈∧g ∈Ai(r0 i )∧c=C(g, φi)} (11) 6 The Multi-Agent Planning Algorithm e.g. collisions, sharing a resource, or cooperation. On the previous description of the coverage problem we did not explicitly consider any kind of interactions. Our idea was (1) to test first the scalability of the MAP algorithm; and (2) to generate, as fast as possible, a valid solution. Robots might occasionally collide at some specific step of the solution plan. However, that collision could be easily resolved during execution by forcing one of the robots to wait until the other robot has left the conflict zone. Then, the stopped robot will continue executing the rest of its plan. On the other hand, there is a subarea of Automated Planning called Planning by Reuse that has been widely employed in areas such as Case-Based Planning [6], or replanning when plan execution fails [15]. Usually, planners that perform plan repair receive three inputs: a domain, a problem and a plan to be fixed. Examples of this kind of planners are LPG- ADAPT [15] or ERRT-PLAN [5]. Therefore, an improvement of our approach is to detect and fix potential collisions right after the individual planning process using an off-the-shelf plan reuse planner. This new feature makes our architecture more robust when executing the solution plan in a real environment. Thus, we slightly changed our PDDL domain to track the collisions by adding a new predicate called occupied. In order to solve the MAP task we use the distributed phase of a previous work of the authors [24]. Our algorithm receives as input a MAP task, which consists of a PDDL domain and problem files (Fig. 6) for each agent in φ′ i ∈′. On the first step, each agent builds its plan individually (line 1). Then the agents’ plans are merged by a simple concatenation of plans in one resulting plan (line 2). If the merged plan is valid, we parallelize the plan (line 4) generated by the merging step. Parallelization is performed in two steps: converting the input total-order plan into a partial-order one by a similar algorithm to [35]; and parallelizing this partial-order plan by ordering actions in the first time step that satisfies all ordering constraints in the partial-order plan. – Occupied (waypoint): indicates that there is a robot on that waypoint. That predicate is set as a new precondition of the navigate action described in Listing 3. This allows the agent to only traverse a connection if the destination way- point is not occupied by a robot. The predicate occupied in combination with the parallelization algorithm avoids two situations: (1) Two robots cannot be on the same waypoint at the same plan step. (2) As we build a parallel plan, neither of them can swap positions during the same plan step. The reason is that in order to move robot1 to y from z, y should be not occupied first. Same happens to z for robot2. Thus, both actions are mutually exclusive and the parallelization algo- rithm does not allow both actions to be performed together. One of the robots would need to move somewhere else first. 1http://icaps-conference.org/index.php/main/competitions 5.3 Multi-Agent Planning Task Generation Then, it assigns each goal g ∈G to the agent φi ∈ that achieves g with the least cost. This strategy avoids, if M′ = ⟨1, ..., n⟩= ⟨D1, P1⟩, ⟨D2, P2⟩, ..., ⟨Dn, Pn⟩ (12) (12) 175 J Intell Robot Syst (2020) 98:165–190 Fig. 6 High level description of the MAP algorithm. Inputs: MAP task (M′), Single-Agent Planner (SAP ). Output: resulting plan (π) or no solution 6 The Multi-Agent Planning Algorithm The main advantage of parallelization is to be able to return a plan in which on each time step, more than one action can be executed at the same time, taking advantage of having multiple robots in the environment as well. As the domain does not have any interactions, there is no need to fix the plan regarding interactions. To set up the algorithm, an off-the-shelf planner had to be chosen. The advantage of this MAP algorithm is that any state-of-the-art planner can be easily included on the distributed planning phase without further code modi- fication. Our configuration employs LAMA-UNIT-COST as SAP . LAMA-UNIT-COST corresponds to the first search that LAMA performs, using greedy-best-first with unit costs for actions [30]. The merged plan is validated using VAL [20], the validator from the International Planning Competition (IPC).1 On Fig. 7 we show a solution example obtained from the MAP algorithm. It corresponds to the scenario called Corridor-High later on the experiments. – Occupied (waypoint): indicates that there is a robot on that waypoint. 7 Dealing with Interactions Real-world robotics environments might imply to deal with potential interactions among robots (at the very least) The new version of our MAP algorithm is described in Fig. 8. As the MAP algorithm starts with the individual 176 J Intell Robot Syst (2020) 98:165–190 Fig. 7 These figures represent the Corridor scenario used in the exper- iments. The waypoint discretization is shown in (a). The resulting path for each robot is shown in (b) to (e), after solving the planning problem using load balance as goal-strategy. Path 1 belongs to the smallest robot. Path 4, to the biggest one using load balance as goal-strategy. Path 1 belongs to the smallest robot. Path 4, to the biggest one using load balance as goal-strategy. Path 1 belongs to the smallest robot. Path 4, to the biggest one Fig. 7 These figures represent the Corridor scenario used in the exper- iments. The waypoint discretization is shown in (a). The resulting path for each robot is shown in (b) to (e), after solving the planning problem using load balance as goal-strategy. Path 1 belongs to the smallest robot. Path 4, to the biggest one represents one orientation. We then using the partial morphological closing operation on each layer, and with that approach we determine individually for each orientation the corresponding actuation space [28]. planning phase, no collisions will be detected at that point (line 1). After concatenation, the solution plan is validated by VAL (line 3). The validator will detect, if any, mutex actions related to occupied positions as explained above. If so, the plan will be invalid. As a result, the M′ task and the invalid plan are sent to the plan reuse planner (line 6). When the plan is fixed, the parallelization step is applied (line 7). Finally, the MAP algorithm runs VAL again (line 8). If the plan is valid, it is returned as the solution. The configuration of our algorithm is the same as the previous version - the Single-Agent Planner is LAMA-UNIT-COST. The plan reuse planner is LPG-ADAPT. First, the algorithm needs images to model both the robot and its actuation capabilities. Both are parametrized by images that can be rotated and scaled to represent any robot. As input, it is also necessary to give the center of the robot and actuation in terms of their model images, and their relative position. 7 Dealing with Interactions Here we assume a quantization of the orientation given by nθ layers. Using the input image for the robot model, we rotate it by θj = 2jπ/nθ, where 0 ≤j < nθ, in order to build a model of the robot for each possible orientation, as shown in Fig. 10. The variable j is used as a layer index. Listing 4 Action Navigate that now checks occupied positions 8 Extending the Approach to Any-Shape Robots In terms of morphological operations, we consider two structuring elements, R and T , to represent the robot and actuation models respectively. After rotating them, we get R(θj) and T (θj), with 0 ≤j < nθ. For the case of non-circular robot footprints (Fig. 9), given the robot model is not rotation invariant, we need to discretize orientation as well. We use a world representation that is composed of multiple layers, where each layer j j Using the structuring element for the robot model, we again apply morphological operations to determine the Listing 4 Action Navigate that now checks occupied positions 177 J Intell Robot Syst (2020) 98:165–190 Fig. 8 MAP algorithm that also includes the plan-reuse phase. Inputs: MAP task (M′), Single-Agent Planner (SAP ), Plan-reuse planner (R). Output: resulting plan (π) or no solution Li(r0 i , θj), represents layer j of the navigation space, which has orientation θj. We show on Fig. 12 different layers of the navigation space for two different robots. free configuration space, now for each possible quantized orientation, dilating the map using a different robot shape model for each layer. We use a circular representation for the layered orientation, where the next layer after layer j = nθ −1 is layer with index 0. For the any-shape robots, a multi-layer representation is used to determine the Actuation Map, representing different orientations. However, in terms of accomplishing goals, we assume it is irrelevant the orientation from which a robot actuates on a waypoint position. Cf ree(θj) = {p ∈G \| p /∈M ⊕R(θj)} ∀0 ≤j < nθ (13) Cf ree(θj) = {p ∈G \| p /∈M ⊕R(θj)} ∀0 ≤j < nθ (13) (13) Therefore, while on the rotation-invariant scenario the domain was discretized in a series of 2D waypoints, for the any-shape case there are two types of waypoints: the 3D waypoints representing (x, y, θ) position, and the 2D waypoints representing (x, y) positions invariant to orientation. In order to model a robot that navigates through waypoints, we need to establish the type of connectivity between points in different layers, such as it is equivalent to the type of motion the robot actually has. As an example, using the connectivity graph from Fig. 11, where one point is connected to all its neighbors in the same layer, and the respective positions in adjacent layers, is equivalent to considering an omnidirectional model of navigation. Fig. 9 Environment and robot models used to test the extended approach to any-shape robots 8 Extending the Approach to Any-Shape Robots The navigability graph now becomes a graph of 3D waypoints connected to each other, modeling the motion capabilities of robots in the world in terms of both rotation and translation, individually or combined, as exemplified for different orientation layers on Fig. 13. Given the connectivity model, it is then possible to find all points in any layer of the configuration space that connect with the starting robot location r0 i , obtaining the navigable sets Li(r0 i , θj), for 0 ≤j ≤nθ layers. The initial orientation is still coded on r0 i , and the navigable set We then use a second dilation operation to the navigable space in each layer to obtain the actuation space for each orientation. The structuring element for this second operation is the one that models the actuation capabilities, T , which dilates the space according with the actuation model. If instead the structuring element R is used again, that would be equivalent to assuming an actuating ability completely coincident with the entire footprint. Thus the actuation space for each layer is given by Fig. 9 Environment and robot models used to test the extended approach to any-shape robots A(r0 i , θj) = Li(r0 i , θj) ⊕T (θj) (14) (14) The actuation space gives the actuation capabilities for each orientation for a given robot shape and starting position. So, if a point belongs to A(r0 i , θj), then it can be actuated by the robot. We show in Fig. 12 the navigable and actuation spaces for different layers, given the robots and map shown in Fig. 9. After determining the actuation space for each layer, we can obtain the overall actuation map in a rotation-invariant Fig. 9 Environment and robot models used to test the extended approach to any-shape robots J Intell Robot Syst (2020) 98:165–190 178 PDDL problem. The 3D to 2D representation is transparent Fig. 10 Example of an image representing the robot footprint, rotated for three different angles, and used as structuring element in the morphological operations applied to the respective orientation layers; robot center shown in red representation by projecting the multiple layers into one single 2D image. PDDL problem. The 3D to 2D representation is transparent to the planning process. The navigate action moves through 3D waypoints, and the actuate action makes 2D waypoints have the actuated predicate. 8 Extending the Approach to Any-Shape Robots The list of goals to solve the prob- lem is still given by a list of 2D waypoints that cover all the space. Thus for the same map, the coverage problem is still the same in terms of goal waypoints and its model- lization, but now we plan for robots to move through the environment and actuate goal positions from some planned orientation. The PDDL domain did not need any further modifications. P(r0 i ) = θj A(r0 i , θj) (15) (15) P(r0 i ) has the same kind of representation we had with the circular robot, where the actuation map is a single 2D image not depending on the orientation. The actuation graph is now a graph of 3D waypoints connected to 2D waypoints, representing the actuation of a rotation-independent position in the projected 2D actuation map, from a 3D robot waypoint location, also shown in Fig. 13. The predicates on the PDDL problem are represented as follows: If we project the multiple layers of the graphs in a 2D image, we can analyze which waypoints are navigable in terms of the robot motion, and which ones are only feasible through an actuation action. As we show in Fig. 15, some of the waypoints are not feasible by any of the robots, and all the feasible waypoints lie inside the Actuation Space (grey region of the images). – Connected (robot, 3Dwaypoint, 3Dwaypoint) – Actuable (robot, 3Dwaypoint, 2Dwaypoint) – Actuable (robot, 3Dwaypoint, 2Dwaypoint) For each 2D waypoint in the circular robot scenario, there is now nθ 3D waypoints in the same (x, y) position, representing the different orientations a robot can have on the same 2D waypoint. As we show in Fig. 14, the two graphs are constructed independently of the initial position, allowing very easily to change the starting location of any robot and solve a different instance of the same problem. Thus, there were no modifications in the modeling of the 9 Experiments and Results In this Section we show the results of the experiments that were designed to test the impact of the preprocessing on two different versions of our algorithm, MAP. First, on the Fig. 11 Three adjancent layers of the discretized orientation, showing in blue the neighbor points of a central orange dot, representing the connectivity/motion model Fig. 11 Three adjancent layers of the discretized orientation, showing in blue the neighbor points of a central orange dot, representing the connectivity/motion model 179 enario describe the five scenarios designed to Then, on Section 9.2 the experiments lem are analyzed. These results were partially included on the previous version of the paper [29]. Finally, on Section 9.3 we show the results on the coverage problem including collision detection. 8:165–190 179 J Intell Robot Syst (2020) 98:165–190 enario Fig. 12 Navigable and Actuation Space for 2 non-circular robots with different sizes, for the scenario shown in Fig. 9 partially included on the previous version of the paper [29]. Finally, on Section 9.3 we show the results on the coverage problem including collision detection. following Section we describe the five scenarios designed to run the experiments. Then, on Section 9.2 the experiments on the coverage problem are analyzed. These results were 180 J Intell Robot Syst (2020) 98:165–190 Fig. 13 The connected and actuable graphs shown in blue and yellow, respectively; as shown for each layer, the yellow actuation graph connects 3D waypoints to the original 2D green waypoints, and the blue connectivity graph connects 3D waypoints not only to neighbors in the same layer, but also in adjacent layers 3 robots move within the horizontal sections, one in each, and their actuation reachabilities are mutually exclusive. Fig. 13 The connected and actuable graphs shown in blue and yellow, respectively; as shown for each layer, the yellow actuation graph connects 3D waypoints to the original 2D green waypoints, and the blue connectivity graph connects 3D waypoints not only to neighbors in the same layer, but also in adjacent layers 9.1 Simulation Description 15 All goal waypoints are shown as spheres on top of the Actuation Map: green represent unfeasible waypoints, in red the ones covered by the connected graph, and in blue the ones only covered by the actuable graph; for the smaller robot 1, the two graphs are the same in the top region, to only 1 being able to reach the bottom. – Extremities: wide open section with three halls depart- ing to different directions, where all 4 robots actuate; at the end of each hall there is a room that can be accessed through an opening, with only one robot reaching the extremity connected with the smallest opening, to three reaching the one connected with the biggest – Rooms: simple floorplan environment with some room- like spaces connected through passages of different sizes as well, used to test the non-circular robot case where they can traverse the passages using only certain orientations. Furthermore, in Table 1 we present the size of each map image, and the number agents and feasible and unfeasible l f h i i bl h id i Rooms: simple floorplan environment with some room- like spaces connected through passages of different sizes as well, used to test the non-circular robot case where they can traverse the passages using only certain orientations. in the top region, to only 1 being able to reach the bottom. in the top region, to only 1 being able to reach the bottom. – Extremities: wide open section with three halls depart- ing to different directions, where all 4 robots actuate; at the end of each hall there is a room that can be accessed through an opening, with only one robot reaching the extremity connected with the smallest opening, to three reaching the one connected with the biggest opening. – Extremities: wide open section with three halls depart- ing to different directions, where all 4 robots actuate; at the end of each hall there is a room that can be accessed through an opening, with only one robot reaching the extremity connected with the smallest opening, to three reaching the one connected with the biggest opening. Furthermore, in Table 1 we present the size of each map image, and the number agents and feasible and unfeasible goals for each scenario. We present in Table 1 the grid size Fig. 16 Maps of the five scenarios used in the experiments. Fig. 16 Maps of the five scenarios used in the experiments. Grey regions represent out-of-reach regions which cannot contain goal waypoints. They are unfeasible for all the robots. Robots are represented with blue circles positioned in the region of their starting position 9.1 Simulation Description Here we describe in detail the scenarios used for running the experiments. We designed five different scenarios, shown in Fig. 16, each one with two levels of waypoint density (H, the higher, and L, the lower density). The scenarios are designed for circular robots except for the last one (called Rooms) that is designed for any-shape robots. – Maze: maze-like scenario with narrow halls and passages with different sizes, resulting in bigger robots not reaching some parts of the maze, or needing to traverse bigger paths to arrive to the same locations as smaller robots. – Corridor: four wide sections with openings of different sizes connecting them; the opening decreases from the top to the bottom, with all 4 robots being able to actuate – Mutual Exclusive: three wide parallel horizontal halls, connected between them by two narrow vertical halls; Fig. 14 The discretized graphs constructed are independent of the ini- tial robot positions, allowing to run the problem from different initial positions; the white regions (navigable space, dependent on initial Fig. 14 The discretized graphs constructed are independent of the ini- tial robot positions, allowing to run the problem from different initial positions; the white regions (navigable space, dependent on initial position) are covered by the graphs, but some black regions (if grey in the respective configuration space, independent of initial position) are also covered by the constructed graphs position) are covered by the graphs, but some black regions (if grey in the respective configuration space, independent of initial position) are also covered by the constructed graphs Fig. 14 The discretized graphs constructed are independent of the ini- tial robot positions, allowing to run the problem from different initial positions; the white regions (navigable space, dependent on initial Fig. 14 The discretized graphs constructed are independent of the ini- tial robot positions, allowing to run the problem from different initial positions; the white regions (navigable space, dependent on initial position) are covered by the graphs, but some black regions (if grey in the respective configuration space, independent of initial position) are also covered by the constructed graphs J Intell Robot Syst (2020) 98:165–190 181 Fig. Fig. 15 All goal waypoints are shown as spheres on top of the Actuation Map: green represent unfeasible waypoints, in red the ones covered by the connected graph, and in blue the ones only covered by the actuable graph; for the smaller robot 1, the two graphs are the same 9.1 Simulation Description Grey regions represent out-of-reach regions which cannot contain goal waypoints. They are unfeasible for all the robots. Robots are represented with blue circles positioned in the region of their starting position Fig. 16 Maps of the five scenarios used in the experiments. Grey regions represent out-of-reach regions which cannot contain goal waypoints. They are unfeasible for all the robots. Robots are represented with blue circles positioned in the region of their starting position 182 J Intell Robot Syst (2020) 98:165–190 Table 1 Number of agents, feasible and unfeasible goals and respective grid size for each problem Scenario Agents Feasible Unfeasible Grid size CorridorH 4 819 118 49x19 CorridorL 4 384 92 33x13 ExtremeH 4 1993 1325 51x63 ExtremeL 4 896 589 34x42 MutExH 3 499 513 45x21 MutExL 3 223 242 30x14 MazeH 3 1389 154 38x38 MazeL 3 672 100 25x25 Rooms2r 2 192 52 13x13 Rooms10r 10 192 52 13x13 Extreme10r 10 1442 589 34x42 Maze10r 10 672 100 25x25 RoomsH* 2 835 182 28x28 RoomsL* 2 131 61 13x13 Scenarios are designed for circular robots except for those marked with (), where robots are any-shape Table 1 Number of agents, feasible and unfeasible goals and respective grid size for each problem Four different configurations of our approach have been set up: – MAP-LB-EC with estimated-cost information (EC). EC refers to the configuration that combines AMs and MAP using the Load-Balance strategy (LB). – MAP-BC-EC with estimated-cost information (EC), also combining AMs and MAP but using instead the Best- cost strategy (BC). – MAP-LB, same as before but without EC information. – MAP-BC same as before but without EC information. As it was mentioned in Section 5.3, the LB strategy helps to minimize the makespan metric. The BC strategy focuses on minimizing the plan length metric. We also run the problems without the preprocessing stage in order to evaluate our impact in terms of computation time and plan quality. Furthermore, the following state-of-the-art planners have been chosen as a comparison baseline: Scenarios are designed for circular robots except for those marked with (), where robots are any-shape – LAMA [30], centralized planer and winner of IPC 2011. – YAHSP [36], a greedy centralized planner and winner of the Agile track of IPC 2014. in terms of the down-sampled grid of waypoints. 2http://agents.fel.cvut.cz/codmap/ 9.1 Simulation Description The orig- inal image had a pixel size approximately 10 times bigger, with a pixel resolution corresponding to 10cm. Therefore, the maps we tested represent environments with a size always bigger than 300 square meters. – ADP [9], a multi-agent planner that performs a decomposition of the domain by following three indicators: (1) dependencies are reduced, (2) goals can be achieved independently and (3) coordination between agents is minimized. The aim is to find the most suitable agentification i.e. identify the agents, in order to divide the problem among the agents. We have generated two problems per scenario, one of them with low density of waypoints (which we identify as L in tables) and the other one with a higher density of waypoints (H). We have also designed versions of Maze, Extremities and Rooms for 10 robots in order to test the behavior of the planners in crowded scenarios. Rooms2r is a similar version of RoomsL but for circular robots. – SIW [26], a multi-agent planner that factorizes the problem into subproblems solving one atomic goal at a time until all atomic goals are achieved jointly. g j y – CMAP [3], a multi-agent planner that employs a centralized approach to solve the problem. For the experiments on this Section, the actuation model is always considered to be equal to the robot footprint. The Actuation Map determination was developed in C++. The three multi-agent planners that have been chosen participated on the 1st Competition of Distributed and Multi-agent Planners (CoDMAP2) and obtained good results on the final classification. 9.2 Experiments on the Coverage Problem ADP reached the memory limit in Maze when planning the solutions before the two hours limit. Even though ADP is a multi-agent planner, the effort of computing plans in big- size environments when all goals are assigned to all agents is very big. LAMA has the same issue as ADP because of its centralized approach (Maze, Extremities, RoomsH). From the set of planners that we chose to compare our approach, SIW is the one that obtains the best results. The maximum time spent on the preprocessing for any scenario was 170 milliseconds, for the Extremities problem with 4 robots. We included the preprocessing times (to generate the AMs) in the GA column of Table 2, and in the total time in Tables 3 and 6. Hardware used for running the planner was IntelXeon 3,4GHz QuadCore 32GB RAM. AMs were computed using a 2.5GHz DualCore 6GB RAM. Table 2 is shown to prove the remarkable impact that information from Actuation Maps (AMs) has in combination with MAP. Goal assignment (GA) times in Table 2 are minimal (MAP-LB-EC) in comparison with the ones of MAP-LB, where it needs to compute the relaxed plans for every goal-agent pair. Even though the individual planning and parallelization time for MAP-LB-EC is similar to MAP-LB, the time gains in GA completely dominate the overall planning time. Before running any problem, MAP performs a MAP compilation of the original problem to generate each agent’s individual problem after goals are assigned (M’ task). Usually this transformation takes seconds and it is included in GA time. However, we observed that given the size and complexity of any-shape scenarios (RoomsH and RoomsL), the compilation time increases considerably and becomes more than half of the time spent on solving the task. This phenomenom is marked with + in column Total time from Table 2. Regarding time results in Table 3, MAP-LB-EC is generally faster if all total times are summed up except in Maze. Also, the impact of combining information from AMs with MAP can be easily appreciated if columns from MAP-LB-EC and MAP-LB are compared. The same Table 4 shows the results regarding the plans’ length and Table 5 the results regarding makespan. 9.2 Experiments on the Coverage Problem Neither of these five planners perform a goal allocation phase separated from the planning process. Thus, we had to test them using equivalent PDDL problems that do not contain unfeasible goals. Also, in order to fairly compare the results of the makespan metric, we had to apply our parallelization algorithm to the resulting plans of ADP and SIW, as they only return the sequential plan. In this Section, we show some experiments that test the impact of the preprocessing in our approach. As it was previously said, we have modeled five different scenarios that include up to four agents with different sizes, and thus different actuation capabilities. Planning results are shown using as metrics the time in seconds, the length of the resulting plan and the makespan. In non-temporal domains, we refer as makespan the length of the parallel plan (number of execution steps, where several actions can be executed at the same execution step). Given that we are dealing with MAP tasks that almost have no interactions, it is expected that agents can execute their actions in parallel whenever possible. Before discussing the results on the tables we need to clarify that a maximum of two hours was given to each planner to solve each scenario. YAHSP results do not appear in the tables because it could not solve any of the scenarios. 183 J Intell Robot Syst (2020) 98:165–190 happens with BC configurations. Our two configurations MAP-LB-EC and MAP-BC-EC solved every problem. There is an exception in RoomsH, in which the parallel plan of both solutions could not be obtained in the remaining time before the 7200 seconds were reached. In general, the easiest scenario to be solved using planning is the Mutual Exclusive (MutExH, MutExL) because it is designed for each robot to traverse a mutual exclusive subset of waypoints. This is the reason why time results are very similar among all planners except for MAP-LB and MAP- BC where the planner needs to compute the relaxed plans for each pair robot-goal. However, CMAP had some trouble during planning in the high density scenario. The circular robot version of Rooms (Rooms2r) is also very easy to solve, even though the number of goals is higher than the any-shape version (RoomsL). If times from Rooms2r and RoomsL are compared, the complexity of just changing from circular to any-shape robots can be empirically appreciated. 9.2 Experiments on the Coverage Problem We have used the words timeout to indicate that a planner consumed the alloted time and could not return a solution, memlimit to Time (s) MAP-LB-EC MAP-LB Scenario Total GA Planning Parallel Total GA Planning Parallel CorridorH 88.07 0.90 68.34 18.83 1748.38 1672.46 58.83 17.09 CorridorL 13.37 0.29 10.54 2.54 484.48 179.19 303.28 2.01 ExtremH 639.54 4.53 427.91 207.10 timeout ExtremL 86.78 1.22 64.11 21.45 5491.00 5377.80 84.64 28.56 MutExH 10.59 0.44 7.36 2.79 1276.18 1265.62 7,78 2.78 MutExL 2.10 0.11 1.64 0.35 103.40 100.72 2.31 0.37 MazeH 1200.37 0.69 944.21 255.47 timeout MazeL 1179.21 0.24 1152.71 26.26 882.93 603.78 240.3 38.85 Rooms2r 0.19 0.05 0.05 0.09 8.31 5.70 1.85 0.76 Rooms10r 4.70 0.19 4.17 0.34 40.54 36.34 3.84 0.36 Extreme10r 93.79 2.97 68.89 21.93 timeout Maze10r 36.37 0.81 28.74 6.82 2121.11 2070.49 39.6 11.02 RoomsH 6180.37+ 9.85 1022.19 1922.87 timeout RoomsL 448.63+ 2.31 2.54 95.05 timeout From left to right total time, goal assignment time, individual planning time and parallelization time. Symbol + indicates that the MAP compilation time is very high From left to right total time, goal assignment time, individual planning time and parallelization time. Symbol + indicates that the MAP compilation time is very high J Intell Robot Syst (2020) 98:165–190 184 Table 3 Total time results in seconds Total Time (s) Scenario MAP-LB-EC MAP-LB MAP-BC-EC MAP-BC SIW ADP CMAP LAMA CorridorH 88.07 1748.38 304.91 1791.85 129.71 484.48 1761.82 95.45 CorridorL 13.37 484.48 33.78 203.00 10.98 85.97 187.57 22.74 ExtremH 639.54 timeout 642.04 timeout 1923.32* 439.58* timeout timeout ExtremL 86.78 5491.00 82.51 5547.00 156.71 402.15 91.24 72.07 MutExH 10.59 1276.18 10.55 1277.47 11.65 6.15 1277.89 6.93 MutExL 2.1 103.40 2.09 97.27 0.81 0.89 96.38 1.06 MazeH 1200.37 timeout 2718.96 timeout 429.87 memlimit 2575.24 2005.42 MazeL 1179.21 882.93 161.03 1213.26 37.92 memlimit timeout 334.8 Rooms2r 0.19 8.31 2.64 8.42 0.77 2.24 7.32 1.54 Rooms10r 4.7 40.54 4.18 36.06 2.26 2.04 34.21 2.55 Extreme10r 93.79 timeout 98.44 timeout 258.72 169.68 timeout 76.56 Maze10r 36.37 2121.11 29.63 2110.11 61.95 20.04 2099.74 29.7 RoomsH 6180.37* timeout 6120.28* timeout timeout timeout timeout timeout RoomsL 448.63 timeout 447.56 timeout 286.34 140.1 timeout 132.02 From left to right MAP with estimated-cost information in Load-balance (LB-EC); MAP without estimated cost information in LB; MAP with estimated cost information in Best-cost (BC-EC); MAP without estimated cost information in BC; ADP, SIW and CMAP are other multi-agent planners and LAMA is a centralized planner. 9.2 Experiments on the Coverage Problem Symbol * indicates that the planner solved the problem but parallelization over-passed the alloted time (7200s) Bold values represent which is the configuration that obtained the best score per scenario Table 3 Total time results in seconds From left to right MAP with estimated-cost information in Load-balance (LB-EC); MAP without estimated cost information in LB; MAP with estimated cost information in Best-cost (BC-EC); MAP without estimated cost information in BC; ADP, SIW and CMAP are other multi-agent planners and LAMA is a centralized planner. Symbol * indicates that the planner solved the problem but parallelization over-passed the alloted time (7200s) B ld l hi h i h fi i h b i d h b i Bold values represent which is the configuration that obtained the best score per scenario indicate that the planner’s memory limit was reached before timeout and parallel to indicate that the planner solved the problem but the parallelization algorithm could not return a solution in the remaining time to reach 7200s (SIW, ADP in ExtemeH; MAP-LB-EC, MAP-BC-EC in RoomsH). The best configurations overall regarding plan length are MAP-BC-EC and SIW. Moreover, MAP-LB-EC configuration is generally the best for reducing makespan. Configurations MAP-LB or SIW also obtain good results in specific scenarios. This issue can be explained by the discretization The best configurations overall regarding plan length are MAP-BC-EC and SIW. Moreover, MAP-LB-EC configuration is generally the best for reducing makespan. Configurations MAP-LB or SIW also obtain good results in specific scenarios. Table 4 Plan length: from left to right MAP with estimated-cost information in Load-balance (MAP-LB-EC); MAP without estimated cost information in LB; MAP with estimated-cost in Best-Cost (MAP-BC-EC); MAP without estimated cost information in BC; SIW, ADP, CMAP and LAMA (7200s) Bold values represent which is the configuration that obtained the best score per scenario 9.2 Experiments on the Coverage Problem This issue can be explained by the discretization Plan length MAP-LB-EC MAP-LB MAP-BC-EC MAP-BC SIW ADP CMAP LAMA CorridorH 1511 1512 1653 1556 1543 3545 1541 1471 CorridorL 727 692 784 791 699 627 746 748 ExtremH 3830 timeout 3830 timeout 3580* 8687* timeout timeout ExtremL 1715 1850 1715 1786 1627 2848 1659 1546 MutExH 658 658 658 658 758 773 658 658 MutExL 301 301 301 301 306 302 301 301 MazeH 3358 timeout 3004 timeout 2570 memlimit 1353 2686 MazeL 1599 1387 1434 1441 1236 memlimit timeout 1345 Rooms2r 142 333 313 315 303 398 302 319 Rooms10r 292 344 287 286 266 318 268 268 Extreme10r 1604 timeout 1627 timeout 1534 2692 timeout 1612 Maze10r 1480 1852 1308 1261 1202 1452 1224 1253 RoomsH 1403* timeout 1337* timeout timeout timeout timeout timeout RoomsL 370 timeout 336 timeout 366 627 timeout 370 Symbol * indicates that the planner solved the problem but parallelization over-passed the alloted time (7200s) Bold values represent which is the configuration that obtained the best score per scenario 185 J Intell Robot Syst (2020) 98:165–190 Makespan MAP-LB-EC MAP-LB MAP-BC-EC MAP-BC SIW ADP CMAP LAMA CorridorH 609 583 1353 862 973 1699 717 698 CorridorL 298 256 657 601 265 1073 444 452 ExtremH 905 timeout 905 timeout parallel parallel timeout timeout ExtremL 376 702 376 1091 450 1378 564 484 MutExH 117 117 117 117 149 155 117 117 MutExL 58 58 58 58 64 58 58 58 MazeH 1631 timeout 1631 timeout 1941 memlimit timeout 1369 MazeL 696 993 1035 1384 837 memlimit 1288 1217 Rooms2r 22 262 264 271 214 342 212 237 Rooms10r 47 55 57 54 59 73 67 67 Extreme10r 258 timeout 258 timeout 214 816 timeout 552 Maze10r 246 367 213 246 212 288 221 243 RoomsH parallel timeout parallel timeout timeout timeout timeout timeout RoomsL 240 timeout 242 timeout 180 510 timeout 177 Bold values represent which is the configuration that obtained the best score per scenario Bold values represent which is the configuration that obtained the best score per scenario errors from Eq. 9, which are greater when the downsampling rate is bigger. When allocating goals, the estimation costs are the only guide for the MAP algorithm. 9.2 Experiments on the Coverage Problem The consequence of having slightly inaccurate cost estimates results in the allocation of some goals to different agents than the ones that the estimated costs from the relaxation of plans would suggest. However, this issue does not have a big impact on makespan and plan length results. In Tables 6 and 7, we refer to MAP&R-LB-EC as running the Fig. 8 using the LB strategy. MAP&R-BC-EC runs the BC strategy instead. We have also compared our approach against the same set of planners as in Section 9.2. The maximum time for each planner to solve each scenario is two hours. YAHSP results are not shown in the tables as it was not able to solve any problem. The aim of this experiment is to analyze the impact of detecting and fixing collisions on makespan and time metrics. Plan length is not relevant on this experiment, as the difficulty relies on the planner’s ability to manage several agents and collision avoidance at the same time. That is a feature that directly affects the makespan result. From the set of planners chosen to compare our approach, SIW obtains the best performance on time, plan length and makespan. SIW is able to solve most of the scenarios due to its serialization of goals. The importance of factorizing a MAP problem is a conclusion that can be extracted after observing Tables 4 and 5, as the planners that do not perform factorization (LAMA, ADP, CMAP, YAHSP) have to solve bigger and more complex tasks. Regarding time results in Table 6, it can be seen that the number of problems solved decreases considerably. Also, time results have increased in all planners. This is due to the collision avoidance effect. On one hand, centralized approaches can deal with it more easily, as the master agent has the whole control of the agents. However, it is still facing the same issue as in the previous experiments: the tasks are harder to solve and now the search space is bigger. Regarding our configurations, MAP-BC-EC and MAP-LB- EC perform better in general than equivalent configurations without estimation costs. On the other hand, the lower the number of agents used to plan, the harder the planning task. Total time in BC is usually worse than in LB configurations on scenarios with higher density of waypoints and multiple robots to plan (CorridorH, MazeH). 9.2 Experiments on the Coverage Problem However, when scenarios are narrowed such as Extremities or Maze, the opposite effect can be given. When using less agents, the task is easier to solve. Our approach is halfway between the centralized and the distributed approach. The first part of our algorithm is distributed while the plan-reuse phase is centralized. Thus, the success of our algorithm depends on the number of collisions and the difficulty of solving them. LPG- ADAPT focus first on reutilizing the greater possible number of the actions from the invalid plan. When a collision is detected, LPG-ADAPT will search for a valid action on the part of the search space that is closer to the invalid action and its current planning state. This causes LPG-ADAPT to 9.3 Experiments Detecting Potential Collisions In this Section we show the results obtained on the same scenarios as in the previous Section but using instead the PDDL domain that detects collisions described in Section 7. J Intell Robot Syst (2020) 98:165–190 186 Table 6 Time in seconds from left to right MAP with estimated-cost information in Load-balance (MAP&R-LB- EC); MAP with estimated-cost in Best-Cost (MAP&R-BC-EC); SIW, ADP, CMAP and LAMA Total Time (s) MAP&R-LB-EC MAP&R-BC-EC SIW ADP CMAP LAMA CorridorH timeout timeout 263.82 timeout timeout 293.11 CorridorL 73.57 81.51 50.64 193.54 202.16 53.24 ExtremH timeout timeout timeout timeout timeout timeout ExtremL timeout timeout 752.5 timeout timeout timeout MutExH timeout timeout 7.61 9.01 1328.77 70.08 MutExL 3.25 3.35 1.35 1.06 104.45 3.25 MazeH timeout timeout 986.17 memlimit timeout timeout MazeL timeout timeout 67.34 memlimit 945.04 timeout Rooms2r 4.78 4.71 1.5 5.85 8.47 2.24 Rooms10r 7.11 7.12 4.86 3.51 40.42 9.62 Extreme10r timeout timeout timeout timeout timeout timeout Maze10r 406.75 timeout 466.03 memlimit 2196.02 560.40 RoomsH timeout timeout timeout timeout timeout timeout RoomsL 3406.21 3092.80 timeout timeout timeout timeout iteratively explore the search space starting from a very concrete section. The exploration distance will be increased as long as the valid action is still not found. This approach works well if the collision requires a small change to be fixed i.e. it only affects to a couple of navigation steps - the solution can be found near the search space of the action and current state. But if the way to avoid the collision affects to a bigger part of the plan i.e. robots have to move back several waypoints and change path directions; LPG-ADAPT might get lost on the search space, as it will try to search first on the space closer to the invalid action and the solution might be far away from there. Thus, timeout will be reached before a solution is found. Scenarios not solved by our approaches on Table 6 fail for that reason. On the other hand, Multi- Agent centralized approaches as SIW solve more problems. Next paragraphs contain a discussion on this aspect. We analyze why this particular situation is given with SIW even though MAP centralized approaches are generally worse in performance on big scenarios. SIW only solves one atomic goal at a time (serialization), which means that goals are not assigned to agents in the first step of the algorithm. The process is interleaved with search. Table 7 Makespan: from left to right MAP with estimated-cost information in Load-balance (MAP&R-LB-EC); MAP with estimated-cost in Best-Cost (MAP&R-BC-EC); SIW, ADP, CMAP and LAMA 9.3 Experiments Detecting Potential Collisions Thus, only one estimation is computed per iteration and current positions of the agents are updated after each goal is reached. Agents work individually but coordination (and thus, collisions) are checked after each iteration. The Makespan MAP&R-LB-EC MAP&R-BC-EC SIW ADP CMAP LAMA CorridorH timeout timeout 1289 timeout timeout 847 CorridorL 801 806 490 1378 452 424 ExtremH timeout timeout timeout timeout timeout timeout ExtremL timeout timeout 704 timeout timeout timeout MutExH timeout timeout 158 211 131 135 MutExL 87 87 80 80 101 95 MazeH timeout timeout 1061 memlimit timeout timeout MazeL timeout 1481 758 memlimit 1139 timeout Rooms2r 293 293 189 465 228 250 Rooms10r 61 61 61 107 77 54 Extreme10r timeout timeout timeout timeout timeout timeout Maze10r 293 timeout 432 memlimit 221 282 RoomsH timeout timeout timeout timeout timeout timeout RoomsL 250 250 timeout timeout timeout timeout J Intell Robot Syst (2020) 98:165–190 187 centralized approach followed by SIW is very efficient on the coverage problem. Collisions can be fully avoided because of solving first only one goal at a time and then updating robots’ positions. Thus, the algorithm obtains good results in number of problems solved and time. However, when the size of the problem increases, as in the any-shape scenarios, SIW has more difficulties to solve the problem in time. This scenario penalizes SIW because the search space is huge in comparison with circular robot scenarios. Planning one goal at a time following a centralized approach now becomes a worse choice. SIW has to usually deal with the following situation when collisions are given: a set of goals has been reached and the next goal on the list cannot be achieved unless the previous part of the plan is partially modified. In any-shape scenarios this aspect takes more time to fix given the size of the search space. true that we lost some performance on collisions, but we have empirically shown that it is also related to the topol- ogy of the scenario and the coverage problem itself. The unexpected advantage of SIW in the experiments detect- ing collisions has also been addressed and analyzed. The serialization of goals and the nature of the coverage problem where interactions are given occasionally, makes any cen- tralized (Multi-agent or Single-agent) planner to behave well. LAMA is closer to SIW and our approaches in that sense, and it is not even a MAP algorithm. 10 Related Work Multi-Agent Planning (MAP) is an active topic within the planning community as shown by the organization of the 1st CoDMAP3 and the wide range of planners that participated [32]. The planners vary from fully distributed to centralized among other features. Our approach uses first a distributed planner and if plan reuse is needed, a centralized phase. A MAP approach that uses a preprocessing step is the automated agent decomposition for multi-robot task planning [10]. In that work there is a preprocessing step, prior to actual search, that exploits decompositions of the problem in domains with a lower level of interaction, boosting the final performance. ADP [9] is also related to that work. Our approach factorizes the problem regarding goals and agents involved, creating independent subtasks for each agent before starting to plan. Regarding makespan results from Table 7, MAP&R-LB- EC and SIW are the two planners that obtain the best results - they also solve most of the problems. Although the RoomsL scenario might seem easier to solve by just looking at Table 1. However, as it works for any-shape robots, the grid of waypoints is bigger and harder to navigate from the planning point of view. The search space is very big and thus centralized approaches are especially penalized. The reason of failing on the Extremities and Maze scenarios is due to the changes on the robots’ paths caused by the collision avoidance or the topology of the scenario. Those scenarios contain narrower areas and large halls where only some robots can reach the end. Thus, robots might spend a lot of time looking for the correct path while at the same time avoiding the rest of the agents. The methodology that uses morphological operations in order to build the AMs was previously introduced by [27]. Moreover, it has recently been shown that similar transformation can be used to obtain Actuation and Visibility Maps for any-shape robots as well [28]. Earlier in 1997, Ambite and Knoblock [1] worked in a post-processing technique which rewrites some planning rules and local search techniques to make efficient the planning process. Thus, they obtained a low-cost plan for problems that were hard to solve from the point of view of planning. As a final conclusion, we would like to discuss the overall performance of our contribution. MAP-LB-EC and MAP-BC- EC clearly complement each other on the set of proposed scenarios. 3http://agents.fel.cvut.cz/codmap/ 9.3 Experiments Detecting Potential Collisions We also want to put in value the scalability of our approach. Through the experiments we have proved that we can successfully deal with different topologies, number of agents, agent’s ori- entation, huge planning tasks, unfeasible goals, independent goal-assignment and pre-processed estimation costs. State- of-the-art planners are not used to satisfy all these features at the same time. Regarding our approach, even though we obtain estima- tion costs through AMs, they are not as effective in guidance as SIW ’s serialization. The estimation of costs is provided to our approach at the beginning. If agents have to modify their route due to collisions, their estimations and assign- ment of goals might not be as useful as in the beginning. It can even penalize the agent’s performance. Also, if an agent needs to change its route several times, it could mean that the original assignment of goals is completely useless. Also, plan reuse planners are not efficiently prepared to perform an extensive search. They would rather prefer to reutilize actions from previous plans, which in the coverage problem results in generating redundant actions around the planning task. We have tried different features of LPG-ADAPT (low memory, speed mode, increasing different fixed constants...) to check if its performance could be improved but neither of them helped. 10 Related Work This is an advantage, as the algorithm can be eas- ily adapted to different situations and environments. It is Another similar problem is the Team Orienteering Prob- lem, where robots maximize the number of covered way- points visited, with constraints on the total travel time of 188 J Intell Robot Syst (2020) 98:165–190 each robot. In order to solve this problem and optimize the overall planning time, an algorithm was proposed that uses a three-tier graph, interleaving the search for optimal way- point assignment, ordering of the waypoints and also con- sidering feasible paths between waypoints while avoiding obstacles [33]. This algorithm guarantees optimal solutions, while in our work we focus on sub-optimal planning for multi-robot teams. The goals were distributed thanks to a goal-allocation algo- rithm and unfeasible goals identified and discarded from the planning task. Then, the planning task was factorized for each robot. They generate their individual paths that result in a maximal space coverage in terms of actuation. We also proposed a new version of the MAP algorithm that is able to fix agents’ interactions after the individual plan- ning phase. On the experiments we have designed a total of eight scenarios, seven for circular robots and one for any-shape robots, which is another contribution to the paper. Another relevant planning problem in robotics is inspection, which searches for robot paths that can perceive a set of sensing targets. Neural network approaches have been proposed [11], and again a preprocessing method was used, answering visibility queries efficiently both in 2D and 3D scenarios [21]. Here the queries ask for visibility from specific positions. In our work we preprocess the environment to find the overall actuation capabilities of the robot from any reachable position. Our approach greatly reduces the GA time, and because GA is one of the bottlenecks of MAP, we were able to also reduce the overall planning time when preprocessed information was provided to the MAP algorithm. The gains in performance depend greatly on the topology of the environment and the characteristics of each robot. Experiments also show that when solving big size multi-agent problems using planning, it is very helpful to first factorize the problem into subtasks. Thus, the total planning time will be smaller than when trying to solve the whole problem at once. Also, factorization is essential when working on problems that explicitly involve agents’ interactions. 10 Related Work Experiments on collision avoidance show the importance of task factorization and the topology of the scenario in order to successfully fix collisions. There are similar environments to our problem defined in previous planning domains. One it is called VisitAll, whose aim is to visit all the waypoints presented in the problem by just navigating through them. It was used for the first time on 7th IPC.4 In our domain we added an actuation action (with an associated actuation range), which was not considered in the VisitAll domain. Another similar problem is the Rovers domain, but in this case each agent (rover) can execute a bigger set of actions when it is placed on a waypoint. Some other examples were mentioned in the Introduction. As future work, we would like to extend the preprocess- ing technique to other domains and consider different -robot or agent- models. Our approach can be easily extended to path planning tasks or real-time strategy videogames. We gave some examples of the former such as surveillance tasks or search and rescue tasks. The latter domain could be inter- esting when designing bots that play automatically. Our approach could improve the player/bot performance when extracting information from the map to decide which goals are more relevant to achieve first. We also want to improve the performance of fixing interactions. Plan reuse works well when collisions only affect to a couple of actions. For biggest plan modifications plan-reuse is not enough. When robots with many degrees of freedom execute successive motions in the same environment, it usually requires many complex planning instances. By applying a preprocessing technique, it is possible to improve efficiency of path planning for those robots [22] Finally, a very common robotic application, coverage path planning, has been widely studied [16]. 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In: Proceedings of the CoDMAP-15, p. 4 (2015) 10. 11 Conclusions and Future Work In this paper we showed how to combine information from Actuation Maps (AMs) with Multi-Agent Planning (MAP) to solve a multi-robot path planning problem more effi- ciently skipping the computation of estimated cost during planning. We used AMs in a preprocessing step to deter- mine the feasibility of pairs robot-goal and to extract an estimated cost. That cost is used later to avoid the com- putation of relaxed plans during Goal-Assignment. The environment map was discretized into a grid of waypoints. Acknowledgements This work has been partially funded by FEDER/ Ministerio de Ciencia, Innovaci´on y Universidades - Agencia Estatal de Investigaci´on/TIN2017-88476-C2-2-R and MINECO/TIN2014- 55637-C2-1-R. I has been also financed by the ERDF – European Regional Development Fund through the Operational Programme for Competitiveness and Internationalisation - COMPETE 2020 Pro- gramme within project <<POCI-01-0145-FEDER-006961>>, and by National Funds through the FCT – Fundac¸˜ao para a Ciˆencia e a Tec- nologia (Portuguese Foundation for Science and Technology) as part of project UID/EEA/50014/2013, and FCT grant SFRH/BD/52158/2013 through Carnegie Mellon Portugal Program. 189 J Intell Robot Syst (2020) 98:165–190 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. 18. 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He has been the Program Co-chair of the International Conference of Automated Planning and Scheduling (ICAPS’13), Conference co-chair of the Symposium of Combinatorial Search (SoCS’12, SoCS’11) and ICAPS’06, and PC member (including Area chair and Senior PC) of the main conferences on Artificial Intelligence (e.g., IJCAI, AAAI, ICAPS, ...). His current research interests lie in goal reasoning, Multi-agent Planning, and Machine Learning applied to Planning. Nerea Luis is a PhD student at the Universidad Carlos III de Madrid. Her thesis is about combining multi-agent planning and plan-reuse to easily adapt to different scenarios. In 2016, she was a visitor student at Manuela Veloso’s CORAL group for 4 months in Carnegie Mellon University (USA). Her current research interests are multi- agent planning, machine learning and robotics. She was awarded with the Women Techmakers scholarship by Google. 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Vidal, V.: YAHSP3 and YAHSP3-MT in the 8th international plan- ning competition. In: In 8th International Planning Competition (IPC-2014), pp. 64–65 (2014) 190 J Intell Robot Syst (2020) 98:165–190 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Daniel Borrajo received his Ph.D. from Universidad Polit´ecnica de Madrid (1990). References degree in ”Logic, Text and Information Technology” (University of Dundee, Scotland, 1991). She worked for 10 years as a software engineer in two private companies, INDRA and Eliop, S.A. She received the Ph.D. degree in Computer Science (Universidad Carlos III de Madrid, 2006). Her current research interest is in Artificial Intelligence, particularly automated planning, machine learning and multi-agent systems. She has participated in several competitive projects related to automated planning, problem solving or agents. She has published more than 20 papers. Ant´onio Moreira is currently an Associated Professor in Electrical Engineering at University of Porto, developing his research within the Robotic and Intelligent Systems Centre of INESC TEC (Centre Coordinator). He graduated with a degree in Electrical Engineering from the University of Porto in 1986. He then pursued graduate studies at the University of Porto, completing a M.Sc. degree in Electrical Engineering - Systems in 1991 and a Ph.D. degree in Electrical Engineering in 1998. From 1986 to 1998 he also worked as an assistant lecturer in the Electrical Engineering Department of the University of Porto. His main research areas are Process Control and Robotics.
https://openalex.org/W4213044763	https://www.cambridge.org/core/services/aop-cambridge-core/content/view/5BE1BF645DED21B3010332B763EBDE73/S0014479721000314a.pdf/div-class-title-measuring-the-value-of-data-governance-in-agricultural-investments-a-case-study-div.pdf	English	null	Measuring the value of data governance in agricultural investments: A case study	Experimental agriculture	2,022	cc-by	11,672	Keywords: Economic evaluation; value creation; data governance Keywords: Economic evaluation; value creation; data governance © The Author(s), 2022. Published by Cambridge University Press. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution and reproduction, provided the original article is properly cited. Experimental Agriculture (2022), vol. 58, e8, 1–20 doi:10.1017/S0014479721000314 RESEARCH ARTICLE RESEARCH ARTICLE Measuring the value of data governance in agricultural investments: A case study Damian Whittard1,* , Felix Ritchie1 , Ruthie Musker2 and Michael Rose2 1Bristol Business School, University of the West of England, Bristol BS16 1QY, UK and 2Centre for Agriculture and Bioscience International, Nosworthy Way, Wallingford, Oxfordshire OX10 8DE, UK Corresponding author. Email: damian2.whittard@uwe.ac.uk 1Bristol Business School, University of the West of England, Bristol BS16 1QY, UK and 2Centre for Agriculture and Bioscience International, Nosworthy Way, Wallingford, Oxfordshire OX10 8DE, UK Corresponding author. Email: damian2.whittard@uwe.ac.uk (Received 28 July 2021; revised 08 December 2021; accepted 28 December 2021) (Received 28 July 2021; revised 08 December 2021; accepted 28 December 2021) (Received 28 July 2021; revised 08 December 2021; accepted 28 December 2021) https://doi.org/10.1017/S0014479721000314 Published online by Cambridge University Press Summary The study at hand measures the value of improving data governance and access in the Supporting Soil Health Interventions (SSHI) project in Ethiopia. We applied two separate but interlinked models, one qualitative and one quantitative, to create a new framework enhancing the traditional cost–benefit analysis. The qualitative analysis provided novel insights into the specific types of value and the mechanisms through which they are generated. These results underpinned the development of an innovative framework to measure this perceived value quantitatively. By combining the quantitative and qualitative framework, the study demonstrated that it is possible to generate plausible and credible quantitative estimates of both costs and benefits of data governance and access. While acknowledging that the estimates are only illustrative, the case study results suggested on a direct cost measure, at a particular point in time, the SSHI data governance activities yielded a negative return. However, indirect social and public benefits are rarely quantified, but this paper shows that relatively few “indirect” benefits (current but unmeasured, or measurable but in the future) are necessary to reverse that view, at least from the point of the economy more generally. Keywords: Economic evaluation; value creation; data governance Introduction Improved data governance and access has the potential to transform the ways we live and work (Ritchie, 2021; KPMG-Atkins, 2021). However, without accurate methods for measuring its value and understanding the mechanisms through which this value is created, the likelihood is that investment in this area will be sub-optimal. This is not a straightforward problem: data gover- nance is an intangible concept that is woven across data management; understanding the value of good data governance requires a breadth and depth of information. g g q p The challenges are heightened for agriculture based in rural communities where traditionally data infrastructure is slower to roll out. This can be further compounded in low- and middle-income countries (LMICs), where national and regional statistical infrastructures are under development. In 2019, an attempt was made to measure the value of improving data governance and access in the GIZ-led, Bill & Melinda Gates Foundation-funded Supporting Soil Health Interventions (SSHI) project in Ethiopia (see Whittard et al., 2021). In order to do so, the study applied two separate but interlinked models to create an innovative framework that provided new insights into how and where value is created. The Five Safes data governance model, widely used in data management but not in evaluation, was used to structure a qualitative investigation. The interviewees identified both tangible and https://doi.org/10.1017/S0014479721000314 Published online by Cambridge University Press Damian Whittard et al. 2 intangible outcomes that should be measured as part of the wider economic and social benefits. This qualitative design was then used to structure a more traditional quantitative case study. This is an important step forward as social and public benefits of data governance have rarely been identified and quantified, particularly in developing countries. Such evidence can then help inform policy, shaping direction and economic growth of the country. By combining the quantitative and qualitative framework, the study demonstrated that it is possible to generate plausible and credible quantitative estimates of both costs and benefits of data governance and access. This study describes the new approach, the challenges faced by the project, and the lessons learned from implementation. Specifically, we This study describes the new approach, the challenges faced by the project, and the lessons learned from implementation. Specifically, we • Develop a theoretical framework for estimating the value of data governance and access in agricultural projects in developing countries. https://doi.org/10.1017/S0014479721000314 Published online by Cambridge University Press Introduction • Develop a theoretical framework for estimating the value of data governance and access in agricultural projects in developing countries. g p j p g • Demonstrate the application of the framework by evaluating the SSHI project in Ethiopia, while identifying the mechanisms through which value is created. • Demonstrate the application of the framework by evaluating the SSHI project in Ethiopia, while identifying the mechanisms through which value is created. The paper concludes by suggesting some potential next steps in order to move this nascent process forward. The paper concludes by suggesting some potential next steps in order to move this nascent process forward. Desk research on previous attempts of valuing data governance and data access A review of research material revealed that the benefits of improving data governance and access are widely acknowledged (European Open Data Portal, 2017; OECD, 2019; HM Treasury, 2018a). For example, McKinsey (2013) reported that open data had the potential to increase global GDP by $3 trillion annually, while Lateral Economics (2014) suggested that more open data could pro- vide over half the G20’s 2% growth target. Although providing exact estimates is challenging, even in developed economies with well-developed statistical systems, the evidence suggests that nations can make considerable economic gains by improving data governance and access. To estimate these gains, economic evaluations normally tend to take the form of a cost– benefit analysis (CBA), following a standard structure (HM Treasury, 2018b). To undertake a detailed CBA, the evaluation needs considerable amounts of well-defined, measurable, high quality data; all of which can be converted to a market price (Baker, 2000; Raitzer, 2003; Whittard et al., 2021). In data related projects, this is potentially problematic. Data, data governance, and data access are intangible in nature and therefore difficult to define and measure (Corrado et al., 2009). Wdowin and Diepeveen (2020) argue that data are neither a good nor a service, are not homoge- nous and, depending on the nature of the data, can be viewed as a “public good” making estimat- ing a market price all the more challenging. Even if it were possible to clearly define and therefore measure all concepts, a robust evaluation would need to be supported by a complex system of data collection, both within and external to the project. The experience of most economic evaluation experts, however, is that the planning for such evaluation starts too late and is carried out too soon after project completion for outcomes and impacts to manifest (Belmana, 2018). Wdowin and Diepeveen (2020) conducted a wide-ranging review to inform understanding of how value of data is generated and what approaches can be taken to measure this value. They concluded that it was important to identify the value for the different stakeholders across the value chain. Bergemann and Bonatti (2018) discuss the importance in market structure (e.g. monopoly, oligopoly, competitive) and resulting pricing strategy in understanding how the value will be shared along the value chain. Experimental Agriculture 3 A number of different approaches have been used to measure value. The value of agriculture projects in LMICs CBA techniques have been used extensively to estimate the economic value of agriculture sector projects in LMICs (Baranchuluun et al., 2014; Gotsch and Burger, 2001; Lemaga et al., 2005; Mittal, 2018; Nkang et al., 2009; Sain et al., 2017). These studies, however, were focused on the value of the project itself and provided no insight into the value of improved data governance and data access or how this was created. Although not specifically focused on improved data governance, there are a number of explicit studies on information, communication and technology (ICT) type projects in LMICs. For exam- ple, Lio and Liu (2006) model aggregate production functions across multiple countries and argue that ICT does provide a positive return on investment (albeit lower in LMICs than in high-income countries). Ngwenyama and Morawczynski (2009) investigate macroeconomic effects of ICT investment in Latin America, using a variety of techniques including econometrics models. They find that the effectiveness of such investment is strongly determined by the existing eco- nomic conditions and infrastructure at the time of the investment. There is also evidence from detailed studies that look at data explicitly. For example, GODAN (2018) used data flows to structure their conceptual analysis of the opportunities from improved data access. GODAN (2016) notes, however, the exporting of data can cause concerns for the farmer who sees potential commercial gains for others rather than his or her own benefit. Regardless of outcome, Carletto et al. (2015)’s detailed study argues that there is a fundamental problem to be considered before the impact of improved data access can be established; this is the lack of quality agricultural data. q y g Overall, the desk research revealed that deriving the value of improved data governance and access is a very challenging, particularly in the absence of a dominant typology. The challenges appear to be amplified in LMICs demonstrated by the lack of work published on the value of data governance and access, part of which may be related to the difficulty in accessing evaluation data. The study attempts to fill this gap by developing and testing a new framework to estimate the value of improved data governance and access. Desk research on previous attempts of valuing data governance and data access PWC (2019) propose that the cost approach can give useful upper and lower valuation bounds. Mawer (2015), however, suggests that data should be viewed as an intangible asset from which an income-based valuation approach can be taken based on estimating future cash flows. A key challenge with this approach is the difficulty of estimating a service length life of such an intangible, and therefore rate of depreciation. The third and most common approach is to use market-based data to approximate value; this can be achieved by either directly using a market price or indirectly taking the market price of an equivalent product. This again can be problematic due to the characteristics of the data. However, what is clear from the litera- ture is that most estimates of valuation are economic and market-based, and there is limited discussion on the social and public evaluation of data (Wdowin and Diepeveen, 2020). https://doi.org/10.1017/S0014479721000314 Published online by Cambridge University Press Defining terms and project selection To collect information on the value of data governance and access, the first task was to agree terminology. A workshop between funders and researchers approved the working definitions: Data governance: • a framework for assigning decision-related rights and duties in order to be able to adequately handle data as an asset (Otto, 2011, p.47). • a framework for assigning decision-related rights and duties in order to be able to adequately handle data as an asset (Otto, 2011, p.47). Value: Value: • The extent to which the project delivered value by meeting its commitments (e.g. project outcomes in relation to cost savings/investments). • The extent to which the project delivered value by meeting its commitments (e.g. project outcomes in relation to cost savings/investments). • The additional value to users. • The additional value to other stakeholders. The second task was to be able to identify which project would make the most appropriate case study. Following an iterative process where various Gates Foundation-funded projects were con- sidered and pilot interviews completed, the SSHI project was selected. The overall objective of the project was to provide proof of concept for the impact of leveraged geo-spatial soil, agronomic, and health data on transformative agricultural development. The specific aims of the project were to: • increase the quality, efficiency, and reach of government and private sector services; • improve national and regional resource allocation and policies; and • increase the quality, availability, and utility of data and evidence. In order to meet the aims, the project was further broken down into six work packages: • WP 0. Conduct an inception phase to analyze data needs and bottlenecks, assess gaps for the “Use Cases,” and mobilize stakeholders. • WP 1. Craft a policy for stakeholders across organizations to share data and collaboratively develop solutions to easing Ethiopian soil system bottlenecks. • WP 2. Provide technical and operational support for, and facilitate inter-institutional con- sultation needed to establish, the Ethiopian Soil Resource Institute as the national IT host for a centralized data repository. y • WP 3. Help convert the Ethiopian Soil Information System (EthioSIS) into a geo-spatial database with online access to serve users from a variety of sectors and with differing needs. • WP 4. Develop IT-assisted use cases to provide proof of concept for the impact of geo-spatial soil, agronomic, or health data. • WP 5. Methodological approach There were three methodological stages to the study There were three methodological stages to the study • Define key terms and project selection • Define key terms and project selection ○Review of project documentation • Define key terms and project selection ○Review of project documentation ○Review of project documentation • Qualitative data collection: I t i ( il t fi t d • Qualitative data collection: ○Interviews (pilot, first and • Qualitative data collection: ○Interviews (pilot, first and second round) ○Interviews (pilot, first and second round) • Quantitative data collection ○Questionnaire. https://doi.org/10.1017/S0014479721000314 Published online by Cambridge University Press Damian Whittard et al. Damian Whittard et al. 4 https://doi.org/10.1017/S0014479721000314 Published online by Cambridge University Press Defining terms and project selection Link up and collaborate with several currently active investments that have comple- mentary initiatives relevant to soil health, fertilizer use efficiency, and transformative agri- cultural development. The final task was to isolate activities undertaken in the SSHI project that were (primarily) focused on improved data governance and access. The final task was to isolate activities undertaken in the SSHI project that were (primarily) focused on improved data governance and access. The final decision on which activities were in scope was taken by the research team following workshops with two partners (i.e. Centre for Agriculture and Bioscience International (CABI) and the Open Data Institute), review of documentation, and feedback from the pilot interviews. Of the six project work packages, Table 1 lists the three work packages and associated activities within each work package that were identified as within scope. https://doi.org/10.1017/S0014479721000314 Published online by Cambridge University Press Experimental Agriculture 5 Table 1. Work packages and activities Work package 0: Inception phase • Activity 1 – Inception workshop • Activity 2 – Sub-grant agreement Work package 1: Craft policy to share data to ease Ethiopian soil system bottlenecks • Activity 1 – Open data sensitization workshop • Activity 2 – Coalition of the willing (CoW)1 • Activity 3 – Workshop on data access and sharing Work package 2: Provide support to establish national IT host for a centralized data repository • Activity 1 – Organizational structure stakeholder consultation workshop • Activity 2 – Human capacity and development • Activity 3 – International exposure visits Source: project documentation. The team reviewed traditional methods for economic evaluation but decided that these fell short for two reasons. First, there was insufficient existing data to carry out a statistical study. Second, there was considerable uncertainty on how to identify the specific governance effects. After alternative methods were explored, the review team proposed a novel solution based upon a two-stage process: a qualitative review employing an innovative structure, which would then inform data collection for a quantitative study using techniques that are more conventional. 1During the open data sensitization workshop, a CoW was created to take forward the idea of enhanced accessibility and sharing of Ethiopian agronomic data and information. The CoW initially consisted of 26 participants from 17 major Ethiopian research institutions. https://doi.org/10.1017/S0014479721000314 Published online by Cambridge University Press Qualitative data collection Qualitative data collection was structured using the “Five Safes” framework. Table 2 provides a brief description of the five safes, with a fuller description on the framework available via the sup- plementary information section. The Five Safes is a recognized framework widely used to structure data governance planning; however, to date it has not been formally applied in evaluations. The framework was chosen to structure discussion as it is designed to allow data governance to be seen as a series of separable but inter-related operating dimensions. p g The initial structure was developed and sent to CABI and its partners for review ahead of its formal application in the interviews. Following feedback, the framework was refined ahead of the first round of interviews. Initially, a pilot semi-structured interview was completed with the two project managers to identify key themes. Within the initial interview, the participants were questioned on an under- standing of the framework. As an initial framework, it elicited little opinion one way or the other, the framework was adopted for all subsequent interviews. A snowballing sample methodology was used to identify further key personnel to interview. Following the pilot, four subsequent interviews with six individuals were completed (two in round one and two in round two). All interviews lasted approximately an hour and were digitally recorded and transcribed with permission from the respondents. The aims of the interviews were: • To test the suitability of the Five Safes as a framing mechanism To test the suitability of the Five Safes as a framing mechanism y g in a deeper understanding of the mechanisms and nuances by which value had been • To gain a deeper understanding of the mechanisms and nuances by which valu created through improved data governance and access in a deeper understanding of the mechanisms and nuances by which value had been d through improved data governance and access • To structure the subsequent quantitative data collection. 1During the open data sensitization workshop, a CoW was created to take forward the idea of enhanced accessibility and sharing of Ethiopian agronomic data and information. The CoW initially consisted of 26 participants from 17 major Ethiopian research institutions. https://doi.org/10.1017/S0014479721000314 Published online by Cambridge University Press Damian Whittard et al. Damian Whittard et al. 6 Table 2. Qualitative data collection The basic Five Safes framework Safe projects The overall aim of the project, intended outcomes, data management, and ethics Safe people The role of people and procedures in effectively using data securely Safe settings The role of IT in facilitating use and protecting data Safe data Identifying the appropriateness level of data detail to balance confidentiality and usefulness Safe outputs Ensuring outputs are valuable while protecting confidentiality Source: adapted from Desai et al. (2016). Table 2. The basic Five Safes framework Table 3. Generic theory of change Term Definition Inputs Resources used to deliver the project Activities What is delivered to the recipient Outputs What the recipient does with the activity Intermediate outcomes The intermediate outcomes of the project produced by the recipient Impacts Wider economic and social outcomes Source: HM Treasury’s Magenta Book (2007). Resources used to deliver the project What is delivered to the recipient What the recipient does with the activity The intermediate outcomes of the project produced by the recipient Wider economic and social outcomes Figure 1. Project specific theory of change. Source: authors’ model. Figure 1. Project specific theory of change. Source: authors’ model. https://doi.org/10.1017/S0014479721000314 Published online by Cambridge University Press Quantitative framework As in a standard CBA, the quantitative evaluation was to be based around developing a project specific theory of change (see Table 3). This would have to be tailored to the project context, depending on who is the recipient in the project context. As in a standard CBA, the quantitative evaluation was to be based around developing a project specific theory of change (see Table 3). This would have to be tailored to the project context, depending on who is the recipient in the project context. The mapping process involves identifying the start- (the challenge) and the endpoint (the over- all objective), and then building a pathway between the two. It should also be tailored to the proj- ect context (i.e. who are the specific beneficiaries). In the absence of a sufficiently detailed theory of change to evaluate each work packages/activity, Figure 1 provides an example of a theory of change created by the project team, to understand how capacity-building investments in human capital could lead to improved downstream economic impacts. p p p This CBA framework was used to guide the approach used to estimate the impact quantitatively. Costs are measured as the financial cost of funding the work package/activity. Benefits relate to measurable outputs, outcomes, and impacts observed from the work package/activities. However, given the limited availability of data generated within the project in relation to the value of data governance and access, it was recognized that any quantitative evaluation would be partial and at best illustrative. https://doi.org/10.1017/S0014479721000314 Published online by Cambridge University Pre Experimental Agriculture 7 Figure 2. Combining the framework. Source: authors’ model. Figure 2. Combining the framework. Source: authors’ model. To improve the quality of the data collection process, the results from the first round of inter- views, which were structured on the Five Safes framework, were mapped against the theory of change developed for each sub-project. To improve the quality of the data collection process, the results from the first round of inter- views, which were structured on the Five Safes framework, were mapped against the theory of change developed for each sub-project. Mapping the two distinct but interlinked frameworks (Five Safes and CBA) provided a more comprehensive overview of the potential impacts of each of the projects/activities and the mech- anisms through which the value is created. Quantitative framework This then helped shape data collection by guiding the remaining interviews and informing the development of the questionnaire. This iterative process, illustrated in Figure 2, ensured that the quantitative and qualitative approaches both informed and were informed by both analytical frameworks. A detailed questionnaire was developed to gather quantitative data on the potential value of data governance. To limit respondent burden, questionnaires were personalized: questions were only asked of respondents on the parts of the project for which they had prior knowledge. Participants were informed any information provided would be confidential, with all question- naires coded for analysis and then destroyed. Results were only reported at the aggregate level. The questionnaire was sent out to 12 experts with knowledge of particular aspects of the pro- gramme; eight were returned. While recognizing that this is a small sample, given that all who were surveyed were experts in at least some aspect of the project, including one project manager, the quality of the data collected was expected to be high. The experts were asked to respond to all questions in their personalized questionnaire, even if uncertain about the estimates. To account for different levels of certainty, the questionnaire asks how confident the respondents are in the accuracy of the answers. This was used to weight any aggregate estimates when combined with other responses. Given the potential language and cul- tural challenges (the questionnaire was written in English but would be completed by mainly Ethiopian experts) the following additional support was provided: • A guide created on how to complete the questionnaire, including worked exam • Questionnaire piloted and supporting guide reviewed by CABI staff in UK • Full questionnaire and support documents reviewed by CABI Ethiopian staff ahead of circulation • You Tube video created with an overview of the project, its aims and instructions for com- pleting the questionnaire • CABI Ethiopian staff provided support on the ground to the experts completing the questionnaire. Safe projects Challenges The full value of the project would only be achieved in the long term: For example, the inter- views revealed that the genesis of the project went back to when the government was preparing its 10-year Soil Health and Fertility Improvement Strategy. In particular, the interviewees identified three main issues that needed to be addressed: • absence of comprehensive datasets to develop evidence-based strategy • absence of comprehensive datasets to develop evidence-based strategy • data collected through public funds remained with researchers and were not necessaril shared • data were collected in different formats, for different purposes, and quality was not consistent. The inference is that, at least from the government perspective, the full value of the project will only be achieved once all three issues are resolved. Value is multifaceted and means different things to different people: The interviews revealed that there was no agreed definition of value and therefore how it should be measured. What was clear, however, was that stakeholders interpreted this concept to be multifaceted and much wider than just financial and economic. Much of the value is generated by intangible mechanisms that do not lend themselves to quantification: For example, interviewees believed that considerable value was being generated due to a change in culture. The new culture that the project was encouraging individuals and organizations to exchange data and consider a more formal process for doing so. It is challenging to set the boundaries of where value is created appropriately: The clearest example of this was when stakeholders identified spill-overs beyond the scope of the original proj- ect, particularly in relation to the wider agriculture sector. The inference is that the indirect value could be considerably larger than the direct value of the project itself. Institutional challenges: Political and institutional instability, delays caused by institutional arrangements, and a stakeholder engagement plan that was too narrow were all cited as potential challenges to maximizing value. For example, there was some evidence of dissatisfaction in plan- ning delays as a result of debates about whether to include data for the whole of the agricultural sector. Eventually, it was agreed that policies were to be limited to the agronomy sector, allowing for lessons learned to be applied later to the broader agricultural sector. https://doi.org/10.1017/S0014479721000314 Published online by Cambridge University Press Safe projects Challenges Value and ethics: In order to maximize the value of this project, while ensuring the project adheres to the highest ethical standards, it is important that there is engagement with a diverse set of stakeholders. The challenge with stakeholder engagement is that it is time consuming and potentially costly. The evidence here, however, is that with more time and with wider engagement, particularly in relation to regional policymakers, practical and ethical concerns can be identified and addressed in order to maximize value. Results: qualitative study The results from the interviews are summarized below under the heading of the Five Safes and are discussed in terms of challenges, best practice, and then implications for quantitative assessment. https://doi.org/10.1017/S0014479721000314 Published online by Cambridge University Press https://doi.org/10.1017/S0014479721000314 Published online by Cambridge University Press Damian Whittard et al. 8 Challenges g Targeted engagement: Interviewees commented on the need to identify and work with high rank- ing officials to champion the project but also highlighted the importance of identifying key “block- ers.” These had the potential to become “powerful advocates for the policy itself” once their issues were addressed. Attendance of the “right people” from donor organizations and international project partners was important in building momentum and attracting a diverse set of local stakeholders. National and regional governance: The issue of federalism was a recurring issue within the interviews. The regional structure in Ethiopia is very important both institutionally and culturally. Training would not only be needed at the national level, but there would also need to be training focused on upskilling researchers in managing local datasets; these can then be combined to piece together the regional and national pictures. Implications for quantitative assessment Implications for quantitative assessment • Recognition that any quantitative assessment will be limited as only certain aspects of value can be quantitatively measured. • Recognition that any quantitative assessment will be limited as only certain aspects of value can be quantitatively measured. q y irect impact of value will need to be tightly defined. y • The direct impact of value will need to be tightly defined. • The direct impact of value will need to be tightly defined. • Indirect impacts should be included while recognizing they may be inaccurately estimated • Indirect impacts should be included while recognizing they may be inaccurately estimated. • The timing of the evaluation means that only the short-term value can be accurately measured. • Indirect impacts should be included while recognizing they may be inaccurately • Indirect impacts should be included while recognizing they may be inaccurately estimated. • The timing of the evaluation means that only the short-term value can be accurately measured. • The timing of the evaluation means that only the short-term value can be accurately measured. • Attempts should be made to quantify the contribution of both tangible (e.g. training events/ workshops) and intangible (e.g. trust) mechanisms that contribute to overall value. This observation is only listed here but is equally supported by the findings from other domains (safe people, safe settings, safe data). • Attempts should be made to quantify the contribution of both tangible (e.g. training events/ workshops) and intangible (e.g. trust) mechanisms that contribute to overall value. This observation is only listed here but is equally supported by the findings from other domains (safe people, safe settings, safe data). https://doi.org/10.1017/S0014479721000314 Published online by Cambridge University Press Best practice Legacy effects: An unexpected spill-over effect was that the informal coalition created had a life beyond the project and was able to contribute to the evidence base needed to address other con- temporary challenges such as the Covid 19 pandemic. This illustrates the difficulty of ascertaining value; a solution to improve data governance in the agronomy sector of Ethiopia found a substan- tial secondary purpose supporting the wider economy. Building a diverse coalition: The project was able to build a coalition of international, national, regional, and local stakeholders which safeguarded that the project adhered to the high- est ethical standards. Experienced government and non-government stakeholders ensured that the correct skills, knowledge, and levels of influence were in place to deliver the project, while https://doi.org/10.1017/S0014479721000314 Published online by Cambridge University Press Experimental Agriculture Experimental Agriculture 9 9 combining experienced international and local institutions ensured that learning from previous projects was also taken into consideration. combining experienced international and local institutions ensured that learning from previous projects was also taken into consideration. Best practice l Local ownership and empowerment: One significant development in the project was the forma- tion of the “Coalition of the Willing” (CoW). Initially, this included 26 participants from 17 major research institutions in Ethiopia; over time, this grew to approaching 100 members. This was set up by researchers to inform the development of the data sharing policy, demonstrating the value of sharing by doing so. This had the direct effect of creating local ownership of the project, improving communication between stakeholders, ensuring buy-in for the project goals, and pro- viding a forum for momentum about the project to be generated. Top-down, bottom-up: The project also drove forward the “top-down” development of the Soil and Agronomy Data Sharing Policy in partnership with the Ethiopian government; tight feed- back loops between these approaches benefited the project. It also helped that the CoW fed directly into the government taskforce. Indeed, several individuals were members of both. This “bottom-up” and “top-down” approach is mutually reinforcing, in a dual approach of incentive- and-sanction working in tandem. The incentive demonstrates what can be achieved when working together; this gives impetus for the sanction (national data sharing policy) to be developed, to encourage those not actively participating. The CoW in particular contributed to the “bottom- up” approach by driving forward localized data sharing initiatives. It worked because there was a clear incentive for individuals to take part, inasmuch as they would be given access to other members’ data. oi.org/10.1017/S0014479721000314 Published online by Cambridge University Press 10 Damian Whittard et al. Building trust: The necessity of building trust (especially between international organizations and local stakeholders), forming strong relationships, and empowering stakeholders was clearly key to developing and potentially implementing the policy. To address long-held cultural and social norms (e.g. in Ethiopia, historically information had been shared as a result of personal relationships rather than through any systematic process), it was important to empower stake- holders to develop their own solutions. Co-created solutions were enabled through an agile and flexible approach to managing the project. The project team also had a significant role in building capacity through the identification of skills gaps and then co-developing materials and workshops to fill such gaps. g Upskilling: Much of the focus on training was for the long-term sustainability of the project which would ensure that individuals were trained to use data effectively and securely. Best practice l In order to do this, the project identified key individuals who could be trained up, who in turn would be able to share the knowledge within their institutions and with future generations. Implications for quantitative assessment g Challenges g Development of culture and systems: The principle of appropriate access was central to the development of the policy. Safe settings are often considered a technical or process matter. However, the evidence here pointed to the need for this to be accompanied by a cultural shift in relation to data access in Ethiopia. Prior to the commencement of the project, data sharing was often only advanced by the use of official letters to request that data be made available for a particular purpose. This process potentially created bottlenecks, particularly for those data- sets where this level of formality may not be required. The evidence was that this project had shifted perceptions of at least some organizations. p p g Supporting framework conditions: The interviewees commented that investment in technol- ogy could automate data sharing efficiently and securely. However, some questioned the scope for automation and suggested the need for further investment in people and skills to build capacity to support and administer the technical solutions. Within the Ethiopian setting, the evidence sug- gested that investment in technology was not the problem, but the relevant framework conditions (e.g. legal, institutional, procedural) require adaptation. https://doi.org/10.1017/S0014479721000314 Published online by Cambridge University Press Implications for quantitative assessment • Potential to estimate the value regionally as oppose to just nationally: given the importance of the regional structure, additional benefit could be generated if regional estimates of the value were provided (but given the limited data this was judged to be beyond the scope of this study). • Potential to estimate the value regionally as oppose to just nationally: given the importance of the regional structure, additional benefit could be generated if regional estimates of the value were provided (but given the limited data this was judged to be beyond the scope of this study). Best practice Technology: Within the project, interviewees suggested that the technology was either already available within Ethiopia or easy to acquire. As such, they stressed the need to look beyond the technical solutions. The suggestion was that in future, the role for CABI in Ethiopia would be to provide advice around infrastructure, connectivity, and the minimum requirements to enable data sharing in line with policy and procedures. Knowledge transfer: One way to develop a safe model of delivery is to learn from other insti- tutions who have similar data handling needs. In support of this aim, the discussion pointed to Members of CoW visiting the Bangladesh Soil Resource Development Institute to learn how it uses a safe setting to manage restricted-access data. Experimental Agriculture Experimental Agriculture 11 Implications for quantitative assessment Implications for quantitative assessment Implications for quantitative assessment • Technology is a considerable cost and can be directly measured, but complementary human skills may be harder to assess. • Technology is a considerable cost and can be directly measured, but complementary human skills may be harder to assess. • There are potentially significant indirect costs in relation to developing the supporting framework conditions; however, these have been judged to be beyond the scope of this analysis. • There are potentially significant indirect costs in relation to developing the supporting framework conditions; however, these have been judged to be beyond the scope of this analysis. Challenges Expertise in data governance: The interviewees highlighted the fact that much of the success of the project was driven through local empowerment; however, much of this expertise was in soil and data science, rather than data governance and access. As such, it was highlighted that it was important to bring in additional (international) expertise to help upskill capacity in relation to the safe management of data. Supporting framework conditions: The supporting framework conditions were highlighted as requiring modification. For example, the lack of consistency in data standards was considered a barrier to sharing data. However, there was some progress on developing harmonized standards and protocols; an example was cited of core treatments being standardized across a number of individual experiments. Some highlighted a lack of trust in institutions, people, and systems as additional barriers; others identified legal and technical issues, while the federal institutional setup posed its own additional challenges. The interviewees recognized the need to shift toward a culture of more open data, and it was suggested that, as a result of the project and its wide engagement, there had been progress in this area. p g Shared understanding: Lack of clarity over terminology was identified as a limitation, partic- ularly around the different dimensions of data (e.g. accuracy, completeness, consistency, timeli- ness, validity, etc.). This prompted suggestions to develop a clear language and clear definitions. This would include key concepts such as what is an identifier and what variables have statistical value. Using internationally accepted frameworks, such as findable, accessible, interoperable, re- usable data, can help develop a shared understanding that can be applied in multiple contexts. Interviewees also pointed to the lack of clarity over those datasets that should be funded, collected, and maintained nationally, compared to those that are only required and collected locally. Being able to clearly identify the former would be helpful. https://doi.org/10.1017/S0014479721000314 Published online by Cambridge University Press Best practice l Local ownership and empowerment: Local empowerment and ownership were highlighted as important factors. For example, a data sharing task force was formed with the primary objective of developing “informal” guidelines for data access and sharing. It was hoped that the task force could add value to the project by advising on issues around data quality, accessibility, and gover- nance procedures, while the group was also seen as being an important influence on the national soil and agronomy data sharing policy. Implications for quantitative assessment nforcing the need for clear definition of terms and agreement on what is/is not measurab Safe outputs Challenges Best practice Top-down, bottom-up: Once again, the top-down, bottom-up approach was recognized as being important for this domain. The interviews discussed a dual approach using both incentives and sanc- tions. Promoting the value of safe outputs can act as the incentive to complement the legal sanction to enforce compliance from those yet to be convinced by the incentive. Interviewees stressed the importance of being able to demonstrate the value of how data collected at a local level can be combined into impor- tant national datasets. For example, workshops were used to present the results of using large datasets. Implications for quantitative assessment • Research outputs (i.e. papers) are directly measurable. • Research outputs (i.e. papers) are directly measurable. • Where possible, it is important to measure activities in relation to developing human capital (e.g. training events/workshops). • Where possible, it is important to measure activities in relation to developing human capital (e.g. training events/workshops). (e.g. training events/workshops). Input costs: data gathered from financial reports The costs presented in Table 4 provide a high-level overview of the inputs for: (1) the CoW and (2) activities undertaken to support the establishment of a national IT host for a centralized data repository (SIRM). Given the uncertainty with the coverage of the data, these input costs should be seen as indicative. Input costs of activities in relation to improving data governance and data access in the SSHI project were approximately US$640 000. To return a positive net benefit, total benefits would need to exceed this figure. Safe outputs g Human capital development: Experience in confidentiality protection was mixed; some reported work on implementation being well advanced, while others suggested that progress was less https://doi.org/10.1017/S0014479721000314 Published online by Cambridge University Press Damian Whittard et al. 12 developed, as they were considered second-order issues. This is potentially an issue, as ensuring that outputs are safe and confidentiality maintained is hugely important to build trust in the process. Given the sensitivities of working with different types of data, it is important for clear data standard controls to be set and enforced. Training in disclosure checking was not practiced, but there was support for training to maximize the value of statistical analysis. Much of this was “on the job” rather than formal training. https://doi.org/10.1017/S0014479721000314 Published online by Cambridge University Press Results: quantitative study The following section details the results of a CBA. In this section, we use this framework to illus- trate how assessments may be made, generating some exploratory estimates as part of the illus- tration (for full details of assumptions made, see Supplementary Table S2). Input costs: data gathered from financial reports Figure 3. Direct internal value by activity. Source: authors’ calculations. Figure 3. Direct internal value by activity. Source: authors’ calculations. 2DIV = estimates of direct internal value; Ps = number of people attending event, by sector (six); HRs = hourly wage rate, by sector; A = number of activities attended; H = hours of attendance. Benefits: data gathered from questionnaire Output: direct internal value p Estimates of direct internal value (DIV) are calculated by multiplying the number of activities (A) undertaken (e.g. workshops) by the number of people from each sector (broken down by six sec- tors) who have engaged with the event (Ps), hourly wage rate for the sector (HRs), and length of time in engagement, measured in hours (H). The assumption that underlies this value is that the expected value from the event is at least as high as the cost of the individual attending the event (measured in this case by paid-for hours). This may be an overestimate as: (1) individuals may be under pressure to attend, (2) individuals are attending to claim “per diem” expenses, (3) individ- uals may not consider the cost to their employer of attendance, and (4) the real value gained may be systematically lower/higher than the direct measure calculated. For example, using this meth- odology activities with large numbers of participants are always recorded as being of greater value than events with small numbers of participants, regardless of the outcome. Experimental Agriculture 13 Equation 1: Direct Internal Value2 DIV X 6 PSs × HRs × A × H Table 4. High level breakdown of input costs, November 2018–July 2020 Expense category Amount USD Capacity building – training $272,253.09 Capacity building – facilities (IT facility for ESRI/SIRM) $157,115.14 Consultants $145,839.49 Workshops $47,648.28 Travel $13,186.81 Grand Total $636,042.82 Source: GIZ. Figure 3. Direct internal value by activity. Source: authors’ calculations. Experimental Agriculture 13 Table 4. High level breakdown of input costs, November 2018–July 2020 Expense category Amount USD Capacity building – training $272,253.09 Capacity building – facilities (IT facility for ESRI/SIRM) $157,115.14 Consultants $145,839.49 Workshops $47,648.28 Travel $13,186.81 Grand Total $636,042.82 Source: GIZ Table 4. High level breakdown of input costs, November 2018–July 2020 https://doi.org/10.1017/S0014479721000314 Published online by Cambridge University Press Equation 1: Direct Internal Value2 DIV X 6 s1 PSs × HRs × A × H Benefits arise from added value over and above that directly funded by the project. The total DIV was therefore allocated between those on the project payroll (labeled as funded) and those external to the project (labeled as unfunded). Benefits arise from added value over and above that directly funded by the project. The total DIV was therefore allocated between those on the project payroll (labeled as funded) and those external to the project (labeled as unfunded). Figure 3 records the DIV of nine activities measured. In total, it shows that they generated over US$353 000 DIV. Over 95% of the DIV was added value to the project as it accrued to individuals external to the project – this is shown by the orange (unfunded) segment of the graph, while 54% of the total overall benefit was generated by just one activity – CoW. Output: indirect internal value 3NTS = net time saved; TSw = time saved, weighted average; ETNw = extra time need, weighted average; AHW = average hourly wage (AHW); HWs = hourly wage, by sector. *All weightings are apportioned by the level of confidence recorded by each respondent in the accuracy of their estimate. Output: indirect internal value Indirect internal value (IIV) was estimated through asking project experts (including some, but not limited to, those individuals interviewed as part of the qualitative framework) to judge how an https://doi.org/10.1017/S0014479721000314 Published online by Cambridge University Press Damian Whittard et al. Damian Whittard et al. 14 Table 5. Indirect internal value Time saving (days) Extra time needed (days) Net time saving (days) Value of time saved (US$) Inception workshop 141 – 141 9,610 Sub-grant agreement 65 – 65 4,453 Open data sensitization workshop 60 – 60 4,082 Coalition of the willing 142 10 132 8,980 Consultation workshop on data access and sharing 105 – 105 7,175 Data sharing task force 84 – 84 5,745 Organizational structure stakeholder consultation workshop 60 – 60 4,082 Human capacity and development 60 – 60 4,082 Total 719 10 709 48 209 Source: authors’ calculations. Table 5. Indirect internal value activity affected the delivery of subsequent activities within the project. They were asked to esti- mate the time saved and/or extra time needed of individuals working on the project as a result of each activity. For example, the experts judged that if the inception workshop had not been held, other work packages would have taken longer to complete – this was estimated to be the equiva- lent of one person working 141 days. activity affected the delivery of subsequent activities within the project. They were asked to esti- mate the time saved and/or extra time needed of individuals working on the project as a result of each activity. For example, the experts judged that if the inception workshop had not been held, other work packages would have taken longer to complete – this was estimated to be the equiva- lent of one person working 141 days. Respondents were then asked to attribute this increase or decrease to one of seven potential reasons for the time saving and/or extra time needed (i.e. trust, champions, blockers, culture, data governance knowledge and skills, consistency of data and other), identified in the qualitative research. https://doi.org/10.1017/S0014479721000314 Published online by Cambridge University Press https://doi.org/10.1017/S0014479721000314 Published online by Cambridge University Press Output: indirect internal value n 2: Indirect Internal Value3 Equation 2: Indirect Internal Value3 NTS TSw ETNw AHW X 6 s1 HWs 6 IIV NTS × AHW IIV NTS × AHW IIV NTS × AHW Due to a lack of responses to this particular question, there were insufficient data to generate IIV estimates for International Exposure Visits; estimates for all other activities are presented in Table 5. Due to a lack of responses to this particular question, there were insufficient data to generate IIV estimates for International Exposure Visits; estimates for all other activities are presented in Table 5. Every category contributed to an indirect time saving, in total equivalent to 719 person-days. The single largest net contributor was the inception workshop, demonstrating the importance of this event in engaging the local community to support the project. The experts were asked to apportion their estimates of the time saved to the mechanism through which it was most likely to occur. Figure 4 provides proportions of the time saved (719 days) attributed to the different mechanisms. As such, the total for all activities totals 100%. In support of the importance suggested in the qualitative interviews, working with “Champions” was judged to accounted for over a quarter (28%) of the IIV time saving estimated in Table 5 – this equated to nearly 200 days. Change in the culture toward more open data and an increase in trust between stakeholders were estimated to account for 22% (156 days) and 15% (106 days) of the IIV, respectively. What is of particular interest about these two indicators is that any https://doi.org/10.1017/S0014479721000314 Published online by Cambridge University Press positive change in culture and trust would be expected to extend past the length of the project and h f l i Thi ill f h diffi l i f h Figure 4. Weighted average of the proportion of time saved allocated to the reason for the time saving. Source: authors’ calculations. Table 6. Publications produced Guidelines Policy report Mapping report Journal article Not specified Total Sub-grant agreement/CoW 2 1 1 15 19 Data sharing task force 1 1 2 Source: authors’ calculations. Experimental Agriculture 15 Experimental Agriculture 15 Table 6. Publications produced Table 6. Publications produced Guidelines Policy report Mapping report Journal article Not specified Total Sub-grant agreement/CoW 2 1 1 15 19 Data sharing task force 1 1 2 Source: authors’ calculations. Output: indirect internal value Total cost and benefits concept, mean that the estimates here should only be considered as illustrative. Given these cav- eats, Table 7 summarizes the following results. concept, mean that the estimates here should only be considered as illustrative. Given these cav- eats, Table 7 summarizes the following results. The case study results suggested that, on a direct cost measure, the SSHI data governance activ- ities yielded a negative net return of US$234 542 (column 3). These figures reflect the monetized direct measurable net benefit for the project. The net return, however, does not include non-monetized benefits (column 4–5), including indirect benefits and longer term spill-over effects. In fact, Whittard et al. (2021) in the full SSHI evaluation report that inclusion of relatively few un-monetized benefits is sufficient to gen- erate a positive return, for example, valuing academic publications (column 4) on cost basis. During the qualitative interviews, the respondents indicated that the overall impact of improved data governance and access was likely to generate wide-ranging spill-over impacts, potentially way beyond the soil and agronomy sector. Given the challenges of estimating a mone- tary value for these, the research team limited their analysis to estimating a long-term impact score based on likelihood and potential impact (column 5) – for more detail see Whittard et al. (2021). Any score over 6.25 indicates a positive spill-over is likely and/or will be impactful, and the strength of the likelihood and/or impact is related to the size of the estimate (25 being the maxi- mum score). Therefore, the results reported in column 5 indicate substantial positive spill-over effects, both within the soil and agronomy sector, but also on the wider economy itself. This would be a pure gain for Ethiopian society from the project if the direct benefit were positive or close to zero. https://doi.org/10.1017/S0014479721000314 Published online by Cambridge University Press Output: indirect internal value Figure 4. Weighted average of the proportion of time saved allocated to the reason for the time saving. Source: authors’ calculations. Figure 4. Weighted average of the proportion of time saved allocated to the reason for the time saving. Source: authors’ calculations. positive change in culture and trust would be expected to extend past the length of the project and therefore generate longer-term impacts. This illustrates one of the difficulties of assessment where structural change is concerned. Intermediate outcomes: external tangible value Given the need for the evaluation to be proportionate and the limited availability of data, the (tangible) outcomes were limited to just measuring publications produced as a result of these activities (some of which will be published in the special edition of Experimental Agriculture). The strength of this approach is that the output is tangible and measurable, and can be directly attributed to the activity. A further step could attempt to monetize the value of the publications, but, given the limited information available on the quality of each publication at the time the evaluation was completed, the authors judged that any such estimation approach would lack validity. As well as not directly monetizing the value of the output, the main weakness with this approach is that it does not capture the full indirect outcomes (e.g., publications leading to changes in policy and practice which result in the generation of additional value). Therefore in terms of a traditional CBA, this approach is an underestimate of the true benefit (assuming outcomes are generally positive). g y p ) Table 6 lists the 21 publications produced as identified by the experts. The challenges in defining and accounting for data governance and access elements of the SSHI projects, and the difficulties of quantifying and monetizing what potentially is an unquantifiable Damian Whittard et al. 16 Table 7. Total cost and benefits Activity Monetized benefits (columns 1–3) Non-monetized additional benefit (columns 4–5) Cost (US$) Benefit (US$ and net time saved) Net cost/ benefit (US$) Additional benefit (number of publications) Long-term impact score (likelihood multiplied by impact) Input: project expenses 636 042 Output: internal direct value 353 291 Output internal indirect value 48 209 (709 days) Intermediate outcomes 21 Impacts: soil and agronomy sector 17 Impacts: wider agricultural sector (excluding agronomy sector) 12 Impacts: other sectors 7 Total 636 042 401 500 - 234 542 21 – Source: authors’ calculations. Table 7. Discussion This report has described both a framework for the evaluation of data governance and the results of applying that framework to a real-world project. The development of the framework was an iterative process, with multiple evaluation structures tried and rejected before the final framework was agreed. The framework was most useful in providing a structure for qualitative data collection. In this phase, the use of experts was essential. Those individuals had in-depth knowledge and so were best placed to provide quantifiable estimates as required. As the experts are likely to have a vested interest in the success of the schemes being evaluated, they may have an incentive to https://doi.org/10.1017/S0014479721000314 Published online by Cambridge University Press 17 Experimental Agriculture over-emphasize the benefits and underplay the costs. In practice, the approach taken attempted to limit this effect as much as possible by asking experts to provide “contextual and factual” infor- mation: what happened, what led to something else, what stopped something happening. This information then allowed the evaluation team to draw the conclusions about costs and benefits. In addition, to encourage open and honest answers the authors expended considerable effort prior to interviews to reassure the experts about the nature of the evaluation: that this was a genuine attempt to explore understand and explain, rather than an exercise in monitoring. The quantitative data analysis followed a more conventional “theory of change model” approach, but with a conscious decision to limit outputs to what was sensible and meaningful to measure. Evaluations need to make much use of assumption and extrapolation, because mea- surement data are missing or impossible to monetize. This is particularly true when the outputs are intangible, the project generates spill-over (indirect) effects, or the benefits are likely to arrive in the future. For evaluating the impact of data and data governance, all three of these challenges are present. p As such, in this type of evaluation the authors could have kept identifying, and monetizing, additional potential benefits, until the end result of the evaluation becomes positive. This would not have necessarily been duplicitous, especially when there is no clear limit as to what is in and out of scope, but inevitably there would be much uncertainty as to the quality of the estimate. Discussion Apart from appeasing funders and certain stakeholders, this approach can be criticized as it pro- vides very little useful information in terms of lessons learned; however, the supportive qualitative approach does this. For this project, the decision to limit the quantitative evaluation to “measurable” items was a conscious choice. This yielded the finding that the SSHI project did not appear to generate a posi- tive return on the investment. The evaluation also identified a very large number of benefits that were not deemed measurable; with a few assumptions, a positive overall return could be generated. By not taking this last step, the evaluation effectively says “These are observable benefits and cost we could identify, and the direct gain or loss; these are additional effects which generate additional but unmeasurable benefits; the choice of whether the project is currently worthwhile can now be considered with reference to this evidence.” This seems a more honest approach when the outputs and impacts are intangible, diffused across society, dynamic, and persistent over time. Moving from a definitive “number” to represent the value of the investment, toward a more nuanced evaluation of what the investment has produced, has one further advantage. It may reduce the incentive for boosting benefits and downplaying costs, as the “value” no longer rests solely on measurable effects. This may also lessen the impact of optimism bias, by placing more stress on interpreting results rather than taking them as definitive. https://doi.org/10.1017/S0014479721000314 Published online by Cambridge University Press Next steps In this paper, we have focused on developing a new methodological approach to the valuation of data governance. Although we did develop specific results, in some ways this project can be seen as a “proof-of-concept.” We propose two measures in particular that would help to develop a prac- tical tool for evaluation and implementation of good data governance. The first is the improved collection of effective information to inform the evaluation. It is a standard lament of every evaluation, that there is “not enough data,” and that more should be done to design in the collection of evaluation data from the project start (Alves et al., 2021). Often this can just mean considering “what inputs/outputs can we measure and cost?” However, this project has demonstrated the limitations of focusing on measurable metrics, the importance of recognizing the limits to measurement and the value of understanding context. We propose the use of expert opinion early on to inform and direct the evaluation strategy, and to identify what is usefully measured and what is important but perhaps unmeasurable. oi.org/10.1017/S0014479721000314 Published online by Cambridge University Press 18 Damian Whittard et al. Second, the Five Safes was originally designed to create a simple language and framework for thinking about data governance decisions. Embedding this into strategic planning would encour- age decision-makers to build on established tools and good practices which use the framework, as well as providing a ready-made structure for evaluation. The use of a recognized framework may also help to encourage trust between data partners as governance arrangements become more transparent. p Third, although the focus of the paper was primarily about developing and testing a framework to estimate the value of data governance and access in agriculture projects in developing countries, by applying the framework to the SSHI project in Ethiopia, inevitably we are also able to comment on the potential to drive this process forward. p p Within Ethiopia, it is clear that there is a sizeable amount of agriculture data, and improved coordination and access to it has the potential to enhance evidence-based research which in turn can lead to better policy decisions. Our evidence shows that building trust between government bodies and researchers is, and will continue to be, central to the success of any such initiative. Next steps Therefore, as well as its usefulness for evaluation purposes, the authorities may wish to consider building capacity regarding the implementation of Five Safes framework. This framework has been adopted worldwide as it is engenders trust by creating an environment to maximize the use of detailed data, while keeping data secure at all times. In terms of operationalizing the Five Safes framework, best practice suggests that the government may wish to establish a review board to evaluate data request applications from potential researchers. This should be supported by the development of survey design and data collection guidelines, in order to ensure the collec- tion of high quality data which is consistent across different surveys. By focusing on improving the coordination between data collection groups, this has the potential to standardize data sources, reduce data redundancy, and increase efficiencies. Indeed, the CADRE project (McEachern, 2021) provides detailed guidance on this. https://doi.org/10.1017/S0014479721000314 Published online by Cambridge University Press Conclusion Work on valuing data and data governance is still in its infancy, and there is no agreed or domi- nant typology. Data governance impacts are intangible: likely to generate many spill-overs; likely to have more effects in the long term than the short; and likely to be very dependent on how they interact with the rest of society. However, this study demonstrates that a novel mixed-methods approach combining a traditional CBA framework with qualitative framing can generate insight- ful and meaningful results. The case study of SSHI demonstrates the new approach is workable, even if there is much to be done on refining it. The use of the Five Safes provided some consistency with the wider data governance literature, but the main value of it was providing a structure for the qualitative review. The expert input, gleaned from interviews, allowed the evaluation team to identify the events and activities that had an impact, and to generate the structure for the quantitative analysis. Throughout both qualitative and quantitative analysis, the emphasis was on identifying impacts, outcomes, and causes, rather than measuring them. This is a break from the traditional cost–benefit approach, where having a final value of whether an investment generates a positive return is the prime driver. However, when dealing with an activity as nebulous as data governance, there is value in limiting the reliance on quantitative analysis and increasing the emphasis on identifying exactly what has happened without the need to monetize it. Some of the challenges of traditional evaluations remain: timing (too late to start data collec- tion, too early to identify benefits), optimism, and other biases, the need to make strong assump- tion even when limiting oneself to measurable activities. The estimates generated for this report are exploratory and tentative, being based on a small number of data points, highly subjective answers, and some very broad assumptions about costs and benefits. Nevertheless, we believe they 19 Experimental Agriculture Experimental Agriculture provide a useful illustration of how something as nebulous as the return on investments in data governance may be measured. There is almost no literature on this to date – which means, amongst other problems, that there are no other studies to compare our findings with – but we expect this will open up opportunities for future research in this area. Conclusion One important finding is the importance of working with organizations, teams, and individuals who are open, prepared for challenge, and ready to explore many pathways, many of which lead nowhere. This approach was embraced by the various teams and individuals involved with the process as we attempted to identify suitable projects and frameworks to test before alighting on our chosen approach. Supplementary Material. For supplementary material for this article, please visit https://doi.org/10.1017/S0014 Acknowledgements. The authors would like to thank all the stakeholders who participated in the development of the evalu- ation framework and assisted in its application. In particular, we would like to thank the many experts who invested their valuable time to take part in the interviews and surveys. Financial Support. This study was supported by the Centre for Agriculture and Bioscience International (CABI) and the Bill & Melinda Gates Foundation. Conflicts of Interest. None. References Alves K., Tava F., Whittard D., Green E., Beata Kreft M. and Ritchie F. (2021). Process and Economic Evaluation of the ODI R&D Programe. London: Open Data Institute. Baker J.L. (2000). 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HM Treasury (2007). The Magenta Book: Guidance Notes for Policy Evaluation and Analysis. London: HM Treasury (Magenta Book Background Papers). HM Treasury (2018a). The Economic Value of Data: A Discussion Paper. Available at https://assets.publishing.service.gov.uk/ government/uploads/system/uploads/attachment_data/file/731349/20180730_HMT_Discussion_Paper_-_The_Economic_ Value_of_Data.pdf (accessed 30 April 2020). HM Treasury (2018b). The Green Book: Central Government Guidance on Appraisal and Evaluation. London: HM Treasury (2018b). The Green Book: Central Government Guidance on Appraisal and Evaluation. London: HM Treasury. KPMG-Atkins (2021). The Value of Information Management in the Construction and Infrastructure Sector. Available at https://www.cdbb.cam.ac.uk/files/cdbb_econ_value_of_im_report.pdf (accessed 20 July 2021). KPMG-Atkins (2021). The Value of Information Management in the Construction and Infrastructure Sector. Available at https://www.cdbb.cam.ac.uk/files/cdbb_econ_value_of_im_report.pdf (accessed 20 July 2021). Lateral Economics (2014). References Open for Business: How Open Data can Help Achieve the G20 Growth Target. Redwood City: Omidyar Network. https://doi.org/10.1017/S0014479721000314 Published online by Cambridge University Press Damian Whittard et al. 20 Lemaga B., Sekitto I., Nsumba J., Natabirwa H., Namutebi A., Owarwi C. and Tindiwensi C. (2005). Cost Benefit Analysis of Sweetpotato-Based On-Farm Enterprises in Central Uganda. Kampala: PRAPACE (The Regional Network for Improvement of Potato and Sweet potato in East and Central Africa). Lio M. and Liu M.-C. (2006). ICT and agricultural productivity: evidence from cross-country data. Agricultural Economics 34, 221–228 Mawer C. (2015). Valuing Data is Hard, Silicon Valley Data Science Blog Post. Available at https://www.svds.com/valuing- data-is-hard/ (accessed 29 October 2020). McEachern S. (2021). 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Information Technology for Development 15, 237–258. Nkang N.M., Ajah E.A., Abang S.O. and Edet E.O. (2009). Investment in cocoa production in Nigeria: a cost and return analysis of three cocoa production management systems in the Cross River State cocoa belt. African Journal of Food Agriculture, Nutrition and Development 9, 713–727. g p OECD (2019). Enhancing Access to and Sharing of Data: Reconciling Risks and Benefits for Data Re-Use Across Societies. Paris: OECD Publishing. Otto B. (2011). Organizing data governance: findings from the telecommunications industry and consequences for large ser- vice providers. Communications of the AIS 29, 45–66. PWC (2019). Putting a Value on Data. Available at https://www.pwc.co.uk/data-analytics/documents/putting-valueon-data. pdf (accessed 29 October 2020). Raitzer D.A. (2003). Benefit-Cost Meta-Analysis of Investment in the International Agricultural Research Centres of the CGIAR. Prepared on Behalf of the CGIAR Standing Panel on Impact Assessment. Available at https://cgspace.cgiar.org/ handle/10947/197 (accessed 30 April 2020). Ritchie F. (2021). Microdata access and privacy: what have we learned over twenty years? Journal of Privacy and Confidentiality 11, 1–8. https://doi.org/10.29012/jpc.766 (accessed 10 February 2021). Cite this article: Whittard D, Ritchie F, Musker R, and Rose M. Measuring the value of data governance in agricultural investments: A case study. Experimental Agriculture. https://doi.org/10.1017/S0014479721000314 https://doi.org/10.1017/S0014479721000314 Published online by Cambridge University Press References Sain G., Loboguerrero A.M., Corner-Dolloff C., Lizarazo M., Nowak A., Martínez-Barón D. and Andrieu N. (2017). Costs and benefits of climate-smart agriculture: the case of the Dry Corridor in Guatemala. Agricultural Systems 151, 163–173. Wd i J d Di S (2020) Th V l f D t C b id B tt I tit t f P bli P li Wdowin J. and Diepeveen S. (2020). The Value of Data. Cambridge: Bennett Institute for Public Polic Whittard D., Ritchie F. and Nyengani J. (2021). Measuring the value of improving data governance and access. Supporting soil health interventions in Ethiopia. A Bill & Melinda Gates Foundation investment case study. Gates Open Research 5, 97. https://doi.org/10.21955/gatesopenres.1116786.1 Cite this article: Whittard D, Ritchie F, Musker R, and Rose M. Measuring the value of data governance in agricultural investments: A case study. Experimental Agriculture. https://doi.org/10.1017/S0014479721000314
https://openalex.org/W2536083757	https://storage.googleapis.com/jnl-sljo-j-sljs-files/journals/1/articles/8116/submission/proof/8116-1-28625-1-10-20150526.pdf	English	null	Neonate with congenital mesoblastic nephroma surviving respiratory failure and pulmonary embolism at laparotomy	Sri Lanka Journal of Surgery/Sri Lanka journal of surgery	2,015	cc-by	1,005	Keywords: Congenital mesoblastic nephroma, pulmonary embolism, disseminated intravascular coagulation encapsulated tumour occupying almost the whole abdomen with the consistency of an “oily sponge cake” seen originating from left kidney. The liver, bladder, uterus and Fallopian tubes were normal. CASE REPORTS CASE REPORTS Introduction Suddenly, generalised bleeding and bradycardia developed suggesting pulmonary air embolism leading to DIC. Blood components were transfused and, the operative field was filled with isotonic saline to facilitate evacuation of pulmonary embolus. Surgery was terminated and tumour remnant over-sutured. Resected tumour mass weighed 350grams. Approximately the same volume of tumour was left in- situ. Congenital mesoblastic nephroma (CMN) first described by Bolande et al in 1967 [1] is the commonest neonatal tumour of renal origin comprising of 3-10% of all childhood tumours [2]. Common modes of presentation include abdominal mass, antenatal diagnosis and features of para-neoplastic syndrome [3]. There are two main histological varieties; classic type where nephroureterectomy is curative and atypical (cellular) type with unpredictable natural history [2,4]. Tumour markers were done as advised by the oncologist (serum βHCG: <0.1miu/ml(<1miu/ml), α fetoprotein: 776.1iu/ml(<25iu/ml). Histology revealed degenerated tumour tissue and undifferentiated spindle cells with no b l a s t e m a l o r e p i t h e l i a l e l e m e n t s . Immunohistochemistry was focally positive for vimentin and, negative for SMA,PCK,CD10 and MyoD1. The final diagnosis of CMN was made(4). We report a two day old developing severe cardio- respiratory failure secondary to CMN, surviving emergency tumour debulking laparotomy that was complicated by pulmonary embolism and disseminated intravascular coagulation (DIC). Subsequent nephroureterectomy with total tumour clearance was performed. To our knowledge there are no reports of CMN with similar presentation in the world literature. Subsequent CT scan of abdomen showed tumour remnant compressing the aorta and inferior vena cava (Figure 1). Neonate with congenital mesoblastic nephroma surviving respiratory failure and pulmonary embolism at laparotomy Neonate with congenital mesoblastic nephroma surviving respiratory failure and pulmonary embolism at laparotomy 1 2 1 1 R. Ranawaka¹, A. Vithanage , P. Rathnayake , K. Hettiarachchi , N. Jayakody 1. Sirimavo Bandaranayake Specialised Children's Hospital, Peradeniya, Sri Lanka 2.Teaching hospital Kandy, Sri Lanka 1 2 1 1 R. Ranawaka¹, A. Vithanage , P. Rathnayake , K. Hettiarachchi , N. Jayakody 1. Sirimavo Bandaranayake Specialised Children's Hospital, Peradeniya, Sri Lanka 2.Teaching hospital Kandy, Sri Lanka 1 2 1 1 R. Ranawaka¹, A. Vithanage , P. Rathnayake , K. Hettiarachchi , N. Jayakody 1. Sirimavo Bandaranayake Specialised Children's Hospital, Peradeniya, Sri Lanka 2.Teaching hospital Kandy, Sri Lanka Keywords: Congenital mesoblastic nephroma, pulmonary embolism, disseminated intravascular coagulation Case An eighteen hour old baby with antenatal diagnosis of “intraabdominal tumour” was transferred on developing laboured breathing. Within hours she required ventilatory support and cardiac inotropes. At re-laparotomy, well encapsulated tumour arising from the lower pole of left kidney with firm and fibrotic consistency found. Rest of the viscera were normal. Left nephroureterectomy with total tumour resection was performed. Tumour weighed 400grams (Figure2). Ultra sound scan detected a large intra-abdominal mass displacing other viscera. The left kidney was not identified. Computerised tomography (CT) scan was not accessible. Histology confirmed classic type CMN confined within excision margins. She recovered completely and remains well three months after surgery. Surveillance USS is planned in three month's time. During emergency laparotomy a bosselated The Sri Lanka Journal of Surgery 2014; 32(3): 44-45 Correspondence: R.Ranawaka E-mail: rravibindu@yahoo.com During emergency laparotomy a bosselated During emergency laparotomy a bosselated 44 Figure 1. CT scan of abdomen after initial tumour debulking laparotomy range from trivial reduction in platelet count and prolongation of clotting time to fulminant DIC [5]. The management includes correction of DIC with transfusion of blood components and, evacuation of pulmonary air embolus by filling the surgical field with isotonic saline while continuing ventilation. Initial surgery leaving a residual tumour bulk could theoretically be considered as tumour upstaging. Adjuvant therapy has a place in metastatic CMN but is not described for classic type. Since complete tumour clearance was confirmed histologically, surveillance for tumour recurrence with regular imaging was planned. The rare experience of successful tumour excision after complicated surgery in a neonate with no proper pre- operative imaging and histology would be informative to surgeons who experience similar surgical emergencies. References Figure 1. CT scan of abdomen after initial tumour debulking laparotomy 1. Bolande PP, Brough AJ, Izant RJ, Congenital mesoblastic nephroma of infancy. Pediatrics 1967;40: 272–279 Figure 2. Specimen after total resection Upper pole of Left Kidney 2. dos Santos LG, de Sousa J, de Carvalho R et al, Cellular congenital mesoblastic nephroma: case report. Jornal Brasileiro de Nefrologoa 2011;33:88-90 3. Minesh K, Horacio O, Michael AS, Congenital Mesoblastic Nephroma Presenting with Neonatal Hypertension. Journal of Perinatology 2005; 25:433–435 4. Argani P, Fritsch M, Kadkol S et al, Detection of the ETV6- NTRK3 chimeric RNA of infantile fibrosarcoma/cellular congenital mesoblastic nephroma in paraffin- embedded tissue: application to challenging pediatric renal stromal tumors. Modern Pathology 2000; 13:29-36 4. Argani P, Fritsch M, Kadkol S et al, Detection of the ETV6- NTRK3 chimeric RNA of infantile fibrosarcoma/cellular congenital mesoblastic nephroma in paraffin- embedded tissue: application to challenging pediatric renal stromal tumors. Modern Pathology 2000; 13:29-36 4. Argani P, Fritsch M, Kadkol S et al, Detection of the ETV6- NTRK3 chimeric RNA of infantile fibrosarcoma/cellular congenital mesoblastic nephroma in paraffin- embedded tissue: application to challenging pediatric renal stromal tumors. Modern Pathology 2000; 13:29-36 Figure 2. Specimen after total resection Figure 2. Specimen after total resection 5. Levi M, Ten Cate H, Disseminated intravascular coagulation. New England Journal of Medicine 1999; 341:586–92. Discussion Pulmonary embolism could occur in surgery during exposure of a large vascular surface. The manifestations Key points:  Pulmonary embolism could occur in surgery during exposure of large vascular surfaces.  Management includes filling the surgical field with isotonic saline while continuing ventilation and correction of DIC.  Management includes filling the surgical field with isotonic saline while continuing ventilation and correction of DIC. The Sri Lanka Journal of Surgery 2014; 32(3): 44-45 45
https://openalex.org/W3210857711	https://www.researchsquare.com/article/rs-842341/latest.pdf	English	null	Support for Lowering Cervical Cancer Screening Age To 25 for Women Living With HIV: Retrospective Cross-Sectional Programmatic Data From Botswana	Research Square (Research Square)	2,021	cc-by	6,969	Support for Lowering Cervical Cancer Screening Age To 25 for Women Living With HIV: Retrospective Cross-Sectional Programmatic Data From Botswana Doreen Ramogola-Masire (  doreen.masire@gma University of Botswana Surbhi Grover Botswana U-Penn Partnership Anikie Mathoma University of Botswana Barati Monare Botswana U-Penn Partnership Lesego Gabaitiri University of Botswana Lisa Bazzett-Matabele University of Botswana G Justus Hofmeyr University of Botswana Chelsea Morroni Botswana-Harvard AIDS Institute Partnership Rebecca Luckett University of Botswana Conclusions: Younger WLWH in Botswana had a significant burden of CIN2+. This finding further supports lowering the screening age for WLWH from 30 to 25. Methods: A retrospective cross-sectional study of 5,714 participants aged 25 through 49 years who underwent VIA screening. VIA-positive women received cryotherapy if indicated or were referred for colposcopy. Known cervical cancer risk factors, screening, and histological results were extracted from the program database. We compared the proportions and association of VIA positivity and CIN2+ by age and HIV status. Background: Women living with human immunodeficiency virus (HIV) tend to develop cervical cancer at a younger age than HIV-negative women. The World Health Organization’s (WHO) new guidelines for screening and treatment of cervical pre-cancer lesions for cervical cancer prevention include a conditional recommendation for initiating screening at age 25 for women living with HIV (WLWH). This recommendation is based on low-certainty evidence, and WHO calls for additional data. We describe the association of age and HIV status with visual inspection with acetic acid (VIA) positivity and cervical intra-epithelial neoplasia grade two or higher (CIN2+) in Botswana. Results: Median age was 35 years [IQR 31-39], and 18% of the women were aged 25-29. Ninety percent were WLWH; median CD4 count was 250 cells/µL [IQR 150-428], and 34.2% were on anti-retroviral treatment (ART). VIA-positivity was associated with younger age (OR 1.48, CI 1.28, 1.72 for 25-29 years versus age 30-49 years ), and HIV-positivity (OR 1.85, CI 1.51, 2.28). CIN2+ was associated with HIV positivity (OR 6.12, CI 3.39, 11.10), and proportions of CIN2+ were similar for both age groups in WLWH (12.1% versus 10.8%). Research Article License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at BMC Women's Health on April 2nd, 2022. See the published version at https://doi.org/10.1186/s12905-022-01680-7. Page 1/15 Page 1/15 HIV procedures Women with unknown HIV status at the time of screening or with documented HIV negative status more than six months prior were referred to an HIV testing center and requested to share their results. Throughout the study period, the Botswana National HIV program initiated anti-retroviral treatment (ART) at a CD4 count of ≤ 350 cells/µL. Outcomes The primary outcome was the association of VIA positivity and age adjusting for cervical cancer risk factors. The secondary outcomes were the association of histopathologically confirmed high-grade pre-cancer and age adjusting for cervical cancer risk factors; HIV-status association with VIA positivity and high-grade pre-cancer; and the proportions of VIA positivity and high-grade pre-cancer by both age and HIV status. Cervical cancer screening procedures All patients underwent a speculum examination of the cervix by a nurse who had participated in the Botswana MOHW VIA training program. Visual assessment was performed after applying 5% acetic acid to the cervix using a cotton swab, and findings were categorized as normal, abnormal with a recommendation for cryotherapy, or abnormal with a recommendation for LEEP. Those with abnormal lesions eligible for cryotherapy were offered same-day treatment and had no histopathology specimen collected. Women with abnormal lesions ineligible for cryotherapy based on appearance, size, or extension into the cervical os, were referred to the colposcopy/LEEP clinic and evaluated by a specialist gynecologist or trained medical officers. The colposcopic appearance of lesions determined diagnostic and treatment decisions. Low-grade appearing lesions were treated with cautery after taking a biopsy; high-grade appearing lesions or those extending into the cervical were treated by LEEP. Histopathology specimens were read by pathologists blinded to VIA findings. Background Low- and middle-income countries (LMICs) carry the highest global burden of cervical cancer incidence and mortality.[1] Cervical cancer is the leading cause of cancer death in women in Southern Africa.[2, 3] While human papillomavirus (HPV) vaccination in young girls offers hope for a significant reduction in cervical cancer in future generations, effective cervical cancer screening services remain essential to reduce morbidity and mortality associated with cervical cancer in women across the globe.[4] Women living with the human immunodeficiency virus (WLWH) have a higher risk of developing pre-invasive cervical disease and cervical cancer.[5–7] Although progression rates from pre-invasive cervical disease to cervical cancer are unknown due to standard intervention for high-grade cervical dysplasia, cervical cancer is diagnosed at younger ages in WLWH compared to HIV-negative women. [5, 8, 9] Guidelines for high-income countries (HICs) recommend cervical cancer screening initiation at an early age of 21.[10–12] Up till recently, guidelines for most LMICs recommended the initiation of cervical cancer screening at the age of 30 despite LMICs having the highest global prevalence of HIV in the reproductive-aged population.[13, 14]. The new WHO guidelines have a conditional recommendation based on low-certainty evidence for initiating screening at age 25 for WLWH, [15] and calls for more data. Further, many LMICs will not be able to change their guidelines straight away due to resource constraints. Botswana has one of the highest HIV prevalences globally, at 25.1% in women aged 15–49.[16] Botswana’s national guidelines prioritize screening in the 30 to 49 year-old age group with either cytology or visual inspection with acetic acid (VIA), regardless of HIV status. While practical, these guidelines may not adequately account for the high prevalence of HIV in Botswana and the higher risk of early cervical cancer progression. There is limited published data from Botswana on the prevalence of pre-invasive disease and the role of screening in younger women. This study describes the association of age and HIV status with VIA positivity and high-grade cervical pre-cancer disease. We aimed to determine how initiating cervical cancer screening at age 25 years, instead of 30 years, in WLWH would improve the identification of high- grade pre-invasive cervical disease without unduly increasing overtreatment of low-grade cervical dysplasia. Data presented here could strengthen the evidence for the WHO recommendation on the target age group for cervical cancer screening in WLWH. Page 2/15 Page 2/15 Study design and patient selection We conducted a retrospective cross-sectional study based on the Botswana Ministry of Health and Wellness (MOHW) National Cervical Cancer Prevention Programme “see-and-treat” pilot programmatic database.[17] The database included women screened with visual inspection after acetic acid (VIA) at Bontleng clinic and those referred to Princess Marina Hospital (PMH) for colposcopy in Gaborone, Botswana, from March 2009 through August 2015. Cervical cancer screening services were initially provided for WLWH as part of comprehensive HIV care and were later extended to HIV-negative women at these sites. Screening services were offered free of charge to all Botswana citizens. Screening services were linked to a physician-led referral colposcopy and loop electrosurgical excision procedure (LEEP) clinic at PMH. Through various channels, women came to screening services, including provider referral, self-referral following sensitization by written materials, and health education talks. Women were excluded from screening if they had previously had a hysterectomy, pelvic radiation for lower genital tract cancer, or a cervical cancer diagnosis. Screening for women who were menstruating heavily, pregnant, or had a persistent vaginal discharge was re-scheduled for after resolution of the condition. Data collection All women undergoing VIA screening completed a questionnaire capturing a limited set of patient-level cervical cancer risk factors, including smoking, age of sexual debut, and parity. HIV status was recorded, and for WLWH, CD4 count at the time of HIV diagnosis and whether on ART at the time of screening was documented. VIA screening outcomes were recorded in the programmatic database. Histology results of women referred for colposcopy/LEEP were extracted from the National Health Laboratory (NHL) electronic medical record when available and entered into the programmatic database. Data analysis The analyzed dataset included only women between the ages of 25 and 49. Patient records with missing data for VIA or histopathology that could not be corrected by cross-reference with primary records were excluded from the primary and secondary analysis, respectively. The sample size for the primary outcome was calculated using a 1-sided alpha of 0.05. To attain a 99% power, we assumed VIA positivity to be 30% in women aged 25 to 29 years and 20% in women aged 30 to 49 years based on previous findings.[17] The sample size required to detect a statistically significant difference in VIA-positivity between the two age groups was 2,076 women (374 women aged 25 to 29 years and 1,702 women aged 30 to 49 years). Page 3/15 Page 3/15 The cervical cancer risk factors adjusted for included: HIV status, parity, smoking, and age of sexual debut. CD4 count and ART were included in the analysis of WLWH. Descriptive statistics for these variables are presented as median [interquartile range (IQR)] and proportions. Continuous variables were categorized into binary variables and compared using the chi-square test. Categorical variables included age groups of younger and older women (25 to 29 years; 30 to 49 years), age of sexual debut (≤18; >18 years), parity (≤2; >2), CD4 count (≤350 cells/µL; >350 cells/µL), and histopathology results (benign or CIN 1 [≤CIN1] for low-grade pre-cancer; CIN2+ for high- grade pre-cancer). Patterns of missing data were described for the study cohort using percentages. Logistic regression models computed unadjusted and adjusted odds ratios (ORs) with 95% confidence intervals (CI). Only exposure variables with a p-value of less than 0.1 for unadjusted ORs were included in the adjusted regression models.[18] A p-value of less than 0.05 was considered to be statistically significant. We used Stata 14.0 (StataCorp LLC, College Station, Texas). Overall patient characteristics The database included 5,724 women aged 25 through 49 years screened with VIA between March 2009 and August 2015 (Fig. 1). Ten women had missing VIA data, leaving 5,714 women for the VIA analysis. As shown in Table 1, the median age was 35 years [IQR 31–39], and 1029 (18%) were between 25 and 29 years of age. Smoking was reported by 285 (5%) of the women. The median age of sexual debut was 18 years [IQR 17–20], and the median parity was two [IQR 1–3]. HIV status was known in 5,583 (98%), and 5,026 (90%) of those with a known status were WLWH. Eight hundred and forty nine (86%) of the women aged 25 to 29 years and 4,177 (91%) of the those aged 30 to 49 years were WLWH. Among the WLWH, the median CD4 count was 250 cells/µL [IQR 150–428], and 1628 (34.2%) were on ART. Missing data was ≤ 5% for all the variables except for CD4 count (11%, n = 551). The level of CD4 count missing data was similar for both age groups (10.5% for 25 to 29 year-olds versus 11.1% for 30 to 49 year-olds). Overall patient characteristics Page 4/15 Table 1 Demographic and clinical characteristics of all study participants Variable All Age 25–29 years Age 30–49 years P-Value for X² test n (%) Median [IQR] n a (%) n a (%) Age 5,714 35 [31, 39] Smoking Yes No Missing 5,661 285 (50.0) 5,376 (94.1) 53 (1.0) 1,018 (18.0) 68 (6.7) 950 (93.3) 4,643 (82.0) 217 (4.7) 4,426 (95.3) 0.008b Sexual debut ≤ 18 > 18 Missing 5,689 3,150 (55.3) 2,539 (44.4) 25 (0.4) 18 [17, 21] 1,024 (18.0) 533 (52.1) 491 (47.9) 4,665 (82.0) 2,617 (56.1) 2,048 (43.9) 0.02b Parity ≤ 2 > 2 Missing 5,612 3,223 (56.4) 2,389 (41.8) 102 (1.8) 2 [1, 3] 1,001 (17.8) 815 (81.4) 186 (18.6) 4,611 (82.2) 2,408 (52.2) 2,203 (47.8) < 0.001b HIV Status Negative Positive Missing 5,583 557 (9.7) 5,026 (88.0) 131 (2.3) 989 (17.7) 140 (14.2) 849 (85.8) 4,594 (82.3) 417 (9.1) 4,177 (90.9) < 0.001b Initial CD4 at HIV Diagnosis c ≤ 350 > 350 Missing 4,475 2,308 (45.9) 2,167 (43.1) 551 (11.0) 250 [150, 428] 760 (16.9) 304 (40.0) 456 (60.0) 3,715 (83.1) 2,004 (53.9) 1,711 (46.1) < 0.001b On ART at time of screening c Yes No Missing 4,766 1,628 (32.4) 3,138 (62.4) 260 (5.2) 802 (16.8) 346 (43.1) 456 (56.9) 3,964 (83.2) 1,282 (32.3) 2,682 (67.7) < 0.001b VIA Results Positive Negative 5,714 1,959 (34.3) 3,755 (65.7) 1,029 (18.0) 428 (41.6) 601 (58.4) 4,685 (82.0) 1,531 (32.7) 3,154 (67.3) < 0.001b Abbreviation: ART, anti-retroviral treatment; CIN, cervical intraepithelial neoplasia; IQR, inter-quartile range; VIA, visual inspection after acetic acid. The number of women in levels of categorical variable may not add up to total “n” because missing category has been removed The overall VIA positivity of the study population was 34.3% (n = 1,959). The proportion was higher in the 25 to 29 year-olds (41.5%, n = 428) than the 30 to 49 year-olds (32.7%, n = 1,531). The WLWH had a higher VIA positivity rate (35.9%, n = 1,841) than HIV-negative women (24.1%, n = 141) (Table 2). Via-positivity a The number of women in levels of categorical variable may not add up to total “n” because missing category has been removed. b for p < 0.05. c for HIV positive patients only. Variable All n (%) VIA Positive n a (%) VIA Negative n a(%) VIA Positivity BVA Odds Ratios (95% CI) P-value VIA Positivity MVA Odds Ratios (95% CI) P-value for X² test CD4 Count at HIV Diagnosis c ≤ 350 > 350 4,475 2,308 (45.9) 2,167 (43.1) 551 (11.0) 1,620 (36.2) 809 (35.1) 811 (37.4) 2,855 (63.8) 1,499 (64.9) 1,356 (62.6) 0.90 (0.80, 1.02) Ref 0.10 0.96 (0.83, 1.10) 0.52 ART at time of screening c No Yes Missing 4,766 3,138 (62.4) 1,628 (32.4) 260 (5.2) 1,710 (35.9) 1,096 (34.9) 614 (37.7) 3,056 (64.1) 2,047 (65.1) 1,014 (62.3) 0.89 (0.78, 1.00) Ref 0.06 0.91 (0.78, 1.05) 0.19 Abbreviations: ART, anti-retroviral therapy; BVA, bivariate analysis; MVA, multivariate analysis; VIA, visual inspection after acetic acid. a The number of women in levels of categorical variable may not add up to total “n” because missing category has been removed. b for p < 0.05. c for HIV positive patients only. In multivariate analyses, VIA positivity was more likely in 25 to 29 year-olds than in 30 to 49 year-olds (OR 1.48, CI 1.28, 1.72), and in WLWH compared to HIV-negative women (OR 1.85, CI 1.51, 2.28). Among WLWH, VIA positivity was not affected by CD4 count (OR = 0.96, CI 0.83, 1.10) or by ART (OR = 0.91, CI 0.78, 1.05) (Table 2). In multivariate analyses, VIA positivity was more likely in 25 to 29 year-olds than in 30 to 49 year-olds (OR 1.48, CI 1.28, 1.72), and in WLWH compared to HIV-negative women (OR 1.85, CI 1.51, 2.28). Among WLWH, VIA positivity was not affected by CD4 count (OR = 0.96, CI 0.83, 1.10) or by ART (OR = 0.91, CI 0.78, 1.05) (Table 2). Via-positivity Page 5/15 Page 5/15 Page 6/15 Table 2 Study participants’ characteristics with bivariate and multivariable odds ratio for VIA positivity Variable All n (%) VIA Positive n a (%) VIA Negative n a(%) VIA Positivity BVA Odds Ratios (95% CI) P-value VIA Positivity MVA Odds Ratios (95% CI) P-value for X² test Age Group 25–29 years 30–49 years 5,714 1,029 (18.0) 4,685 (82.0) 1,959 (34.3) 428 (41.6) 1,531 (32.7) 3,755 (65.7) 601 (58.4) 3,154 (67.3) 1.47 (1.28, 1.69) Ref < 0.001b 1.48(1.28, 1.72) < 0.001b Smoker No Yes Missing 5,661 5,376 (94.1) 285 (5.0) 53 (0.9) 1,937 (34.2) 1,825 (33.9) 112 (39.3) 3,724 (65.8) 3,551 (66.1) 173 (60.7) Ref 1.27(1.00, 1.62) 0.053 1.14(0.89, 1.46) 0.31 Age Sexual debut ≤ 18 > 18 missing 5,689 3,150 (55.1) 2,539 (44.4) 25 (0.5) 1,950 (34.3) 1,075 (34.1) 875 (34.5) 3,739 (65.7) 2,075 (65.9) 1,664 (65.5) 0.99(0.88, 1.10) Ref 0.79 N/A N/A Parity ≤ 2 > 2 Missing 5,612 3,223 (56.40) 2,389 (41.81) 102 (1.79) 1,924 (34.3) 1,151 (35.7) 773 (32.4) 3,688 (65.7) 2,072 (64.3) 1,616 (67.6) Ref 0.86 (0.77, 0.96) 0.009b 0.88 (0.79, 0.99) 0.04b HIV Status Negative Positive Missing 5,583 557 (9.74) 5,026 (87.96) 131 (2.30) 1,941 (34.8) 134 (24.1) 1,807 (35.9) 3,642 (65.2) 423 (75.9) 3,219 (64.1) Ref 1.77 (1.45, 2.17) < 0.001b 1.85 (1.51, 2.28) < 0.001b Page 7/15 Abbreviations: ART, anti-retroviral therapy; BVA, bivariate analysis; MVA, multivariate analysis; VIA, visual inspection after acetic acid. a The number of women in levels of categorical variable may not add up to total “n” because missing category has been removed. b for p < 0.05. c for HIV positive patients only. Page 7/15 Variable All n (%) VIA Positive n a (%) VIA Negative n a(%) VIA Positivity BVA Odds Ratios (95% CI) P-value VIA Positivity MVA Odds Ratios (95% CI) P-value for X² test CD4 Count at HIV Diagnosis c ≤ 350 > 350 Missing 4,475 2,308 (45.9) 2,167 (43.1) 551 (11.0) 1,620 (36.2) 809 (35.1) 811 (37.4) 2,855 (63.8) 1,499 (64.9) 1,356 (62.6) 0.90 (0.80, 1.02) Ref 0.10 0.96 (0.83, 1.10) 0.52 ART at time of screening c No Yes Missing 4,766 3,138 (62.4) 1,628 (32.4) 260 (5.2) 1,710 (35.9) 1,096 (34.9) 614 (37.7) 3,056 (64.1) 2,047 (65.1) 1,014 (62.3) 0.89 (0.78, 1.00) Ref 0.06 0.91 (0.78, 1.05) 0.19 Abbreviations: ART, anti-retroviral therapy; BVA, bivariate analysis; MVA, multivariate analysis; VIA, visual inspection after acetic acid. In multivariate analyses, CIN2 + was associated with a positive HIV status (aOR 6.12, CI 3.39, 11.10), but not with age (OR 1.07, CI 0.75– 1.52 for 25 to 29 year-olds compared to 30 to 49 year-olds). In WLWH, neither CD4 count nor ART was associated with CIN2+ (Table 4). High-grade Pre-cancer The majority of the VIA-positive lesions were ineligible for treatment with cryotherapy (68%, n = 1,330); this was similar for both the 25 to 29 year-olds and the 30 to 49 year-olds (67.1% versus 68.1%, respectively). Of the 1,330 women referred to colposcopy/LEEP, 878 (66%) attended and had recorded histopathology results (58.5% for 25 to 29 year-olds, and 68.1% for 30–49 year-olds). The overall population CIN2 + point prevalence was 10.1% (10.9% for 25 to 29 year-olds and 9.9% for 30 to 49 year-olds) (Fig. 1 &Table 3). Page 8/15 Page 8/15 Table 3 Table 3 VIA and histological outcomes by age group and HIV status Figures for a and b do not add up to 5,714 due to missing HIV status in 131 records. Table 3 VIA and histological outcomes by age group and HIV status Page 9/15 Page 9/15 Table 4 All Study participants’ characteristics with bivariate and multivariable odds ratio for CIN2+ Variable All n (%) ≥CIN2+ n a (%) ≤CIN1 n a (%) CIN2+ BVA Odds Ratios (95% CI) P-value CIN2+ MVA Odds Ratios (95% CI) P-value for X² test Age Group 25–29 years 30–49 years 878 168 (19.1) 710 (80.9) 575 (65.5) 112 (66.7) 463 (65.2) 303 (34.5) 56 (33.3) 247 (34.8) 1.07(0.75, 1.52) Ref 0.72 N/A N/A Smoker No Yes Missing 867 817 (93.1) 50 (5.7) 11 (1.2) 566 (65.3) 530 (64.9) 36 (72.0) 301 (34.7) 287 (35.1) 14 (28.0) Ref 1.38(0.74, 2.62) 0.30 N/A N/A Age Sexual debut ≤ 18 > 18 missing 873 505 (57.5) 368 (41.9) 5 (0.6) 572 (65.5) 338 (66.9) 234 (63.6) 301 (34.5) 167 (33.1) 134 (36.4) 1.16(0.87, 1.54) Ref 0.31 N/A N/A Parity ≤ 2 > 2 Missing 861 517 (58.9) 344 (39.2) 17 (2.0) 566 (65.7) 328 (63.4) 238(69.2%) 295 (34.3) 189 (36.6) 106 (30.8) Ref 1.29(0.97, 1.73) 0.08 1.30(0.96, 1.75) 0.09 Table 4 All Study participants’ characteristics with bivariate and multivariable odds ratio for CIN2+ Variable All n (%) ≥CIN2+ n a (%) ≤CIN1 n a (%) CIN2+ BVA Odds Ratios (95% CI) P-value CIN2+ MVA Odds Ratios (95% CI) P-value for X² test Age Group 25–29 years 30–49 years 878 168 (19.1) 710 (80.9) 575 (65.5) 112 (66.7) 463 (65.2) 303 (34.5) 56 (33.3) 247 (34.8) 1.07(0.75, 1.52) Ref 0.72 N/A N/A Smoker No 867 817 (93.1) 50 (5.7) 11 (1.2) 566 (65.3) 530 (64.9) 301 (34.7) 287 (35.1) 14 (28.0) Ref 1.38(0.74, 2.62) 0.30 N/A N/A Yes Missing 36 (72.0) Age Sexual debut ≤ 18 > 18 missing 873 505 (57.5) 368 (41.9) 5 (0.6) 572 (65.5) 338 (66.9) 234 (63.6) 301 (34.5) 167 (33.1) 134 (36.4) 1.16(0.87, 1.54) Ref 0.31 N/A N/A Parity ≤ 2 > 2 Missing 861 517 (58.9) 344 (39.2) 17 (2.0) 566 (65.7) 328 (63.4) 238(69.2%) 295 (34.3) 189 (36.6) 106 (30.8) Ref 1.29(0.97, 1.73) 0.08 1.30(0.96, 1.75) 0.09 36 (72.0) Page 10/15 Abbreviations: ART, anti-retroviral therapy; BVA, bivariate analysis; MVA, multivariate analysis; VIA, visual inspection after acetic acid. a The number of women in levels of categorical variable may not add up to total “n” because missing category has been removed. b for p < 0.05. c for HIV positive patients only. Table 3 VIA and histological outcomes by age group and HIV status VIA and histological outcomes by age group and HIV status VIA outcomes All participants HIV positive a HIV negative b 25–29 Age group 30–49 Age group 25–29 Age group 30–49 Age group 25–29 Age group 30–49 age group Number Screened with VIA N = 1029 N = 4685 n = 849 N = 4177 N = 140 N = 417 VIA Results Negative Positive 601(58.4%) 428(41.6%) 3154(67.3%) 1531(32.7%) 472(55.6%) 377(44.4%) 2747(65.8%) 1430(34.2%) 95(67.9%) 45(32.1%) 328(78.7%) 89(21.3%) Eligible for cryotherapy Not eligible for cryotherapy 141(32.9%) 287(67.1%) 488(31.9%) 1043(68.1%) 121(32%) 256 (68%) 477(33.4%) 953(66.6%) 18(40%) 27(60%) 34(38.2%) 55(61.8% Histology outcomes Not eligible for cryotherapy, arrived at Colposcopy with histology results N = 168 N = 710 n = 149 n = 659 N = 16 N = 46 ≤CIN 1 ≥CIN2+ 56(33.3%) 112(66.7%) 247(34.8%) 463(65.2%) 46((30.9%) 103(69.1%) 209(31.7%) 450(68.3%) 8(50%) 8(50%) 36(78.2%) 10(21.8%) ≥CIN2 + in screened population 112/1029 (10.9%) 463/4685 (9.9%) 103/849 (12.1%) 450/4177 (10.8%) 8/140 (5.7%) 10/417 (2.4%) Abbreviation: CIN, cervical intraepithelial neoplasia; VIA, visual inspection after acetic acid. Figures for a and b do not add up to 5,714 due to missing HIV status in 131 records. In multivariate analyses, CIN2 + was associated with a positive HIV status (aOR 6.12, CI 3.39, 11.10), but not with age (OR 1.07, CI 0.75– 1.52 for 25 to 29 year-olds compared to 30 to 49 year-olds). In WLWH, neither CD4 count nor ART was associated with CIN2+ (Table 4). In multivariate analyses, CIN2 + was associated with a positive HIV status (aOR 6.12, CI 3.39, 11.10), but not with age (OR 1.07, CI 0.75– 1.52 for 25 to 29 year-olds compared to 30 to 49 year-olds). In WLWH, neither CD4 count nor ART was associated with CIN2+ (Table 4). Table 3 VIA and histological outcomes by age group and HIV status Page 10/15 Abbreviations: ART, anti-retroviral therapy; BVA, bivariate analysis; MVA, multivariate analysis; VIA, visual inspection after acetic acid. a The number of women in levels of categorical variable may not add up to total “n” because missing category has been removed. b for p < 0.05. c for HIV positive patients only. Page 10/15 Variable All n (%) ≥CIN2+ n a (%) ≤CIN1 n a (%) CIN2+ BVA Odds Ratios (95% CI) P-value CIN2+ MVA Odds Ratios (95% CI) P-value for X² test HIV Status Negative Positive Missing 870 62 (7.1) 808 (92.0) 8 (0.9) 571 (65.6) 18 (29.0) 553 (68.4) 299 (34.4) 44 (71.0) 255 (31.6) Ref 5.30(2.96, 9.49) < 0.001b 6.12(3.39, 11.10) < 0.001b CD4 Count at HIV Diagnosis c ≤ 350 701 336 (41.6) 494 (70.5) 234 (69.6) 260 (71.2 207 (29.5) 102 (30.4) 105 (28.8) 0.93(0.67, 1.28) Ref 0.65 N/A N/A Variable All n (%) ≥CIN2+ n a (%) ≤CIN1 n a (%) CIN2+ BVA Odds Ratios (95% CI) P-value CIN2+ MVA Odds Ratios (95% CI) P-value for X² test HIV Status Negative Positive 870 62 (7.1) 808 (92.0) 8 (0.9) 571 (65.6) 18 (29.0) 553 (68.4) 299 (34.4) 44 (71.0) 255 (31.6) Ref 5.30(2.96, 9.49) < 0.001b 6.12(3.39, 11.10) < 0.001b Missing CD4 Count at HIV Diagnosis c ≤ 350 701 336 (41.6) 494 (70.5) 234 (69.6) 260 (71.2 207 (29.5) 102 (30.4) 105 (28.8) 0.93(0.67, 1.28) Ref 0.65 N/A N/A Missing CD4 Count at HIV Diagnosis c ≤ 350 > 350 Missing 701 336 (41.6) 365 (45.2) 107 (12.2) 494 (70.5) 234 (69.6) 260 (71.2 207 (29.5) 102 (30.4) 105 (28.8) 0.93(0.67, 1.28) Ref 0.65 N/A N/A N/A Page 11/15 Abbreviations: ART, anti-retroviral therapy; BVA, bivariate analysis; MVA, multivariate analysis; VIA, visual inspection after acetic acid. a The number of women in levels of categorical variable may not add up to total “n” because missing category has been removed. b for p < 0.05. c for HIV positive patients only. Page 11/15 Abbreviations: ART, anti-retroviral therapy; BVA, bivariate analysis; MVA, multivariate analysis; VIA, visual inspection after acetic acid. a The number of women in levels of categorical variable may not add up to total “n” because missing category has been removed. b for p < 0.05. c for HIV positive patients only. Discussion WLWH aged 25 through 29 years attending routine cervical cancer screening in our national program had the same odds of having high- grade cervical pre-cancer as women aged 30 to 49 years. Prior research has indicated a link between younger age and cervical cancer among WLWH.[5, 8, 9] Our findings confirm the presence of a significant level of cervical cancer precursors requiring intervention in women as young as 25 years, particularly in WLWH, thus supporting the new WHO recommendation to lower the age of initiation of cervical cancer screening from 30 to 25 years in WLWH. A concern about lowering the cervical cancer screening age is that clinically insignificant lesions from transient HPV infections would be intervened upon unnecessarily, resulting in overtreatment of young women.[19, 20] Although women in this cohort aged 25 through 29 years had higher rates of VIA positivity than women aged 30 to 49 years, similar proportions were referred for the excisional procedure. The histopathology results indicate that the proportions of CIN2 + detected and appropriately treated were similar for the two age groups in WLWH. The proportion of CIN2 + was more than three times lower in HIV-negative women than in WLWH, and younger women were twice more likely to have CIN2+. If overtreatment did occur, it would primarily have occurred in the group of women treated with cryotherapy, a treatment that ultimately has minimal side effects.[21] We had expected to find a correlation between patient age and high-grade dysplasia because older women would have had a longer time to progress from HPV infection to cervical pre-cancer without opportunities for intervention.[22] However, our data do not support this hypothesis. Instead, younger women overall and in WLWH had a similar proportion of CIN2 + to older women. Data is limited on HPV progression to cervical pre-cancer and cancer in women aged 20 to 29 years. Adolescent WLWH are more likely to have HPV co-infections and coexisting abnormalities, albeit low-grade, relative to their HIV-negative counterparts.[23] The shorter timeline from HPV infection to the development of pre-cancer in adolescent WLWH is in line with our finding of high rates of high-grade pre-cancer in the 25 to 29 year- olds because of the likey accelerated timeframe of progression of pre-cancer from low- to high-grade. Our analysis has limitations because it utilized a programmatic database that collected limited patient-level demographic and risk factor data. Via-positivity And High-grade Pre-cancer By Age And Hiv-status In WLWH, the 25 to 29 year-olds were more likely to be VIA positive than the 30 to 49 year-olds (44.41%, n = 377 versus 34.2%, n = 1430, respectively). We observed a similar pattern for HIV-negative women (32.1%, n = 45 for 25 to 29 year-olds, compared to 21.3%, n = 89 for 49 year-olds). The overall proportion of CIN2 + in WLWH was 11% (12.1% for the 25 to 29 year-olds and 10.8% for the 30 to 49 year-olds). The overall proportion of CIN2 + in HIV-negative women was 3.2% (5.7% for 25 to 29 year-olds and 2.4% for 30 to 49 year-olds) (Table 3). Table 3 VIA and histological outcomes by age group and HIV status Page 11/15 Page 11/15 Variable All n (%) ≥CIN2+ n a (%) ≤CIN1 n a (%) CIN2+ BVA Odds Ratios (95% CI) P-value CIN2+ MVA Odds Ratios (95% CI) P-value for X² test ART at time of screening c No Yes Missing 773 455 (56.3) 318 (39.4) 35 (4.3) 527 (68.2) 315 (69.2) 212 (66.7) 246 (31.8) 140 (30.8) 106 (33.3) 1.13(0.83, 1.53) Ref 0.45 N/A N/A Abbreviations: ART, anti-retroviral therapy; BVA, bivariate analysis; MVA, multivariate analysis; VIA, visual inspection after acetic acid. a The number of women in levels of categorical variable may not add up to total “n” because missing category has been removed. b for p < 0.05. c for HIV positive patients only. Discussion CD4 count had 11% missing data; however, this was similar for both age groups, and we doubt that it would have had a significant effect on the outcomes. The rate of cryotherapy ineligibility was high, but high rates have been observed in other high HIV burden areas. [24] This high rate could be related to lack of prior screening and high rates of cervicitis.[25] Documentation of colposcopy/LEEP referral Page 12/15 Page 12/15 appointment attendance was not recorded, and thus histopathology results may not represent the entire cohort of women who had a colposcopic evaluation. However, the population point prevalence of the screened cohort was similar for the two age groups when computed using the available histology results proportions. Key HIV-related variables, including viral load and timing of HIV treatment, were not collected, and therefore, the full extent of the immune status of WLWH could not be assessed. Finally, the determination of the proportion of women with high-grade dysplasia was further limited by the lack of histopathology data in women who underwent cryotherapy and the sensitivity of VIA. The accuracy of VIA positivity is affected by the prevalence of cervicitis related to sexually transmitted infections, and high rates of cervicitis have been reported in WLWH in Botswana.[25] appointment attendance was not recorded, and thus histopathology results may not represent the entire cohort of women who had a colposcopic evaluation. However, the population point prevalence of the screened cohort was similar for the two age groups when computed using the available histology results proportions. Key HIV-related variables, including viral load and timing of HIV treatment, were not collected, and therefore, the full extent of the immune status of WLWH could not be assessed. Finally, the determination of the proportion of women with high-grade dysplasia was further limited by the lack of histopathology data in women who underwent cryotherapy and the sensitivity of VIA. The accuracy of VIA positivity is affected by the prevalence of cervicitis related to sexually transmitted infections, and high rates of cervicitis have been reported in WLWH in Botswana.[25] Abbreviations ART: Anti-retroviral treatment; CI: Confidence interval; CIN: Cervical intraepithelial neoplasia; HIC: High-income country; HIV: Human immunodeficiency virus; HPV: Human papillomavirus; HRDC: Health research and development committee; LEEP: Loop electro-surgical excision procedure; LMIC: Low-middle income county; IQR: Interquartile range; MOHW: Ministry of health and wellness; NHL: National health laboratory OR: Odds ratio; PMH: Princess Marina hospital; VIA: Visual inspection after acetic acid; WHO: World Health Organization; WLHV: Women living with HIV Conclusions Despite the limitations of this study, we present new evidence of the significant burden of CIN2 + in younger WLWH in Botswana. Until the population-level effects of HPV vaccination and universal ART to improve overall immune competence in WLWH are realized,[26] the reduction in cervical cancer in LMICs will depend on effective, comprehensive screening programs for WLWH. This additional evidence further supports the current WHO conditional recommendation for initiating screening at age 25. Ethics approval and consent to participate The Health Research and Development Committee (HRDC) of the Botswana Ministry of Health and Wellness (MOHW) approved this analysis (HPDME-13/181). Patient consent was waived as this was a secondary analysis of routinely collected programmatic data. All methods were performed in accordance with the Declaration of Helsinki. Acknowledgments We acknowledge with thanks the contributions of Harvey Friedman, Nicola Zetola, Ari Ho-Foster, Balladia Kizoto, and Kadimo Khutsafalo. We acknowledge with thanks the contributions of Harvey Friedman, Nicola Zetola, Ari Ho-Foster, B Author contributions Study conception and design: DRM, RL, SG; Acquisition of data: BM, DRM; Analysis and interpretation of data: DRM, RL, SG, GJH, AM, CM, LG ; Drafting of manuscript: DRM, RL, SG; Critical revision: GJH, CM, LBM, AM. All authors approved the manuscript before submission. Study conception and design: DRM, RL, SG; Acquisition of data: BM, DRM; Analysis and interpretation of data: DRM, RL, SG, GJH, AM, CM, LG ; Drafting of manuscript: DRM, RL, SG; Critical revision: GJH, CM, LBM, AM. All authors approved the manuscript before submission. Acknowledgments Availability of data and materials The datasets generated during the current study are available from the corresponding author on reasonable request. The datasets generated during the current study are available from the corresponding author on reasonable request. Competing interests No competing interests declared by the authors No competing interests declared by the authors The development of the ‘see and treat’ pilot program database was undertaken as part of the PEPFAR-supported Botswana-UPenn Partnership Technical assistance for in-service training for health care providers in Botswana (U2GPS001949) References A case-control study of HIV infection and cancer in the era of antiretroviral therapy in Rwanda. International journal of cancer Journal international du cancer. 2018;143(6):1348–55. 9. Mpunga T, Znaor A, Uwizeye FR, et al. A case-control study of HIV infection and cancer in the era of antiretroviral therapy in Rwanda. International journal of cancer Journal international du cancer. 2018;143(6):1348–55. 10. Cancer screening in the European Union. Cancer screening in the European Union. Report on the implementation of the council recommendation on cancer. 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Kelly H, Weiss HA, Benavente Y, et al. Association of antiretroviral therapy with high-risk human papillomavirus, cervical intraepithelial neoplasia, and invasive cervical cancer in women living with HIV: a systematic review and meta-analysis. Lancet HIV. 2018;5(1):e45- e58. 22. Kelly H, Weiss HA, Benavente Y, et al. Association of antiretroviral therapy with high-risk human papillomavirus, cervical intraepithelial neoplasia, and invasive cervical cancer in women living with HIV: a systematic review and meta-analysis. Lancet HIV. 2018;5(1):e45- e58. Page 14/15 Page 14/15 23. Moscicki AB, Ellenberg JH, Crowley-Nowick P, et al. References Risk of high-grade squamous intraepithelial lesion in HIV-infected adolescents. The Journal of infectious diseases. 2004;190(8):1413–21. 23. Moscicki AB, Ellenberg JH, Crowley-Nowick P, et al. Risk of high-grade squamous intraepithelial lesion in HIV-infected adolescents. The Journal of infectious diseases. 2004;190(8):1413–21. 23. Moscicki AB, Ellenberg JH, Crowley-Nowick P, et al. Risk of high-grade squamous intraepithelial lesion in HIV-infected adolescents. The Journal of infectious diseases. 2004;190(8):1413–21. 24. Parham GP, Mwanahamuntu MH, Kapambwe S, et al. Population-level scale-up of cervical cancer prevention services in a low-resource setting: development, implementation, and evaluation of the cervical cancer prevention program in Zambia. PloS one. 2015;10(4):e0122169. 24. Parham GP, Mwanahamuntu MH, Kapambwe S, et al. Population-level scale-up of cervical cancer prevention services in a low-resource setting: development, implementation, and evaluation of the cervical cancer prevention program in Zambia. PloS one. 2015;10(4):e0122169. 25. Painter H, Erlinger A, Simon B, et al. Impact of cervicitis on performance of cervical cancer screening using HRHPV testing and visual evaluation in women living with HIV in Botswana. 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https://openalex.org/W3104365465	https://www.confer.cz/metal/2020/download/3552-deposition-of-ultra-black-nickel-phosphorus-coatings.pdf	English	null	Deposition of ultra-black nickel phosphorus coatings	Metal ...	2,020	cc-by	3,014	Abstract Absorbing surfaces of solar receivers are coated with black chrome, which provides the surface of steel or aluminum tubes with good absorption properties. However, the chrome coating does not protect well against corrosion because of its high porosity and high internal stresses, which cause a cracking of coatings. In this paper, we investigated two methods for formation of black coatings on chemically formed Ni-P layers: etching in nitric acid and anodic treatment in a phosphoric acid solution. It was found that in the course of processing the black coatings, their roughness and porosity increase. It was shown that nickel oxides NiO and Ni2O3 and nickel phosphates are presented in the coating composition. It was established that the heat treatment increases the wear resistance and protective ability of black coatings. Keywords: Chemical nickel plating, ultra-black nickel-containing coatings, steel surface treatment, heat treatment of black coatings, Ni-P coatings, protective properties, roughness, XPS analysis of the composition May 20th - 22nd 2020, Brno, Czech Republic, EU https://doi.org/10.37904/metal.2020.3552 https://doi.org/10.37904/metal.2020.3552 https://doi.org/10.37904/metal.2020.3552 1MUCTR - D. Mendeleev University of Chemical Technology of Russia, Moscow, Russia, katerina.sherbin@mail.ru 1MUCTR - D. Mendeleev University of Chemical Technology of Russia, Moscow, Russia, katerina.sherbin@mail.ru https://doi.org/10.37904/metal.2020.3552 2. EXPERIMENTAL MATERIALS We used chemical reagents of grades “pur”, “ p.a.” and distilled water to prepare working solutions. We used chemical reagents of grades “pur”, “ p.a.” and distilled water to The sheets of cold rolled steel measuring 3x4 cm were used as samples. The sheets of cold rolled steel measuring 3x4 cm were used as samples. The process of applying black coatings took place in two stages. At the first stage, a Ni-P coating from a solution of the following composition (g / l) was applied to steel samples: NiSO4•7H2O – 35; NaH2PO2*H2O – 25; СH3COONa –18; С3Н6О3 – 34; C6H8O7 – 5. The duration of the process is 3 hours at a temperature of 90- 92 ° C. At the second stage, the Ni-P coating was blackened due to etching in nitric acid or anodic treatment in phosphoric acid. The abrasion resistance of nickel-containing coatings was studied using a modernized Taber Elcometer 5135 rotational abrasimeter. Coated flat samples were pressed against a felt disk with the same external load on both arms equal to 3.5 N / cm2. The abrasion of the samples was carried out during rotation of the felt disk with a rotation speed of 60 rpm. The abrasion resistance of the samples was evaluated by the number of cycles until the first signs of abrasion appeared. One cycle amounted to one full circle of felt disk [18]. Corrosion testing of coated samples was carried out in an Ascott S450iP salt spray chamber (UK) in accordance with ASTM B117. Morphological studies of the surface of coatings and determination of their roughness were carried out using an MPLAPONLEXT 100 objective using a LEXT – OSL 4100 confocal laser microscope. The composition of the surface layers was studied via X-ray photoelectron spectroscopy (XPS) [19]. Coated samples were fixed in a holder and placed in the pre-evacuation chamber of an OMICRON ESCA+ XPS unit (Germany). The samples were then transferred to the analyzer chamber, where the pressure was maintained at a level no higher than 8 × 10−10 mbar. MgKα radiation was used (1253.6 eV; power, 252 W). The pass energy of the analyzer was 20 eV. To allow for the charge of the samples, the position of the XPS peaks was standardized with respect to the C 1s peak of hydrocarbon impurities from the atmosphere; bonding energy Eb of the peak was assumed to be 285.0 eV. 2. EXPERIMENTAL MATERIALS The spectra were decomposed after subtracting the background determined using the Shirley method [20]. The peak position was determined with an accuracy of ±0.1 eV. Component ratios were calculated using integrated intensities under the peaks while allowing for photoionization cross sections σ of the respective electron shells [21]. Using the integrated intensity of the peaks and the MultiQuant software program [22], the thicknesses of the layers formed on the surface were calculated with allowance for the mean free path of electrons λ, determined using the formula of Cumpson and Seah [24]. When analyzing the obtained spectra, it was considered that the analyzing beam penetrates the material to the depth of 5 nm, i.e. the resulting spectra correspond to a layer of material of 5 nm thick. 3. EXPERIMENTAL WORK According to the literary recommendations, the formation of black Ni-containing coatings takes place in two stages: at the first stage, the Ni-P film is deposited, and then in one of two ways it becomes black: - etching in nitric acid (HNO3); - anode treatment in phosphoric acid (H3PO4). 1. INTRODUCTION Galvanic black coatings are used to absorb light in optical devices and collectors, which convert light energy into heat, as well as protective and decorative coatings for various products [1-3]. In most cases, black chrome and nickel coatings are used for these purposes [4-10]. Known disadvantages of chromium plating processes are the difficulty of implementation and high energy intensity, due to the need to heat solutions and losses due to the side reaction of hydrogen evolution. To obtain a chrome coating, you need to spend 3 times more electricity than for applying other galvanic coatings. In addition, the chrome coating poorly protects the steel from corrosion as it has high porosity, high internal stresses, leading to cracking of the coating [1]. Known processes of black nickel plating are less energy-intensive and easier to implement, but they have disadvantages such as low levels of protective ability, wear resistance, and poor adhesion to the substrate, especially to steel. As a rule, during the deposition of a black nickel-containing coating, a sublayer of copper or brushed nickel is first applied, on which a black coating is formed, consisting of hydroxides and sulfides of zinc and nickel, which are dispersed in the mass of metallic nickel. The thickness of such coatings does not exceed 0.5-1.0 μm, and the corrosion resistance is determined mainly by the corrosion resistance of the sublayer [3-5]. A possible alternative to the process of electrodeposition of black chrome and nickel coatings is the process of chemical nickel plating [11-17]. A process feature is the application of uniform in thickness coatings onto complex parts. The resulting coatings are characterized by high corrosion resistance and hardness, which can be significantly increased by subsequent heat treatment. Therefore, the aim of this work is to study the technical processes of the formation of black coatings on the surface of chemically deposited nickel. May 20th - 22nd 2020, Brno, Czech Republic, EU 3.1. Determination of the parameters of etching process of Ni-P coatings in nitric acid It was found that the maximum black color on Ni-P coatings can be obtained at a nitric acid concentration of 6 mol / L or higher, and the processing time is at least 5-5.5 minutes. As expected, the roughness during etching increased from 0.504 to 0.707 microns. Raising the temperature of the solution above 45 ° C has a bad effect as the color does not become deep enough. It is important to note that at a temperature below 35 ° C a black coating is not formed. May 20th - 22nd 2020, Brno, Czech Republic, EU Photographs of the surface obtained with a confocal microscope indicate that this process is accompanied by the formation of microscopic conical pores perpendicular to the surface. Pore diameter, depth and distance vary from a fraction of a micrometer to several micrometers or from about one to several light wavelengths. Consequently, pores create a black appearance capturing any incident light in a wide spectral range. The photographs show the Ni-P coating before etching had a thickness of 30 μm. After etching, the thickness decreased to 7 μm, 2-3 of which is the formed black layer (Figure 1). Over 20 μm of the coating obtained in the first stage were etched. Figure 1 Photographs of cross sections of Ni-containing coatings before and after etching in nitric acid Basic Basic Black coating Figure 1 Photographs of cross sections of Ni-containing coatings before and after etching in nitric acid Using X-ray photoelectron spectroscopy, it was found that oxides of divalent and trivalent nickel, as well as compounds of nickel with phosphorus, are present on the surface (Figure 2). Figure 2 The individual XPS spectrum of Ni Binding energy, eV Intensity, units Binding energy, eV Figure 2 The individual XPS spectrum of Ni Figure 2 The individual XPS spectrum of Ni Based on the information received, we assume that the following reactions occur in the process, written using chemical equations (1), (2), (3), (4), (5) and (6). 3.1. Determination of the parameters of etching process of Ni-P coatings in nitric acid Ni + NO3– → NiO + NO2– (1) 2Ni + 3NO3– → Ni2O3 + 3NO2– (2) 3P + 5HNO3 + 2H2O → 3H3PO4 + 5NO↑ (3) Ni2+ + 2H3PO4 → Ni(H2PO4)2 + 2H+ (4) Ni2+ + H3PO4 → NiHPO4 + 2H+ (5) 3Ni2+ + H3PO4 → Ni3PO4 + 3H+ (6) Ni + NO3– → NiO + NO2– 2Ni + 3NO3– → Ni2O3 + 3NO2– 3P + 5HNO3 + 2H2O → 3H3PO4 + 5NO↑ Ni2+ + 2H3PO4 → Ni(H2PO4)2 + 2H+ Ni2+ + H3PO4 → NiHPO4 + 2H+ 3Ni2+ + H3PO4 → Ni3PO4 + 3H+ (6) (6) The black coatings were obtained using the treatment in nitric acid, but the significant drawback of this process is the large loss of non-ferrous metal during etching. The black coatings were obtained using the treatment in nitric acid, but the significant drawback of this process is the large loss of non-ferrous metal during etching. - anode treatment in phosphoric acid (H3PO4). - anode treatment in phosphoric acid (H3PO4). - anode treatment in phosphoric acid (H3PO4). May 20th - 22nd 2020, Brno, Czech Republic, EU 2. Determination of process parameters for anodizing Ni-P coatings in phosphoric acid The dependence of the coating quality on the concentration of phosphoric acid and the voltage on the bath is investigated. It was experimentally found that the blackest coatings are formed at a concentration of 2.7 M and a voltage of 1.1-1.3 V for a duration of 9 minutes at a room temperature. Roughness indices were also obtained after anodic treatment in phosphoric acid. The roughness during anodization increases from 0.504 to 0.733 microns. It was found that after anodizing the Ni-P layer, a greater surface development is observed than in the case of etching in nitric acid. The number of pores is greatly increased. The coating thickness is etched less from 30 to 12 microns. The thickness of the black layer is only 0.4 μm (Figure 3). Figure 3 Photos of cross sections of Ni-containing coatings before and after processing in H3РO4 Black coating Basic Basic gure 3 Photos of cross sections of Ni-containing coatings before and after processing in H3РO4 The individual nickel spectrum showed that the composition of the black film also includes NiO, Ni2O3 and nickel phosphates (Figure 4). Figure 4 The individual XPS spectrum of Ni Binding energy, eV Intensity, units Binding energy, eV So, we can assume that the following reactions take place, which can be written using chemical equations (7), (8), (9), (10) and (11). Ni+PO43-→NiO+PO33- (7) 2Ni+3PO43-→Ni2O3+ 3PO33- (8) Ni2+ + 2H3PO4 → Ni(H2PO4)2 +2H+ (9) Ni2+ + H3PO4 → NiHPO4 + 2H+ (10) 3Ni2+ + 2H3PO4 → Ni3(PO4)2+ 6H+ (11) Ni+PO43-→NiO+PO33- (7) 2Ni+3PO43-→Ni2O3+ 3PO33- (8) Ni2+ + 2H3PO4 → Ni(H2PO4)2 +2H+ (9) Ni2+ + H3PO4 → NiHPO4 + 2H+ (10) 3Ni2+ + 2H3PO4 → Ni3(PO4)2+ 6H+ (11) Ni+PO43-→NiO+PO33- 2Ni+3PO43-→Ni2O3+ 3PO33- Ni2+ + 2H3PO4 → Ni(H2PO4)2 +2H+ Ni2+ + H3PO4 → NiHPO4 + 2H+ 3Ni2+ + 2H3PO4 → Ni3(PO4)2+ 6H+ (11) May 20th - 22nd 2020, Brno, Czech Republic, EU 4. CONCLUSION As a result, methods have been developed for the formation of black layers on chemical coatings in the following ways: As a result, methods have been developed for the formation of black layers on chemical coatings in the following ways: - 6М HNO3, Т (solution) = 40°С и τ process 5-7 min - 2,7М H3PO4, U=1,1-1,3 V, Т (solution) = 20-40°С It was revealed that in the process of processing black coatings, an increase in roughness and porosity occurs. It was shown that the composition of the coating includes nickel oxides NiO, Ni2O3 and nickel phosphates. It was found that heat treatment increases the wear resistance and protective ability of coatings. 3.3. Tests of the coatings It should be noted that black coatings formed by these methods were characterized by smearing. The possibility of eliminating this phenomenon with the help of subsequent heat treatment of coatings is investigated. It is shown that coatings cease to be smeared during heat treatment in the indicated ranges. For nitric acid, this is 220 ° C for more than half an hour. And for coatings blackened using anodic treatment in phosphoric acid, it is 190 ° C for more than an hour and a half. In addition, it was revealed that during the heat treatment, the roughness of the coatings increases in both cases. When anodizing in phosphoric acid, the surface development is less than when etching in nitrogen: Ra = 0.865 and 1.212 μm, respectively. The wear resistance of the coatings was investigated. The number of cycles was evaluated until the first signs of abrasion appeared. Table 1 shows that coatings obtained by anodizing in phosphoric acid with subsequent heat treatment have the highest wear resistance. Corrosion tests have shown that these coatings also have the greatest protective ability. Table 1 Comparison of the functional characteristics of the resulting coatings Process Wear resistance of black coatings (number of cycles until the first signs of loss of black color appear) The protective ability of black coatings (Time until the appearance of corrosion products of the base, hour) Etching in HNO3 850 39 Etching in HNO3 + heat treatment 11750 98 Anodization in H3PO4 1050 45 Anodization in H3PO4 + heat treatment 16000 130 Electrodeposition of black nickel- containing coating 3000 50 Electroplating black chrome coating 13000 120 Table 1 Comparison of the functional characteristics of the resulting coatings This table allows you to compare the studied coatings with black electroplated nickel and chrome coatings. It can be seen that the films obtained by anodic treatment in a solution of phosphoric acid followed by heat treatment turn out to be the best in both terms of wear resistance and protective ability. REFERENCES [1] RUDENKO M.F., KRAVTSOV E.E., IDIATULIN S.A. Effective surfaces of solar receivers. Himicheskoe i neftegazovoe mashinostroenie. 1998, no. 7, pp. 33-35. [2] MESHALKIN V.P., ABRASHOV A.A., VAGRAMYAN T.A., GRIGORYAN N.S., UTOCHKINA D.S. Development of Composition and Investigation of Properties of a New, Environmentally Friendly Molybdenum-Containing Decorative Protec-tive Conversion Coating on Zinс-Plated Surfaces. Doklady Chemistry. 2018, vol. 480, no. 5, pp. 555–558. [3] TAKADOUM J. Black coatings: a review. The European physical journal applied physics. 2010, no. 52, pp 30401 (p1-p7). [4] SOMASUNDARAM S., PILLAI A.M., RAJENDRA A., SHARMA A.K. High emittance black nickel coating on copper substrate for space applications. Journal of Alloys and Compounds. 2015, vol. 643, pp. 263-269. [5] ESTRELLA-GUTIÉRREZ M.A., LIZAMA-TZEC F.I., ARÉS-MUZIO O., OSKAM G. Inﬂuence of a metallic nickel interlayer on the performance of solar absorber coatings based on black nickel electrodeposited onto copper. 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Project Number Х-2020-027» «The work was supported by the Mendeleev University of Chemical Technology of Russia. Project Number Х-2020-027» May 20th - 22nd 2020, Brno, Czech Republic, EU REFERENCES 2010, vol. 40, pp. 333. [15] GUO Z., KEONG K.G., SHA W. Physical and chemical properties of electroless nickel–phosphorus alloys. Journal of alloys and coatings 2003, vol. 358, pp. 112–119. [16] CHEONG W.J., LUAN B.L., SHOESMITH D.W. Optimization of nanostructured black coatings. Corrosion Science. 2007, vol. 49, pp. 1777–1798. [17] BROWN R.J.C., BREWER P.J., MILTON M.J.T. The physical and chemical properties of electroless nickel– phosphorus alloys and low reflectance nickel–phosphorus black surfaces. Journal of Materials Chemistry. 2002, vol. 10, pp. 2749-2754. [18] GRIGORYAN N.S., ABRASHOV A.A., VAGRAMYAN T.A., KOSTIUK A.G. Passivation of ferrous and non-ferrous metals in a solution based on gallic acid esters. Chemistry and technology of organic substances. 2017, no 4, pp. 55-63. [19] Shirley, D.A. High-resolution X-ray photoemission spectrum of the valence bands of gold. Phys. Rev. 1972. B 5, 4709-4713. [20] Scofield H., J. Hartree-Slater subshell photoionization cross-sections at 1254 and 1487 eV. Electron Spectrosc. Relat. Phenom. 1976, vol. 8, pp. 129-137. [21] MOHAI, M. XPS MultiQuant: multimodel XPS quantification software. Surf. Interface Anal. 2004, vol. 36, pp. 828– 832.
https://openalex.org/W2999594613	https://discovery.ucl.ac.uk/10092801/8/Guo_Reversible%2C%20Two-Step%20Single-Crystal%20to%20Single-Crystal%20Phase%20Transitions%20between%20Desloratadine%20Forms%20I%2C%20II%2C%20and%20III_VoR.pdf	English	null	Reversible, Two-Step Single-Crystal to Single-Crystal Phase Transitions between Desloratadine Forms I, II, and III	Crystal growth & design	2,020	cc-by	11,663	pubs.acs.org/crystal Computational spectroscopy, using periodic DFT-D phonon calculations, reproduces the experimental variable-temperatureTHz-Raman spectra and rules out the possibility of the phase transformations occurring via any classical soft mode. A combination of variable-temperature powder X-ray diﬀraction, solid-state NMR, and computational spectroscopy provides a detailed molecular description of the phase transitions, indicating a ﬁrst-order diﬀusionless process between I →II and II →III, wherein both conformational changes and lattice distortions occur simultaneously in the crystal lattice. The study indicates that a nucleation and growth mechanism is compatible with concerted movements producing a conformational change in organic molecular crystals. ABSTRACT: Single-crystal to single-crystal polymorphic transformations in molecular solids are relatively rare, with changes in crystal structure more commonly leading to destruction of the parent crystal. However, the structural basis for such transitions is of considerable interest given the changes in material properties that can result. The antihistamine desloratadine displays a two-step, reversible single-crystal to single-crystal phase transition during heating/cooling cycles between three conformational polymorphs: the low temperature form I, a polytypic intermediate form II, and the high temperature form III. The two-step transition involves a sequential ﬂipping of the piperidine rings of desloratadine molecules in the crystals, which induce reversible micrometer-scale contraction on heating and expansion on cooling of the largest face of a desloratadine single crystal. Distinct, slow-moving phase boundaries, originating on the (001) face of the crystal, were observed sweeping through the entire crystal in hot-stage microscopy, suggesting a single nucleation event. Computational spectroscopy, using periodic DFT-D phonon calculations, reproduces the experimental variable-temperatureTHz-Raman spectra and rules out the possibility of the phase transformations occurring via any classical soft mode. A combination of variable-temperature powder X-ray diﬀraction, solid-state NMR, and computational spectroscopy provides a detailed molecular description of the phase transitions, indicating a ﬁrst-order diﬀusionless process between I →II and II →III, wherein both conformational changes and lattice distortions occur simultaneously in the crystal lattice. The study indicates that a nucleation and growth mechanism is compatible with concerted movements producing a conformational change in organic molecular crystals. pubs.acs.org/crystal The antihistamine desloratadine displays a two-step, reversible single-crystal to single-crystal phase transition during heating/cooling cycles between three conformational polymorphs: the low temperature form I, a polytypic intermediate form II, and the high temperature form III. The two-step transition involves a sequential ﬂipping of the piperidine rings of desloratadine molecules in the crystals, which induce reversible micrometer-scale contraction on heating and expansion on cooling of the largest face of a desloratadine single crystal. Distinct, slow-moving phase boundaries, originating on the (001) face of the crystal, were observed sweeping through the entire crystal in hot-stage microscopy, suggesting a single nucleation event. Computational spectroscopy, using periodic DFT-D phonon calculations, reproduces the experimental variable-temperatureTHz-Raman spectra and rules out the possibility of the phase transformations occurring via any classical soft mode. A combination of variable-temperature powder X-ray diﬀraction, solid-state NMR, and computational spectroscopy provides a detailed molecular description of the phase transitions, indicating a ﬁrst-order diﬀusionless process between I →II and II →III, wherein both conformational changes and lattice distortions occur simultaneously in the crystal lattice. The study indicates that a nucleation and growth mechanism is compatible with concerted movements producing a conformational change in organic molecular crystals. ABSTRACT: Single-crystal to single-crystal polymorphic transformations in molecular solids are relative ABSTRACT: Single-crystal to single-crystal polymorphic transformations in molecular solids are relatively rare, with changes in crystal structure more commonly leading to destruction of the parent crystal However the structural basis for such transitions is of ABSTRACT: Single-crystal to single-crystal polymorphic transformations in molecular solids are relatively rare, with changes in crystal structure more commonly leading to destruction of the parent crystal. However, the structural basis for such transitions is of considerable interest given the changes in material properties that can result. The antihistamine desloratadine displays a two-step, reversible single-crystal to single-crystal phase transition during heating/cooling cycles between three conformational polymorphs: the low temperature form I, a polytypic intermediate form II, and the high temperature form III. The two-step transition involves a sequential ﬂipping of the piperidine rings of desloratadine molecules in the crystals, which induce reversible micrometer-scale contraction on heating and expansion on cooling of the largest face of a desloratadine single crystal. Distinct, slow-moving phase boundaries, originating on the (001) face of the crystal, were observed sweeping through the entire crystal in hot-stage microscopy, suggesting a single nucleation event. This is an open access article published under a Creative Commons Attribution (CC-BY) License, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited. This is an open access article published under a Creative Commons Attribution (CC-BY) License, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited. This is an open access article published under a Creative Commons Attribution (CC-BY) License, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited. © 2020 American Chemical Society https://dx.doi.org/10.1021/acs.cgd.9b01522 Cryst. Growth Des. 2020, 20, 1800−1810 © 2020 American Chemical Society Article pubs.acs.org/crystal Reversible, Two-Step Single-Crystal to Single-Crystal Phase Transitions between Desloratadine Forms I, II, and III Published as part of a Crystal Growth and Design virtual special issue Remembering the Contributions and Life of Prof. Joel Bernstein f f J Vijay K. Srirambhatla, Rui Guo, Daniel M. Dawson, Sarah L. Price, and Alastair J. Florence* Cite This: Cryst. Growth Des. 2020, 20, 1800−1810 Read Online ACCESS Metrics & More Article Recommendations * sı Supporting Information ABSTRACT: Single-crystal to single-crystal polymorphic transformations in molecular solids are relatively rare, with changes in crystal structure more commonly leading to destruction of the parent crystal. However, the structural basis for such transitions is of considerable interest given the changes in material properties that can result. The antihistamine desloratadine displays a two-step, reversible single-crystal to single-crystal phase transition during heating/cooling cycles between three conformational polymorphs: the low temperature form I, a polytypic intermediate form II, and the high temperature form III. The two-step transition involves a sequential ﬂipping of the piperidine rings of desloratadine molecules in the crystals, which induce reversible micrometer-scale contraction on heating and expansion on cooling of the largest face of a desloratadine single crystal. Distinct, slow-moving phase boundaries, originating on the (001) face of the crystal, were observed sweeping through the entire crystal in hot-stage microscopy, suggesting a single nucleation event. Computational spectroscopy, using periodic DFT-D phonon calculations, reproduces the experimental variable-temperatureTHz-Raman spectra and rules out the possibility of the phase transformations occurring via any classical soft mode. A combination of variable-temperature powder X-ray diﬀraction, solid-state NMR, and computational spectroscopy provides a detailed molecular description of the phase transitions, indicating a ﬁrst-order diﬀusionless process between I →II and II →III, wherein both conformational changes and lattice distortions occur simultaneously in the crystal lattice. The study indicates that a nucleation and growth mechanism is compatible with concerted movements producing a conformational change in organic molecular crystals. Downloaded via UNIV COLLEGE LONDON on March 4, 2020 at 11:11:22 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. Supporting Information ABSTRACT: Single-crystal to single-crystal polymorphic transformations in molecular solids are relatively rare, with changes in crystal structure more commonly leading to destruction of the parent crystal. However, the structural basis for such transitions is of considerable interest given the changes in material properties that can result. Crystal Growth & Design DSC measure- ments were performed on polycrystalline material using a Netzsch DSC214 Polyma diﬀerential scanning calorimeter. The samples were weighed into aluminum DSC pans and crimped with a pinhole in the lid. The samples were analyzed over the temperature range RT to 450 K at heating rates of 5, 10, 15, and 40 K per minute. g p 2.4. In-Situ Variable Temperature Powder X-ray Diﬀraction. Powder X-ray diﬀraction data of the polycrystalline material were collected using a Bruker AXS D8-diﬀractometer. Finely ground DES form I was loaded into a 0.7 mm borosilicate glass capillary and mounted on the diﬀractometer operating in transmission geometry, equipped with a Johansson monochromator using Cu Kα radiation (λ = 1.5406 Å) and Lynxeye detector. An Oxford Cryosystems Cryostream was used to control the temperature of the sample prior to data collection. Variable temperature diﬀraction data were collected in the temperature range 150−380 K. Data were collected over the angular range 3 ≤2θ/° ≤50, in 0.01° steps, at a counting time of 1 s/frame increments of detector position. Quantiﬁcation of polymorphs by Rietveld analysis38 was performed using TOPAS-V 5.0. Desloratadine (DES) is an antihistamine drug commonly used in the treatment of allergic reactions.24 The molecular structure of DES contains a piperidine ring attached to a N- substituted dibenzazepine (NDBA) moiety (Figure 1). Figure 1. Molecular structure of DES. 2.5. In-Situ Variable Temperature Single-Crystal X-ray Diﬀraction. The single crystal X-ray diﬀraction data were collected using Mo−Kα radiation (λ = 0.7107 Å), on a Bruker Apex-II diﬀractometer equipped with a CCD detector, controlled using APEX3 software. An Oxford Cryosystems cryostream was used to control the temperature of the crystals prior to data collection. Data integration and reduction were performed using the SAINT software (version 8.34A).39 The crystal structures were solved by direct methods using the program SHELXS,40 and subsequent Fourier calculations and least-squares reﬁnements were performed on F2 using the program CRYSTALS.41 All non-hydrogen atoms were reﬁned with anisotropic displacement parameters. All hydrogen atoms bonded to the carbon atoms were placed geometrically and reﬁned with the isotropic displacement parameter ﬁxed at 1.5 times Ueq of the atoms to which they are attached. Protons involved in hydrogen bonding were located directly via inspection of diﬀerence Fourier maps and were reﬁned isotropically. The crystallographic data of DES polymorphs I, II, and III are provided in Supporting Information, Section 1.2.1. 1. INTRODUCTION key material properties that can result. Conversion between polymorphs can change numerous physical properties including density, solubility, electric conductivity, color, mechanical strength, and morphology. Depending on the application, it may be necessary to control such trans- formations to ensure consistent performance or to deliberately exploit them to realize structures with desirable functions. Understanding polymorphism and phase transformations in molecular crystals is crucial to enable the eﬀective manufacture and exploitation of this important class of materials. Poly- morphism1,2 describes the ability of a chemical compound to adopt diﬀerent crystal packing arrangements. Conformational polymorphs contain molecular conformations that approximate diﬀerent isolated molecule conformational minima.3−5 Poly- morphism is ubiquitous4 and has been reported for foodstuﬀs,6 pigments,7 agrochemical products,8,9 and pharmaceuticals.10 While single-crystal to single-crystal transformations in Received: November 13, 2019 Revised: January 14, 2020 Published: January 16, 2020 00 https://dx.doi.org/10.1021/acs.cgd.9b01522 Cryst. Growth Des. 2020, 20, 1800−1810 Received: November 13, 2019 Revised: January 14, 2020 Published: January 16, 2020 Received: November 13, 2019 Revised: January 14, 2020 Published: January 16, 2020 Received: November 13, 2019 Revised: January 14, 2020 Published: January 16, 2020 While single-crystal to single-crystal transformations in molecular solids are relatively rare, the structural basis for such transitions is of considerable interest given the impact on © 2020 American Chemical Society © 2020 American Chemical Society 1800 1800 pubs.acs.org/crystal Crystal Growth & Design Article However, despite ongoing research, it remains challenging to predict speciﬁc occurrences of polymorphism,11 let alone know which polymorphs can readily interconvert, for example, by a single-crystal to single-crystal polymorphic transformation. eight low-energy conformations of DES were determined by optimization at PBE0/6-31G(d,p) level using GAUSSIAN0928 and the charge density analyzed by GDMA2.029 to give a distributed multipole model for the electrostatic interactions. These conforma- tions were used to generate hypothetical structures using CrystalPredictor2.1,30 and the crystal structures were reﬁned using Crystal Optimizer2.4,31 using the ab initio relative molecular energies, the distributed multipoles, and the FIT exp-6 potential32 to allow all the bond angles and torsions to respond to the packing forces. The term “dynamic molecular crystals” has been applied to polymorphs where changes in physical properties can be controlled by external stimuli such as heat, light, or electric ﬁeld.12 Despite the discovery of a number of molecular crystals that change structure under external stimuli,12−14 the design of new crystalline materials that can transmit molecular-scale structural changes to desirable macroscopic changes in crystal shape, while keeping their crystallinity throughout the transition,15,16 remains a signiﬁcant challenge in organic crystal engineering. In particular, the transformation mechanisms involved are generally ill-deﬁned and poorly understood.17 This is largely because direct observation of the molecular processes at or near the critical transition point is challenging, owing to the absence of suitable analytical techniques with suﬃcient spatial and temporal resolution. The thermody- namic,18 structural,19 and other classiﬁcations20 for phase transitions were largely developed for inorganic materials, and there is increasing evidence that they do not adequately capture the phenomena occurring in molecular crystals,21,22 particularly for molecules that have very nonspherical shapes, anisotropic intermolecular interactions, and suﬃcient ﬂexibility to change conformation.23 g p p g Periodic density functional calculations of lattice energy opti- mizations were performed with the PBE functional,33 with various dispersion corrections,34−36 using CASTEP17.2.2.37 The harmonic phonons were calculated by ﬁnite displacements using the PBE-TS functional and using supercells to sample the Brillouin zone. g 2.2. Crystallization of DES. DES (8-chloro-11-(piperidin-4- ylidene)-6,11-dihydro-5H-benzo[5,6]cyclohepta[1,2-b]pyridine) and anhydrous ethyl acetate was procured from Sigma-Aldrich. Single crystals of DES were obtained by dissolving ∼100 mg of DES in ∼2− 3 mL of ethyl acetate solvent and allowing the solution to evaporate slowly at RT. Colorless crystals that appeared after 5−8 days were used for subsequent analysis. q y 2.3. Diﬀerential Scanning Calorimetry (DSC). Crystal Growth & Design Face indexing of the DES crystals was performed using the Index Crystal Faces routine of Apex 3 software. Figure 1. Molecular structure of DES. Reported solid forms of DES include form I25 (CSD refcode: GEHXEX) and a benzoate salt (CSD refcode: DEGNOV).26 While the existence of a high temperature form is known,27 no crystal structure has thus far been reported. In the present work, reversible conformational phase transitions of DES are investigated using an array of experimental and computational methods to probe the structural and energetic changes in this system. Hot-stage optical microscopy has also revealed unusual features associated with the mechanism of transformation as well as macroscopic and reversible shape change in DES crystals undergoing phase transformations. The striking and unusual nature of phase transformations in DES is detailed while also highlighting the complexity associated with understanding solid−solid phase transformations in industrially relevant organic molecular materials. 2.6. Temperature-Resolved Terahertz Raman Spectroscopy. Variable temperature Raman measurements was performed using the Ondax THz-Raman Spectroscopy Systems using a 785 nm laser. Variable temperature THz-Raman data on a single crystal of DES were collected between RT and 380 K during heating and cooling cycles in the extended spectral range of 0−1700 cm−1. The sample temperature was controlled using a Linkam LST 420 hot-stage. 2.6. Temperature-Resolved Terahertz Raman Spectroscopy. Variable temperature Raman measurements was performed using the Ondax THz-Raman Spectroscopy Systems using a 785 nm laser. Variable temperature THz-Raman data on a single crystal of DES were collected between RT and 380 K during heating and cooling cycles in the extended spectral range of 0−1700 cm−1. The sample temperature was controlled using a Linkam LST 420 hot-stage. 2. EXPERIMENTAL SECTION 2.1. Crystal Structure Prediction and Periodic DFT-D Calculations. The complete details of crystal structure prediction studies are given in Supporting Information, Sections 2.2 and 2.3. The g g 2.7. Hot-Stage Optical Microscopy. Temperature-resolved microscopic measurements were performed using a Leica DM6000 1801 https://dx.doi.org/10.1021/acs.cgd.9b01522 Cryst. Growth Des. 2020, 20, 1800−1810 Figure 2. DSC curves of powder DES I, with a heating cycle in green. The insert shows the cooling cycle in red. Data collected at 5 K/min. The black lines indicate area under the curve. pubs.acs.org/crystal Article Crystal Growth & Design pubs.acs.org/crystal FS microscope, equipped with Leica DFC 310 FX camera. A Linkam LTS 420 hot-stage, equipped with T95-Linkam control pad and T95 Linkam temperature controller unit, and an optional LNP95 liquid nitrogen cooling pump system were used to control the temperature of the crystals. y 2.8. Temperature-Resolved Solid State NMR Spectroscopy. Solid-state NMR spectra were recorded on a Bruker Avance III spectrometer equipped with a wide-bore 9.4 T superconducting magnet (Larmor frequencies of 400.13 and 100.61 MHz for 1H and 13C, respectively). The sample was packed into a standard 4 mm zirconia rotor and rotated at the magic angle at 12.5 kHz. Spectra were acquired with cross-polarization (CP) from 1H with a spin lock (ramped for 1H) of 5 ms. High-power (ν1 ≈100 kHz) TPPM-15 decoupling of 1H was applied during acquisition. Signal averaging was carried out for 24 transients with a recycle interval of 300 s. The sample temperature was controlled using a Bruker BVT control unit, equipped with BCU-II chiller and BVTB-3000 heater booster. The sample was heated to the target temperature and then allowed to reach thermal equilibrium for 5 min before data acquisition. The sample temperature was calibrated using the hexagonal to fcc phase transition of DABCO (351.1 K)42 as a ﬁxed temperature point and the temperature dependence of the 87Rb chemical shift of RbCl to calibrate temperatures across the relevant range.43 13C Chemical shifts are reported in ppm relative to (CH3)4Si using the CH3 resonance of L-alanine (δ = 20.5 ppm) as a secondary solid reference. Figure 2. DSC curves of powder DES I, with a heating cycle in green. The insert shows the cooling cycle in red. Data collected at 5 K/min. The black lines indicate area under the curve. observed before melting, the polymorphs are related enantiotropically. Repeated experiments between RT and 375 K with diﬀerent heating and cooling rates (at 5−40 K/ min) resulted in similar thermal data conﬁrming the reversible and reproducible nature of the phase transformations (Supporting Information, Figures S16−S19). The DSC data suggest that DES form I is the thermodynamically stable form at room temperature (RT) with II and III being enantiotropi- cally related. While the exact onset of DES I →II and II →III phase transitions in the heating cycle are not clear, DSC analysis indicates a thermal hysteresis of ∼20 K based on the observed onset temperatures. 3. RESULTS As part of the investigation into polymorphism of DES, a crystal structure prediction (CSP)44 study was performed (Supporting Information, Section 2.2). The progress in CSP methods in recent years11,45 has reached the stage where it is starting to inform experimental screening approaches46,47 to allow targeted crystallization of computationally predicted polymorphs.48−51 Conformational analysis of a DES molecule in the gas phase resulted in eight conformational minima within 4 kJ/mol of the global minimum (Supporting Information, Section 2.1). These were used in the CSP search, which was also constrained to only consider Z′ = 1 structures. The search (Supporting Information, Section 2.2) revealed two densely packed, dispersion bound structures, the most stable being form I and a higher energy structure that was later shown to be form III. The other low energy structures were less dense, and most were based on N−H···N hydrogen-bonded molecular chains. Alternative evaluations of the relative stability of the CSP generated structures suggested that the hydrogen-bonded structures were thermodynamically less likely than form I and III (Supporting Information, Section 2.3). Hence, the CSP study did not suggest any further conformational or packing polymorphs to be targeted. 3.2. In-Situ Variable Temperature Powder X-ray Diﬀraction (VT-PXRD). A ﬁnely ground sample of recrystal- lized DES I was used to perform in situ VT-PXRD analysis. PXRD data were collected between 150 and 400 K for both the heating and cooling cycles. Representative X-ray diﬀraction data for forms I, II, and III are shown in Figure 3 with the Figure 3. Selected powder XRD patterns at diﬀerent temperatures during a cooling cycle. At 340 K (DES III, blue), 320 K (DES II, red), and at 300 K (DES I, black). g g 3.1. Diﬀerential Scanning Calorimetry (DSC). DSC analysis of polycrystalline DES I exhibits two endothermic transitions upon heating with peak temperatures of 338.6 and 431.3 K (Figure 2). While the second endotherm is consistent with the melting of the compound, the ﬁrst endotherm with an onset of 334.8 K can be attributed to successive solid−solid phase transformations, i.e., overlapping signals from the conversion of DES I →II and II →III (based on diﬀraction data) (Section 2.2 and 2.3). Indeed, upon cooling from 375 K, the thermogram exhibits two exothermic transitions with onset temperatures of 326.5 and 313.9 K corresponding to polymorphic transformations of III →II and II →I. 3. RESULTS transformations in the powder between forms I to III extend over ∼15 K in the heating cycle and ∼25 K during the cooling cycle, consistent with the DSC and variable temperature solid- state NMR (VT-SS-NMR Supporting Information, Section 1.6) measurements. More quantitative measurements (see Section 2.5) indicate that phase transformations between forms I and III occur across a wider temperature range of over 40 K. N interactions (C···N = 3.369 (2) and 3.360 (3) Å), which arrange to form 1D H-bonded chains extending along the b axis. Form II was always observed in the presence of either form I (on the heating cycle) or form III (on the cooling cycle), suggesting that fragments of the crystal had not yet transformed at the measurement temperature. N interactions (C···N = 3.369 (2) and 3.360 (3) Å), which arrange to form 1D H-bonded chains extending along the b axis. Form II was always observed in the presence of either form I (on the heating cycle) or form III (on the cooling cycle), suggesting that fragments of the crystal had not yet transformed at the measurement temperature. p On heating above 335 K, the DES form II crystal subsequently transforms to a high temperature phase, form III. Structure determination at 350 K in space group P21 yields one molecule in the asymmetric unit in the ASE conformation, highlighting that all molecules in the lattice have changed conformation. Upon cooling from 350 K, form III crystals transform back to form II at 320 K, which subsequently transforms to form I at 293 K conﬁrming that both transformations are reversible. The VT-SXRD results show the transition between forms I, II, and III is a completely reversible, two-step single-crystal to single-crystal, solid-state transformation. A number of SXRD measurements with crystals of diﬀerent sizes resulted in similar observations with form II as the intermediate structure. The single particle transformation mechanisms and transitions are consistent with those observed from the bulk samples. It is also notable from both single crystal and PXRD data for DES II that diﬀraction from DES I (heating) or III (cooling) was always present. This is likely due to hysteresis in nucleation of individual crystallites in the polycrystalline samples or untransformed domains within the single crystal. 3.3. In-Situ Variable Temperature Single Crystal X-ray Diﬀraction. Variable temperature single crystal X-ray diﬀraction (VT-SXRD) was used to track the speciﬁc structural changes in individual crystals. 3. RESULTS The enthalpy changes associated with the transformations III →II and II →I during the cooling cycle are 0.4 ± 0.1 and 0.3 ± 0.1 kJ/mol. According to the heat of transition rule proposed by Burger and Ramberger,52 if an endothermic transition is Figure 3. Selected powder XRD patterns at diﬀerent temperatures during a cooling cycle. At 340 K (DES III, blue), 320 K (DES II, red), and at 300 K (DES I, black). complete data sets in Supporting Information, Section 1.3. The PXRD data indicate that, upon heating, form I transforms to II and then to III. Upon cooling, the reverse transformations of III to II and then to I were observed. All three phases are observed to coexist over a range of temperatures: visual inspection of the VT-PXRD data indicates the phase 1802 https://dx.doi.org/10.1021/acs.cgd.9b01522 Cryst. Growth Des. 2020, 20, 1800−1810 Figure 4. (a) Superimposition of asymmetric units of form I (green Z′ = 1), II (gray Z′ = 2), and III (red Z′ = 1), (b) overlay of the molecular conformations AAA (blue) and ASE (red) in DES I and DES III, (c) crystal packing arrangements of DES I (P21, a = 7.061 Å b = 12.070 Å c = 9.559 Å, β = 108.2°), II (P21, a = 14.818 Å, b = 12.223 Å, c = 9.631 Å β = 113.3°), and III (P21, a = 7.754 Å b = 12.139 Å c = 9.608 Å β = 108.2°), with the a axis in red, b in green, and c in blue. The hydrogens in (b) and (c) are omitted for clarity. Crystal Growth & Design pubs.acs.org/crystal Article pubs.acs.org/crystal Article Crystal Growth & Design Article Figure 4. (a) Superimposition of asymmetric units of form I (green Z′ = 1), II (gray Z′ = 2), and III (red Z′ = 1), (b) overlay of the molecular conformations AAA (blue) and ASE (red) in DES I and DES III, (c) crystal packing arrangements of DES I (P21, a = 7.061 Å b = 12.070 Å c = 9.559 Å, β = 108.2°), II (P21, a = 14.818 Å, b = 12.223 Å, c = 9.631 Å β = 113.3°), and III (P21, a = 7.754 Å b = 12.139 Å c = 9.608 Å β = 108.2°), with the a axis in red, b in green, and c in blue. The hydrogens in (b) and (c) are omitted for clarity. https://dx.doi.org/10.1021/acs.cgd.9b01522 Cryst. Growth Des. 2020, 20, 1800−1810 Crystal Growth & Design Crystal Growth & Design pubs.acs.org/crystal Article piperidines (Figure 4b). Thus, the three DES structures reported here are clearly conformational polymorphs3−5 with form II being a polytypic53 arrangement of alternating layers of I and III. Owing to the close similarity in crystal packing arrangements of all the three forms, there is a close orientational relationship between the unit-cell parameters of forms I, II, and III as measured with the TOPO software54 (Supporting Information, Section 1.2.5). A direct comparison of unit cell parameters of the three polymorphs shows that the a axis doubles, the b axis expands, while the c axis insigniﬁcantly contracts during the I →II phase transformation (Supporting Information, Section 1.2.4). Also, during the II → III transformation the a axis halves, while the b and c axes expand over this temperature range. Additionally, the angle β sequentially increases from form I to II to III (108.08°, 113.38°, and 118.78° respectively). The reﬁned Flack parameter values (Supporting Information, Section 1.2.1) at diﬀerent temperatures indicate that the reversible trans- formation occurs without a change in the handedness of the crystals. The mosaicity (∼0.59 ± 0.02) of the crystal also remains constant over the heating and cooling cycles, suggesting no signiﬁcant change in individual crystal quality. Section 1.4). The appearance and disappearance of vibrational modes conﬁrm the reversible nature of the phase trans- formation. The spectrum of form I shows that several modes shift to lower frequency on heating, which reverses on cooling, which may be attributed to the changes in thermal vibrations.56 There is no evidence from the THz-Raman data of any transient amorphous or disordered intermediate phases during the heating/cooling cycles.57 While the THz Raman data are rich in information corresponding to both inter- and intramolecular vibrations, extracting relevant information is challenging without exploit- ing the recent development of computational methods.58,59 Theoretical harmonic phonon dispersion curves along high- symmetry paths of the Brillouin zone were calculated using periodic electronic structure (PBE-TS) methods (see Support- ing Information, Section 2.4). Allowing for the expected oﬀset between the calculated and observed modes in the THz region, the calculated and experimental modes match reasonably well (Figure 5), particularly considering the elevated temperatures at which the THz spectra were measured. The free energy diﬀerences between the three forms were also estimated from the calculated modes and lattice energies (Supporting Information, Section 2.4). Crystal Growth & Design Form I was found to be the most thermodynamically stable form at lower T, with form II being the next most stable in lattice energy, but the thermal contributions stabilize form III, such that form III becomes the most stable phase above about 380 K. The energy diﬀerences between form I and III are in reasonable agreement with the DSC measurements, given the uncertainty in the lattice energies and the use of the harmonic approximation without allowance for thermal expansion.60 Form II is very thermodynamically competitive with the other forms around the transition region (Supporting Information, Figure S28). 61 3.4. Variable Temperature THz Raman. Lattice dynamics are of central importance to understand the mechanism of solid−solid phase transformations and material properties. Terahertz (THz) spectroscopic55 techniques can readily measure the phonon modes at the Brillouin zone center, and so temperature resolved THz-low frequency Raman measurements on a single crystal of DES were performed to investigate the mechanism of reversible phase transformation. The evolution of the low-frequency Raman spectra as a function of temperature is measured under nitrogen atmosphere during the heating and cooling cycles over the extended spectral range 0−1700 cm−1 (Supporting Information, Section 1.4). The representative low-frequency Raman spectra (below 210 cm−1) of DES I, II, and III (Figure 5) show substantial diﬀerences. Additionally, there is a progressive change in the Raman spectra with change in temperature accompanying the transformation of form I to form II and subsequently to form III (Supporting Information, Visual examination of the calculated THz modes in Jmol61 indicates strong mixing of inter- and intramolecular vibrations, and there is no single phonon mode that can be seen as a pathway for the phase transformations. The calculated phonon modes also indicate that all phonon frequencies are positive across the entire Brillouin zone (Supporting Information, Figure S27), implying that all three forms are stable and ruling out the possibility of either of the two phase transformations (I →II and II →III) occurring via a “soft” mode mechanism.62 This contrasts with the order−disorder phase transformation observed in camphor, where it was shown that one single low- frequency mode was responsible for the phase trans- formation.63 Figure 5. Low-frequency regions of the measured THz-Raman spectra of crystalline DES polymorphs I, II, and III. The markings at the bottom of each spectrum are the calculated Γ-point phonons of the respective polymorphs (Supporting Information, Section 2.5). 3. RESULTS Starting with a single crystal of form I, single crystal X-ray diﬀraction data were obtained at 80 K, 293 K, 320 K, 335 K, and 350 K during heating and then at 320 K, 315 K, and 293 K during cooling to explore the sequence of structural transitions. q Investigating the reciprocal lattice planes at diﬀerent temperatures indicated that additional reﬂections that do not index with form I unit cell parameters appear at ∼320 K (Supporting Information, Section 1.2.3), before the onset of phase transformation to II expected at ∼335 K from DSC. The intensity and number of these reﬂections increase on approaching the onset of the DSC derived transition temperature. Structure determination at 335 K demonstrates that single crystal has transformed with two molecules in the asymmetric unit and the length of the “a” axis doubling in size. One molecule is in the AAA conformation, and the other is in the ASE conformation (where the piperidine ring and the C6 carbon of the NDBA moiety are in the “syn” orientation), which is nearly equi-energetic with the AAA conformation (Supporting Information, Section 2.1). The two molecules in the asymmetric unit of form II are connected by weak C−H··· g Despite the conformational change of the piperidine ring involved in each phase transformation, all three polymorphs contain similar packing arrangements. Overlays of the asymmetric units (molecules) in the three structures indicate a conformational change analogous to a chair inversion in 1803 https://dx.doi.org/10.1021/acs.cgd.9b01522 Cryst. Growth Des. 2020, 20, 1800−1810 https://dx.doi.org/10.1021/acs.cgd.9b01522 Cryst. Growth Des. 2020, 20, 1800−1810 Crystal Growth & Design In the ﬁrst stage, form III transforms to form II with the accompanying transformation of some ASE con- formers to AAA, while some form II may have transformed to form I by the end of this stage. However, at 315 K, form II is the main component (64%) of the sample, with about 27% of untransformed form III and 9% completely transformed to form I. This is presumably down to diﬀerent nucleation rates in diﬀerent individual crystals in the sample and may result from diﬀerences in particle size and quality. At the end of the ﬁrst stage (315 K), THz Raman showed a slow-down of conformation change, followed quickly by the second stage of transformation (from II →I), matching the second exothermic peak in DSC (Figure 2), along with any remaining III →II →I transformation resulting in the complete transformation of the sample to form I (and AAA conformation). Figure 6. Comparison of (bottom) experimental THz-Raman spectra at two temperatures and (top) scaled calculated Raman spectra for an isolated molecule of DES in the AAA and ASE conformations. The peak (observed at 987.3 cm−1) marked with an asterisk is unique to the AAA conformation. 3.6. In-Situ Hot-Stage Microscopy. Crystals of DES form I were immersed in silicon oil and examined by hot-stage microscopy during heating and cooling between RT and 360 K. Upon heating, the crystal visibly changes shape (Figure 8, Supporting Information, Figures S1 and S3, Supporting Information Video_S1 and S2), and a continuous transverse wavefront can be observed, initiating from the (001) face, that moves through the crystal as growth of the emergent phase proceeds. In the heating cycle, it has not been possible to attribute the observed optical phenomenon to either of the two possible transformations (I →II or II →III), due to the narrow temperature diﬀerence between successive trans- formations. Viewed perpendicular to the (101̅) face, the width of the crystal between faces (100) and (001) reduces by ∼9.5% at the phase transition on heating and returns to its original dimensions on cooling back to RT (see Supporting Information, Video_S1 and S2). During the cooling cycle between the temperatures (360−330 K), corresponding to the III →II transition, limited changes are observed. Crystal Growth & Design The intensities and frequencies of several optical modes in the mid-infrared region of the Raman spectra also diﬀer between the forms. These are in good agreement with the computed Raman spectra for the isolated molecule in the AAA and/or ASE conformations (Figure 6). The main diﬀerences in the Raman spectra are associated with the piperidine ring conformation. Comparison of the calculated Raman spectra suggests that a peak at 987.3 cm−1 can be used unambiguously as the ﬁngerprint of the AAA conformation (Supporting Information, Section 2.6). This peak was used to quantify the proportion of the AAA conformation during heating and cooling and the accompanying phase transitions (Figure 7). g p y g p g 3.5. Quantifying and Interpreting DES Phase Trans- formations through Temperature Cycles. The progress of DES phase transformations through temperature cycles, as monitored through the percentage of molecules in the AAA conformation, from variable temperature Raman single-crystal Figure 5. Low-frequency regions of the measured THz-Raman spectra of crystalline DES polymorphs I, II, and III. The markings at the bottom of each spectrum are the calculated Γ-point phonons of the respective polymorphs (Supporting Information, Section 2.5). Figure 5. Low-frequency regions of the measured THz-Raman spectra of crystalline DES polymorphs I, II, and III. The markings at the bottom of each spectrum are the calculated Γ-point phonons of the respective polymorphs (Supporting Information, Section 2.5). Figure 5. Low-frequency regions of the measured THz-Raman spectra of crystalline DES polymorphs I, II, and III. The markings at the bottom of each spectrum are the calculated Γ-point phonons of the respective polymorphs (Supporting Information, Section 2.5). https://dx.doi.org/10.1021/acs.cgd.9b01522 Cryst. Growth Des. 2020, 20, 1800−1810 1804 Figure 6. Comparison of (bottom) experimental THz-Raman spectra at two temperatures and (top) scaled calculated Raman spectra for an isolated molecule of DES in the AAA and ASE conformations. The peak (observed at 987.3 cm−1) marked with an asterisk is unique to the AAA conformation. Crystal Growth & Design Crystal Growth & Design Crystal Growth & Design Crystal Growth & Design pubs.acs.org/crystal Article endothermic peak from DSC upon heating (Figure 2). During cooling, THz Raman revealed two stages for the molecular conformation changes, separated by an evident slow-down just before the AAA percentage reached 50% (black lines in Figure 7b), again matching the observed transition temperatures by DSC (T(III →II)onset = 326.5 K and T(II→I)onset = 313.9 K) (Figure 2). Crystal Growth & Design (c) Crystal packing arrangement on the (100) face. Figure 9. (a) Distance between chlorine atoms (dCl−Cl) in the (101̅) plane measured in form I and form III. (b) Photographs of crystal deformation at low and high temperatures. The crystal contracts by around 9.5% on the (101̅) face, which is a consequence of the change in dCl−Cl distance in the (101̅) plane. upon further cooling through the II →I transformation between 320 and 300 K, the crystal shape changes visibly as the wavefront propagates through the crystal with a change in relative intensity of the light transmitted through the crystal. The propagation of a discernible wavefront occurred in a number of crystals of diﬀerent sizes at a range of heating/ cooling rates. The speed of the propagating wavefront during both the heating (I →II and/or II →III) and cooling cycles (II−I) was estimated to be in the range of 0.17−0.35 μm/s, which showed some dependence on the heating/cooling rate as well as the crystal size. The speed of the wavefront of transformation in all cases is several orders of magnitude slower than the displacive second order transformations64 observed in metals and alloys.65 The reversible phase transitions and shape changing eﬀects occur with complete retention of the crystal orientation and morphology.23 upon further cooling through the II →I transformation between 320 and 300 K, the crystal shape changes visibly as the wavefront propagates through the crystal with a change in relative intensity of the light transmitted through the crystal. The propagation of a discernible wavefront occurred in a number of crystals of diﬀerent sizes at a range of heating/ cooling rates. The speed of the propagating wavefront during both the heating (I →II and/or II →III) and cooling cycles (II−I) was estimated to be in the range of 0.17−0.35 μm/s, which showed some dependence on the heating/cooling rate as well as the crystal size. Crystal Growth & Design However, spectra, correlates well with that shown from the quantitative Rietveld analysis of variable temperature PXRD observations in Section 2.2 (Figure 7), Along with the other measurements on macroscopic samples including DSC (Section 2.1, Supporting Information, Section 1.5) and SS-NMR (Supporting Informa- tion, Section 1.6), this oﬀers a molecular-level interpretation of diﬀerent stages of DES phase transitions in both bulk powder and single crystal samples. It is clear that during heating form I ﬁrst transforms to form II with the appearance of the ASE conformation. On the basis of the PXRD data, Form II reaches a maximum composition in the powder sample at 335 K (28%) after which the sample rapidly and completely converts to form III above 350 K. This indicates that during the DSC heating measurements (Figure 2), the two phase transitions (I →II and II →III) overlap, probably due to hysteresis, resulting in a single observable endotherm (Tonset = 334.8 K). The temperature range over which conformational change is observed (black lines in Figure 7a) from the Raman measurements is in good agreement with the observed Figure 7. Percentage of polymorphs I (blue), II (magenta), and III (red) of DES as a function of temperature, from quantitative variable temperature PXRD, compared to and percentage of AAA conformations analyzed by THz-Raman during the (a) heating and (b) cooling cycle. https://dx.doi.org/10.1021/acs.cgd.9b01522 Cryst. Growth Des. 2020, 20, 1800−1810 1805 Figure 7. Percentage of polymorphs I (blue), II (magenta), and III (red) of DES as a function of temperature, from quantitative variable temperature PXRD, compared to and percentage of AAA conformations analyzed by THz-Raman during the (a) heating and (b) cooling cycle. 1805 https://dx.doi.org/10.1021/acs.cgd.9b01522 Cryst. Growth Des. 2020, 20, 1800−1810 Figure 8. (a) Microscopic image of a DES crystal immersed in silicon oil on cycling through the phase transitions. The red dashed line indicates the movement of the visible phase boundary between DES II →I. (b) SXRD face indexing of representative single crystal used in hot-stage analysis. (c) Crystal packing arrangement on the (100) face. Crystal Growth & Design pubs.acs.org/crystal Article Crystal Growth & Design Article pubs.acs.org/crystal Figure 8. (a) Microscopic image of a DES crystal immersed in silicon oil on cycling through the phase transitions. The red dashed line indicates the movement of the visible phase boundary between DES II →I. (b) SXRD face indexing of representative single crystal used in hot-stage analysis. Crystal Growth & Design The speed of the wavefront of transformation in all cases is several orders of magnitude slower than the displacive second order transformations64 observed in metals and alloys.65 The reversible phase transitions and shape changing eﬀects occur with complete retention of the crystal orientation and morphology.23 y p gy If the crystals are heated when exposed in air, cracks appear accompanied by a visible propagation of a transformation interface in the bulk of the crystal, followed by small movements of the crystal as it transforms to form III (Supporting Information Figure S2, Supporting Information, Video_S3). There are again no observable optical phenomen- on during the cooling III →II transformation. However, upon further cooling, a transverse wavefront was observed during the II →I transformation. The numerous cracks observed during the heating cycle are possibly due to strains from temperature gradients, arising from the less uniform heat transfer in the absence of silicon oil. The estimated speed of the wavefront in air-exposed crystals is generally faster (∼37 μm/s) compared to crystals immersed in silicon oil. The slight movement of the crystal (see Supporting Information Video_S3) is a demon- stration of the thermosalient eﬀect, although is not as strong as those observed in other jumping crystals,66−68 where crystals jump oﬀthe surface due to the sudden release of strain or elastic energy following a phase transformation. Figure 9. (a) Distance between chlorine atoms (dCl−Cl) in the (101̅) plane measured in form I and form III. (b) Photographs of crystal deformation at low and high temperatures. The crystal contracts by around 9.5% on the (101̅) face, which is a consequence of the change in dCl−Cl distance in the (101̅) plane. distance between two chlorine atoms (dCl−Cl) decreases by 0.976 Å or 9.8%, from 9.959(2) Å in form I to 8.983 Å in form III (Figure 9a). This change in dCl−Cl distance following the conformational ﬂipping of the piperidine ring in DES results in a contraction in the crystal size by ∼9.5% (from 182.5 →165.2 μm; Figure 9b and Supporting Information, Figure S3) as observed in hot-stage microscopy. The transformed crystal at high temperature reverts back to the original shape when cooled to RT. While molecular crystals that reversibly change shape due to order−disorder69 phase transformations14,70−73 have been reported, DES is the ﬁrst case of macroscopic shape- changing organic crystal resulting from signiﬁcant conforma- tional changes. https://dx.doi.org/10.1021/acs.cgd.9b01522 Cryst. Growth Des. 2020, 20, 1800−1810 5. CONCLUSION The transformations in DES provide a rare chance to explore and understand the mechanism of a reversible, two-step single- crystal-to-single-crystal phase transition between conforma- tional polymorphs. The transition is seen to initiate on speciﬁc facets of the crystal, suggesting a single surface nucleation event that propagates the transformation as an observable wavefront across the crystal. The observations contrast vividly with phase transitions observed in inorganic or metallic crystals, or indeed rigid molecular crystals where displacive positional shifts bring about phase transitions. While DES bears some similarities to transformation in ciclopirox,22 the conformational change observed in these DES phase transitions is unique. The ring inversion process in DES is too complex to occur via a low-energy phonon softening as observed in organic jumping crystals of 1,2,4,5-tetrabromo- benzene.83,84 Employing a hierarchy of experimental techni- ques coupled with computational approaches, we clearly demonstrate that the two reversible phase transformations observed in DES are ﬁrst-order transformations involving the concerted movement of molecules within the crystal. The phase transitions of DES highlight the limitations in current classiﬁcations of solid-state phase transitions, historically derived from inorganic systems, and call for special care when these are applied to the rich and diverse behavior of solid−solid phase transitions of organic compounds.23,85 The single-crystal to single-crystal transformations in DES are ﬁrst-order processes proceeding via nucleation and growth, manifested as heat-transfer events in DSC and their considerable hysteresis. The sluggish-moving wave fronts observed during the phase changes also do not ﬁt into any mechanism involving a concerted movement within the whole crystal. The wave fronts in DES always initiate on the (100/ 001) face and propagate in the [101] direction of the crystal, suggesting a single nucleation event initiated by the ﬂipping of the piperidine moiety of DES molecules on the (001) face. Additionally, there is a consistent orientation relationship between the parent and daughter lattices, probably the result of epitaxial growth of the latter.49,50,75,76 Previous examples of concerted movements in molecular crystals have been linked to martensitic type transformations. Anwar et al. used molecular dynamics to report concerted bilayer displacements in the β → α topotactic phase transformation in DL-norleucine.77 Yao et al. Crystal Growth & Design The macroscopic and reversible shape deformation in DES crystals is not instantaneous, as would be observed in second-order shuﬄe-type transformations; rather, it is induced locally and consistently via nucleation on distance between two chlorine atoms (dCl−Cl) decreases by 0.976 Å or 9.8%, from 9.959(2) Å in form I to 8.983 Å in form III (Figure 9a). This change in dCl−Cl distance following the conformational ﬂipping of the piperidine ring in DES results in a contraction in the crystal size by ∼9.5% (from 182.5 →165.2 μm; Figure 9b and Supporting Information, Figure S3) as observed in hot-stage microscopy. The transformed crystal at high temperature reverts back to the original shape when cooled to RT. While molecular crystals that reversibly change shape due to order−disorder69 phase transformations14,70−73 The reversible shape change in the I →II →III transition is most apparent at the dominant (101̅) face of the DES crystal, deﬁning the width of the crystal as viewed in Figure 9. The crystal packing arrangement in the (101̅) plane shows that the 1806 https://dx.doi.org/10.1021/acs.cgd.9b01522 Cryst. Growth Des. 2020, 20, 1800−1810 pubs.acs.org/crystal Crystal Growth & Design Article sate transitions.82 The ability of DES to undergo the conformational change of ﬂipping the piperidine ring in the solid state, presumably aided by the two steps (I →II →III) in a reversible shape-changing manner, shows how remarkably accommodating weakly bound pharmaceutical crystals can be to structural transformation. the (001) face, and growth spreads gradually over the entire crystal within a small temperature window via propagation of the transformation wavefront. While the exact cause of the reproducible directional wavefront remains unknown, the sluggishness of the wavefront and the associated shape changing eﬀect can be attributed to the changes in molecular conformation and intermolecular interactions in the crystal. The thermally induced transformations between the three polymorphs, investigated by several in-situ variable-temper- ature studies, revealed the ease with which the polymorphic conversions can occur in DES. The transformation temper- atures identiﬁed for transition to DES II and III are routinely accessible during the drying and granulation processing steps, for example. If the drug product was exposed to temperatures above the transition temperature, the reversible shape change of the crystallites could aﬀect tablet integrity and associated performance attributes. 5. CONCLUSION reported cooperative movements of [NiII(en)3]2+ cations due to 90° rotation of the oxalate ion in [NiII(en)3](ox) complex.78 Martensitic type transformations were also reported in superelastic crystals of terephthalamide79 and 3,5-diﬂuoroben- zoic acid.80 While DES polymorphs exhibit an orientational relationship and the crystals display reversible shape changing eﬀects, the transformation speed in DES, which depends on the crystal size and quality, and its environment (air/silicon oil), is several orders of magnitude slower than the speed of elastic waves observed in martensitic type and thermosalient phase transformations.66−68,81 This is consistent with the phonon mode calculations on DES showing that it is not a phase transition mediated by a soft mode. These observations emphasize that cooperative motion, most commonly asso- ciated with second-order transitions, is compatible with nucleation and growth theory for the mechanism of solid- 4. DISCUSSION The experimental observations have structurally characterized the high-temperature form (form III) of DES as a conforma- tional polymorph and have shown that there is an ordered intermediate phase (form II) with CSP showing that further polymorphs are unlikely. Periodic DFT-D calculations put the lattice energy and density of form II midway between those of form I and III. The three polymorphs have nearly identical packing with a deﬁnite orientational relationship between the parent and daughter phases (Supporting Information, Section 1.2.5). While there are signiﬁcant conformational changes in the piperidine moiety of DES, there are only relatively subtle changes in the intermolecular distances between neighboring molecules, preserving their relative orientation and packing. This is reminiscent of diﬀusionless phase transformations71 which in metals and inorganics preserves the interatomic distances between successive phases. However, unlike atoms in metal crystals, which only need to be speciﬁed with interatomic distances, molecules in molecular crystals can change their conformations and orientations. Attempts to ﬁt these transitions neatly into any existing categories of diﬀusionless transformations74 remain diﬃcult. ■ASSOCIATED CONTENT * sı Supporting Information * sı Supporting Information The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.cgd.9b01522. Full details of all the experimental and computational modeling (PDF) Full details of all the experimental and computational modeling (PDF) Videos of hot-stage microscopy of DES phase trans- formation (MP4-1, MP4-2, MP4) Videos of hot-stage microscopy of DES phase trans- formation (MP4-1, MP4-2, MP4) Crystal Growth & Design pubs.acs.org/crystal Article (5) Nangia, A. Conformational polymorphism in organic crystals. Acc. Chem. Res. 2008, 41, 595−604. the transformation matrices [1 0 1 0 1 0 1̅ 0 0] and [1̅ 0 0 0 1̅ 0 1 0 1] respectively to have consistent orientation between all three polymorphs. (6) Sanphui, P.; Goud, N. R.; Khandavilli, U. B. R.; Bhanoth, S.; Nangia, A. New polymorphs of curcumin. Chem. Commun. 2011, 47, 5013−5015. ■ACKNOWLEDGMENTS We thank Profs. Pantelides and Adjiman for the use of CrystalPredictor and CrystalOptimizer codes, Prof. Keith Refson for help with using CASTEP for phonon calculations, and Prof. Bruce M. Foxman for fruitful discussion and for the TOPO v 4.40 code. (19) Buerger, M. J. In Phase Transformations in Solids; Smoluchowski, R.; Mayer, J. E.; Weyl, W. A., Eds.; John Wiley & Son: New York, 1951. (20) Ubbelohde, A. R. Thermal transformations in solids. Q. Rev., Chem. Soc. 1957, 11, 246−272. ■ABBREVIATIONS (21) Herbstein, F. On the mechanism of some first-order enantiotropic solid-state phase transitions: from Simon through Ubbelohde to Mnyukh. Acta Crystallogr., Sect. B: Struct. Sci. 2006, 62, 341−383. DES, desloratadine; DSC, diﬀerential scanning calorimetry; VT-PXRD, variable temperature X-ray powder diﬀraction; VT- SS-NMR, variable temperature solid state nuclear magnetic resonance spectroscopy; THz, terahertz; NDBA, N-substituted dibenzazepine; VT-SXRD, variable temperature single crystal X-ray diﬀraction; DABCO, 1,4-diazabicyclo[2.2.2]octane DES, desloratadine; DSC, diﬀerential scanning calorimetry; VT-PXRD, variable temperature X-ray powder diﬀraction; VT- SS-NMR, variable temperature solid state nuclear magnetic resonance spectroscopy; THz, terahertz; NDBA, N-substituted dibenzazepine; VT-SXRD, variable temperature single crystal X-ray diﬀraction; DABCO, 1,4-diazabicyclo[2.2.2]octane (22) Brandel, C.; Cartigny, Y.; Couvrat, N.; Eusebio, M. E. S.; Canotilho, J.; Petit, S.; Coquerel, G. Mechanisms of Reversible Phase Transitions in Molecular Crystals: Case of Ciclopirox. Chem. Mater. 2015, 27, 6360−6373. (23) Byrn, S. R.; Curtin, D. Y.; Paul, I. C. X-ray crystal structures of the yellow and white forms of dimethyl 3,6-dichloro-2,5-dihydrox- yterephthalate and a study of the conversion of the yellow form to the white form in the solid state. J. Am. Chem. Soc. 1972, 94, 890−898. Notes The authors declare no competing ﬁnancial interest. (18) Ehrenfest, P. Phase conversions in a general and enhanced sense, classified according to the specific singularities of the thermodynamic potential. P. K. Akad. Wet-Amsterd. 1933, 36, 153− 157. Corresponding Author Corresponding Author Alastair J. Florence −Strathclyde Institute of Pharmacy and Biomedical Sciences and EPSRC Future Continuous Manufacturing and Advanced Crystallisation Hub, University of Strathclyde, Glasgow G4 0RE, U.K.; orcid.org/0000-0002- 9706-8364; Email: alastair.ﬂorence@strath.ac.uk (8) Braga, D.; Grepioni, F.; Chelazzi, L.; Nanna, S.; Rubini, K.; Curzi, M.; Giaffreda, S. L.; Saxell, H. E.; Bratz, M.; Chiodo, T. Bentazon: Effect of Additives on the Crystallization of Pure and Mixed Polymorphic Forms of a Commercial Herbicide. Cryst. Growth Des. 2014, 14, 5729−5736. f g y , y f Strathclyde, Glasgow G4 0RE, U.K.; orcid.org/0000-0002- 9706-8364; Email: alastair.ﬂorence@strath.ac.uk (9) Yang, J.; Zhu, X.; Hu, C. T.; Qiu, M.; Zhu, Q.; Ward, M. D.; Kahr, B. Inverse Correlation between Lethality and Thermodynamic Stability of Contact Insecticide Polymorphs. Cryst. Growth Des. 2019, 19, 1839−1844. Funding (16) Naumov, P.; Chizhik, S.; Panda, M. K.; Nath, N. K.; Boldyreva, E. Mechanically Responsive Molecular Crystals. Chem. Rev. 2015, 115, 12440−12490. This work was supported by EPSRC (EP/K039229/1). ARCHER time was provided via our membership of the UK’s HEC Materials Chemistry Consortium, which is funded by EPSRC (EP/L000202). (17) Smets, M.; Pitak, M.; Cadden, J.; Kip, V.; de Wijs, G.; van Eck, E.; Tinnemans, P.; Meekes, H.; Vlieg, E.; Coles, S.; Cuppen, H. The Rich Solid-State Phase Behavior of DL-Aminoheptanoic Acid: Five Polymorphic Forms and Their Phase Transitions. Cryst. Growth Des. 2018, 18, 242−252. Authors Vijay K. Srirambhatla −Strathclyde Institute of Pharmacy and Biomedical Sciences and EPSRC Future Continuous Manufacturing and Advanced Crystallisation Hub, University of Strathclyde, Glasgow G4 0RE, U.K.; orcid.org/0000-0002- 4492-7567 (10) Ting, V. P.; Schmidtmann, M.; Wilson, C. C.; Weller, M. T. Cisplatin: Polymorphism and Structural Insights into an Important Chemotherapeutic Drug. Angew. Chem., Int. Ed. 2010, 49, 9408− 9411. Rui Guo −Department of Chemistry, University College London, London WC1H 0AJ, U.K.; orcid.org/0000-0002-1129- 4716 (11) Price, S. L. Is zeroth order crystal structure prediction (CSP_0) coming to maturity? What should we aim for in an ideal crystal structure prediction code? Faraday Discuss. 2018, 211, 9−30. Daniel M. Dawson −School of Chemistry, EaStCHEM and St Andrews Centre for Magnetic Resonance, University of St Andrews, North Haugh KY16 9ST, U.K. (12) Sato, O. Dynamic molecular crystals with switchable physical properties. Nat. Chem. 2016, 8, 644−644. (13) Vogelsberg, C. S.; Garcia-Garibay, M. A. Crystalline molecular machines: function, phase order, dimensionality, and composition. Chem. Soc. Rev. 2012, 41, 1892−1910. Sarah L. Price −Department of Chemistry, University College London, London WC1H 0AJ, U.K.; orcid.org/0000-0002- 1230-7427 (14) Ahmed, E.; Karothu, D. P.; Naumov, P. Crystal Adaptronics: Mechanically Reconfigurable Elastic and Superelastic Molecular Crystals. Angew. Chem., Int. Ed. 2018, 57, 8837−8846. Complete contact information is available at: Complete contact information is available at: https://pubs.acs.org/10.1021/acs.cgd.9b01522 Complete contact information is available at: https://pubs.acs.org/10.1021/acs.cgd.9b01522 https://pubs.acs.org/10.1021/acs.cgd.9b01522 y g (15) Browne, W. R.; Feringa, B. L. Making molecular machines work. Nat. Nanotechnol. 2006, 1, 25−35. ■AUTHOR INFORMATION (7) Yu, L. Polymorphism in Molecular Solids: An Extraordinary System of Red, Orange, and Yellow Crystals. Acc. Chem. Res. 2010, 43, 1257−1266. (1) Bernstein, J. Polymorphism in Molecular Crystals; Clarendon Press: Oxford, 2002. (2) Brittain, H. G. Polymorphism in Pharmaceutical Solids; Marcel Dekker AG: Basel, Switzerland, 1999. (3) Cruz-Cabeza, A. J.; Bernstein, J. Conformational Polymorphism. Chem. Rev. 2014, 114, 2170−2191. (4) Cruz-Cabeza, A. J.; Reutzel-Edens, S. M.; Bernstein, J. Facts and fictions about polymorphism. Chem. Soc. Rev. 2015, 44, 8619−8635. Accession Codes Accession Codes CCDC 1963618−1963621 (DES I at 80 K, DES III at 350 K, DES I at 293 K, DES II at 335 K, respectively) contain the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/ data_request/cif, or by emailing data_request@ccdc.cam.ac. uk, or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033. The reduced unit cell parameters of DES II and III were transformed to the reported values in CIF ﬁles using 1807 https://dx.doi.org/10.1021/acs.cgd.9b01522 Cryst. Growth Des. 2020, 20, 1800−1810 (1) Bernstein, J. Polymorphism in Molecular Crystals; Clarendon Press: Oxford, 2002. (4) Cruz-Cabeza, A. J.; Reutzel-Edens, S. M.; Bernstein, J. Facts and fictions about polymorphism. Chem. Soc. Rev. 2015, 44, 8619−8635. Crystal Growth & Design A., Jr; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. 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https://openalex.org/W2625097671	https://digitalcommons.usf.edu/context/kip_articles/article/5894/viewcontent/K26_00009.pdf	English	null	Resource capture and competitive ability of non-pathogenic Pseudogymnoascus spp. and P. destructans, the cause of white-nose syndrome in bats	PloS one	2,017	cc-by	10,037	University of South Florida University of South Florida Digital Commons @ University of Digital Commons @ University of South Florida South Florida KIP Articles KIP Research Publications June 2017 Resource capture and competitive ability of non-pathogenic Resource capture and competitive ability of non-pathogenic Pseudogymnoascus spp. and P. destructans, the cause of white- Pseudogymnoascus spp. and P. destructans, the cause of white- nose syndrome in bats nose syndrome in bats Michael B. Wilson Benjamin W. Held Amanda H. Freiborg KIP Research Publications Follow this and additional works at: https://digitalcommons.usf.edu/kip_articles Recommended Citation Recommended Citation Wilson, Michael B.; Held, Benjamin W.; and Freiborg, Amanda H., "Resource capture and competitive ability of non-pathogenic Pseudogymnoascus spp. and P. destructans, the cause of white-nose syndrome in bats" (2017). KIP Articles. 4895. https://digitalcommons.usf.edu/kip_articles/4895 This Article is brought to you for free and open access by the KIP Research Publications at Digital Commons @ University of South Florida. It has been accepted for inclusion in KIP Articles by an authorized administrator of Digital Commons @ University of South Florida. For more information, please contact digitalcommons@usf.edu. RESEARCH ARTICLE Michael B. Wilson1, Benjamin W. Held2, Amanda H. Freiborg1, Robert A. Blanchette2, Christine E. Salomon1* Michael B. Wilson1, Benjamin W. Held2, Amanda H. Freiborg1, Robert A. Blanchette2, Christine E. Salomon1* Michael B. Wilson1, Benjamin W. Held2, Amanda H. Freiborg1, Robert A. Blanchette2, Christine E. Salomon1* 1 Center for Drug Design, University of Minnesota, Minneapolis, Minnesota, United States of America, 2 Department of Plant Pathology, University of Minnesota, Saint Paul, Minnesota, United States of America a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 * csalomon@umn.edu * csalomon@umn.edu OPEN ACCESS Citation: Wilson MB, Held BW, Freiborg AH, Blanchette RA, Salomon CE (2017) Resource capture and competitive ability of non-pathogenic Pseudogymnoascus spp. and P. destructans, the cause of white-nose syndrome in bats. PLoS ONE 12(6): e0178968. https://doi.org/10.1371/journal. pone.0178968 Editor: Michelle L. Baker, CSIRO, AUSTRALIA Received: January 13, 2017 Accepted: May 21, 2017 Published: June 15, 2017 Copyright: © 2017 Wilson et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All gene sequences used for phylogenetic analysis have been deposited in GenBank (accession numbers are provided in the Supplemental data S1). All other relevant data are provided within the paper and its Supporting Information files. Abstract White-nose syndrome (WNS) is a devastating fungal disease that has been causing the mass mortality of hibernating bats in North America since 2006 and is caused by the psy- chrophilic dermatophyte Pseudogymnoascus destructans. Infected bats shed conidia into hibernaculum sediments and surfaces, but it is unknown if P. destructans can form stable, reproductive populations outside its bat hosts. Previous studies have found non-pathogenic Pseudogymnoascus in bat hibernacula, and these fungi may provide insight into the natural history of P. destructans. We compared the relatedness, resource capture, and competitive ability of non-pathogenic Pseudogymnoascus isolates with P. destructans to determine if they have similar adaptations for survival in hibernacula sediment. All non-pathogenic Pseu- dogymnoascus isolates grew faster, utilized a broader range of substrates with higher effi- ciency, and were generally more resistant to antifungals compared to P. destructans. All isolates also showed the ability to displace P. destructans in co-culture assays, but only some produced extractible antifungal metabolites. These results suggest that P. destruc- tans would perform poorly in the same environmental niche as non-pathogenic Pseudogym- noascus, and must have an alternative saprophytic survival strategy if it establishes active populations in hibernaculum sediment and non-host surfaces. Resource capture and competitive ability of non-pathogenic Pseudogymnoascus spp. and P. destructans. states and five Canadian providences, including two endangered and one threatened species [10,11]. Even though WNS is now wide-spread, P. destructans is of a single clonal genotype in North America [12,13]. WNS is an important national economic issue because bats are esti- mated to perform $22.9 billions worth of agricultural pest control in the U.S. annually [14]. collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist WNS-positive bats shed infectious conidia into the hibernaculum environment where they can persist [15], but it is unclear if P. destructans can form stable, reproductive populations in sediment and surfaces that can act as permanent infectious reservoirs. Understanding the nat- ural history of P. destructans on hibernaculum surfaces is critical for WNS management because it affects the feasibility of some intervention strategies. Bats in hibernacula with envi- ronmental populations of P. destructans would be at constant risk of infection regardless of individual treatment, culling, or reintroduction after population collapse. Since the emergence of WNS, researchers have discovered many non-pathogenic Geomyces and Pseudogymnoascus fungi in bat hibernacula with culture-based methods [16,17]. These species complicated WNS diagnosis because their internal transcribed spacer (ITS) region sequences of the rRNA gene complex can be very similar to P. destructans [16,18]. However, more recent methods targeting the intergenic spacer (IGS) region can resolve P. destructans from its close relatives [19]. It is hypothesized that P. destructans emerged from non-patho- genic ancestors [16,20,21], so these closely related species may provide a comparative platform to study P. destructans’s adaptions for survival in hibernacula sediment. To the authors’ knowl- edge there are no explicit reports of culture-independent fungal community analyses in bat hibernacula, therefore the natural abundance of non-pathogenic Pseudogymnoascus and Geo- myces is unknown in this environment. Previous work has demonstrated that P. destructans can grow on complex substrates found in hibernacula [22] and at least transiently in sterilized cave sediments in vitro [20], but has greatly reduced saprophytic enzyme activity and growth compared to non-pathogenic Pseudo- gymnoascus [20,23]. The initial magnitude of P. destructans growth in cave sediments was posi- tively correlated with sediment total organic carbon, but P. destructans viability decreased after 28 days in 4 out of 5 sediments tested [20]. P. Introduction White-nose syndrome (WNS) is an invasive mycosis of hibernating bats caused by the psy- chrophilic fungus Pseudogymnoascus destructans, formerly Geomyces destructans [1–3]. P. destructans was completely unknown before the mass mortality of North American bats began in 2006, but is now recognized as an invasive pathogen from Europe [4–6]. While P. destruc- tans does not cause mass mortality in its native range [5], up to 6.7 million North American bats died of WNS by 2012 [7] with the possibility of widespread or local extinctions in the future [8,9]. At least seven North American bat species are affected by WNS throughout 29 Funding: Funding for this project was provided by the Minnesota Environment and Natural Resources Trust Fund, M.L. 2013, Chp. 52, Sec. 2, Subd. 03 (http://www.lccmr.leg.mn/index.html) to CES and RAB. The funders had no role in study design, data PLOS ONE \| https://doi.org/10.1371/journal.pone.0178968 June 15, 2017 1 / 18 PLOS ONE \| https://doi.org/10.1371/journal.pone.0178968 June 15, 2017 destructans was recoverable from all sediments after 238 days [20], but its life cycle and stages of differentiation were not monitored. Little is known about P. destructan’s saprophytic adaptations, but a recent gene duplication of a high affinity nitrate transporter in P. destructans may contribute to its ability to survive on cave sed- iments [21]. The capacity to grow on substrates in vitro is not wholly reflective of the challenges that P. destructans must overcome as a saprophyte, as it must also compete with other microbes for space and utilizable resources. Since P. destructans is unlikely to avoid competition by quickly colonizing resources due to its slow growth [24], it may have some combination of offensive and defensive strategies to colonize saprophytic resources [25–27]. These may include the pro- duction of or resistance to diffusible antifungal molecules [27]. P. destructans is susceptible to amphotericin B, itraconazole, and ketoconazole [28], and it is likely to encounter antifungal metabolites in hibernaculum environments. Discovering how P. destructans fits into the com- petitive hierarchy of the heterogeneous and variable microbial communities of caves and mines [29] will help us understand if it can plausibly form self-sustaining populations in hiber- naculum sediment. Depending on the resource capture and competitive ability of P. destructans, biological control may be an effective way to reduce the conidial load of hibernaculum substrates and prevent the perpetual contamination of infected hibernacula. One strain of Trichoderma poly- sporum isolated from William Preserve Mine in New York has already been shown to outcom- pete P. destructans in autoclaved cave sediment, likely due to the production of diffusible antifungals [30]. Any treatment in caves or mines will be logistically challenging due to their PLOS ONE \| https://doi.org/10.1371/journal.pone.0178968 June 15, 2017 2 / 18 Resource capture and competitive ability of non-pathogenic Pseudogymnoascus spp. and P. destructans. size and inaccessibility, but biological control has an advantage over other interventions because it is potentially self-perpetuating and self-spreading. There is a risk that biological con- trol agents will affect the microbial communities in hibernacula, but this is true of any WNS treatment. The risk of disturbing native microbial communities is mitigated because these communities in WNS-positive hibernacula have likely already shifted in response to the intro- duction of an invasive fungus and the loss of bats. Theoretically, a good generalist saprophyte can utilize a diverse array of carbon sources and defend those resources from other microbes [25,27]. This study assessed the resource capture and competitive ability of P. destructans relative to non-pathogenic Pseudogymnoascus isolates from the Soudan Iron Mine (SM) in Tower, MN to explore the possibility that P. destructans and SM Pseudogymnoascus isolates share a suite of adaptations for survival in hibernaculum sediment. These experiments offer more insight into the basic biology of P. destructans and its potential to be a sustained risk in hibernaculum substrates and surfaces. Pseudogymnoascus spp. collection and propagation All relevant protocols for bat sampling were approved by the University of Minnesota IACUC (protocol ID: 1508-32924A). Wall surface swabs, wood samples, and sediment were collected on seven levels of the Soudan Underground Mine State Park from 2013 to 2015, which was prior to the detection of WNS in Minnesota. Samples were streaked or placed onto 1.5% malt extract agar (BD Difco–Franklin Lakes, NJ) amended with 0.01 g/L of streptomycin sulfate and incubated at 10 or 22˚C. P. destructans type-strain MYA-4855 was acquired from the American Tissue Type Culture Collection (ATCC), which was originally isolated from a little brown bat (Myotis lucifugus) in Williams Hotel Mine, NY. All Pseudogymnoascus spp. were maintained on Sabouraud dextrose agar (SDA), with SM Pseudogymnoascus isolates main- tained at room temperature (~22˚C) and P. destructans maintained at 10 or 15˚C. Conidia were prepared for experiments by adding 2 mL of 15% glycerol to conidiated cultures, gently scraping colonies to release conidia, and then filtering the resulting conidial suspension through a sterile 5 mL pipette tip packed with cotton. PLOS ONE \| https://doi.org/10.1371/journal.pone.0178968 June 15, 2017 Fungal DNA extraction, PCR, and phylogenic analysis Phylogenic analysis was performed with the same three genes used in previous studies [19,31]. DNA was isolated from pure cultures grown on malt agar (15 g malt extract, 15 g agar, 1 L de- ionized water) using a CTAB extraction procedure described previously [32]. ITS1F/ITS4, 983F/2218R, and fRPB2-7cf/RPB2-3053br primers were used to amplify the internal tran- scribed spacer (ITS) region of the rRNA gene clusters, tef1, and rpb2, respectively, via PCR [33–36]. Each 25 μl PCR reaction contained ~12 ng of DNA template, 0.25 μM forward primer, 0.25 μM reverse primer, 0.05 μg/μl BSA, 1X GoTaq1 green mastermix, and nuclease- free sterile water. The thermocycler programs for amplification were as follows: ITS– 94˚C for 5 min, then 35 cycles of 94˚C for 1 min, 50˚C for 1 min, and 72˚C for 1 min, and a final exten- sion at 72˚C for 5 min; rpb2–94 C for 5 min, then 47 cycles of 94˚C for 1 min, 55.2˚C for 2 min, 72˚C for 2:10 min, and a final extension at 72˚C for 10 min; tef1–94˚C for 2 min, then 47 cycles of 94˚C for 1 min, 55˚C for 1 min, 72˚C for 1:10 min, and a final extension at 72˚C for 10 min. Amplicons were verified by gel electrophoresis on 1% agarose with a SYBR green 1 prestain and imaged with a Dark Reader DR45 (Clare Chemical Research–Denver, CO). PCR products were sequenced using the same primers as above on an ABI 3730xl DNA sequencer (Applied Biosystems–Foster City, CA). Consensus sequences were assembled using Geneious 9.0 [37], and BLASTn was used to identify closely related sequences in GenBank for PCR products were sequenced using the same primers as above on an ABI 3730xl DNA sequencer (Applied Biosystems–Foster City, CA). Consensus sequences were assembled using Geneious 9.0 [37], and BLASTn was used to identify closely related sequences in GenBank for PLOS ONE \| https://doi.org/10.1371/journal.pone.0178968 June 15, 2017 3 / 18 Resource capture and competitive ability of non-pathogenic Pseudogymnoascus spp. and P. destructans. phylogenetic analysis. Geneious 9.0 was also used to determine phylogenetic relationships among sequences, with the MAFFT v7.222 and MrBayes 3.2.6 plugins [38,39] used for sequence alignment and Bayesian analysis, respectively. jModelTest 2.1.10 [40] was used to determine the appropriate model (JC69) for Bayesian analysis. Fungal DNA extraction, PCR, and phylogenic analysis 1.1 x 106 MCMC generations were used for analysis with a sampling frequency every 200 generations, with the first 10% of sampled trees discarded as burn in. The resulting tree topologies were congruent for the analy- ses of each gene region, therefore sequences were concatenated and analyzed using the same methods as individual gene regions. Substrate utilization Filamentous fungi (FF) phenotype microarrays (Biolog–Hayward, CA; Cat# 1006) were used to compare the ability of Pseudogymnoascus spp. to metabolize different carbon sources per the manufacture’s protocol. These arrays are 96-well microplates that contain 95 different sub- strates including amines, amino acids, sugars, carboxylic acids, various polymers, and one no-substrate control. In addition to a substrate, each well supplies essential micronutrients in- cluding nitrogen, phosphorous, potassium, and sulfur. Briefly, a swab was wetted with Biolog FF-IF inoculation fluid and rolled over Pseudogymnoascus spp. colonies on malt extract agar to remove conidia and mycelium fragments. Swab contents were diluted to 0.14 OD600 in inocu- lation fluid and 200 μL were transferred to each well in triplicate FF arrays. Arrays were incu- bated and shaken at 15˚C. Growth was evaluated at OD750 using an EL800 microplate reader (Bio-Tek–Winooski, VT) after seven days for SM Pseudogymnoascus isolates and after 14 days for P. destructans. Values were restricted to 0.0–2.0 OD750 in all analyses to account for the linear range of the microplate reader. It was empirically determined that 7 and 14 days would result in a reason- able comparison since SM Pseudogymnoascus isolates grow approximately twice as fast as P. destructans at 10˚C. Niche width was defined as the number of substrates utilized in the FF arrays, while growth efficiency was defined as the mean OD750 of wells with utilized substrates [41]. Since some growth occurred in the no-substrate control wells, pairwise one-tailed Welch’s t-tests were used to determine a utilization threshold on a substrate-by-substrate basis instead of an abso- lute OD750 cutoff applied to all substrates. A one-tailed test was appropriate because growth promotion, not growth inhibition, is relevant to establishing a utilization threshold and maxi- mizes statistical power for a given alpha. A substrate was considered “utilized” if the mean growth on a substrate was statistically greater than mean control growth (p < 0.05). Pair-wise, two tailed Welch’s t-tests were used to compare growth efficiency (mean growth) among Pseu- dogymnoascus spp. at the 95% confidence level. Pseudogymnoascus colony expansion SDA petri plates were spotted with ~2,500 Pseudogymnoascus spp. conidia in 10 μL of sterile water and incubated at 4˚C in a walk-in cold room, at 10˚C in a Danby #DBC120BLS incuba- tor, or at room temperature in the lab (~22˚C). Each Pseudogymnoascus sp. was spotted in qua- druplicate for each temperature. Colonies were established for four days at room temperature and 10˚C, and for eight days at 4˚C. Thereafter, colony diameter was measured daily for 30 days by measuring length twice at right angles and taking the average. Susceptibility testing Disk-diffusion assays were performed on 50 mL SDA in 150 x 15 mm petri plates. Sterile filter paper disks were prepared with 6, 3, 1.5, 0.375, and 0.18 μg of amphotericin B (MP 4 / 18 PLOS ONE \| https://doi.org/10.1371/journal.pone.0178968 June 15, 2017 Resource capture and competitive ability of non-pathogenic Pseudogymnoascus spp. and P. destructans. Biomedicals–Santa Ana, CA), caspofungin, itraconazole, or ketoconazole (LTK Laboratories– St. Paul, MN) in methanol. These antifungals were selected to represent different classes of sys- temic antifungals found in nature (polyenes, echinocandins, and azoles). Duplicate plates were spread-inoculated with ~4,000 Pseudogymnoascus spp. conidia in 200 μL of sterile water and allowed to dry. Antifungal disks were placed on dry plates and plates were incubated at 4 or 15˚C. Minimum inhibitory concentrations (MIC) of each antifungal were determined visually after two and three weeks for SM Pseudogymnoascus isolates and P. destructans at 15˚C, respec- tively, and after three and five weeks for SM Pseudogymnoascus isolates and P. destructans at 4˚C, respectively. HPLC analysis and anti-P. destructans activity of SM Pseudogymnoascus extracts SM Pseudogymnoascus isolates were grown on rice (10g of rice autoclaved in 12 mL water) at 10˚C for 30 days. Cultures were extracted overnight with two rounds of 100 mL methanol fol- lowed by two rounds of 100 mL ethyl acetate. Extracts from the same culture were combined and dried under reduced pressure. Supelco 3 mL DSC-18 (C18) solid-phase extraction columns (Sigma-Aldrich–St. Louis, MO) were used to clean up samples prior to disk diffusion and HPLC analysis. Extract residues were re-dissolved in 6 mL 5% methanol and bound to pre- equilibrated columns, washed with two volumes of 5% methanol, and eluted with two volumes of 100% methanol. Eluted metabolites were dried and re-suspended to 1 mg/mL in methanol for reversed-phase HPLC analysis on an Agilent 1200 system with an Eclipse XDB-C18 col- umn (5 μm, 4.6 x 150 mm). Metabolites were eluted at 1 mL/min with 10% acetonitrile for 1.5 mins, followed by a linear gradient from 10% to 95% acetonitrile over 20 mins, a hold at 95% acetonitrile for 2 mins, a linear gradient from 95% to 100% acetonitrile over 3 mins, and finally re-equilibration to 10% acetonitrile over 2 mins. Chromatographic peaks were detected by diode array from 200 to 600 nm in 10 nm increments. Disk-diffusion assays were performed on 15 mL SDA in 100 x 10 mm petri plates to assess the anti-P. destructans activity of SM Pseudogymnoascus rice culture extracts. Filter paper disks were prepared with 0.5 mg SPE-cleaned extract, placed on plates spread inoculated with ~2,500 P. destructans conidia, and incubated at 10˚C for three weeks. Assays were performed in triplicate. Competition assays between SM Pseudogymnoascus isolates and P. destructans Agar plugs (5 mm3) were cut from the edge of established Pseudogymnoascus spp. colonies and placed ~1 cm apart on SDA and incubated at 10˚C for four weeks in duplicate. Inhibition of P. destructans growth was qualitatively evaluated by visually assessing the size of control and competing P. destructans colonies. Resource capture and competitive ability of non-pathogenic Pseudogymnoascus spp. and P. destructans. SM isolates group with Genbank Pseudogymnoascus sequences reported by other investigations in New Hampshire, New York, and Minnesota. Posterior probabilities >50 are shown at branch nodes. Geomyces auratus served as the outgroup. SM isolates group with Genbank Pseudogymnoascus sequences reported by other investigations in New Hampshire, New York, and Minnesota. Posterior probabilities >50 are shown at branch nodes. Geomyces auratus served as the outgroup. https://doi.org/10.1371/journal.pone.0178968.g001 https://doi.org/10.1371/journal.pone.0178968.g001 SM14-10-3-2, SM13-9-3-2. However, short branch lengths indicate minor base changes among these isolates. All gene sequences used for phylogenic analysis have been deposited in Genbank (S1 Table). SM14-10-3-2, SM13-9-3-2. However, short branch lengths indicate minor base changes among these isolates. All gene sequences used for phylogenic analysis have been deposited in Genbank (S1 Table). SM Pseudogymnoascus colonies expand faster and at higher temperature than P. destructans colonies Expansion rates were similar among SM Pseudogymnoascus isolates at all temperatures, and these organisms are subsequently referred to as one group (Fig 2A–2C). SM Pseudogymnoascus colonies reached diameters of ~60 mm after 30 days at room temperature, with no P. destruc- tans growth as expected [24] (Fig 2A). SM Pseudogymnoascus colonies reached diameters of ~50 mm at 10˚C after 30 days on SDA, while P. destructans colonies achieved a diameter of 25 mm (Fig 2B). Differences in growth were minimized at 4˚C, with SM Pseudogymnoascus colo- nies reaching diameters ~25 mm and P. destructans colonies reaching diameters of 13 mm (Fig 2C). SM Pseudogymnoascus are closely related to other Pseudogymnoascus spp. isolated from hibernacula A three-gene phylogeny of Pseudogymnoascus species shows close similarity among isolates from the different mine shaft levels (Fig 1). SM isolates group with the previously described P. verrucosus complex, matching isolates from NH, NY and MN hibernacula [31]. Overall, the SM isolates separated into two clades with SM13-7-5-2, SM14-12-9-2, SM14-8-4-4, SM13-11- 1-1, SM14-10-3-3, SM14-12-8-2 separate from SM13-15-4-2, SM14-8-4-5, SM13-11-7-3, 5 / 18 PLOS ONE \| https://doi.org/10.1371/journal.pone.0178968 June 15, 2017 Resource capture and competitive ability of non-pathogenic Pseudogymnoascus spp. and P. destructans. Fig 1. Phylogenetic tree of Pseudogymnoascus isolates obtained from the Soudan Iron Mine (SM) in Min Bayesian analysis of combined ITS, EF-1 and RPB2 gene regions from Pseudogymnoascus species. Soudan Mi Fig 1. Phylogenetic tree of Pseudogymnoascus isolates obtained from the Soudan Iron Mine (SM) in Minnesota. Tree was constructed using Bayesian analysis of combined ITS, EF-1 and RPB2 gene regions from Pseudogymnoascus species. Soudan Mine (SM) isolates are indicated in bold. Fig 1. Phylogenetic tree of Pseudogymnoascus isolates obtained from the Soudan Iron Mine (SM) in Minnesota. Tree was constructed using Bayesian analysis of combined ITS, EF-1 and RPB2 gene regions from Pseudogymnoascus species. Soudan Mine (SM) isolates are indicated in bold. PLOS ONE \| https://doi.org/10.1371/journal.pone.0178968 June 15, 2017 6 / 18 PLOS ONE \| https://doi.org/10.1371/journal.pone.0178968 June 15, 2017 SM Pseudogymnoascus isolates utilized more substrates more quickly than P. destructans Biolog filamentous fungi (FF) phenotype microarrays were inoculated with SM Pseudogym- noascus isolates or P. destructans to compare their substrate utilization capacities. Each well in the plate contains a single carbon source with essential micronutrients, and the capacity to uti- lize different substrates can be determined by measuring fungal growth in each well. The utili- zation of different sugars, amino acids, carboxylic acids, and polymers can be summarized by ‘niche width’ (the total number of utilized substrates, ranging 0–95) and ‘growth efficiency’ (mean growth in wells with utilized substrates) [41]. The full Biolog FF array dataset can be found in S1 Text. Niche widths were ~87 for all SM Pseudogymnoascus isolates after 7 days of growth, com- pared to a niche width of 7 for P. destructans after 14 days (Fig 3). SM Pseudogymnoascus iso- lates grow approximately twice as fast as P. destructans at 10˚C (Fig 2), therefore 7 and 14 days were chosen as comparable endpoints. The niche width of P. destructans increased to 19 after 30 days of growth; however, this was still well below the niche widths achieved by the SM Pseu- dogymnoascus isolates after only 7 days. Growth efficiencies of all SM Pseudogymnoascus isolates were significantly higher than the efficiency of 14 day-P. destructans (p < 0.05) (Fig 3, S2 Table). The growth efficiency of P. destructans greatly increased over time, with the efficiency of 30 day-P. destructans matching SM 10-3-3 and significantly exceeded that of SM 7-5-2, SM 8-4-5, SM 9-3-2, and SM 10-3-2 (p < 0.05) (Fig 3, S2 Table). The growth efficiency of 30-day P. destructans was still lower than that of SM 12-9-2 (p < 0.05) (Fig 3, S2 Table). Fig 4 displays the utilization of specific substrates in the FF arrays by Pseudogymnoascus spp. SM Pseudogymnoascus isolates utilized a wide, but very similar, variety of sugars, carbox- ylic acids, amino acids, and other substrates with no clear metabolic preferences (Fig 4). The notable exception to their similar utilization profiles was L-fucose, which was only utilized by SM 7-5-2 and 10-3-3 (Fig 4). Growth efficiencies on particular substrates among the SM Pseu- dogymnoascus isolates differed greatly, with SM 12-9-2 typically being the most efficient isolate (Fig 4). 7 / 18 PLOS ONE \| https://doi.org/10.1371/journal.pone.0178968 June 15, 2017 Resource capture and competitive ability of non-pathogenic Pseudogymnoascus spp. and P. destructans. Resource capture and competitive ability of non-pathogenic Pseudogymnoascus spp. and P. destructans. SM Pseudogymnoascus isolates utilized more substrates more quickly than P. destructans OD750 of wells with utilized substrates is displayed, where the mean OD750 is each strain’s growth efficiency. SM Pseudogymnoascus isolates were evaluated after 7 days of growth, while P. destructans was evaluated after 14 and 30 days of growth. Statistical differences (p < 0.05) are indicated by * and # for pair-wise Welch’s t- tests against 14 day and 30 day-P. destructans, respectively. Pd = P. destructans. https://doi.org/10.1371/journal.pone.0178968.g003 https://doi.org/10.1371/journal.pone.0178968.g003 P. destructans utilized only simple sugars, lipid-like compounds, and amino acids after 14 days (Fig 4). P. destructans expanded its utilization of simple sugars and amino acids after 30 days, but also started to utilize small carboxylic acids and amino sugars (Fig 4). Even though P. destructans had a much smaller niche width than the SM Pseudogymnoascus isolates (Fig 3), it was the exclusive utilizer of N-acetyl-D-galactosamine (Fig 4). The lipid-like compounds (Tween 80, sebacic acid, and amygdalin) were the only substrates where P. destructans’s growth efficiency matched or exceeded those of the SM Pseudogymnoascus isolates after 14 days (Fig 4). SM Pseudogymnoascus isolates utilized more substrates more quickly than P. destructans Fig 2. Colony expansion of Pseudogymnoascus spp. on SDA over 30 days. Diameter of Pseudogymnoascus spp. colonies at various temperatures over time (a) 22˚C, (b) 10˚C, (c) 4˚C. Error bars represent standard error of averaged replicates. https://doi.org/10.1371/journal.pone.0178968.g002 rnal.pone.0178968 June 15, 2017 8 / 18 Fig 2. Colony expansion of Pseudogymnoascus spp. on SDA over 30 days. Diameter of Pseudogymnoascus spp. colonies at various temperatures over time (a) 22˚C, (b) 10˚C, (c) 4˚C. Error bars represent standard error of averaged replicates. https://doi.org/10.1371/journal.pone.0178968.g002 PLOS ONE \| https://doi.org/10.1371/journal.pone.0178968 June 15, 2017 8 / 18 Fig 2. Colony expansion of Pseudogymnoascus spp. on SDA over 30 days. Diameter of Pseudogymnoascus spp. colonies at various temperatures over time (a) 22˚C, (b) 10˚C, (c) 4˚C. Error bars represent standard error of averaged replicates. https://doi.org/10.1371/journal.pone.0178968.g002 PLOS ONE \| https://doi.org/10.1371/journal.pone.0178968 June 15, 2017 8 / 18 Resource capture and competitive ability of non-pathogenic Pseudogymnoascus spp. and P. destructans. Fig 3. Niche width and growth efficiency of Pseudogymnoascus spp. at 15˚C. The number of substrates utilized by each strain (niche width) in Biolog FF phenotype mircoarrays is indicated in black boxes. OD750 of wells with utilized substrates is displayed, where the mean OD750 is each strain’s growth efficiency. SM Pseudogymnoascus isolates were evaluated after 7 days of growth, while P. destructans was evaluated after 14 and 30 days of growth. Statistical differences (p < 0.05) are indicated by * and # for pair-wise Welch’s t- tests against 14 day and 30 day-P. destructans, respectively. Pd = P. destructans. https://doi org/10 1371/journal pone 0178968 g003 Fig 3. Niche width and growth efficiency of Pseudogymnoascus spp. at 15˚C. The number of substrates utilized by each strain (niche width) in Biolog FF phenotype mircoarrays is indicated in black boxes. OD750 of wells with utilized substrates is displayed, where the mean OD750 is each strain’s growth efficiency. SM Pseudogymnoascus isolates were evaluated after 7 days of growth, while P. destructans was evaluated after 14 and 30 days of growth. Statistical differences (p < 0.05) are indicated by * and # for pair-wise Welch’s t- tests against 14 day and 30 day-P. destructans, respectively. Pd = P. destructans. Fig 3. Niche width and growth efficiency of Pseudogymnoascus spp. at 15˚C. The number of substrates utilized by each strain (niche width) in Biolog FF phenotype mircoarrays is indicated in black boxes. PLOS ONE \| https://doi.org/10.1371/journal.pone.0178968 June 15, 2017 PLOS ONE \| https://doi.org/10.1371/journal.pone.0178968 June 15, 2017 SM Pseudogymnoascus outcompetes P. destructans in co-culture SM Pseudogymnoascus outcompetes P. destructans in co-culture All SM Pseudogymnoascus colonies limited the expansion of P. destructans colonies after four weeks of incubation at 10˚C (Fig 5). P. destructans colonies expanded more against SM 8-4-5, 9-3-2, and 12-9-2 colonies than against 7-5-2, 10-3-2, and 10-3-3 colonies (Fig 5). All SM Pseudogymnoascus colonies limited the expansion of P. destructans colonies after four weeks of incubation at 10˚C (Fig 5). P. destructans colonies expanded more against SM 8-4-5, 9-3-2, and 12-9-2 colonies than against 7-5-2, 10-3-2, and 10-3-3 colonies (Fig 5). P. destructans is more susceptible to antifungals than SM Pseudogymnoascus isolates, but susceptibility varies with temperature P. destructans was susceptible to amphotericin B, itraconazole, and ketoconazole (Table 1) [28]. While its susceptibility to itraconazole and ketoconazole did not change (MIC < 0.18 and MIC = 1.5 μg, respectively), its susceptibility to amphotericin B increased at higher tem- perature (MIC = 1.5 μg at 4˚C vs. MIC < 0.18 μg at 15˚C) (Table 1). P. destructans was resis- tant to caspofungin within our concentration range (MIC > 6 μg) as previously reported [28], and there was no noticeable change in susceptibility with temperature. SM Pseudogymnoascus isolates were more resistant to amphotericin B, itraconazole, and ketoconazole relative to P. destructans at 4 and 15˚C (Table 1). Unlike P. destructans, SM Pseudogymnoascus susceptibility to amphotericin B did not noticeably change with tempera- ture Pseudogymnoascus(Table 1). SM Pseudogymnoascus isolates were most susceptible to PLOS ONE \| https://doi.org/10.1371/journal.pone.0178968 June 15, 2017 9 / 18 Resource capture and competitive ability of non-pathogenic Pseudogymnoascus spp. and P. destructans. 10 / 18 PLOS ONE \| https://doi.org/10.1371/journal.pone.0178968 June 15, 2017 Resource capture and competitive ability of non-pathogenic Pseudogymnoascus spp. and P. destructans. Fig 4. Heatmap of Pseudogymnoascus spp. substrate utilization at 15˚C. Rows correspond to average growth (OD750) in wells of Biolog FF phenotype microarrays, while columns correspond to different Pseudogymnoascus spp. SM Pseudogymnoascus isolates were evaluated after 7 days of growth, while P. destructans was evaluated after 14 and 30 days of growth. Rows are clustered by k-means to display patterns of utilization. itraconazole, but their MICs were at least an order of magnitude higher than those observed for P. destructans (Table 1). SM isolates 10-3-2 and 10-3-3 were exceptions to this trend with MIC = 0.375 μg against itraconazole (Table 1). Azole susceptibility differentially varied with temperature for some SM Pseudogymnoascus isolates, with itraconazole and ketoconazole showing opposite trends (Table 1). SM isolates 8-4-5, 10-3-2, and 10-3-3 were less susceptible to itraconazole at higher temperature (MIC = 1.5 or 0.375 μg at 4˚C vs. MIC = 3 μg at 15˚C), but itraconazole susceptibility did not change for SM 7-5-2, 9-3-2, or 12-9-2 (MIC = 1.5 μg) (Table 1). SM isolates 9-3-2 and 12-9-2 were more susceptible to ketoconazole at higher tem- perature (MIC > 6 μg at 4˚C vs. MIC = 3 μg at 15˚C), with SM 7-5-2 and 8-4-5 showing poten- tially smaller increases in ketoconazole susceptibility (MIC > 6 μg at 4˚C vs. MIC = 6 μg at 15˚C) (Table 1). MICs were determined as the lowest concentration of antifungal that did not produce a visible zone of inhibition in disk-diffusion assays. Plates incubated at 4˚C were evaluated after three weeks for SM Pseudogymnoascus isolates and after ﬁve weeks for P. destructans. Plates incubated at 15˚C were evaluated after two weeks for SM Pseudogymnoascus isolates and after three weeks for P. destructans. https://doi.org/10.1371/journal.pone.0178968.t001 P. destructans is more susceptible to antifungals than SM Pseudogymnoascus isolates, but susceptibility varies with temperature Ketoconazole susceptibility did not noticeably change for SM 10-3-2 or 10-3- 3 (MIC = 6 and > 6 μg, respectively) (Table 1). All SM Pseudogymnoascus isolates were more susceptible to caspofungin at lower temperatures (Table 1). itraconazole, but their MICs were at least an order of magnitude higher than those observed for P. destructans (Table 1). SM isolates 10-3-2 and 10-3-3 were exceptions to this trend with MIC = 0.375 μg against itraconazole (Table 1). Azole susceptibility differentially varied with temperature for some SM Pseudogymnoascus isolates, with itraconazole and ketoconazole showing opposite trends (Table 1). SM isolates 8-4-5, 10-3-2, and 10-3-3 were less susceptible to itraconazole at higher temperature (MIC = 1.5 or 0.375 μg at 4˚C vs. MIC = 3 μg at 15˚C), but itraconazole susceptibility did not change for SM 7-5-2, 9-3-2, or 12-9-2 (MIC = 1.5 μg) (Table 1). SM isolates 9-3-2 and 12-9-2 were more susceptible to ketoconazole at higher tem- perature (MIC > 6 μg at 4˚C vs. MIC = 3 μg at 15˚C), with SM 7-5-2 and 8-4-5 showing poten- tially smaller increases in ketoconazole susceptibility (MIC > 6 μg at 4˚C vs. MIC = 6 μg at 15˚C) (Table 1). Ketoconazole susceptibility did not noticeably change for SM 10-3-2 or 10-3- 3 (MIC = 6 and > 6 μg, respectively) (Table 1). All SM Pseudogymnoascus isolates were more susceptible to caspofungin at lower temperatures (Table 1). SM Pseudogymnoascus isolates produce more extractable metabolites in culture, with some having anti-P. destructans activity destructans colonies are on the left of each panel, while SM Pseudogymnoascus colonies are on the right. The control was a P. destructans colony grown in the presence of an uninoculated agar block. https://doi.org/10.1371/journal.pone.0178968.g005 https://doi.org/10.1371/journal.pone.0178968.g005 https://doi.org/10.1371/journal.pone.0178968.g005 SM Pseudogymnoascus isolates produce more extractable metabolites in culture, with some having anti-P. destructans activity Most SM Pseudogymnoascus isolates had a similar diversity of extractable semi-polar metabo- lites when grown on rice at 10˚C (Fig 6, S2 Fig). SM isolate 7-5-2 and P. destructans did not appear to produce any extractable metabolites under the experimental conditions (Fig 6, S2 Fig). Three SM Pseudogymnoascus extracts showed anti-P. destructans activity in disk diffusion assays (Table 2). Extracts from SM isolates 10-3-2 and 10-3-3 produced small zones that were sustained over three weeks, while the 12-9-2 extract produced a large initial zone that receded by week three (Table 2). Pictures of disk-diffusion assays can be found in S1 Fig. Table 1. Antifungal susceptibility of Pseudogymnoascus spp. Table 1. Antifungal susceptibility of Pseudogymnoascus spp. Pseudogymnoascus spp. MIC (μg) P. destructans SM 7-5-2 SM 8-4-5 SM 9-3-2 SM 10-3-2 SM 10-3-3 SM 12-9-2 Antifungal 4˚C 15˚C 4˚C 15˚C 4˚C 15˚C 4˚C 15˚C 4˚C 15˚C 4˚C 15˚C 4˚C 15˚C Caspofungin > 6 > 6 3 > 6 3 > 6 6 > 6 3 > 6 3 > 6 1.5 > 6 Amphotericin B 1.5 < 0.18 > 6 > 6 > 6 > 6 > 6 > 6 > 6 > 6 > 6 > 6 > 6 > 6 Itraconazole < 0.18 < 0.18 1.5 1.5 1.5 3 1.5 1.5 0.375 3 0.375 3 1.5 1.5 Ketoconazole 1.5 1.5 > 6 6 > 6 6 > 6 3 6 6 > 6 > 6 > 6 3 MICs were determined as the lowest concentration of antifungal that did not produce a visible zone of inhibition in disk-diffusion assays. Plates incubated at 4˚C l t d ft th k f SM P d i l t d ft ﬁ k f P d t t Pl t i b t d t 15˚C l t d PLOS ONE \| https://doi.org/10.1371/journal.pone.0178968 June 15, 2017 11 / 18 Resource capture and competitive ability of non-pathogenic Pseudogymnoascus spp. and P. destructans. Fig 5. Competition of SM Pseudogymnoascus and P. destructans colonies. Plug competition assay after four weeks of growth on SDA at 15˚C. P. destructans colonies are on the left of each panel, while SM Pseudogymnoascus colonies are on the right. The control was a P. destructans colony grown in the presence of an uninoculated agar block. https://doi.org/10.1371/journal.pone.0178968.g005 Fig 5. Competition of SM Pseudogymnoascus and P. destructans colonies. Plug competition assay after four weeks of growth on SDA at 15˚C. P. PLOS ONE \| https://doi.org/10.1371/journal.pone.0178968 June 15, 2017 Discussion White-nose syndrome has spurred research on the microbiome of bat hibernacula to better understand the biology of P. destructans. Many Pseudogymnoascus and Geomyces species have been discovered in WNS-positive hibernacula [16,31], but our Pseudogymnoascus isolates rep- resent the SM fungal community prior to the invasion of P. destructans. SM isolates separated into two distinct clades and grouped with other Pseudogymnoascus from the P. verrucosus complex in a three-gene phylogeny, although short branch lengths indicated only minor differ- ences among the isolates (Fig 1). We collected physiological data from a subset of each clade, but there was no clear association between clades and our physiological data. P. destructans was both genetically and physiologically distinct from all SM Pseudogymnoascus isolates. The first important distinction between the SM Pseudogymnoascus isolates and P. destruc- tans is their ability to grow at room temperature (Fig 2). While P. destructans is psychrophilic with a growth optimum of ~15˚C and upper critical temperature of ~19˚C [24], the SM Pseudogymnoascus isolates were psychrotolerant with the ability to grow at both room and low temperature (Fig 2). Even though all SM Pseudogymnoacus isolates grew best at room White-nose syndrome has spurred research on the microbiome of bat hibernacula to better understand the biology of P. destructans. Many Pseudogymnoascus and Geomyces species have been discovered in WNS-positive hibernacula [16,31], but our Pseudogymnoascus isolates rep- resent the SM fungal community prior to the invasion of P. destructans. SM isolates separated into two distinct clades and grouped with other Pseudogymnoascus from the P. verrucosus complex in a three-gene phylogeny, although short branch lengths indicated only minor differ- ences among the isolates (Fig 1). We collected physiological data from a subset of each clade, but there was no clear association between clades and our physiological data. P. destructans was both genetically and physiologically distinct from all SM Pseudogymnoascus isolates. The first important distinction between the SM Pseudogymnoascus isolates and P. destruc- tans is their ability to grow at room temperature (Fig 2). While P. destructans is psychrophilic with a growth optimum of ~15˚C and upper critical temperature of ~19˚C [24], the SM Pseudogymnoascus isolates were psychrotolerant with the ability to grow at both room and low temperature (Fig 2). Even though all SM Pseudogymnoacus isolates grew best at room PLOS ONE \| https://doi.org/10.1371/journal.pone.0178968 June 15, 2017 12 / 18 Resource capture and competitive ability of non-pathogenic Pseudogymnoascus spp. and P. destructans. Discussion Resource capture and competitive ability of non-pathogenic Pseudogymnoascus spp. and P. destructans. Fig 6. Reversed-phase HPLC chromatograms of Pseudogymnoascus spp. rice culture extracts. All extracts were normalized to 1 mg/mL, and chromatographic peaks at 220 nm are displayed. The control was an extract of uninoculated rice. https://doi org/10 1371/journal pone 0178968 g006 Fig 6. Reversed-phase HPLC chromatograms of Pseudogymnoascus spp. rice culture extracts. All extracts were normalized to 1 mg/mL, and chromatographic peaks at 220 nm are displayed. The control was an extract of uninoculated rice. https://doi.org/10.1371/journal.pone.0178968.g006 https://doi.org/10.1371/journal.pone.0178968.g006 temperature (~22˚C), they still outgrew P. destructans at 4 and 10˚C (Fig 2). For reference, temperatures where most bats can be found in the Soudan Mine (level 12 and 27) are generally 2–10˚C during the hibernation period. Even though their growth rates were clearly different, neither P. destructans nor SM isolates displayed expansion that could be characterized as “fast”. In general, slow growth may be an important adaptation in oligotrophic environments, such as caves [42]. P. destructans is an opportunistic or facultative pathogen of hibernating bats that takes advantage of its host’s decreased immune function during torpor [43]. Since torpor is a reliably reoccurring state, P. destructans may have metabolic requirements more like an obligate patho- gen because immunosuppression due to torpor is not an abnormal or rare host state. If P. destructans is a facultative pathogen in the environment, it may have resource capture traits similar to non-pathogenic Pseudogymnoascus spp. The results presented here show P. destruc- tans had a reduced niche width compared to SM Pseudogymnoascus isolates in substrate utili- zation assays (Fig 3), suggesting it is more specialized than its non-pathogenic relatives. This conclusion is further supported by previous reports that P. destructans has reduced saprophytic enzyme activity compared to non-pathogenic Pseudogymnoascus spp. [23]. Since the nutrients available to microbes in different caves and mines are highly variable and heterogeneously dis- tributed in space and time [29], it is difficult to generalize which nutrients are ecologically Table 2. Anti-P. destructans activity of SM Pseudogymnoascus extracts. Extract SM 7-5-2 SM 8-4-5 SM 9-3-2 SM 10-3-2 SM 10-3-3 SM 12-9-2 Zone size (2 wk) - - - + + +++ Zone size (3 wk) - - - Minimal Minimal + Disk diffusion assays were performed in triplicate to test the activity of SM Pseudogymnoascus extracts against P. destructans. Disk diffusion assays were performed in triplicate to test the activity of SM Pseudogymnoascus extracts against P. destructans. Inhibitory activity was quantiﬁed as a zone of inhibition, with (+) indicating zones 3–20 mm in diameter and (-) indicating no inhibition. Disks contained 0.5 mg of rice culture extracts from SM isolates. Plates were incubated at 15˚C and evaluated after two and three weeks. Pictures of disk diffusion assays can be found in S1 Fig. Discussion destructans drastically increased its growth efficiency over time (Figs 3 and 4) and utilized its limited panel of substrates as effectively as the SM Pseudogymnoascus isolates after 30 days. Even though the SM Pseudogymnoascus iso- lates were genetically distinct (Fig 1), they had very similar utilization profiles and growth effi- ciencies (Figs 3 and 4). This is good evidence these isolates are occupying similar nutritional niches within the mine. Overall, P. destructans is unlikely to occupy the same generalist sapro- phyte role presumably occupied by non-pathogenic Pseudogymnoascus spp. due to its com- pressed niche width and much slower conversion of substrates to biomass. P. destructans can grow and reproduce on some complex substrates in vitro [22], but it certainly faces competition for these resources in hibernacula sediment. Attack and defense strategies are important to displace other microbes from contested resources or to avoid dis- placement, respectively [27]. Since P. destructans grows very slowly relative to other fungi [24] (Fig 2A–2C), it is unlikely to avoid attack by rapidly colonizing resources and may require robust defense strategies in hibernaculum substrates. The plasma membrane is a major anti- fungal target that changes composition in response to temperature, therefore environmental temperatures are important considerations in microbial defense. Using Geomyces pannorum as a comparative model in the literature, we expected increased susceptibility to polyenes and azoles at higher temperature when ergosterol is more abundant in the plasma membrane [44]. However, this trend was only apparent for amphotericin B vs. P. destructans and ketoconazole vs. some SM Pseudogymnoascus isolates (Table 1). The efficiency of resistance mechanisms may also vary with temperature, which could explain the decrease in itraconazole susceptibility observed for some SM Pseudogymnascus isolates at higher temperature and the lack of azole response in P. destructans. To the authors’ knowledge, changes in the cell wall are not a known adaptation of psychrophilic or psychrotolerant fungi [45,46], so we did not expect the observed change in susceptibility to caspofungin (Table 1). Just considering these variations in antifun- gal susceptibility, P. destructans may be more easily displaced by polyene and azole-producing microbes compared to the SM Pseudogymnoascus isolates, but is better defended against echi- nocandin-producing organisms at low temperature. Future work could focus on the interplay between temperature, membrane/cell wall structure, and resistance mechanisms of Pseudo- gymnoascus spp. P. destructans and other Pseudogymnoascus spp. Discussion Inhibitory activity was quantiﬁed as a zone of inhibition, with (+) indicating zones 3–20 mm in diameter and (-) indicating no inhibition. Disks contained 0.5 mg of rice culture extracts from SM isolates Plates were incubated at 15˚C and evaluated after two and three weeks Pictures of disk diffusion assays can be found in S1 Fig Table 2. Anti-P. destructans activity of SM Pseudogymnoascus extracts. Extract SM 7-5-2 SM 8-4-5 SM 9-3-2 SM 10-3-2 SM 10-3-3 SM 12-9-2 Zone size (2 wk) - - - + + +++ Zone size (3 wk) - - - Minimal Minimal + Table 2. Anti-P. destructans activity of SM Pseudogymnoascus extracts. Disk diffusion assays were performed in triplicate to test the activity of SM Pseudogymnoascus extracts against P. destructans. Inhibitory activity was quantiﬁed as a zone of inhibition, with (+) indicating zones 3–20 mm in diameter and (-) indicating no inhibition. Disks contained 0.5 mg of rice culture extracts from SM isolates. Plates were incubated at 15˚C and evaluated after two and three weeks. Pictures of disk diffusion assays can be found in S1 Fig. Disk diffusion assays were performed in triplicate to test the activity of SM Pseudogymnoascus extracts against P. destructans. Inhibitory activity was quantiﬁed as a zone of inhibition, with (+) indicating zones 3–20 mm in diameter and (-) indicating no inhibition. Disks contained 0.5 mg of rice culture extracts from SM isolates. Plates were incubated at 15˚C and evaluated after two and three weeks. Pictures of disk diffusion assays can be found in S1 Fig. PLOS ONE \| https://doi.org/10.1371/journal.pone.0178968 June 15, 2017 13 / 18 Resource capture and competitive ability of non-pathogenic Pseudogymnoascus spp. and P. destructans. relevant to saprophytic survival. The Biolog substrates provide a general view of what sub- strates may be utilized, but additional study is needed to identify compounds/nutrients being utilized in specific caves and mines. The overall conversion of substrate to biomass should be maximized at 15˚C for P. destruc- tans [24], but not for the SM Pseudogymnoascus isolates (Fig 2). Lipid-like compounds (Tween 80, sebacic acid, and amygdalin) were the only substrates used by P. destructans after 14 days with efficiencies comparable to those of the SM Pseudogymnoascus isolates (Fig 4), which sup- ports its nutritional preference for lipids [22]. P. PLOS ONE \| https://doi.org/10.1371/journal.pone.0178968 June 15, 2017 Discussion may encounter each other in hibernaculum substrates and compete for space, if not resources. All SM Pseudogymnoascus isolates outcom- peted P. destructans on nutrient-rich media (Fig 5), but this was not surprising based on their faster growth and superior resource capture ability. A slower growing, more specialized organ- ism could use attack strategies to outcompete faster growing generalists, but this was not true for P. destructans under our conditions. Assays on artificial media are not always reflective of competition in the environment [27], so field studies are needed to confirm this behavior. It is important to consider how interactions among P. destructans and non-pathogenic Pseudogym- noascus spp. occur in an oligotrophic subterranean environment. Competition assays could not be used to determine if growth inhibition was a result of direct antagonism or indirect nutrient limitation, so each SM Pseudogymnoascus isolate was extracted to assess the production of anti-P. destructans metabolites. All SM Pseudogymnoascus PLOS ONE \| https://doi.org/10.1371/journal.pone.0178968 June 15, 2017 14 / 18 Resource capture and competitive ability of non-pathogenic Pseudogymnoascus spp. and P. destructans. extracts (except 7-5-2) produced similar HPLC profiles (Fig 6), but only extracts of SM 10-3-2, 10-3-3, and 12-9-2 inhibited P. destructans growth (Table 2) demonstrating these isolates have some offensive mechanisms effective against P. destructans. Interestingly, the activity of these extracts was transient (Table 2), indicating low stability of active metabolites or delayed defen- sive mechanisms by P. destructans. P. destructans is likely susceptible to biological control agents in hibernacula sediments con- sidering its demonstrated growth (Fig 2), resource capture (Figs 3 and 4), and competitive ability (Tables 1 and 2, Fig 5). Several Trichoderma isolates from cave sediments capable of pre- venting P. destructans conidia from germinating in cave sediments have been identified [30], and non-pathogenic Pseudogymnoascus spp. may be another common community member that can help control P. destructans. A multi-species pool of biological control candidates is useful because it is unlikely a single organism will provide a good solution for all hibernacula due to differing mineral compositions, nutrient availability, and moisture levels. However, the WNS community needs to better understand the risk of P. destructans infection from envi- ronmental reservoirs to correctly implement any biological control scheme. It is important to consider that P. destructans and non-pathogenic Pseudogymnoascus spp. are part of larger microbial communities with complex interactions that may facilitate or prevent the establish- ment of P. destructans. Discussion This work focused on non-pathogenic Pseudogymnoascus spp. because they may reveal traits important for P. destructans survival in hibernaculum substrates, but the relative importance of their interactions within the microbial community is unknown. We initially hypothesized that SM Pseudogymnoascus isolates could be used as a proxy for P. destructans in antifungal assays due to their close genetic relationship and ability to grow rapidly at room temperature. However, our phenotypic studies revealed that P. destructans is remarkably different from SM Pseudogymnoascus isolates. These phenotypic differences are likely reflections of traits that help non-pathogenic Pseudogymnoascus spp. thrive as generalist saprophytes and traits that allow P. destructans to thrive as a bat pathogen. While P. destructans may have the capacity to obtain some saprophytically-derived nutrients [22,23] (Figs 3 and 4), it was more specialized and less competitive than SM Pseudogymnoascus isolates (Tables 1 and 2, Fig 5). This does not mean that P. destructans cannot or does not form stable populations in hibernacula sediment and surfaces, but that it must do so differently than its non-pathogenic relatives. Acknowledgments We thank Gerda Nordquist, Jim Essig, and Tony Zavodnik from the Minnesota Department of Natural Resources for their assistance at the Soudan Underground Mine State Park; Zoja Germuskova, Josh Kielsmeier-Cook, Garrett Beier, Sam Redford, and Camille Schlegel for assistance in the laboratory and during sample collection at the mine. Supporting information S1 Fig. Pictures of disk diffusion plates summarized in Table 2. Disks contained 0.5 mg of HPLC-ready extract and plates were incubated at 15˚C and evaluated after (a) two and (b) three weeks. ( ) S2 Fig. Reversed-phase HPLC of Pseudogymnoascusrice culture extracts. Chromatographic peaks were detected by diode array. The control was an extract of uninoculated rice, and all extracts were normalized to 1 mg/mL. All SM Pseudogymnoascus produced more detectible semi-polar metabolites in rice culture compared to P. destructans, with the exception of SM 7- 5-2. (PDF) S2 Fig. Reversed-phase HPLC of Pseudogymnoascusrice culture extracts. Chromatographic peaks were detected by diode array. The control was an extract of uninoculated rice, and all extracts were normalized to 1 mg/mL. All SM Pseudogymnoascus produced more detectible semi-polar metabolites in rice culture compared to P. destructans, with the exception of SM 7- 5-2. (PDF) S1 Table. GenBank numbers of Pseudogymnoascusisolates used in antagonism studies and phylogenetic analysis (in bold) and those obtained from GenBank for phylogenetic com- parison. (PDF) S1 Table. GenBank numbers of Pseudogymnoascusisolates used in antagonism studies and phylogenetic analysis (in bold) and those obtained from GenBank for phylogenetic com- parison. (PDF) 15 / 18 PLOS ONE \| https://doi.org/10.1371/journal.pone.0178968 June 15, 2017 Resource capture and competitive ability of non-pathogenic Pseudogymnoascus spp. and P. destructans. PLOS ONE \| https://doi.org/10.1371/journal.pone.0178968 June 15, 2017 S2 Table. Pair-wise Welsh’s t-test results for comparisons of growth efficiency. See the materials and methods for t-test details, values are rounded to the nearest non-zero integer. (PDF) S2 Table. Pair-wise Welsh’s t-test results for comparisons of growth efficiency. See the materials and methods for t-test details, values are rounded to the nearest non-zero integer. 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https://openalex.org/W3095741880	https://www.researchsquare.com/article/rs-67722/v1.pdf?c=1599090332000	English	null	The prospects for sustaining evidence-based responses to the US opioid epidemic: state leadership perspectives	Substance abuse treatment, prevention, and policy	2,020	cc-by	8,255	The Prospects for Sustaining Evidence-based Responses to the US Opioid Epidemic State Leadership Perspectives P 1/22 The Prospects for Sustaining Evidence-based Responses to the US Opioid Epidemic State Leadership Perspectives Lauren Caton Stanford University School of Medicine https://orcid.org/0000-0002-4376-1251 Mina Yuan Stanford University School of Medicine Dexter Louie Stanford University School of Medicine Carlos Gallo Northwestern University Feinberg School of Medicine Karen Abram Northwestern University Feinberg School of Medicine Lawrence Palinkas University of Southern California Suzanne Dworak-Peck School of Social Work C Hendricks Brown Northwestern University Feinberg School of Medicine Mark McGovern (  mpmcg@stanford.edu ) Stanford University School of Medicine https://orcid.org/0000-0002-9736-573X Research Keywords: Sustainment, sustainability, opioid use disorder treatment, barriers and facilitators, health policy, grant funding Posted Date: September 2nd, 2020 DOI: https://doi.org/10.21203/rs.3.rs-67722/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published on November 4th, 2020. See the publishe version at https://doi.org/10.1186/s13011-020-00326-x. Research Version of Record: A version of this preprint was published on November 4th, 2020. See the published version at https://doi.org/10.1186/s13011-020-00326-x. Page 1/22 Abstract Background: The US 21st Century Cures Act provided $7.5 billion in grant funding to states and territories for evidence-based responses to the opioid epidemic. Currently, little is known about optimal strategies for sustaining these programs beyond this start-up funding. Methods: Using an inductive, conventional content analysis, we conducted key informant interviews with former and current state leaders (n=16) about barriers/facilitators to sustainment and strategies for sustaining time-limited grants. Results: Financing and reimbursement, service integration, and workforce capacity were the most cited barriers to sustainment. Status in state government structure, public support, and spending flexibility were noted as key facilitators. Effective levers to increase chances for sustainment included strong partnerships with other state agencies, workforce and credentialing changes, and marshalling advocacy through public awareness campaigns. Conclusions: Understanding the strategies that leaders have successfully used to sustain programs in the past can inform how to continue future time-limited, grant-funded initiatives. Methods This is a qualitative study featuring key informant interviews with eight former and eight current state agency leaders who oversee substance use prevention and treatment services within their respective states. This sample size, though small, represents 16% (out of 50) of all possible individuals actively holding these positions. Interviews were oriented to ascertain perceptions on sustaining time-limited programs. All methods and reporting are in alignment with the consolidated criteria for reporting qualitative research (COREQ) guidelines (27). Background delineate identified barriers and facilitators to sustainment of the opioid response or similar time- limited programs; and 2. suggest how strategies might be tailored to address typical barriers and facilitators to support sustainment. Our method featured semi-structured interviews of a representative sample of current and former state leaders. We organized the discourse using the EPIS framework (10). It is imperative we improve our capacity to sustain evidence-based programs in healthcare delivery systems that are launched by federal funding. The overarching goal of the present study is to describe key levers to sustainment of programs that have been implemented in response to the US opioid epidemic. Background Opioid overdose death is a major public health concern (1). In response, the 21st Century Cures Act provided $7.5 billion in start-up funding to 57 states and territories administered through the Substance Abuse and Mental Health Services Administration (SAMHSA) (2). This bolus in funding aimed to reduce overdose deaths through expanding access to evidence-based treatment; including the FDA-approved medications of methadone, buprenorphine, and naltrexone. States employed a variety of evidence-based programs with this funding, ranging from peer navigators to post-overdose emergency department connection programs (3). With this record funding comes a growing concern about the continuation, or sustainment (4), of these grant-financed programs (5). This anxiety is reflected in public health service contexts, where scant research examines sustainment of evidence-based programs at the systems level (6–12). Given the recent FY 2021 renewal of these grants, it is both timely and imperative to understand how policymaking state leaders attempt to ensure sustainment of these, and similar, programs. Optimal strategies for sustainment are presently unknown. Emerging advancements in implementation science – the study of integrating evidence-based interventions into practice (4) – have developed sustainability definitions and frameworks (13–19). One implementation science heuristic that conceptualizes sustainment is the four-phase Exploration, Preparation, Active Implementation, and Sustainment (EPIS) framework (9, 10). The framework delineates an inner and outer context, referring to the intra-organizational and systems-wide levels respectively, within which factors involved in the four phases of implementation may operate (10). Applications of EPIS focus on the first three phases within clinic and provider settings, not addressing the fourth phase - sustainment - or exploring its impact across systems-level contexts (23). Additionally, pragmatic research in policy settings has yet to investigate Page 2/22 Page 2/22 factors that promote and undermine sustainment (7, 12, 20–22). Beyond recent scientific support for tailoring strategies to identified barriers and facilitators, scant research investigates the applicability of tailoring for sustainment ventures (24, 25, 26). Lack of research and tools leaves leaders without support for increasing the likelihood of sustainment. Documenting determinants (i.e. barriers and facilitators) that systems leaders face may aid in scientific understanding of sustainment in policy settings, and in developing potential strategies. To address this issue, we sought to: Documenting determinants (i.e. barriers and facilitators) that systems leaders face may aid in scientific understanding of sustainment in policy settings, and in developing potential strategies. To address this issue, we sought to: 1. Interview Guides & Process The authors, skilled in qualitative research design, created two semi-structured interview guides, available upon request from the corresponding author. All systems leaders (participants) were asked to identify 1) examples of programs successfully sustained beyond the grant, 2) barriers and facilitators to continuing these programs, 3) strategies they employed in order to successfully sustain programs, and 4) recommendations for types of training or content they would find beneficial for continuing to sustain programs in the future. This study primarily reports on questions 2 and 3. Additionally, the current leaders were asked the same questions in regard to their work in sustaining programs within their purview, including but not limited to current opioid response-funded programs. Participants received a copy of the study abstract and IRB consent forms in advance, but not a copy of the interview guide unless requested. Interviews lasted approximately one hour and were conducted by the male senior author. The interviewer holds a PhD in psychology, has expertise in clinical and mixed methods interviewing, and is a tenured professor at the parent institution. All interviews took place following introductions, explanation of purpose, and confidentiality agreements (aligned with exempt status from Stanford University Institutional Review Board). They were audio recorded via a secure videoconference platform where participant video was optional; the interviewer enabled video and audio. No repeat interviews were conducted. The interviewer took anonymized field notes during interviews and interviewee identities were blinded and de-identified for transcription and coding. Transcripts were not returned to participants for comment or correction, but transcriptions were verbatim with the exception of eliminated named identities. Participants Leaders from the research team and a national organization of state agency directors identified state agency leaders with authority over federal grants for substance use treatment. From these recommendations, nine former and nine current leaders were invited via email with intentional sampling diversity in US state region, geographic size, and population density. Eight from each groups agreed, with non-participants citing full schedules, making a final sample of sixteen state leaders. Former leaders were not included for comparative purposes, but to provide a tenured and historical perspective compared to individuals currently managing statewide programs. We did not match current or former leaders by state, and only 25% of the sample included leaders from the same state. Page 3/22 Data Analysis An inductive, conventional content analysis approach was employed to examine the transcripts for leaders’ perceptions of and experiences sustaining grant-funded programs (28). Transcripts were organized into analyzable paragraph sections, first coded a priori using the interview guide open-ended questions for topic categories (barriers, facilitator, strategies employed) (29). These sections were then analyzed for emergent themes through open coding (30), a process by which units of text are qualified by concepts and added to a working codebook for continual reference (29). After all sections were coded by one author, a second coded the material separately for comparison; any differences were discussed to the point of consensus (31). Codes were then crosscut and analyzed for context and relationship to each other to solidify themes, a process known as axial coding (29,31). Coder agreement ranged from 68 to 92% with a median score of 81%, falling in the very good to excellent range for standard qualitative research metrics (32-34). All analysis was completed using NVIVO 12 Pro. Once these codes were determined, they were organized within the outer and inner contexts of the EPIS sustainability framework, which reflects differential-level factors that influence adoption of evidence-based practice within a system (Figure 1) (10). For this analysis, the contexts include the following: Page 4/22 Page 4/22 i. Outer context: For this analysis, this includes state-level systems and all public, government, and legislative entities. Subdomains include sociopolitical and systems-level dynamics such as the service environment (funding mechanisms) and inter-organizational characteristics (external leadership and public-academic partnerships) that influence adoption of a practice. ii. Inner context: For this analysis, this pertains to the state agency within which the interviewed authority operates. Subdomains include intra-organizational characteristics (staffing and management) and organizational-level factors that facilitate adoption of a practice. Fig 1. EPIS Framework with Inner and Outer Context Subdomains. a“Individual” within the inner context of the EPIS framework is interpreted as unit-level factors influencing adoption of a practice within a system. For our analysis, the inner context unit is at the organizational-level. Adapted from Aarons, et al. (9) and Aarons & Green (11). Participant Demographics Table 1 Page 6/22 Consumer Support/Advocacy External pressure/public support 2 (25.0) 3 (37.5) Service-Environment Flexibility in spending 2 (25.0) 2 (25.0) Technical assistance 3 (37.5) 1 (12.5) Inner Context Intra-organizational Strategic planning 6 (75.0) 4 (50.0) Demonstration of outcomes 1 (12.5) X X: Not identified as a theme among interviewees in this group. Note. This table only reflects responses to the barrier and facilitators prompt, in-text citation may differ when referencing separate prompt. †“Individual” within the inner context of the EPIS framework is interpreted as unit-level factors influencing adoption of a practice within a system. For our analysis, the inner context unit is at the organizational- level. Participant Demographics Of the sixteen leaders who were interviewed, the majority (68.7%) were female. The leaders most commonly led from Census designated Southern states (5) followed by the Northeast (4), Southwest (3), West (2), and Midwest (2). The majority (87.5%) of both leadership groups were housed in Medicaid expansion states, which adjusted eligibility to uninsured individuals at or below 138% of the federal poverty level. This is slightly over-representative of the national Medicaid expansion proportion of 74% of states (35). Seventy-five percent of the current leaders were housed within their states’ department of health, with 50% of those within divisions of behavioral and mental health and 25% in substance use departments, exclusively. The remaining two leaders led from within departments of child welfare and human services, respectively. The leaders had an average of 5.34 years of experience in the role of state agency authority and an average of 12.85 years in the substance use field. Seven of the eight current leaders reported directly to an appointee of the governor or legislature and half were also appointees themselves. Emergent themes from the responses to the a priori questions on barriers and facilitators to sustaining federal programs are listed in alignment with the EPIS framework (Table 1). Page 5/22 Table 1 Emergent themes: Barriers and Facilitators categorized within EPIS framework. Theme responses by leader type Current (n= 8) Former (n = 8) n (%) n (%) SUSTAINMENT BARRIERS Outer Context Service-Environment Medicaid coverage & reimbursement 4 (50.0) 4 (50.0) Rely on continued funding 3 (37.5) X Infrastructure 2 (25.0) 3 (37.5) Inter-organizational Reduced timeframes 4 (50.0) X Opioid-specific funding cliff 3 (37.5) X Opioid-specific data collection 2 (25.0) X Inner Context Intra-organizational Costs to integrate services 3 (37.5) 4 (50.0) Lack of adequate workforce 2 (25.0) 3 (37.5) Individual† Adopter Characteristics Competing priorities 3 (37.5) 5 (62.5) Shifting priorities X 2 (25.0) SUSTAINMENT FACILITIATORS Outer Context Inter-organizational Positioning within the state 8 (100.0) 4 (50.0) Access to governor office 3 (37.5) X Access to Medicaid office 6 (75.0) X Professional networks X 3 (37.5) Table 1 Emergent themes: Barriers and Facilitators categorized within EPIS framework. Current leader 5, Medicaid expansion state Current leader 5, Medicaid expansion state A major non-funding related barrier that emerged among both current (n=2) and former (n=3) leaders was lack of ability or allocation by the grant itself to support projects that develop infrastructure. Former leader 3 described this difficulty to “build infrastructure on funding that’s going to go away… what was most badly needed is to expand both our treatment and prevention infrastructure.” Leaders often followed this up with an example of a project that addressed an “immediate” need – such as Naloxone kits (current leader 5) – in the absence of developing treatment capacity. Outer Context: Inter-organizational Another major theme identified by half of the current leaders (n=4) interviewed was the impact of condensed grant timelines on project selection and ability to integrate sustainability planning. Current leader 8 questioned, “When do you have time to build in the sustainability” for projects with time-limits when it “potentially loses the sustainability factor if you aren’t given the time to plan.” Current leader 2 touched on how shorter time to implement a project can make agencies “a little more cautious and risk adverse if you know you only have two years to do something – [which] also impacts sustainability.” Current leadership recognized that the limited period of grant funding was immediately impactful on projects funded through the opioid response grant mechanisms. Many current leaders (n=3) described concerns that as the impending funding cliff was at odds with the non-fleeting nature of the epidemic. For example, current leader 3 described substance use disorders as “[a] chronic illness, it’s not like this is an epidemic that’s going to end” and that without continuation of funding for “some of these services I see people returning back to illicit opioids.” One quarter of current leaders (n=2) also expressed concerns about data collection and the inability to create infrastructure within the time limit. Current leader 4 said, “the only way I can do that is I can build something internally. This is two-year money … I'm still probably a year away from having a system to actually gather this information.” Outer Context: Service Environment Outer context barriers included Medicaid and reimbursement challenges for programs requiring continued funding beyond the allocated period. Half of the current (n=4) and former (n=4) leaders interviewed identified reimbursement, both within and outside of Medicaid expansion, as one of the most prominent barriers to sustaining grant-funded programs. Former leader 2 (Medicaid expansion state) described their experience attempting to get a Screening, Brief Intervention, and Referral to Treatment (SBIRT) program “reimburs[ed] through Medicaid and Medicaid commercial insurance” and their inability to “sustain that by making it kind of [a] billable insurance service.” Some leaders acknowledged this was particularly true for differential reimbursements across formularies of addiction medication (medications for addiction treatment [MAT]). Current leader 1 (Medicaid expansion state) described an inability to “have Medicaid coverage for MAT in our state…particularly for Methadone”, while current leader 8, also from an expansion state, experienced similar difficulty “[on] the injectable Naltrexone side … getting that medication paid for patients.” Current leaders (n=3) pointed out that certain treatment-based programs Page 7/22 Page 7/22 which relied on additional funding inherently posed challenges to sustainment beyond the life of the grant. One reported: “So, for those initiatives that are new like sober living… or recovery support services through… the discretionary grants [make] sense. But, treatment is largely funded now in this country like healthcare is funded and if we're going to look at expansions and treatment services - providers need sustainable payer resources… When the money's gone. It's gone.” Inner Context: Intra-organizational There was high recognition among current (n=3) and former leaders (n= 4) that the impending grant end would leave certain programs unfunded, a cost that would have to be subsumed and integrated with other entities in order to continue. Current leader 6 questioned - in relation to the community behavioral Page 8/22 Page 8/22 clinics they served - “what is going to be billable, do they have the staff resources they need to keep things going?”. This concern was often mentioned in the context of integrated care between service entities. Current leader 8 mentioned the particular need within “hospital settings… if they [could] bill … services to Medicaid and find a way to absorb the costs of some of the ancillary services.” Former leaders also highlighted addressing the barrier of dis-integrated care in order to sustain programs past the funding period. Former leader 8 referenced a need to replicate the accomplishments during the HIV epidemic, when “agencies came together and … every funding stream [paid] for what they can pay for.” Former leader 4 also highlighted the lack of joint care prioritized in the grants themselves “as we see more and more focus on just integration in general, whether it’s integration of state agencies… integration of behavioral health with physical health… there should be this focus on integration of the funding.” Many former (n=3) and current (n=2) leaders discussed lack of adequate workforce, both with the skillsets and availability, to continue projects started within grant funding. Current leader 4 described this impact on system capabilities as “the lack of workforce, that’s really what constrains the expanding [sic] provider capacity.” Another, former leader 8, introduced that even if funding may exist to continue a program past the grant period, “the fact that we had so few people who were specifically qualified to treat adolescents and young adults, that was a problem for other funders.” Specific workforce challenges related to substance use, such as provider stigma, were also presented when current leader 4 described “everything from bias within the practice setting against this population and fear that it’s going to alienate their other patients.” Capacity, qualifications, and stigma were all workforce components that were acknowledged by leaders as prohibitive factors in sustaining grant funded programs. Outer Context: Inter-organizational Across both leaders, the most commonly cited outer context facilitator was one’s political positioning within the state. All current leaders (n = 8) and half of the former leaders (n = 4) discussed how their standing within state government either contributed to, or hampered, their efforts to enact change. They noted that there was a “political reality” (current leader 5) which officials felt obliged to operate within. One, former leader 6, indicated that support “from above… makes it more likely that the work can continue” after a grant has ended. Furthermore, respondents pointed to multiple players they felt required to negotiate and collaborate with, including Medicaid, the governor’s office, and others. As a sub-theme of positioning within the state, collaboration with the governor’s office was mentioned by some current leaders (n = 3). Current leader 6 pointed out the importance of the governor’s support, saying “I certainly think that having people buy in at every level… from the governor's office, all the way down, we have an absolute buy-in about the need for this kind of work.” More noticeably however, current leaders (n = 6) endorsed that their relationship with the Medicaid office was an important facilitator. Those leaders who enjoyed a “good relationship” Furthermore, respondents pointed to multiple players they felt required to negotiate and collaborate with, including Medicaid, the governor’s office, and others. As a sub-theme of positioning within the state, collaboration with the governor’s office was mentioned by some current leaders (n = 3). Current leader 6 pointed out the importance of the governor’s support, saying “I certainly think that having people buy in at every level… from the governor's office, all the way down, we have an absolute buy-in about the need for this kind of work.” More noticeably however, current leaders (n = 6) endorsed that their relationship with the Medicaid office was an important facilitator. Those leaders who enjoyed a “good relationship” (current leader 8) were able to collaborate by virtue of state bureaucratic infrastructure. Inner Context: Individual Adopter Characteristics Competing priorities between funded projects was overwhelmingly identified by both the former (n=5) and current leaders (n=3) as the most prominent barrier to sustaining programs; spanning time, resources, and projects. Current leader 2 discussed this strain on time and staffing, saying “there’s a competition between my time… I didn’t get extra staff to implement these grants, and they’re so fast.” Former leader 4 contextualized this as their agencies’ differential “attention [paid] to the fast block grants rather than some of the smaller ones.” Current leaders (4 and 7) noted differences that exist between state and federal agency priorities as some “organizations will get funding from SAMHSA, and create a whole new program that may not be on the state priority list” in which later on the project may “not make the cut and thing[s] will not continue.” A small number of former leaders (n=2) contextualized a shift in prioritization as the substance use landscape transitions from one substance to another, creating a “moving target” of different approaches and resources: “I would also add the moving target of problems –for example the opioid money … as the problem shifts to cocaine or methamphetamine or marijuana, whatever works for one thing doesn’t always work for another.” Former leader 7 Page 9/22 Page 9/22 Outer Context: Inter-organizational As current leader 7 noted: “we have a really strong partnership with our Medicaid agency…some of that is the fact that this is a separate state agency that's part of the governor's cabinet that works on addiction issues and that it's not merged together with mental health and/or with health, generally.” These kinds of bureaucratic structures seemed to matter insofar as they also impacted the ability of officials to work seamlessly. For example, (current leader 8) were able to collaborate by virtue of state bureaucratic infrastructure. As current leader 7 noted: “we have a really strong partnership with our Medicaid agency…some of that is the fact that this is a separate state agency that's part of the governor's cabinet that works on addiction issues and that it's not merged together with mental health and/or with health, generally.” These kinds of bureaucratic structures seemed to matter insofar as they also impacted the ability of officials to work seamlessly. For example, “The further removed you are from the Office of Medicaid, the harder it is to influence Medicaid policy. So being in the same department, having the director of Medicaid as a peer… facilitates being able to integrate the benefit in meaningful ways.” Current leader 4 Similarly, some former leaders (n = 3) also cited professional networks more broadly, not necessarily solely within government, as being important outside facilitators. As one former leader reported: “One thing that helped me accomplish one of the goals in the state ... was that I knew who to contact… So I called a colleague in public health who then figured out who I could access that was external to government [who] helped us… [So] getting people that are external – that’s the facilitator. Having a leader, Page 10/22 Page 10/22 having someone in the leadership team that has good connections with people outside of the state… getting expertise from outside of the industry, this was definitely something that I encouraged.” having someone in the leadership team that has good connections with people outside of the state… getting expertise from outside of the industry, this was definitely something that I encouraged.” Former leader 7 Outer Context: Service-Environment Some current leaders (n = 2) identified flexibility in spending as a facilitator. Current leader 2 noted that “traditionally funding services [are] so siloed” but – in relation to the opioid response grants - “this type of funding [opioid response grants] that is so flexible, umm you know, I think, ultimately, it really does help with removing silos.” Current leaders (n = 3) also reported that knowledge retention, sometimes in the form of technical assistance from outside contractors, was a helpful resource. Outer Context: Consumer Support/Advocacy Current (n=2) and former (n=3) leaders pointed to public support and external pressure as outer facilitators as they put pressure on governments to increase resources. According to former leader 7, “getting that public support is important…particularly if what you wanted to do with that public funding… is going to require new funds to continue things moving forward.” Former leader 4 also agreed, noting that this is critical given the scope and heightened public awareness of the opioid crisis: “We all have, I think, recognized now that the opioid epidemic has gotten everyone’s attention, which, I think we can use to our advantage as a substance use disorder treatment community. We’ve been wanting for years for others to recognize the chronic… nature of addiction… there’s a lot of pressure for providers to demonstrate quality, to demonstrate outcomes, and to position themselves within the true healthcare community, if you will.” Former leader 4 Inner Context: Intra-organizational Among former leaders (n=4), strategic planning was the most frequently mentioned facilitator. Former leader 7 created and continually adapted a strategic plan “with the goal that within our resources, [the program] was already something we were going to maintain,” thus bringing sustainment into the conversation earlier. Another added that strategic planning involved viewing time-limited grants not as Page 11/22 isolated initiatives, but as “pilot or demonstration projects” (former leader 9) whose results could be collected and built upon for future endeavors. Current leaders (n=6) also reported that strategic planning was helpful in past sustainment efforts, although none identified planning as a facilitator of current project sustainment. Four of the six who acknowledged planning as important cited the benefits gap or needs analyses. One, current leader 2, said because of gap analysis, “we [already] knew where we wanted to go. So when the funding came… it was just like, ‘Okay, we know that we have an issue in this area, so we’re going to put funding over here’.” Preemptively gaining an understanding of the most urgent needs, pre-existing coverage, and the “policy landscape and… infrastructure,” current leader 5 added, enabled leaders to “try to build and grow and sustain and create good policy… [even] if the funding goes away.” When asked about program sustainment supports, both former and current leaders also discussed the importance of a program’s ability to demonstrate outcomes, with current leader 6 characterizing demonstration of outcomes as “building a business case for return on investment… pushing agendas and making sure that people see… return on investment exists.” Although only one current leader explicitly identified demonstration of outcomes as a facilitator, several current leaders (n=3) expressed interest in receiving training on it and many former leaders (n=4) recommended discussing the topic with current leaders (not listed in Table 1). Commonly cited was the need to collect and present program data in formats understandable by state legislatures. Current leader 6, who identified this as a facilitator, said they intentionally use of data showing “impact and… [returns on investment]” to potentially catch the attention of “an interested lawmaker who looks to us for… the newest, latest, and greatest thing that needs to be supported and sustained.” Emergent Themes: Sustainment Strategies When prompted to discuss strategies taken to sustain current or former programs within their tenure, leaders often highlighted initiatives that directly addressed the aforementioned barriers, which are listed in alignment with the EPIS framework (Table 2). Page 12/22 Page 12/22 Table 2 Emergent Themes: Strategies categorized within EPIS framework Theme responses by leader type Current (n= 8) Former (n = 8) n (%) n (%) Outer Context Inter-organizational Involving stakeholders 6 (75.0) X Technical assistance 3 (37.5) X Creating buy-in 2 (25.0) X Selecting champions 1 (12.5) X Engaging leadership 3 (37.5) 8 (100.0) Enacting policy change 1 (12.5) 2 (25.0) Consumer Support/Advocacy Raising awareness 4 (50.0) X Inner Context Intra-organizational Workforce/credentialing 5 (62.5) 3 (37.5) Capacity development 6 (75.0) 1 (12.5) Demonstrating outcomes 2 (25.0) X Primary prevention initiatives 2 (25.0) X Table 2 X: Not identified as a theme among interviewees in this group Outer Context: Inter-organizational A major strategy employed by the current leaders (n=6) included involving stakeholders early on in discussions and initiatives to create ownership for sustaining programs. For many who noted this, it included technical assistance activities (n=3), creating buy-in (n=2), and selecting champions (n=1). Current leader 2 described the importance of creating this buy-in by “talking about sustainability … as we’re reaching across silos of different systems – how do we get them to understand and own the problem, too?” A related strategy strongly employed by both current (n=3) and all former (n=8) leaders was engaging other state department leaders and offices and using one’s position to partner with and advocate for policy and funding changes. Former leader 3 described this inter-agency connectivity “…as a Cabinet-level official, I was able to work with the Physician General, and we called health insurers all over the state, and raised a half a million dollars for naloxone for police.” Another described this role positioning as a key component of change as “the biggest thing … where they [the leader] are in the hierarchy and where their sphere of influence actually is” (former leader 7). Enacting policy changes was another avenue employed by leaders [former (n=2), current (n=1)]. Former leader 7 described their plan to change licensing policies as “one of the plans to sustain was to change the contract language or changing … the rules and regulations.” Another, former leader 5, explicitly described rewriting “bundle payments differently for OTPs… so even though this money will go away, their rate for all other state and federal money will support the program.” Another strategy acknowledged by current leaders (n=4) was raising awareness through information campaigns –categorized within the “consumer support/advocacy” subdomain of the outer context (Table 2). Outer Context: Service Environment Financing was a major strategy employed among the current leaders (n=5) and ranged from “working with our legislature to get them to approve funding for putting methadone on the formulary, for Medicaid” (current leader 1) to “[writing] a new 1115 waiver that included … behavioral health advancements” (current leader 2) to adding “Medicaid as a reimbursement structure for peer support” (current leader 4). Current leader 2 specifically mentioned financial reimbursement strategies’ increased impact on Page 13/22 Page 13/22 sustaining programs in that “with some of the other short-term funding … the sustainability is really impacted because you’re not going to do projects necessarily increasing access.” Although not a stated strategy, there was also a high recognition among current leaders (n=3) of wanting to receive training or content knowledge on healthcare financing, a separate question. sustaining programs in that “with some of the other short-term funding … the sustainability is really impacted because you’re not going to do projects necessarily increasing access.” Although not a stated strategy, there was also a high recognition among current leaders (n=3) of wanting to receive training or content knowledge on healthcare financing, a separate question. Inner Context: Intra-organizational Several strategies were stated by former (n=3) and current leaders (n=5), respectively, to address barriers related to workforce capacity. This included prioritizing “co-location models” (current leader 8) to “hiring people in government [with] business experiences,” because “learning about insurance and about how the healthcare world works … never used to be so essential as it is now” (former leader 6). Former leader 8 even discussed the intentionality of workforce capacity as a key component of sustainability, providing an example of “credentialing over 300 folks to be peer specialists … purposefully put into both the [opioid response] grants to encourage that sustainability.” Page 14/22 Page 14/22 The majority of current leaders (n=6) and one former leader mentioned employment of capacity development initiatives to build content knowledge among practitioners, lasting beyond the grant-funding period. This included training initiatives on diversion and medication-assisted therapy within jail settings (current leader 2), hospital training collaboratives, and telehealth technology among primary care providers (current leader 4). Current leader 8 contextualized using these grant funds as an opportunity to “to be paying for activities that put things in place like … a good knowledge basis and some training … that they can build from any kind of workforce development.” Other minor strategies listed by current leaders included the importance of demonstrating outcomes (n=2) and engaging in primary prevention initiatives (n=2). Main Findings States received unprecedented funding through the recent 21st Century Cures Act opioid response legislation. Our findings have particular relevance to the current grants and document levers to sustain their impact beyond their allotted funding timeline. The analysis revealed financing and reimbursement, service integration, and workforce capacity as the most commonly cited barriers to sustainment. Status in state government structure, public support, and spending flexibility were noted as key facilitators. The most commonly cited barriers – “costs and integration of services” and “Medicaid coverage and reimbursement” – aligned with the intra-organizational domain of the EPIS inner context and the service- environment funding domain of the outer context, respectively (Table 1). The most commonly cited facilitators – “positioning within state government" and “external pressure/public support” – fell into the inter-organizational domain of the EPIS outer context. Initiatives to help states sustain programs may benefit from orientation to outer context components (leadership, policies, funding, and collaborations), given their prevalence as stated barriers (8). Several themes were salient across the identified barriers, facilitators, and sustainment strategies. The most highly cited strategy for sustainment, “engaging leadership,” might be interpreted as the actionable version of the most commonly cited outer sociopolitical facilitator theme, “positioning within the state” (Table 2). This may point to the political nature of the leaders’ roles, and the applicability of sustainment strategies targeting sociopolitical dynamics. It also points to a need to develop leadership capacity in support of implementation through networking, collaboration, and engagement of stakeholders – another stated strategy. Other key facilitators identified among leaders were “external pressure/public support” and “raising awareness,” which as stated strategies reiterate both the high visibility and urgency of the opioid response landscape. Financing was often identified as a barrier to sustainment for both former and current leaders, but only cited as a common strategy among current leaders. This might indicate an increased priority placed on financing in the post-expansion landscape, but leaders still may lack the resources and expertise to navigate this burgeoning field. Overall, while leaders noted their advancements and an obligation to sustainment, they consistently reiterated their lack of resources to execute them Page 15/22 Page 15/22 effectively. A commitment to sustainment is of little value if leadership lacks the tools to make it happen. In understanding these barriers to better target sustainment strategies (25), policy practitioners can create targeted supports and tools for states to increase longevity of substance use treatment programs. Main Findings effectively. A commitment to sustainment is of little value if leadership lacks the tools to make it happen. In understanding these barriers to better target sustainment strategies (25), policy practitioners can create targeted supports and tools for states to increase longevity of substance use treatment programs. These findings align with a recent literature review on implementation determinants in the policy realm (36). Here, organizational climate, actor relationships within networks, and public awareness and knowledge – all outer context components – were the most highly cited determinants of successful policy (36). It aligns with one study recognizing outer setting contexts as a driver of evidence-based practice uptake within the adoption phase of implementation (37). Many of the themes identified in the interviews align with quantitative studies which identified political support, funding stability, partnerships, and strategic planning as important domains for sustainability, generally (8, 38). Additionally, the practice of using identified facilitators to provide guidance on sustainability of programs after grant funding has been investigated by community-coalition projects (24, 39). Themes in this study heavily aligned with the outer context, similar to the emerging literature on policy determinants (8, 36, 37). However, a recent systematic review of the EPIS framework review found it makes up a lower proportion of implementation research overall (23). The methods in this study and corresponding results answer a call for more exploratory qualitative and mixed-methods research on factors affecting sustainment (11, 40, 41, 42). It is the first to qualitatively identify barriers and facilitators among policy-facing leaders within the sustainment phase and in-relation to opioid epidemic funding (6, 11). It is also among the first to present sustainment strategies in alignment with a stated sustainability framework (6, 11). Consent for publication All participants consented to having their anonymized responses appear in publication. Authors can provide copies of the consent and confidentiality form upon request. Ethics approval and consent to participate Interviews were deemed exempt by Stanford University’s Institutional Review Board. Interviews took place following confidentiality and consent agreements approved by the IRB and recorded via a secure videoconference platform. The interviewee and named identities were blinded and de-identified for transcription and coding. Competing interests The authors declare that they have no competing interests. Limitations And Next Steps This study has several key limitations - notably the small, nonrandom sample of state authority leaders. While our study represents a significant percentage of individuals who hold these roles, this limits the ability to generalize results to leaders in different government roles or health service fields. The study encountered common restrictions of qualitative work, i.e. the difficulty of measuring contextual determinants (ex: specific strategies employed by states) while preserving participant confidentiality. Although we allowed themes to emerge inductively, they were grouped by a priori prompts of barriers, facilitators, and strategies. The responses elicited by the interviewees may therefore be limited to the questions that were asked, though standardized across the study. Additionally, the nature of qualitative content analysis does not allow causal inferences from the data, but can inform initial and iterative framework and strategy development (43). Continued research must focus on improved understanding of organizational and political drivers of sustainment (4, 5, 23, 44). The field of addiction policy needs sustainment strategies developed from an understanding of key drivers (24, 41, 44, 45). The present analysis provides insight into effective sustainment strategies deployed by policy-facing state leaders to extend time-limited programs. It adds to the growing literature supporting identification of barriers and facilitators in implementation, which must Page 16/22 Page 16/22 Page 16/22 be strategically linked to sustainment ventures (24, 25, 26). Utilizing these findings may help in the development of feasible, tailored, and effective strategies that state health care authorities can use (4, 24, 25). As state systems leaders work with temporarily extended 21st Century Cures Act funding, it is important to make sustainment a priority. Failure to plan how to sustain effective programs for persons with opioid use disorders will cost lives. Availability of data and materials The transcripts generated and/or analyzed during the current study are not publicly available due to interviewee confidentiality. Though anonymized, information provided by participants may make them identifiable given the small sample pool. However, they may be made available from the corresponding author on reasonable request. Abbreviations Substance Abuse and Mental Health Services Administration (SAMHSA), U.S. Food and Drug Administration (FDA), Exploration Preparation Active Implementation and Sustainment framework (EPIS), SSA (Single State Authority), Medications for Addiction Treatment (MAT), OTPs (Opioid Treatment Programs) Authors’ contributions Lauren Caton oversaw the investigation, methodology, formal analysis, and writing of the manuscript. Mina Yuan and Dexter Louie contributed equally to the formal qualitative analysis and manuscript writing. Drs. Carlos Gallo, Karen Abram, C Hendricks Brown and Mark McGovern assisted in manuscript writing, data acquisition, interview guide creation, and funding acquisition for the project. Dr. Lawrence Palinkas assisted in manuscript drafts and developed the guiding qualitative methodology and outline of the paper. Funding Page 17/22 This paper was supported by National Institute on Drug Abuse (NIH/NIDA) grants (R01DA037222 and R01DA037222-05S1, McGovern PI, and P30DA027828, P30DA027828-10S1, Brown PI). This paper was supported by National Institute on Drug Abuse (NIH/NIDA) grants (R01DA037222 and R01DA037222-05S1, McGovern PI, and P30DA027828, P30DA027828-10S1, Brown PI). Acknowledgements We thank the sixteen former and current state leaders for their public service and for their participation in this study. We also acknowledge Barbara Cimaglio, Rick Harwood, and Melanie Whitter and others who extended their expertise in the development of interview guides and participant recruitment. We are grateful to our colleagues Drs. Helene Chokron Garneau and Mehret Assefa for their support in manuscript preparation. 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Prev Chronic Dis. 2014;11:130184. doi:10.5888/pcd11.130184 Figures Figure 1 EPIS Framework with Inner and Outer Context Subdomains Figure 1 EPIS Framework with Inner and Outer Context Subdomains EPIS Framework with Inner and Outer Context Subdomains Page 22/22 Page 22/22
https://openalex.org/W2146389983	https://bmcbioinformatics.biomedcentral.com/counter/pdf/10.1186/1471-2105-11-S6-S7	English	null	PEPPI: a peptidomic database of human protein isoforms for proteomics experiments	BMC bioinformatics	2,010	cc-by	9,500	© 2010 Chen et al; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. PROCEEDINGS Open Access Open Access Ao Zhou1,3†, Fan Zhang1,3†, Jake Y Chen1,2,3* From Seventh Annual MCBIOS Conference. Bioinformatics: Systems, Biology, Informatics and Computation Jonesboro, AR, USA. 19-20 February 2010 * Correspondence: jakechen@iupui.edu † Contributed equally 1School of Informatics, Indiana University, Indianapolis, IN 46202, USA Full list of author information is available at the end of the article Abstract Background: Protein isoform generation, which may derive from alternative splicing, genetic polymorphism, and posttranslational modification, is an essential source of achieving molecular diversity by eukaryotic cells. Previous studies have shown that protein isoforms play critical roles in disease diagnosis, risk assessment, sub-typing, prognosis, and treatment outcome predictions. Understanding the types, presence, and abundance of different protein isoforms in different cellular and physiological conditions is a major task in functional proteomics, and may pave ways to molecular biomarker discovery of human diseases. In tandem mass spectrometry (MS/MS) based proteomics analysis, peptide peaks with exact matches to protein sequence records in the proteomics database may be identified with mass spectrometry (MS) search software. However, due to limited annotation and poor coverage of protein isoforms in proteomics databases, high throughput protein isoform identifications, particularly those arising from alternative splicing and genetic polymorphism, have not been possible. Results: Therefore, we present the PEPtidomics Protein Isoform Database (PEPPI, http://bio.informatics.iupui.edu/ peppi), a comprehensive database of computationally-synthesized human peptides that can identify protein isoforms derived from either alternatively spliced mRNA transcripts or SNP variations. We collected genome, pre- mRNA alternative splicing and SNP information from Ensembl. We synthesized in silico isoform transcripts that cover all exons and theoretically possible junctions of exons and introns, as well as all their variations derived from known SNPs. With three case studies, we further demonstrated that the database can help researchers discover and characterize new protein isoform biomarkers from experimental proteomics data. Conclusions: We developed a new tool for the proteomics community to characterize protein isoforms from MS- based proteomics experiments. By cataloguing each peptide configurations in the PEPPI database, users can study genetic variations and alternative splicing events at the proteome level. They can also batch-download peptide sequences in FASTA format to search for MS/MS spectra derived from human samples. The database can help generate novel hypotheses on molecular risk factors and molecular mechanisms of complex diseases, leading to identification of potentially highly specific protein isoform biomarkers. Zhou et al. BMC Bioinformatics 2010, 11(Suppl 6):S7 http://www.biomedcentral.com/1471-2105/11/S6/S7 Zhou et al. BMC Bioinformatics 2010, 11(Suppl 6):S7 http://www.biomedcentral.com/1471-2105/11/S6/S7 PEPPI: a peptidomic database of human protein isoforms for proteomics experiments Ao Zhou1,3†, Fan Zhang1,3†, Jake Y Chen1,2,3* Background Identifying disease- related protein isoforms using tandem mass spectrome- try, therefore, can provide hope for improving both the sensitivity and the specificity of candidate disease bio- markers, because proteomics identification, instead of quantification, of the same set of protein isoforms is often sufficient to distinguish between disease samples and controls. In this paper, we describe the development of a Pepti- deomics Database of Protein Isoforms (PEPPI), which consists of systematically generated virtual peptides that cover alternative splicing events and known SNP varia- tions, for identifying protein isoforms in large-scale pro- teomics results. In the PEPPI database, we introduce a peptidomics approach to integrating genome, transcrip- tome, proteome and SNP information for human proteo- mics studies. The database contains a comprehensive set of peptides derived from all known annotated human genes in the Genome Reference Consortium Human gen- ome build 37 by generating alternative splicing events and incorporating non-synonymous SNPs. It is the first comprehensive database that can be used to characterize novel protein isoforms derived from alternative splicing and SNP variations in MS spectra. The database has a web user interface that allows its users to query a gene/ protein and compare all its above-mentioned types of protein isoforms and associated virtual peptides online. However, identifying protein isoforms using current MS proteomics search databases and software tools has been challenging, primarily because of the smaller size of known or common alternatively spliced protein iso- forms relative to several orders of magnitude larger size of MS search databases, which makes exhaustive novel peptide identification computationally inefficient for routine proteomics studies. Up to 80% of all MS spectra peaks in a typical proteomics experiment may remain uncharacterized when searched against a standard MS database with little protein isoform information. Such standard MS search databases include: the IPI database [13], the NCBI-nr database, and the UniProt knowledge base [14]. These databases integrate more than a dozen public protein and DNA sequence databases into a non- redundant list of both known and predicted protein sequences, with only publicly known splice variant tran- scripts represented. MS search software such as SEQUEST [15], Mascot [16], X!Tandem [17], and OMSSA [18]. may further allow customized identifica- tion of limited types of PTM-derived peptides and pro- teins. Background Different forms of a protein may be produced from related genes such as single-nucleotide polymorphisms (SNPs) or may arise from the same gene by alternative splicing or post-translational modifications (PTM). Alternative splicing and SNPs expands the number of messenger RNAs to about 88,000 mRNA variants during transcription of these genes. About 8% of these protein isoforms are generated from mRNA transcripts affected by alternative splicing or SNPs, whereas over 90% of protein isoforms are created through post-translational Human cells benefit from elaborate mechanisms to modify proteins, creating many protein variants (iso- forms), both to increase the diversity of functions and to regulate the activities of proteins. A protein isoform is any of several different forms of the same protein. * Correspondence: jakechen@iupui.edu † Contributed equally 1School of Informatics, Indiana University, Indianapolis, IN 46202, USA Full list of author information is available at the end of the article Zhou et al. BMC Bioinformatics 2010, 11(Suppl 6):S7 http://www.biomedcentral.com/1471-2105/11/S6/S7 Page 2 of 13 (mostly expressed sequence tags, ESTs) to a genomic sequence, some limitations exist in all these methods mostly due to the sequence errors frequently occurring in ESTs and to the repetitive structure of the genome sequence. Moreover, all the databases mentioned con- tain a rather small set of alternatively spliced peptides because they are either manually curated or literature- based data sets, as well as poor annotation of splice events, which are inadequate for the identification of alternatively spliced protein isoforms. To explore the huge solution space of all possible alternatively spliced combination of exons and potentially coding introns, one must generate virtual peptides exhaustively so that uncharacterized MS spectra can be searched against them. In addition, the database of virtual peptides should be expanded to accommodate the amino acid alterations introduced by each SNP. modifications (PTMs) after the mRNA is translated into a protein [1]. Recent studies have shown that the identi- fication, analysis and characterization of these individual protein isoforms (Alternative Splicing, SNPs and PTMs) could improve understanding of diseases improve dis- ease diagnosis or interventions [2-11]. Recent advances in clinical proteomics technology, particularly liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS), have enabled biomedi- cal researchers to characterize thousands of proteins in parallel in biological samples[12]. Background However, these protein sequence databases do not contain information about alternatively spliced tran- scripts or theoretically possible “mis-spliced” protein iso- forms; nor do they contain peptide variants arising from SNPs that result in amino acid changes. Therefore, they are ill-suited for comprehensive protein isoform identifi- cation purposes. Database content Drawn from Ensembl’s genomic data [27], the PEPPI database contains a comprehensive set of peptides derived from all known human protein-coding genes and was constructed by generating both annotated and hypothetical alternative splicing events and incorporat- ing non-synonymous SNPs. In addition to representing an in-frame peptide for each exonic region (EXON_KB) of human proteins, four types of PEPPI splice junctions are also captured for all possible combinations of each coding sequence of gene: annotated exon-exon junctions (E_E_KB type), hypothetical exon-exon junc- tions (E_E_TH type), hypothetical exon-intron junctions (E_I_TH type), and hypothetical intron-exon junctions (I_E_TH type). An exonic region or a splice junction is defined as a peptide region. For each peptide region, we Although there are several publicly available alterna- tive splicing mRNA transcript databases and SNP data- bases including ASTD [19], EID [20,21], ASPicDB [22], ECgene [23], MutDB [24,25], and dbSNP [26], none of these databases can be readily used for identification of novel peptides derived from uncharacterized protein iso- forms. Since predictions of gene splicing patterns in all the methods are based on alignments of transcript data Zhou et al. BMC Bioinformatics 2010, 11(Suppl 6):S7 http://www.biomedcentral.com/1471-2105/11/S6/S7 Page 3 of 13 study we show how to browse the PEPPI peptide regions, PEPPI peptides and related information within gene PRH1. Users will start from the Search Home (Fig- ure 1A), go through the Gene View (Figure 1B), Region View (Figure 1C), and finally navigate to the Peptide View (Figure 1D). also include hypothetical peptides translated with each known non-synonymous SNP. By cataloguing each pep- tide configurations in the PEPPI database, users can study alternative splicing events such as exon skipping, alterna- tive donor site, alternative accepter site, and intron reten- tion at the proteome level. They can also batch-download the peptide annotation and sequences in FASTA format for MS data searching. The current PEPPI database includes human data only. As of April 2010, it is com- prised of 7,848,236 PEPPI peptide entries derived from 23,491 protein-coding genes and 66,384 proteins, incor- porating 150,054 non-synonymous SNPs (Table 1). By searching with gene PRH1 in the standard query box provided at the PEPPI database home page, users can retrieve all peptide regions corresponding to this gene (Figure 2). In the Gene View, the PEPPI database visualizes all the peptide regions that can be mapped to this gene. General online features In Figure 1, we show the user interfaces of the web- based online version of the PEPPI database. It allows searching by Ensembl Gene ID, gene symbol, UniProt ID, IPI AC, peptide sequence, PEPPI Peptide Region ID and PEPPI Peptide ID. With the cross-links users can easily link to Ensembl [27], IPI [13], UniProt [28], HAPPI [29] and HPD [30] and get access to much more detailed information about genes, proteins, protein-pro- tein interactions and human pathways. The peptide annotations and sequences are freely available for batch- download in FASTA format on the download page. The Region View (Figure 3) displays detailed informa- tion of the peptide region and the peptide sequences within this region. In the “Peptide Region Overview” section, the exon coordinate is the chromosome coordi- nate of the source exon, and the segment coordinate is the coordinate of the flanking sequence beside the splice site. The peptide without SNP is displayed on the top of the “cDNA/Peptide Sequence” section, and the peptides with SNPs are displayed below. In the sequences, black and blue are used to color different exons/introns. An amino acid residue overlapping a splice site is colored in red. SNPs are highlighted by green and light cyan. By clicking on the highlighted SNPs, the SNP ID will be shown along with the nucleotide change and amino acid change. A link to the corresponding page in dbSNP is also provided. By clicking on a PEPPI peptide ID, e.g., Database content The “Location” section shows this gene is located on chromosome 12, from 11,033,560 bp to 11,036,883 bp. Links to Ensembl are provided on the gene ID and location. A scale of chromosome coordi- nate is provided on the top and bottom of the visualiza- tion. The arrow on the chromosome coordinate scale shows this gene is located on the reverse strand, so the 5’ end of the gene should be the right end. Peptide regions are displayed in five categories, including EXON_KB, E_E_KB, E_I_TH, I_E_TH and E_E_TH. The color of the region indicates the protein translation open reading frame (ORF) of the corresponding cDNA. By clicking on the peptide region REG005324254, the browser will be re-directed to the Region View. A peptide-protein mapping is also captured for compar- ing the MS search results derived with the PEPPI and con- ventional protein sequence databases. In total 613,591 peptides are mapped to 66,384 IPI [13] proteins (Table 1). Case studies We show three case studies of increasing complexity and biological significance to demonstrate that the data- base can help researchers discover and characterize new protein isoform biomarkers from experimental proteo- mics data. Case study 1: browsing PEPPI peptides and relating information based on a query gene Case study 1: browsing PEPPI peptides and relating information based on a query gene For users who would like to review all the peptide regions and peptides within a gene of interest, we pro- vide the standard gene search procedure. In this case Table 1 Database Content Statistics Region Type Regions / Peptides without SNP Peptides with SNP All Peptides of the Region Type EXON_KB 264,599 140,777 405,376 E_E_KB 259,329 146,016 405,345 E_I_TH 400,571 126,678 527,249 I_E_TH 108,754 41,977 150,731 E_E_TH 4,291,289 2,068,246 6,359,535 Total 5,324,542 2,523,694 7,848,236 Mapped Genes: 23,516 Mapped Proteins: 66,384 Non-synonymous SNP: 150,054 Table 1 Database Content Statistics Zhou et al. BMC Bioinformatics 2010, 11(Suppl 6):S7 http://www.biomedcentral.com/1471-2105/11/S6/S7 Page 4 of 13 Figure 1 Web Interface Structure (A) Search Home: main search page allowing five types of query string: Ensembl gene ID, IPI protein accession number, peptide sequence, PEPPI region ID and peptide ID. (B) Gene View: search result page visualizing peptide regions within a gene. (C) Region View: search result page displaying peptides within a peptide region. (D) Peptide View: PEPPI peptide information page. (E) Protein View: search result page of PEPPI peptides mapped to an IPI protein. (F) Sequence Search: search result page of PEPPI peptides mapped to a query peptide sequence. Figure 1 Web Interface Structure (A) Search Home: main search page allowing five types of query string: Ensembl gene ID, IPI protein accession number, peptide sequence, PEPPI region ID and peptide ID. (B) Gene View: search result page visualizing peptide regions within a gene. (C) Region View: search result page displaying peptides within a peptide region. (D) Peptide View: PEPPI peptide information page. (E) Protein View: search result page of PEPPI peptides mapped to an IPI protein. (F) Sequence Search: search result page of PEPPI peptides mapped to a query peptide sequence. Figure 1 Web Interface Structure (A) Search Home: main search page allowing five types of query string: Ensembl gene ID, IPI protein accession number, peptide sequence, PEPPI region ID and peptide ID. (B) Gene View: search result page visualizing peptide regions within a gene. (C) Region View: search result page displaying peptides within a peptide region. (D) Peptide View: PEPPI peptide information page. (E) Protein View: search result page of PEPPI peptides mapped to an IPI protein. (F) Sequence Search: search result page of PEPPI peptides mapped to a query peptide sequence. “PEP007847820”, the browser will be navigated to the Peptide View. Case study 1: browsing PEPPI peptides and relating information based on a query gene we provided a peptide sequence search function (Figure 1F) and the Protein View (Figure 1E). In this case study we demonstrate that the PEPPI database can help iden- tify the genomic origins of peptides detected from MS data, and can help characterize the alternative splicing events related to these peptides. In the Peptide View (Figure 4), detailed information of the peptide region and a peptide-protein mapping is shown in the “Peptide Region Overview” section. The cDNA and peptide sequence is displayed in the same pattern as the Region View. The “Protein Mapping” sec- tion lists the proteins mapped to the current peptide. The result shows that IPI00847261 is the only protein mapped to the peptide PEP007847820. The annotation on IPI states that IPI00847261 is one of the protein pro- ducts of PRH1. Since the peptide PEP007847820 con- tains a mutant non-synonymous SNP allele, we can infer that the mapped protein IPI00847261 is not the wild-type. The MS peptides can be derived from Healthy Human Individual’s Integrated Plasma Proteome Database (HIP- 2) [31] by inputting its protein ID. For this example, by entering “IPI00023636” as the query, a mapping table with several MS peptides identified by the MS data ana- lysis program will be returned (Figure 5A). To identify the genomic region that encodes a specific peptide sequence, we can search the peptide sequence on the PEPPI database’s search home. Case study 2: identifying genomic origins and alternative splicing events from peptides detected in MS experiments For users, especially MS proteomics scientists, who want to start the query from a peptide sequence or a protein, As shown in Figure 5B and 5C, peptide 1 is mapped to “PEP000841715”, which is an E_E_KB peptide, and peptide 2 is mapped to “PEP000841692” which is an EXON_KB peptide. This indicates peptide 1 is coded by Zhou et al. BMC Bioinformatics 2010, 11(Suppl 6):S7 http://www.biomedcentral.com/1471-2105/11/S6/S7 Page 5 of 13 Figure 2 Gene View (A) Gene scale. Shows the user the chromosome coordinates and strand of the gene. With the gene scale, users can read the approximate position of the peptide regions. (B) Peptide regions. Shows the user which five types of peptide regions within current given gene that the peptide belong to. The coloring of the peptide regions indicates the ORF (Red: 0, Green: 1, Blue: 2). The solid bars indicate exons, and the blank bars indicate introns. Case study 1: browsing PEPPI peptides and relating information based on a query gene The curve between two exons means the exons are spliced with each other. Figure 2 Gene View (A) Gene scale. Shows the user the chromosome coordinates and strand of the gene. With the gene scale, users can read the approximate position of the peptide regions. (B) Peptide regions. Shows the user which five types of peptide regions within current given gene that the peptide belong to. The coloring of the peptide regions indicates the ORF (Red: 0, Green: 1, Blue: 2). The solid bars indicate exons, and the blank bars indicate introns. The curve between two exons means the exons are spliced with each other. peptide mapping of the wild-type and the third iso- form of MP2K7_HUMAN. In the wild-type MP2K7_HUMAN (Figure 5D), “PEP000841715” con- tains the splice junction of two exons (PEP000841690 and PEP000841692), and peptide 1 just crosses the splice site. Nevertheless, in the MP2K7_HUMAN iso- form 3 (Figure 5E), we found a unique cassette exon (PEP000841691) spliced between the two exons, which hampered the coding of sub-sequence map- pable to peptide 1. Meanwhile, peptide 2 is only mapped to a single exon (PEP000841692), which exists in all five proteins and unaffected by any splice events. Thus we have confirmed the suspicion that a cassette exon event caused the protein mapping dif- ference between two MS peptides, and have shown the PEPPI database’s ability to help infer alternative splicing events from peptides detected from MS experiments. an exon-exon junction, and peptide 2 is coded by a sin- gle exon. To study the related alternative splicing events, we then compared the number of proteins which can be mapped to these peptides. By clicking on the peptide ID, we can get access to the proteins mapped to each peptide. We found 4 proteins mapped to peptide 1, and 5 proteins mapped to peptide 2. Interestingly, only one protein (IPI00745806) was differentially mapped. By looking up the protein information in IPI, we found that the proteins involved are five different alternatively spliced isoforms of MP2K7_HUMAN, and IPI00745806 is the third isoform. Therefore, it is likely that only a specific alternative splicing event that takes place is annotated and can be mapped onto the protein sequence IPI00745806. To verify our suspicion on the existence of the alternative splicing event, we compared the protein- Zhou et al. Case study 1: browsing PEPPI peptides and relating information based on a query gene BMC Bioinformatics 2010, 11(Suppl 6):S7 http://www.biomedcentral.com/1471-2105/11/S6/S7 Page 6 of 13 Figure 3 Region View (A) The different colors of the cDNA and peptide sequences indicate two different exons, or an exon and an intron. The amino acid letter colored in red overlaps with the splice site. (B) Green and light cyan backgrounds are used to indicate SNP in cDNA and peptide sequences. (C) By clicking on a SNP in sequence, users can see the details of the SNP. A link to the dbSNP database is provided. Figure 3 Region View (A) The different colors of the cDNA and peptide sequences indicate two different exons, or an exon and an intron. The amino acid letter colored in red overlaps with the splice site. (B) Green and light cyan backgrounds are used to indicate SNP in cDNA and peptide sequences. (C) By clicking on a SNP in sequence, users can see the details of the SNP. A link to the dbSNP database is provided. Figure 4 Peptide View (A) The cDNA and peptide sequences that the current PEPPI peptide is mapped to. Same color theme is used in the region view. By clicking on SNP in sequence, users can access detailed information of the SNP. (B) In the protein mapping list, the view displays all the proteins mapped to the current PEPPI peptide. The peptide sequence is highlighted from within the protein sequence. Figure 3 Region View (A) The different colors of the cDNA and peptide sequences indicate two different exons, or an exon and an intron. The amino acid letter colored in red overlaps with the splice site. (B) Green and light cyan backgrounds are used to indicate SNP in cDNA and peptide sequences. (C) By clicking on a SNP in sequence, users can see the details of the SNP. A link to the dbSNP database is provided. Figure 3 Region View (A) The different colors of the cDNA and peptide sequences indicate two different exons, or an exon and an intron. The amino acid letter colored in red overlaps with the splice site. (B) Green and light cyan backgrounds are used to indicate SNP in cDNA and peptide sequences. (C) By clicking on a SNP in sequence, users can see the details of the SNP. A link to the dbSNP database is provided. Case study 1: browsing PEPPI peptides and relating information based on a query gene (E) The search result of the 3rd isoform of MP2K7_HUMAN, the protein that is mapped to the peptide 2 but not mapped to peptide 1. That is because the insertion of a cassette exon (PEP000841691) changed the sequence of the protein. Figure 5 Identifying The Genomic Origin of MS Detected Peptides and The Relating Alternative Splicing Event (A) The HIP-2 search result page of protein IPI00023636, displaying the evidence peptides detected in MS experiments. (B) The PEPPI sequence search result page of peptide 1, indicating the query peptide is produced from an exon-exon combination region. The corresponding PEPPI peptide can be mapped to 4 proteins. (C) The sequence search result page of peptide 2, indicating the peptide comes from an exon, and can be mapped to 5 proteins. (D) The search result of the wild-type MP2K7_HUMAN, showing the regions mapped by the peptides. Peptide 1 crosses the splice site of two exons (PEP000841690 and PEP000841692). Peptide 2 is produced from a single exon, PEP000841692. (E) The search result of the 3rd isoform of MP2K7_HUMAN, the protein that is mapped to the peptide 2 but not mapped to peptide 1. That is because the insertion of a cassette exon (PEP000841691) changed the sequence of the protein. Figure 5 Identifying The Genomic Origin of MS Detected Peptides and The Relating Alternative Splicing Event (A) The HIP-2 search result page of protein IPI00023636, displaying the evidence peptides detected in MS experiments. (B) The PEPPI sequence search result page of peptide 1, indicating the query peptide is produced from an exon-exon combination region. The corresponding PEPPI peptide can be mapped to 4 proteins. (C) The sequence search result page of peptide 2, indicating the peptide comes from an exon, and can be mapped to 5 proteins. (D) The search result of the wild-type MP2K7_HUMAN, showing the regions mapped by the peptides. Peptide 1 crosses the splice site of two exons (PEP000841690 and PEP000841692). Peptide 2 is produced from a single exon, PEP000841692. (E) The search result of the 3rd isoform of MP2K7_HUMAN, the protein that is mapped to the peptide 2 but not mapped to peptide 1. That is because the insertion of a cassette exon (PEP000841691) changed the sequence of the protein. Case study 1: browsing PEPPI peptides and relating information based on a query gene Figure 4 Peptide View (A) The cDNA and peptide sequences that the current PEPPI peptide is mapped to. Same color theme is used in the region view. By clicking on SNP in sequence, users can access detailed information of the SNP. (B) In the protein mapping list, the view displays all the proteins mapped to the current PEPPI peptide. The peptide sequence is highlighted from within the protein sequence. Figure 4 Peptide View (A) The cDNA and peptide sequences that the current PEPPI peptide is mapped to. Same color egion view. By clicking on SNP in sequence, users can access detailed information of the SNP. (B) In the protein mapping l ll the proteins mapped to the current PEPPI peptide. The peptide sequence is highlighted from within the protein sequen Figure 4 Peptide View (A) The cDNA and peptide sequences that the current PEPPI peptide is mapped to. Same color theme is used in the region view. By clicking on SNP in sequence, users can access detailed information of the SNP. (B) In the protein mapping list, the view displays all the proteins mapped to the current PEPPI peptide. The peptide sequence is highlighted from within the protein sequence. Zhou et al. BMC Bioinformatics 2010, 11(Suppl 6):S7 http://www.biomedcentral.com/1471-2105/11/S6/S7 Page 7 of 13 Figure 5 Identifying The Genomic Origin of MS Detected Peptides and The Relating Alternative Splicing Event (A) The HIP-2 search result page of protein IPI00023636 displaying the evidence peptides detected in MS experiments (B) The PEPPI sequence search result page of Figure 5 Identifying The Genomic Origin of MS Detected Peptides and The Relating Alternative Splicing Event (A) The HIP-2 search result page of protein IPI00023636, displaying the evidence peptides detected in MS experiments. (B) The PEPPI sequence search result page of peptide 1, indicating the query peptide is produced from an exon-exon combination region. The corresponding PEPPI peptide can be mapped to 4 proteins. (C) The sequence search result page of peptide 2, indicating the peptide comes from an exon, and can be mapped to 5 proteins. (D) The search result of the wild-type MP2K7_HUMAN, showing the regions mapped by the peptides. Peptide 1 crosses the splice site of two exons (PEP000841690 and PEP000841692). Peptide 2 is produced from a single exon, PEP000841692. Case study 1: browsing PEPPI peptides and relating information based on a query gene Case study 3: identifying new peptide isoforms for human Human fetal liver can evolve into a major site of embryonic hematopoiesis; therefore, protein profiling may help researchers understand how the interaction between hepatic and hematopoietic systems and the migration of the hematopoietic system during mamma- lian development take place. We collected four human fetal liver cytoplasm proteome data sets from the human fetal liver project (http://hlpic.hupo.org.cn /dblep). SDS-PAGE with different cross-linking percen- tages 15%, 10%, and 7.5% was used for protein separa- tion to obtain a full representation of proteins ranging from 5 kDa to more than 300 kDa. After these gels were stained with Colloidal Coomassie Blue R250 and the gel lanes were manually excised from loading posi- tion to the bottom of the gel, the extracted peptide mix- tures were loaded onto nanoscale LC-ESI-Q-TOF MS or micro-LC-ion trap MS systems for protein identification [32]. In order to show that the PEPPI database can be used to identify additional novel peptide isoforms than the traditional protein database, we downloaded the protein database IPI and created three datasets using the PEPPI database: 1) annotated exonic peptides and exon-exon combinations without SNP (PEPPI_KB), 2) all PEPPI peptides without SNP (PEPPI_without_SNP), and 3) all PEPPI peptides including peptides with SNP (PEPPI_- with_SNP). PEPPI_KB consists of peptides of both the EXON_KB and E_E_KB region types without additional SNP permutations; PEPPI_without_SNP consists of pep- tides of the EXON_KB, E_E_KB, E_I_TH, I_E_TH, and E_E_TH region types without additional SNP permuta- tions; PEPPI_with_SNP consists of peptides of all types, with or without SNPs, in the PEPPI database. We also created four corresponding inverse sequence datasets to evaluate the false discovery rate with a target-decoy search strategy [33]. The four peak list files of human fetal liver from LC-ESI-Q-TOF MS or micro-LC-ion Zhou et al. BMC Bioinformatics 2010, 11(Suppl 6):S7 http://www.biomedcentral.com/1471-2105/11/S6/S7 Page 8 of 13 trap MS raw files were searched by OMSSA [18] against the four databases and their four inverse databases in order to compare the results among them. and target PEPPI hits all increase while the correspond- ing FDRs remained approximate. The overlap of genes identified by each database is shown graphically by Venn diagram in Figure 6. The results show that the PEPPI database can be used to identify more peptides/ proteins under the same false positive rate than the tra- ditional IPI database. p g OMSSA reports hits ranked by E-value. Case study 1: browsing PEPPI peptides and relating information based on a query gene An E-value for a hit is a score that is the expected number of ran- dom hits from a search library to a given spectrum, such that the random hits have an equal or better score than the hit. For example, a hit with an E-value of 1.0 implies that one hit with a score equal to or better than the hit being scored would be expected at random from a sequence library search [18]. The search results with OMSSA can vary substantially with differing search parameters, sequence libraries, and samples [33]. There- fore, we adopted the MS/MS false discovery rate (FDR) instead of E-value as scoring criterion for evaluating the four databases, and this method is based on commonly used scoring methodologies and the target-decoy search strategy [33]. All other OMSSA search parameters [18] for the four databases are the same. To increase identifi- cation accuracy, only peptides/proteins with at least two hits of different samples was recognized as true pep- tides/proteins. From the four human fetal liver MS data sets, we identified 63 peptides which mapped to 74 PEPPI pep- tides and 9 SNP events using PEPPI_with_SNP (See additional file 1). Among the 74 PEPPI peptides, 55 EXON_KB type peptides were also annotated previously in IPI, and 19 peptides were novel peptides uniquely identified with the PEPPI database (13 E_E_KB type peptides, 2 E_E_TH type peptides, 1 I_E_TH type pep- tide, and 3 E_I_TH type peptides). The peptide hit matrix shows the number of PEPPI peptides mapped to the peptides detected from the sam- ples, and the number of samples (N) in which the pep- tide is detected (See additional file 2). Discussion and perspectives A comparison of search results against four MS data- bases, i.e., IPI, PEPPI_KB, PEPPI_without_SNP, and PEPPI_with_SNP, is shown in Table 2. Results are shown only at a commonly used 1% MS/MS FDR for each database. Compared to the traditional IPI database, the elapsed time for PEPPI_KB decreased although the dataset size increased by two and a half times. And with the increase of sizes, the elapsed time increases signifi- cantly linearly from PEPPI_KB to PEPPI_without_SNP to PEPPI_with_SNP (Intercept = 8.17021, slope=0.04738 , and adjusted R2= 0.9975). We created a comprehensive PEPPI database of both annotated and hypothetical peptides representing human protein isoforms for MS analysis. The PEPPI database made it possible for high-throughput identifica- tion of gene variations, exon expression, and alternative splicing events at the proteome level. We also con- structed a web-server for searching and visualizing the peptides. With the user-friendly interface and powerful search functions, users can easily study the alternative splicing events and gene variations related to any gene, protein, or peptide sequence of interest. Under the criteria of MS/MS FDR 0.01, the target MS/MS hits markedly increases with the increase of database size, and target peptide hits, target protein hits, A comparison between the PEPPI database and con- ventional MS methods is shown in Table 3. An MS approach with the PEPPI database uses the same Table 2 Summary of Search Results When MS/MS FDR ≤0.01 Table 2 Summary of Search Results When MS/MS FDR ≤0.01 IPI PEPPI_KB PEPPI_without_SNP PEPPI_with_SNP Size (M) 47 115.2 1181.1 1769.7 Elapsed Time(h:mm:ss) 0:11:05 0:10:39 1:02:32 1:33:04 MS2 FDR 0.01000 0.00992 0.00996 0.00999 Target MS2 hits 22685 21473 68137 116363 Decoy MS2 hits 114 107 341 584 Peptide FDR 0.01575 0.01527 0.01242 0.01571 Target peptide hits 125 129 159 188 Decoy peptide hits 2 2 2 3 Protein/gene FDR 0.02797 0.02778 0.02439 0.02339 Target protein/gene hits 368/141 142 162 169 Decoy protein/gene hits 5/2 2 2 2 PEPPI FDR 0.01376 0.01283 0.01376 Target PEPPI hits 433 1549 2886 Decoy PEPPI hits 3 10 20 Zhou et al. BMC Bioinformatics 2010, 11(Suppl 6):S7 http://www.biomedcentral.com/1471-2105/11/S6/S7 Page 9 of 13 Figure 6 Overlap of Peptides/Genes Identified by Four Search Databases. (A) Peptides identified from two or more samples. (B) Proteins identified from two or more samples. Figure 6 Overlap of Peptides/Genes Identified by Four Search Databases. (A) Peptides identified from two or more samples. (B) Proteins identified from two or more samples. Discussion and perspectives samples, equipments and analysis software as a conven- tional MS approach. To use the PEPPI database, users just need to set the PEPPI database or a subset of the PEPPI database as the user defined sequence database in the MS search software. With the PEPPI database, users can gain information on the expression of exons, alter- native splicing events, SNPs, and protein existence from the proteome, while the conventional MS approach can only derive the protein existence information. Users can opt to use different subsets of the PEPPI database for different study purposes. The computational cost of adopting the PEPPI database approach over a conven- tional approach is kept low, due to the use of computing cycles without human intervention. In the future, we plan to enhance the user interface of the web application such as replacing the static down- load page with an interactive batch-download interface, where users can specify the peptide type, gene IDs and protein IDs. We also plan to improve the performance of peptide sequence search with parallel computing and a more powerful database server. Genome data source The whole data generation process was divided into three steps: (A) deriving the genome data from Ensembl; (B) pre-processing of the data to select protein-coding genes and SNPs within coding exons; and (C) generation of peptide regions and PEPPI peptides. The result datasets are colored in red. Figure 7 Data Generation Process. The whole data generation process was divided into three steps: (A) deriving the genome data from Ensembl; (B) pre-processing of the data to select protein-coding genes and SNPs within coding exons; and (C) generation of peptide regions and PEPPI peptides. The result datasets are colored in red. and nucleotide shift. The PEPPI database incorporated 44,285 genes and 16,489,577 SNPs. considered as non-coding genes. The Coding Gene Sequence and Coding Gene Structure table was derived after filtering, and 21,351 protein-coding genes were captured. Genome data source The PEPPI peptides were generated from the human genome. The source genome data was downloaded from Ensembl Version 55 [27] with BioMart[35]. As shown in Figure 7A, four tables, including Un-Translated Region (UTR), Gene Sequence, Gene-Protein Mapping and Gene Structure, were pulled from the Ensembl Homo sapiens Genes Dataset. The UTR table describes the coordinates for all transcript UTRs. The Gene Sequence table contains chromosome coordinates and sequences in FASTA format. The Gene Structure table contains the exon annotation information, including Exon ID, Gene ID, Transcript ID, as well as the genome coordi- nate and translation phase of the exons. The SNP table was derived from the Ensembl Homo sapiens Variation Dataset, and contains the SNP’s chromosome coordinate Since the database incorporated a large number of hypothetical peptides, it is possible that the search result contains false positives due to the noise. To solve this problem, we will design an optimized routine for MS data analysis. For example, users may analyze several samples at one time, and only keep the peptides detected in more than one sample. On the other hand, a recently published article [34] reported tissue dependent splicing patterns, which make it possible to generate tis- sue specific PEPPI peptides and reduce the chance of incorporating false positives. Table 3 Comparison with Conventional MS Methods MS with PEPPI Conventional MS Methods Sample Same as right Ordinary proteome samples Spectrum Same as right Ordinary MS equipments Software Same as right SEQUEST, Mascot, OMSSA etc. Sequence Database PEPPI or subset of PEPPI Conventional protein sequence databases (IPI, UniProtKB, e.g.) Detection Ability 1. Exonic region 2. Exon-Exon combinations (annotated transcripts) 3. Exon-Intron combinations 4. Intron-Exon combinations 5. Hypothetical Exon-Exon combinations 6. SNP peptides Only proteins Configuration User configurable Pre-defined by database producer Cost Same as right Not very expensive Table 3 Comparison with Conventional MS Methods Zhou et al. BMC Bioinformatics 2010, 11(Suppl 6):S7 http://www.biomedcentral.com/1471-2105/11/S6/S7 Page 10 of 13 Figure 7 Data Generation Process. The whole data generation process was divided into three steps: (A) deriving the genome data from Ensembl; (B) pre-processing of the data to select protein-coding genes and SNPs within coding exons; and (C) generation of peptide regions and PEPPI peptides. The result datasets are colored in red. Figure 7 Data Generation Process. Data pre-processing Then we compiled a C program with the SQLite3 library to extract the annotated transcription informa- tion from the Coding Gene Structure table, and pro- duced two tables. The Exon Knowledgebase table describes all the protein-coding exons, and the Exon Comb Knowledgebase table describes all the exon-exon combinations found in the annotated transcripts. Then the SNP table was searched against the Exon A data pre-processing procedure (Figure 7B) was imple- mented to remove non-coding genes and SNPs which are not in exonic regions. Firstly we imported all the source tables into a SQLite3 database with the SQLite3 command-line inter- face, and then we used SQL statements to remove non- coding genes. The Gene-Protein Mapping was utilized as a filter, and genes not mapped to proteins were Zhou et al. BMC Bioinformatics 2010, 11(Suppl 6):S7 http://www.biomedcentral.com/1471-2105/11/S6/S7 Page 11 of 13 Table 4 Data Generation Protocols cDNA Sequence Length Open Reading Frame (ORF) Stop Codon Toleration EXON_KB Whole Length Annotated ORF Only Not Tolerated E_E_KB ≤240 bp Annotated ORF Only Not Tolerated E_I_TH ≤240 bp All 3 ORFs In 3’ end segment I_E_TH ≤240 bp All 3 ORFs Not Tolerated E_E_TH ≤240 bp All 3 ORFs In 3’ end segment We limited the lengths of the combination transcripts to 240 bp, with a flanking sequence of 120 bp on each side of the splice site. Table 4 Data Generation Protocols Knowledgebase table, and 390,539 SNPs within the annotated coding exons were retrieved for peptide generation. cDNA sequence was captured from the gene sequence. Then the exon’s cDNA sequences were translated into peptides according to the annotated ORF. In the peptide estimation process, if a stop codon existed anywhere except the end of the peptide, the corresponding region was considered invalid and was discarded. Peptide region generation We compiled a pipeline program with C and the SQLite3 library to generate peptide regions (Figure 7C). The program first generated the wild-type cDNA sequences of the peptide region, and then translated the cDNA sequences into peptides. The derived peptides were estimated by the program according to a set of protocols, and un-qualified peptides and the corre- sponding region were discarded. Different cDNA gen- eration procedures and peptide estimation protocols were implemented on different types of peptide regions (Table 4). Similar to the EXON_KB type, the chromosome coor- dinates of the two exons in the E_E_KB type were derived directly from the Exon Comb Knowledgebase table. Then the derived cDNA sequences were translated into peptides according to the annotated ORF of the exon on the 5’ end. The same peptide estimation proto- col used with the EXON_KB type was applied to the E_E_KB type. For the E_I_TH and I_E_TH type, the program derived the chromosome coordinates of exons from the Exon Knowledgebase table and spliced them with the adjacent introns. The cDNA flanking sequence on both side of the splice site was limited to 120 nucleotides. If For the EXON_KB type, the chromosome coordinate of the exons were derived directly from the Exon Knowledgebase table, and the whole length of the exon Figure 8 The UML of Database Backend The datasets derived by the data generation pipeline are colored in red, and the datasets derived from other databases are colored in black. Figure 8 The UML of Database Backend The datasets derived by the data generation pipeline are colored in red, and the datasets derived from other databases are colored in black. Zhou et al. BMC Bioinformatics 2010, 11(Suppl 6):S7 http://www.biomedcentral.com/1471-2105/11/S6/S7 Page 12 of 13 mapping tables for genes and proteins enable users to query the database with different IDs. the exon/intron is shorter than 120 nucleotides, the pro- gram will pull out the actual sequence. This limit was set according to the longest peptide in the HIP-2 data- base [31], which had a length of 80 amino acids and corresponded to 240 nucleotides in the cDNA sequence. This represents the length of the longest peptide that can be identified from an MS experiment. In this way we made sure that any MS identified peptide that crosses the splice site can be captured by the PEPPI database. Peptide region generation In the peptide estimation process, a stop codon is tolerated in the intron of E_I_TH, but not tol- erated anywhere expect the 3’ end in I_E_TH. Author details 1 After the generation of peptide region, PEPPI peptides were produced by inserting non-synonymous SNPs into the wild-type peptide of the corresponding region (Fig- ure 7C). All the non-synonymous SNPs within a peptide region were first captured in a list, and then inserted into the wild-type cDNA according to their chromo- some coordinates. Each cDNA sequence with SNP was then translated into peptides. The peptides were then estimated according to the peptide estimation protocol of its own region type, and invalid ones were discarded. During peptide generation, a table of Peptide-SNP Map- pings was also generated. The wild type peptides were also deposited in the PEPPI Peptide table. 1School of Informatics, Indiana University, Indianapolis, IN 46202, USA . 2Department of Computer and Information Science, School of Science, Purdue University, Indianapolis, IN 46202, USA . 3Indiana Center for Systems Biology and Personalized Medicine, Indianapolis, IN 46202, USA . Authors’ contributions JYC conceived the initial work, designed the method for the database construction. AZ implemented the design, generated the datasets, and developed the database backend and the web-based interface from the generated datasets. FZ collected and analyzed the MS data, performed the statistical analyses of the case studies. All authors are involved in the drafting and revisions of the manuscript. Acknowledgements g This work was supported by a grant from the National Cancer Institute (U24CA126480-01), which is a part of NCI’s Clinical Proteomic Technologies Initiative (http://proteomics.cancer.gov). The grant was awarded to Dr. Fred Regnier (PI) and Dr. Jake Chen (co-PI). We thank the support of Indiana Center for Systems Biology and Personalized Medicine. We especially thank David Michael Grobe from UITS at Indiana University for thoroughly proofreading the manuscript. We also thank two anonymous reviewers for comments that helped us improve this manuscript. This article has been published as part of BMC Bioinformatics Volume 11 Supplement 6, 2010: Proceedings of the Seventh Annual MCBIOS Conference. Bioinformatics: Systems, Biology, Informatics and Computation. The full contents of the supplement are available online at http://www.biomedcentral.com/1471-2105/11?issue=S6. This work was supported by a grant from the National Cancer Institute (U24CA126480-01), which is a part of NCI’s Clinical Proteomic Technologies Initiative (http://proteomics.cancer.gov). The grant was awarded to Dr. Fred Regnier (PI) and Dr. Jake Chen (co-PI). We thank the support of Indiana Center for Systems Biology and Personalized Medicine. We especially thank David Michael Grobe from UITS at Indiana University for thoroughly proofreading the manuscript. We also thank two anonymous reviewers for comments that helped us improve this manuscript. This article has been published as part of BMC Bioinformatics Volume 11 Supplement 6, 2010: Proceedings of the Seventh Annual MCBIOS Conference. Bioinformatics: Systems, Biology, Informatics and Computation. The full contents of the supplement are available online at http://www.biomedcentral.com/1471-2105/11?issue=S6. Additional material Additional File 1: 63 Peptides Identified by PEPPI Additional File 2: Peptide Hit MatrixN: number of samples in which the peptide is detected The 2nd to 9th columns are 8 human fetal liver samples marked by Pride accession numbers. The digits in the 8 columns represent the numbers of PEPPI peptides mapped to the peptides detected from the samples. Additional File 1: 63 Peptides Identified by PEPPI Additional File 2: Peptide Hit MatrixN: number of samples in which the peptide is detected The 2nd to 9th columns are 8 human fetal liver samples marked by Pride accession numbers. The digits in the 8 columns represent the numbers of PEPPI peptides mapped to the peptides detected from the samples. When producing the E_E_TH type of peptide regions, all possible exon-exon combinations were generated and searched against the Exon Comb table. Any combina- tions that cannot be found in the Exon Comb table were captured as an E_E_TH type candidate. Then each E_E_TH type cDNA was translated in all 3 ORFs, and if a stop codon was found in the 5’ end exon, the peptide was discarded. Note if more than one E_E_TH peptide derived from the 3 ORFs were considered valid, then a peptide region was created for each ORF. Competing interests The authors declare that they have no competing interests. The authors declare that they have no competing interests. 6. Venables JP, Klinck R, Bramard A, Inkel L, Dufresne-Martin G, Koh C, Gervais- Bird J, Lapointe E, Froehlich U, Durand M, et al: Identification of alternative splicing markers for breast cancer. Cancer Res 2008, 68(22):9525-9531. Additional File 1: 63 Peptides Identified by PEPPI Additional File 2: Peptide Hit MatrixN: number of samples in which the peptide is detected The 2nd to 9th columns are 8 human fetal liver samples marked by Pride accession numbers. The digits in the 8 columns represent the numbers of PEPPI peptides mapped to the peptides detected from the samples. 1. Kim P, Kim N, Lee Y, Kim B, Shin Y, Lee S: ECgene: genome annotation for alternative splicing. Nucleic Acids Res 2005, 33(Database issue):D75-79. 2. Lixia M, Zhijian C, Chao S, Chaojiang G, Congyi Z: Alternative splicing of breast cancer associated gene BRCA1 from breast cancer cell line. J Biochem Mol Biol 2007, 40(1):15-21. 3. Zhu Z, Xing S, Cheng P, Zeng F, Lu G: Modification of alternative splicing of Bcl-x pre-mRNA in bladder cancer cells. J Huazhong Univ Sci Technolog Med Sci 2006, 26(2):213-216. 4. Ku TH, Hsu FR: Mining colon cancer specific alternative splicing in EST database. AMIA Annu Symp Proc 2005, 1012. 5. Ogawa T, Shiga K, Hashimoto S, Kobayashi T, Horii A, Furukawa T: APAF-1- ALT, a novel alternative splicing form of APAF-1, potentially causes impeded ability of undergoing DNA damage-induced apoptosis in the LNCaP human prostate cancer cell line. Biochem Biophys Res Commun 2003, 306(2):537-543. 6. Venables JP, Klinck R, Bramard A, Inkel L, Dufresne-Martin G, Koh C, Gervais- Bird J, Lapointe E, Froehlich U, Durand M, et al: Identification of alternative splicing markers for breast cancer. Cancer Res 2008, 68(22):9525-9531. Zhou et al. BMC Bioinformatics 2010, 11(Suppl 6):S7 http://www.biomedcentral.com/1471-2105/11/S6/S7 Koscielny G, Texier VL, Gopalakrishnan C, Kumanduri V, Riethoven JJ, Nardone F, Stanley E, Fallsehr C, Hofmann O, Kull M, et al: ASTD: The Alternative Splicing and Transcript Diversity database. Genomics 2008. 19. Koscielny G, Texier VL, Gopalakrishnan C, Kumanduri V, Riethoven JJ, Nardone F, Stanley E, Fallsehr C, Hofmann O, Kull M, et al: ASTD: The Alternative Splicing and Transcript Diversity database. Genomics 2008. 20. Shepelev V, Fedorov A: Advances in the Exon-Intron Database (EID). Brief Bioinform 2006, 7(2):178-185. 21. 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Nucleic Acids Res 2004, 32(Database issue): D115-119. 29. Zhou et al. BMC Bioinformatics 2010, 11(Suppl 6):S7 http://www.biomedcentral.com/1471-2105/11/S6/S7 30. Chowbina SR, Wu X, Zhang F, Li PM, Pandey R, Kasamsetty HN, Chen JY: HPD: an online integrated human pathway database enabling systems biology studies. BMC Bioinformatics 2009, 10(Suppl 11):S5. 31. Saha S, Harrison SH, Shen C, Tang H, Radivojac P, Arnold RJ, Zhang X, Chen JY: HIP2: an online database of human plasma proteins from healthy individuals. BMC Med Genomics 2008, 1:12. 32. Ying W, Jiang Y, Guo L, Hao Y, Zhang Y, Wu S, Zhong F, Wang J, Shi R, Li D, et al: A dataset of human fetal liver proteome identified by subcellular fractionation and multiple protein separation and identification technology. Mol Cell Proteomics 2006, 5(9):1703-1707. 33. Balgley BM, Laudeman T, Yang L, Song T, Lee CS: Comparative evaluation of tandem MS search algorithms using a target-decoy search strategy. 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Gyorgy B, Toth E, Tarcsa E, Falus A, Buzas EI: Citrullination: a posttranslational modification in health and disease. Int J Biochem Cell Biol 2006, 38(10):1662-1677. y 33. Balgley BM, Laudeman T, Yang L, Song T, Lee CS: Comparative evaluation of tandem MS search algorithms using a target-decoy search strategy. Mol Cell Proteomics 2007, 6(9):1599-1608. 11. Wang JZ, Grundke-Iqbal I, Iqbal K: Glycosylation of microtubule-associated protein tau: an abnormal posttranslational modification in Alzheimer’s disease. Nature medicine 1996, 2(8):871-875. 12. Klampfl CW: Review coupling of capillary electrochromatography to mass spectrometry. J Chromatogr A 2004, 1044(1-2):131-144. 34. Barash Y, Calarco JA, Gao W, Pan Q, Wang X, Shai O, Blencowe BJ, Frey BJ: Deciphering the splicing code. Nature 465(7294):53-59. 35. Smedley D, Haider S, Ballester B, Holland R, London D, Thorisson G, Kasprzyk A: BioMart–biological queries made easy. BMC Genomics 2009, 10:22. 13. 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Koenig T, Menze BH, Kirchner M, Monigatti F, Parker KC, Patterson T, Steen JJ, Hamprecht FA, Steen H: Robust prediction of the MASCOT score for an improved quality assessment in mass spectrometric proteomics. J Proteome Res 2008, 7(9):3708-3717. 17. Bjornson RD, Carriero NJ, Colangelo C, Shifman M, Cheung KH, Miller PL, Williams K: X!!Tandem, an improved method for running X!tandem in parallel on collections of commodity computers. J Proteome Res 2008, 7(1):293-299. 18. Geer LY, Markey SP, Kowalak JA, Wagner L, Xu M, Maynard DM, Yang X, Shi W, Bryant SH: Open mass spectrometry search algorithm. J Proteome Res 2004, 3(5):958-964. 19. References The online version of PEPPI database is a typical 3-tier web application, with a MySQL database at the backend database service layer, Apache/PHP server scripts to the middleware application web server layer, and CSS driven web pages presented on the browser. The Javascript library uuCanvas (http://uupaa-js-spinoff.googlecode. com/svn/trunk/uuCanvas.js/) is used to render the real- time data visualizations in the gene view and the protein view. 1. Kim P, Kim N, Lee Y, Kim B, Shin Y, Lee S: ECgene: genome annotation for alternative splicing. Nucleic Acids Res 2005, 33(Database issue):D75-79. 1. Kim P, Kim N, Lee Y, Kim B, Shin Y, Lee S: ECgene: genome annotation for alternative splicing. Nucleic Acids Res 2005, 33(Database issue):D75-79. 2. Lixia M, Zhijian C, Chao S, Chaojiang G, Congyi Z: Alternative splicing of breast cancer associated gene BRCA1 from breast cancer cell line. J Biochem Mol Biol 2007, 40(1):15-21. 3. Zhu Z, Xing S, Cheng P, Zeng F, Lu G: Modification of alternative splicing of Bcl-x pre-mRNA in bladder cancer cells. J Huazhong Univ Sci Technolog Med Sci 2006, 26(2):213-216. 4. Ku TH, Hsu FR: Mining colon cancer specific alternative splicing in EST database. AMIA Annu Symp Proc 2005, 1012. 5. Ogawa T, Shiga K, Hashimoto S, Kobayashi T, Horii A, Furukawa T: APAF-1- ALT, a novel alternative splicing form of APAF-1, potentially causes impeded ability of undergoing DNA damage-induced apoptosis in the LNCaP human prostate cancer cell line. Biochem Biophys Res Commun 2003, 306(2):537-543. The result tables derived from the data generation step were imported into the MySQL database (Figure 8). The chromosome coordinate information was deposited in the Peptide Region table, and the sequence informa- tion was deposited in the PEPPI Peptide table. The ID 6. Venables JP, Klinck R, Bramard A, Inkel L, Dufresne-Martin G, Koh C, Gervais- Bird J, Lapointe E, Froehlich U, Durand M, et al: Identification of alternative splicing markers for breast cancer. Cancer Res 2008, 68(22):9525-9531. Zhou et al. BMC Bioinformatics 2010, 11(Suppl 6):S7 http://www.biomedcentral.com/1471-2105/11/S6/S7 Page 13 of 13 Zhou et al. 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W4376474974.txt	https://jgu.garmian.edu.krd/article_171100_f0bd847f470d3628b337e6133d1274e5.pdf	en		Controlling volatility and its relationship to professional fitness among kindergarten managers in Sulaimani	Govarî zankoî germîan/Govarî zankoy Germyan	1,999	cc-by-sa	14,564	‫‪Available online at http://jgu.garmian.edu.krd‬‬ ‫‪Journal of University of Garmian‬‬ ‫‪10.24271/jgu.2023.1010014‬‬ ‫ی‬ ‫ی‬ ‫ی‬ ‫ی‬ ‫ر‬ ‫کۆنتۆڵکردن هەڵچوون و پەیوەندی بە گونجان پیشەییەوە الی بەرێوەبەران‬ ‫ا‬ ‫باخچەی منداڵن لە شاری سلێمانیدا‬ ‫ی‬ ‫مصطف ‪ ،‬ژینۆ عزیز محمد‬ ‫صابر بکر‬ ‫ا‬ ‫ی‬ ‫ی‬ ‫کۆلیی پەروەردەی بنەڕەت‪ ،‬زانکۆی سلێمان‬ ‫بەش باخچەی منداڵن‪،‬‬ ‫پوختە‬ ‫ئ ا ااام ا ااانە لە ریتیینەوە ە‪ ،‬بنی پەە لە زانپو پەۆنەن ا اادی نچنان کۆنی دن ە چنون و‬ ‫ا‬ ‫پەۆنەن اادی بە انن ااان ە الا ا ا ا ااەۆەەوە می بەرتنەبەران ب اااخچەی من ااداڵن‪ ،‬ەروە ااا زانپو‬ ‫ا‬ ‫ئاسا ا ا ا ااو کۆنی دن ە چنون می بەرتنەبەران باخچەی منداڵن‪ ،‬لە ەمان اردا زانپو‬ ‫ا‬ ‫اەن می بەرتنەبەران باخچەی منداڵن بە ەەو ەریەڕ لە اۆڕاوە ان‬ ‫ئاس ا ااو انن ان ە ال ا ا ر‬ ‫[رەاەز‪ -‬جۆری با اااخچە ون ا و نا اااون ایه‪ ،‬بۆ بەدۆ چنا ااان ئە ئا اااما ااان ا ااانە ریت ەران‬ ‫مپتۆدی وەسا ا ا ا ااب پەۆنەنا اادۆەا ااان بە ا ااار چنا اااوە‪ ،‬لە بەرئەوەی لەاە ئا اااما ااانە و و ا ا ا ا ااو‬ ‫ریتیینەوەکە انن اوە و بەدۆ چنەری ئامان ە ان ریتیینەوە ەکە ە‪.‬کۆمە گەی ریتیینەوەکە‬ ‫ا‬ ‫ەێک ارنوە لە س ااەرجە بەرتنەبەران اااری س االێمان بۆ س ااا خیتندن ‪2022-2021‬ی‪ ،‬کە‬ ‫ژمارە ان ‪78‬ی بەرتنەبەرەو ریت ەران سااەرجە کۆمە گەی ریتیینەوەکە ان وەرا ت‪ ،‬ئامزازی‬ ‫راایتااییاانەوەکە باانیاتااو باانو لە بااۆڕر ڕاوەراا ی بااۆ ەریەڕ لە اااۆڕاوە ا ااان کااۆناای ا ا دن‬ ‫اەنی‪ ،‬ەچنەری کۆنی دن ە چنون لە ‪42‬ی بزاە ەێک اربنو‬ ‫ە چنون‪ -‬انن ان ە الا ا ا ا ا ر‬ ‫اەن لە ‪48‬ی‬ ‫ا‬ ‫ا‬ ‫ا‬ ‫ا‬ ‫ا‬ ‫ال‬ ‫ە‬ ‫ان‬ ‫انن‬ ‫ەچنەری‬ ‫ابنو‪،‬‬ ‫ئامادەک‬ ‫کنردی‬ ‫زمان‬ ‫ولە م ەن زبی ی یفی بە‬ ‫ر‬ ‫بزاە ەێک اربنو و لە م ەن وس ا ا ا ا ااا عدادی بە زمان عەرەن ئامادەک ابنو‪ .‬لە ا نگیین ئەوە‬ ‫ئامزازە ئاماریەانەی کە بە ار ارنون بنیتی لە ناوەندی ە‬ ‫ژمی رەن‪ ،‬مدان ەچنەری‪ ،‬اوکۆل ەی‬ ‫پەۆنەندی ەەیساۆن‪ ،‬رایەک دنەوەی ‪ T-test‬ی و ‪ ANOVA‬ی‪ .‬لەپا د نەابنو لە ڕاساو و‬ ‫جێگیی ەچنەرە ا ااان ئەوە بە سا ا ا ا ااەر نمنونەی ریتیینەوەکەدا جپ ەجێک ا‪ .‬بەمەبەس ا ا ا ا ااو‬ ‫چارەس ااەری ئاماری و ااەک دنەوەی دارا و زانەاریەە ان‪ ،‬ریت ەران ە ا ار ئاماری ‪SPSS‬یی‬ ‫بە ااار چن اااوە‪ ،‬لە کۆر اااۆە اادا ریتیینەوەکە اەاال ا ا ا ا ا نە ئە ئەن ااام ااانە‪،‬ئ اااسا ا ا ا ااو کۆنی دن‬ ‫ا‬ ‫ە چنون لە ئاسا ا ا ا ا ێ بەرزدا ە می بەڕتنەبەران باخچەی منداڵن ا ا ا اااری س ا ا ا االێمان بە‬ ‫ا ا ااچنە ەج االا ا ااو و جەاوازی بە گەداری ئاماری نەبنو بۆ ئاسا ا ااو کۆنی دن ە چنون‬ ‫ا‬ ‫منداڵن ا ا ا ا اااری سا ا ا ا االێمان بە ەەو اۆڕاوی ڕەاەزی ەنی و ە‬ ‫ری و‬ ‫می بەڕتنەبەران باخچەی‬ ‫جە اااوازی بە گەداری ئ ااام اااری نەبنو بۆ ئ اااسا ا ا ا ااو کۆنی دن ە چنون می بەڕتنەبەران‬ ‫ا‬ ‫الەن لە ئاس ێ‬ ‫باخچەی منداڵن اری سلێمان بە ەەو جۆری باخچە‪ .‬ئاسو انن ان ە‬ ‫ر‬ ‫ا‬ ‫بەرزدا ە می بەڕتنەبەران باخچەی منداڵن ا اااری سا االێمان بە ا ااچنە ەج االا ااو جەاوازی‬ ‫ا‬ ‫اەن می بەڕتنەبەران باخچەی منداڵن‬ ‫بە گەداری ئاماری نەبنو لە ئاسا ا ا ا ااو انن ان ە الا ا ا ا ا ر‬ ‫ا ا ا اااری س ا ا ا االێمان بە ەەو اۆڕاوی ڕەاەزی ەنی و ە‬ ‫ری ‪ .‬جەاوازی بە گەداری ئاماری نەبنو لە‬ ‫ا‬ ‫ە‬ ‫اەن می بەڕتنەبەران باخچەی منداڵن ا اااری سا االێمان بە ەو جۆری‬ ‫ئاسا ااو انن ان ە الا ا ر‬ ‫باخچە‪ .‬پەۆنەند ەج ڕاسا ا ااتەوانەی مواز ە ە لە نچنان کۆنی دن ە چنون و انن ان‬ ‫ا‬ ‫الەن می بەڕتنەبەران باخچەی منداڵن اری سلێمان‪.‬‬ ‫ە ر‬ ‫کێشەی توێژینەوەکە‬ ‫ەر ریتیینەوە ەڕ بنی پەە لە بنون ک ال ا ا ا ا ااە ەڕ‪،‬‬ ‫دەس ا ا ا ا ا ەێ‬ ‫د اردە ەڕ‪ ،‬ان بابەرێک کە سا ا ا ا ااەرنک ریت ەر ڕادەک ال ا ا ا ا ا و‬ ‫بیو زەی داگی دە ات‪ ،‬بە جۆرتک‪ ،‬کە ئەن امدان ریتیینەوە‬ ‫‪Article Info‬‬ ‫‪Received: November, 2022‬‬ ‫‪Revised: January, 2023‬‬ ‫‪Accepted: April, 2023‬‬ ‫‪S‬‬ ‫‪Keywords‬‬ ‫کۆنی دن ە چنون‪ ،‬بەرتنەبەری باخچەی‬ ‫ا‬ ‫ا‬ ‫الەن‪ ,‬باخچەی منداڵن‪.‬‬ ‫منداڵن‪ ,‬انن ان ە ر‬ ‫‪Corresponding Author‬‬ ‫دەربارەی ئەو بابەرە بە ا نگ و ەچییسا ا ا ا ا ا دەزان بۆ ئەمە‬ ‫ئەو ک ال ا ا ا ا ااە ە‪ ،‬ااان ئەو د اااردە ە لە ژت ناااون الا ا ا ا ا ااان ک ال ا ا ا ا ااەی‬ ‫ریتیینەوە دەخارەڕوو‪ .‬ک الااەی سااەرەج ئە ریتیینەوە ە بنی ەە‬ ‫لە ئا ا ااا ا ا ااا کااۆناای ا ا دن ە اچاانون پەۆاانەنا ا اادی بە ااانن ا ا ا ااان‬ ‫‪152‬‬ ‫‪Journal of the University of Garmian 10 (1), 2023‬‬ ‫ا‬ ‫دروس دەب ‪ ،‬بەڵ بە بنون ئە انن انە ئەوە ئامان ە ان‬ ‫بە جنان بەد اادێن و کە ا ا ا ا ااێ ئ ااارا لە ژینگەی ب اااخچەکەدا‬ ‫اەن و کۆنی دن ە چنون‬ ‫دروسا ا دەب ‪ .‬انن ان ە ال ا ر‬ ‫لە پەۆنەند دان لەاە ەڕ‪ ،‬بەو مانا ەی کە راە اەر بتنان‬ ‫کۆنی دن ە چنونە ا ا ااان خۆی ب ا ا ااات بە ەەو وێس ا ا ا ا ااتگە‬ ‫جاەا ا اااوازە ا ا ااان کە ڕووبەروی دەبایااتەوە ئەوە دەکانتا ا ا لەاە‬ ‫ە الەکەاالەدا سازان ەب ‪.‬‬ ‫لەبەر ئە ۆ ارانە و لە ئەن ار ریبینەک دن ریت ەر بۆ ااچنازی‬ ‫اری بەرتنەب دن کە ماوە ەڕ لەو ەۆس ااتەدا اری ک دووە‪ ،‬بایە‬ ‫ا‬ ‫ریت ەر ەو داوە کە لە بارە ەوە ریتیینەوە ئەن ا بدات و و‬ ‫ا‬ ‫وەڵمێ زانس ەانە بۆ ک الەی سەرەج ریتیینەوەکە بد زتتەوە‪،‬‬ ‫کە بنی ەە لە‪-:‬‬ ‫ئا ا ااا ا ا ااا پەۆ اانەنا ا اادی ە ە لە ن اچ اانان ک ااۆن اای ا ا دن ە اچ اانون‬ ‫ا‬ ‫اەن می بەرتنەبەران باخچەی منداڵن ا ا اااری‬ ‫وانن ان ە ال ا ا ا ر‬ ‫سلێمان‬ ‫‪:‬‬ ‫بە ە ان ە سەاری سەرەج ک الەی ریتیینەوەکە ‪-‬‬ ‫ئا ا ااا ا ا ااا ک ااۆن اای ا ا دن ە اچ اانون می بەرت اانەبەران‬ ‫‪-1‬‬ ‫ا‬ ‫باخچەی منداڵن اری سلێمان لە ئاس ێکدا ە‬ ‫اەن می بەرتنەبەران ب اااخچەی‬ ‫‪-2‬‬ ‫ئ ااا ااا انن ااان ە ال ا ا ا ا ا ر‬ ‫ا‬ ‫منداڵن اری سلێمان لە ئاس ێکدا ە‬ ‫ی‬ ‫گرنگ توێژینەوەکە‪:‬‬ ‫ەر ریتیینەوە ەج زانس ا ا ا ا ااو بە ەەو و ا ا ا ا ااو خۆی و ب ااابەن‬ ‫ی‬ ‫ریتیینەوەکەی ا ن ر اااۆ ەت بە خۆی ە ە‪ .‬ریت ەر لە رت ااای‬ ‫ئەن ااام اادان ریتیینەوەوە ەو اادەدات ک ال ا ا ا ا ااە و ئ اااریال ا ا ا ا ااە ااان‬ ‫بناس ا ا ا ا ااینێ و بە دوای ئاسا ا ا ا ا ا ‪ ،‬ۆ ار و چارەس ا ا ا ا ااەرە ان ئە‬ ‫ک ال ا ا ااانەدا بگەرت و لەاە ئەوە ا ا اادا بە ریتیینەوە ە ریت ەر‬ ‫دەرنان ا ا س ا ا ا ا ااەرنک ئەوانەی ئا ااارەزووی ا اااروبا اااری پەروەردە‬ ‫دەکەن بنروژتنێ ا ‪ ،‬لەوانە بەرپ س ا ا ا ا ا ااان و س ا ا ا ا ااەن ااد ااا ااان و‬ ‫ی‬ ‫ریت ەران‪ ،‬بۆ ەریەکێ ا ااک لە ا ن ئە دوو اۆڕاوە کە ریت ەر‬ ‫اری لە س ا ا ا ااەر دە ات‪ ،‬لە م ەج ر ەوە زانپو ک ال ا ا ا ااە ان اری‬ ‫ا‬ ‫بەڕتاانەبەری با ا اااخااچەی مانا ا ااداڵن لە نا ا اااوەڕ ج ەا ال ا ا ا ا ااەکەدا و‬ ‫ا‬ ‫ەروە ا ااا زانپو داوا ا اااریە ا ااانەا ااان جا ااا داوا ا اااری کۆمەڵ ەن بن‬ ‫اەن‬ ‫وەڕ خاننوبەرە و با ا ا ا اایک دن منوچە‪ ،‬ان داوا اری ە ال ا ا ا ا ر‬ ‫وەڕ با ااا ا ا ا ا اایک دن با ااارود ن ا ااارک دن و چا ااام ک دن‪ ...‬ا ااان‬ ‫پەروەردەن باان بااۆ با ا ااا ا ا ا ا ا اایک ا دن م ەنە پەروەردەو‬ ‫داوا ا ا اااری‬ ‫ر‬ ‫ە‬ ‫بیک دن‪ .‬ەروە ا س ا ا ااەرەڕای ب رەن ریتیینەوە ان لە س ا ا ااەر دوو‬ ‫الەن کە ئەن ا‬ ‫اۆراوی کۆنی دن ە چنون و انن ان ە ر‬ ‫ا‬ ‫دراوە بەڵ نەبەسا ا ا ا ا ا ا ا اایاوەرەوە‪ ،‬بە بەڕتا ا ا اانەبەرەوە‪ ،‬ئەمە‬ ‫ا‬ ‫وا اک ا دووە ئە ر اایت اایی اانەوە ە ب اب اچ ااتە خا ا ااا دەسا ا ا ا ا ا ا اە اێا ا ااک ب ااۆ‬ ‫ل اێ اکااۆ اپاانەوە ا ا ااان دا ا ا اااراانو‪ ،‬بە را ا اااۆ ا ەت بااۆ ژی انااگەی ەرتااا‬ ‫کنردستان و ع اق بە االو‪.‬‬ ‫ە ال ا ا ا ا ااەۆەەوە ە ە لەمی بەرتنەبەران باااخچەوە ەکێااک لەو‬ ‫ۆ ااارانەی کە وای ک د ریت ەر ەسا ا ا ا ا ا ێ ا بە ئەن اااماادان ئە‬ ‫ریتیینەوە ە‪ ،‬ۆ اری خندی بنو‪ ،‬وارە وەز و خنلەای ریت ەر بۆ‬ ‫بابارەکە و ئەن امدان ە سااەکە بە االااو‪ ،‬و ەروە ا ۆ اری‬ ‫بااابەن بنو‪ ،‬بەو مااانااا ەی ریت ەر ەو دەدات ئەوە بزان ا لە‬ ‫ا‬ ‫سااەردەر جەن ا و ە الااکەورنەدا ە چنونە ان ر لەان ە ە‬ ‫لەوەی انن ا ااان رووب ا اادات لە دونە ا ااای ا ااارو ە ال ا ا ا ا ااەدا‪ ،‬و بە‬ ‫ەێچەوانە ا ا ا ا ااەوە دونەای ارو ە الا ا ا ا ااە اریگەریەج دەب لە‬ ‫سەر ە چنونە ان م ڤ‪.‬‬ ‫ە‬ ‫لە ڕاس ا ا ەدا ئەوە ڕون و ئا ا ااک ا ە ژیان ئەم ن ە الا ااە و ار‬ ‫ا‬ ‫ن اااب ا ا ‪ ،‬وارە ەر ر ااا ێ ااک بە جۆرت ااک لە جۆرە ااان رێکەڵوی‬ ‫ا‬ ‫دونەااای ااارو ە ال ا ا ا ا ااە دەب ا ‪ ،‬بایە لە رێکەڵو بنونەدا دەب ا‬ ‫بتناننتا انن ااان بیتەدی لەاە ااارەکەدا‪ .‬ە ال ا ا ا ا ااە بە ا ا ا ا ااێ‬ ‫س ا ا ا ا ااەرەج ژیااان ەمنو رااا ێکە‪ ،‬بنون بە بەرتنەبەری باااخچەی‬ ‫ا‬ ‫منا ا ااداڵن یک ەێگە ەج ا ا ااارک دنە کە را ا ااا بەرتنەبەر بۆئەوەی‬ ‫بتنان ئامان ە راۆ ەت و االا ا ا ا ا ا ەە ان پەروەردە بەدۆب چنێ‬ ‫دەب ا ا انن ااان ەب ا ا لەاە ااارەکە اادا و دەب ا ا رت ەرو‬ ‫ڕابەرت ااارامەو اااریگەر ب ا و کەسا ا ا ا ا اێااک دابیت ا کە رنانااا و‬ ‫اتەن انن ا اااوی ا ااان ە ە و لە اە م ا ااا ەەن اۆرانە‬ ‫ا ا ا ا ا ا ااااس ا ا ا ا ا ر‬ ‫پەروەردەن و ە‬ ‫بی اااریە ااان اااوئااا ەنگەااان ەب ا ‪ ،‬ەلیەوە بەوە‬ ‫ر‬ ‫دەاەۆن کە ە‬ ‫بڵی دەب ا ا انن ا ااان ە ال ا ا ا ا ااەۆەا ااان ەب ا ا ‪ ،‬بەو‬ ‫اەن بنن ەب ا ئەوە ئااامااان ە‬ ‫وارااا ەی کە اەر انن ااان ە ال ا ا ا ا ا ر‬ ‫ە‬ ‫د اریک اوە ان پەروەردە و ە‬ ‫بیک دن وەدی دێن و لیەدا کە کەش‬ ‫بەرتنەبەر بۆئەوەی ئەو انن ااانە ب چنچتە ئ اااراوە وا ەچنس ا ا ا ا ا ا‬ ‫دە ات کە رتکخسی بۆ ە چنونە ان ب ات بە جۆرتک بە ەەوە‬ ‫ە یێس ا ا ا ا ااتە جەاااوازە ااان و مااامە ە جەاااوازە ااان کە ڕووبەڕووی‬ ‫دەبیتەوە بتنان دروسا اایین و با ا اایین رەبتار ن الا ااان بدات را‬ ‫د خە ااان بە با ی‬ ‫ااش کۆنی ب ااات‪ .‬ەلیەدا ە ا نگە بزاننت ا کە‬ ‫بەرتنەبەر ر ااا چەن ااد رنان ااای ر ک دن ە ە لە اە ئە د خە‬ ‫اەن لە کیتدا ە‪ ،‬لە بەرئەوەی‬ ‫جەاوازنە و ئاس ا ا ا ااو انن ان ە ال ا ا ا ا ر‬ ‫ئەن ا دان ەر رەبتارتک بە ا ا ا ا ااچنەی ئۆرۆ اریگەری دەخارە‬ ‫س ا ا ا ا ااەر ەمنو دەوروبەری ئەو بەرتنەبەرە‪ ،‬ەر لە دەس ا ا ا ا ااتەی‬ ‫ا‬ ‫ااار ە‬ ‫اییەوە بەگ ە رااا ەمنو مااامۆسا ا ا ا ا اتااا ااان و منااداڵن باااخچە و‬ ‫ەریەکە لە خ ا ا ا ەایان ەم ا ا اانو ئەم ا ا ااانە ‪ ،‬لەبەر ئەوەی ئەو‬ ‫ەێگە ەی کە اااری بەرتنەب دن رێ اادا ئەن ااا دەدات اااریگەری‬ ‫ەمە م ەن دادەن ا و ەچییس ا ا ا ا ااو بە ە یێس ا ا ا ا ا ا و رەبتاااری‬ ‫بەرپ سانە ە ە‪.‬‬ ‫اەن نەبنو لە می راە وەڕ بەرتنەبەر‬ ‫اەر ارنو انن ان ە ال ا ا ر‬ ‫ە‬ ‫پەروەردەن و بی اااریە ااان دەس ا ا ا ا ااتەبەر ن اااب ا ا ‪،‬‬ ‫ئەوە ئ ااام ااان ە‬ ‫ر‬ ‫ا‬ ‫ە‬ ‫ون ااارایی ب اااخچەی من ااداڵن ئەرکە ااان خای ااان‬ ‫ەروە ااا ا ر‬ ‫ی‬ ‫بەباش ڕ ران نا ەن‪ ،‬وارە بە ااچنە ەج االااو ااچناندن و نارت‬ ‫‪153‬‬ ‫‪Journal of the University of Garmian 10 (1), 2023‬‬ ‫ی‬ ‫ا ن ئە ریتیینەوە ە لەوەدا ە کە ئە ارامەۆەە کۆنی دن‬ ‫ە چنونی ااریگەری جۆراوجۆری ە ە لە م ەنە جەااوازە اان‬ ‫ژیان م ڤ‪ ،‬ارلێکە ان نچنان کەسە ان‪ ،‬رەندروسو دەروون‬ ‫اتەن م ڤ ‪Dunham ,other‬‬ ‫و رەندروس ا ااو بیی و جەس ا ا ر‬ ‫‪:2008:248‬ی ە چنونە ا ااان لە ژیا ااان را ااا ا ادا چەنا اادۆن ڕ و‬ ‫ە‬ ‫دەایت ‪ ،‬وەڕ بە ەیک دن پەۆنەندی نچنان کەسە ان و‬ ‫ئەرڕ‬ ‫با ا ا اایک دن رەندروس ا ا ااو و اردانەوە چۆنا ەریەە ان بە راۆ ەت‬ ‫مەعنیب‪ ،‬بەو ەیەەی ڕتگە و ا ا ااچنازی جۆراوجۆر ە ە کە راە‬ ‫دەرنان لە ڕتگە ەوە ەس ااتە ان ڕتک خات و دروسا ا ک دن‬ ‫وتن ا ااا ەج زەۆو و ەو ا اادان بۆ اە ا ا ا ا اای بە ەمنو ئ ا ااام ا ااان ە‬ ‫ڕتکخ اوە را اااە و کۆ ا ااان لە ا اااری بەرتنەب دنا اادا‪ ،‬ەچیێس ا ا ا ا ااو بە‬ ‫ی‬ ‫ی‬ ‫ا ا اااوئا ا ااا ەن ا ا ا و کا ا ااۆرەن ا ا ا را ا ااا بەرتا ا اانەبەر ە ە لە کا ا ااۆی‬ ‫اااروبااارە ااانەاادا و بۆ وەدەۆ چنااان ئەمە ەچیێس ا ا ا ا ا ا بە زەۆنێ‬ ‫رتکخ او و ئە نەن ە ە‪ ،‬بە جۆرتا ا ااک بتناننتا ا ا ئا ا اااما ا ااان ە‬ ‫ە‬ ‫ەدێبی و ئەوەی ەچیێس ا ا ا ا ا ا ب ا ئەن ااا‬ ‫د اااریک اوە ااان ااار ب‬ ‫ی‬ ‫بدرت کە ەمنو ئەمانە ڕون و ئا ک ا ە ا ن کۆنی دن‬ ‫ە چانون دەردەخەن‪ ،‬ەروەڕ چۆن ە چانون بە ا ا ا ا ااێ‬ ‫و ا ا ا ا ا اااریا ا ا ا ا ااگەری خا ا ا ا ا ااۆی‬ ‫دانەب ا ا ا ا ا ازاوی م ا ا ا ا ا ا ە و ڕ‬ ‫ە ە ‪Purnamaningsih,2017:53‬ی بە ا ااچنە ە اریگەری‬ ‫ەسا ا ا ا ا و س ا ا ا ااۆزە نەرتنپەە ان دەردەکەوت ئەاەر لە ڕتگەی‬ ‫ارییەە ئەرتنپەە ااانەوە بە ب ا ی‬ ‫س ا ا ا ا اای ی‬ ‫ااش ڕت ااک نەخنتن‪ ،‬کە دەبیتە‬ ‫ۆی جێگیبنون لە بەرام ەر رەوەدد ارە اندا و بەدەسا اات چنان‬ ‫سەرکەورنە پەروەردەۆەە ان)‪ Fredrickson,2003 :2015‬ی‪.‬‬ ‫ی‬ ‫ریتیین ا وە ولێكۆ پن ا وە جەاااوازەكااان ج ا ختەااان ل ا س ا ا ا ا ا ا ر ا ن‬ ‫اەن د ر وە ل دوو م ن س ا ا ااەرەکەەوە‪ ،‬ەکە‬ ‫انن ان ە ال ا ا ا ر‬ ‫ە‬ ‫اەن‬ ‫ب رمان ر‪ :‬ئ م‬ ‫ب ریامان دەربارەی انن ان ە ال ا ا ا ا ا ر‬ ‫دە خ ااارا ا ن اااو دەروون ر ااا ا ا وە لا ا ئەن ااار اە ااان ما ا عنا ا وی‬ ‫ب ا رزەوە و ەری دن ەچییسا ا ا ا ا ا ە ا كااان ا وە دروس ا ا ا ا ا ا دەب ا و لە‬ ‫دواۆاەا ا اادا س ا ا ا ا ااەردەکا ال ا ا ا ا ا ا ا ا ا بااۆ اچاای ە‬ ‫ج ودا اچانا ا ااان وچا ا ااام‬ ‫سامەە‪35:2020،‬ی‪ .‬و دووە ب ر م چنان‪ :‬ركارێك انن ان‬ ‫اەن زیادی د‪ ،‬ئ وكات ەو و کۆ ا اایک ئەن ا دەدرت‬ ‫ە ال ا ا ر‬ ‫بۆ دەستکەوت و بەر ەم چنان زیار ‪ .‬منش‪65:2010 ،‬ی‪.‬‬ ‫ئا ا ا ا ا ااک ا ە کە انن ان وارە ڕازیبنون لەخند و ەبنون ئارار‬ ‫دەروون و د نە ااابنون‪ .‬ەسا ا ا ا ا اتک دن بە رنان ااای خۆانن ااان اادن‬ ‫لەاە ژینگە و ا ا ااارلێ ک دن لەاە کەسا ا ا ا ا ا ا ااان د کەدا و ئەو‬ ‫ڕەوا ەی کە کۆمە گااا دا اادەن ا ‪ ،‬بە لەبەرچاااوا رو ژینگە وەڕ‬ ‫ئام ازتک بۆ ە س ا ا ا ا ااەی ەریک دن ەێداویسا ا ا ا ا ا پەە ان راە ارێک‬ ‫اەن‬ ‫پا ا ااا نەرە ا ا ااانەا ا ااان دەوروژت ا ا ا ‪ .‬ەروە ا ا ااا انن ا ا ااان ە ال ا ا ا ا ا ر‬ ‫ا نگەەکەی لە دوو م ەن دەردەکەوتا ا ا ەکەمەا ا ااان با ا ااا تەر‬ ‫ۆ ا اااری کە ا نگپەەج ز ری ە ە لە انن ا ااان ە ال ا ا ا ا ااەۆەا اادا‪،‬‬ ‫چننکە ئەل ام ەخالە لە را دا بە مۆڕا ێ بەرز بۆ بەدەست چنان‬ ‫ان دەس ااتکەوت ە‬ ‫وریک دن ەێداویسا ا پەە ان‪ ،‬ئەمە پا نەرتکە‬ ‫بۆ دەس ە الخەری و دا چنان ک دن‪ .‬و دووەمەان دەرئەن ا ‪ :‬را‬ ‫ە الا ا ا ا ا اەکە انن اااوی زی ااار ب ا ‪ ،‬زی ااار دەبیتە ۆی زی ااادبنون‬ ‫بەر ەما ا ا ا ا ا ا اچا ا ا ان ا ا ااان‪ ،‬لە ڕووی چ ا ا ااۆن ا ا ااا ەن و لە ڕووی‬ ‫چەن اادا ەریەەوە‪ ،‬ەروە ااا پ ااا ا ا ا ا ااەکەورک دن ەو و ااات و‬ ‫رێچنون‪ .‬الزبە اادی‪320:2018،‬ی کەوارە بە ەەو ئە دەربنین ااانە‬ ‫ی‬ ‫الەن بۆ را ێک کە بەرتنەبەری باخچەی‬ ‫ب ا ن انن ان ە ر‬ ‫ا‬ ‫منداڵنە ڕوون و ئا ااک ا ە‪ .‬ەروەڕ چۆن بەرتنەبەرا ەرەە ان‬ ‫ا‬ ‫پەروەردەی ە یک ینرااابخااانە باااخچەی منااداڵنی لە و ا ا ا ا ااو‬ ‫یۆنا ااام رەمەن منا اادا ەوە س ا ا ا ا ااەرچا اااوە دەانتا ا ‪ .‬ئە یۆنا ااا ە‬ ‫ەکێکە لە ەرە ا نگ و مەر سەداررنین سەردەر ژیان م ڤ‪،‬‬ ‫بایە ەچییس ا ا ا ااو بە چاودێ ی راۆ ەت ە ە بەلەبەرچاوا رو ئەو‬ ‫بااارود خەی ەێاادا ریدەر دەب ا ‪ ،‬ەروە ااا جەاااوازی رااا ااا ەن لە‬ ‫ی‬ ‫ا‬ ‫نچنان من ا ااداڵن ا اادا لەبەرچ ا اااو دەانتا ا ا ‪ ،‬ئە یۆن ا ااا ە ا ن‬ ‫راۆ ەن خۆی ە ە عدس‪ (366:2009.،‬بەرتنەبەری باخچەی‬ ‫ا‬ ‫ا‬ ‫اەڵن ااار ە‬ ‫ایی دە ااات لە‬ ‫من ااداڵن نیتنەرا ەن بەرزرنین دەس ا ا ا ا ا‬ ‫ا‬ ‫اوەن لە ناااو باااخچەی منااداڵناادا‪،‬‬ ‫چنارچچنەی ەێک ااارەی ڕتکخ ر‬ ‫بایە بەرپ ش ەکەمە لە ڕادەی بەدۆ چناان ئەو ئااماان اانەی کە‬ ‫ا‬ ‫ا‬ ‫سا ا ا ا ا اە اااس ا ا ا ا ااەن خیتن اادن ب اااڵ دا اادەن ا ا بۆ ب اااخچەی من ااداڵن‬ ‫ا‬ ‫ەروە ا بەرپ سە لە پەرەەێدان بەرنامە ان باخچەی منداڵن‪،‬‬ ‫ەروە ا بەرپ س ااە لە ئاس ااو ئەدای ئەندامان لە ەر ارێکدا کە‬ ‫ا‬ ‫ب اکاانتا ا ا ‪ ،‬بەرتاانەبەران با ا اااخااچەی م انا ا ااداڵن دەزاناان کە ئەوان‬ ‫بەرپ س اان لە پنندانان و دەر چنان و کۆنی دن ە س ااەکە بۆ‬ ‫ئەوەی بەرپ سا ا ا ا ا اەاااربن لە ئەن اااماادان پەروەردەک دن مناادا بە‬ ‫ئا ا اااما ا ااان ااک پەروەردەک ا دن اەا ا ااان بە ە ا ر ە‬ ‫او ب اانەما ا اااپەروەردەۆ ااەە ا ا ااان‬ ‫وەمە‪9:2021،‬ی‬ ‫راایت ا ەر وای دەب ای ان اێا ا ا کە ئە راایتااییاانەوە ە ا ا نااگە بااۆ زان اپااو‬ ‫ب ااارود ن ئەدای بەڕتنەبەرە ااان لە ااارە ااانە ااان و ەروە ااا بۆ‬ ‫ا‬ ‫بینپو وایەیع پەروەردە لە باخچەی منداڵن اااری ساالێمان و‬ ‫ەروە ا ا ااا لە نا اازیا ااکەوە بەرکەورا اان بە ە ا ا ال ا ا ا ا ااەی بەڕتا اانەبەری‬ ‫ا‬ ‫با ا اااخااچە ا ا ااان م انا ا ااداڵن و زان اپااو ا ا ن اگااییاان ئا ا اااس ا ا ا ا ا ااتەنا ا ااگ و‬ ‫ئاس ا ا ا ااتەنگە ان بەردە بەڕتنەبەر لە ان ئەن امدان ارە انەدا‪،‬‬ ‫بەراۆ ەت نەبنون ڕوون و ا ا ا ااەباە س ا ا ا ااەبارەت بەوەی کە لە‬ ‫سەردەمەدا اۆڕان اریەە پەروەردەۆەە ان ئەمز رن دەخنازرت ‪.‬‬ ‫ی‬ ‫لە ژت ر ا ا ا ا ا ان ا ا راان س ا ا ا ا ااەرچ ا اااوەو ریتیینەوە ا ااان ا اادا ریت ەر ا ن‬ ‫ا‬ ‫ئەن امدان ریتیینەوەکە لە خاڵنەدا دەخارە ڕوو‪-:‬‬ ‫لە ریتیینەوە ەدا ەچنەرتک کە ە گ ی راۆ ەرمەندی‬ ‫‪-1‬‬ ‫اەن‬ ‫س ا ا ااا کۆمەرنیە بۆ کۆنی دن ە چنون او انن ان ە ال ا ا ا ر‬ ‫دەخنتتە ڕوو بۆ بەرتنەبەران باخچەی منداڵن‪.‬‬ ‫ریتیینەوەکە ا نگە بۆ ریت ەران بناری پەروەردەی و‬ ‫‪-2‬‬ ‫دەروون لە دا ارنودا ریت ەران ئە بنارانە س ا ااندی لچنەردەا ن‬ ‫بۆ ئەن امدان ریتیینەوەی او چنە‪.‬‬ ‫‪154‬‬ ‫‪Journal of the University of Garmian 10 (1), 2023‬‬ ‫ا‬ ‫ە چنونەە ا ااان‪ ،‬و وەڵم ا اادانەوەی ە چنونە ا ااان بەرام ەر ‪،‬‬ ‫ەروە ا ااا کۆنی دن ە چنون دە گنتتەوە ۆ ا ا ا ا ا اەا اااری و‬ ‫رێگەاالا ااتو ە چنونە ان و یەبن ک دنەان‪ .‬و رنانای بایاردانە‬ ‫لە س ا ا ا ا ااەر پ ااا نەرە ااان‪ ،‬و رنان ااای بە ااار چن ااان س ا ا ا ا اای ی‬ ‫ارییە ااان‬ ‫ا‬ ‫کۆنی دن ە چنون بۆ چااا سا ا ا ا ا ااازی ک دن لە وەڵماادانەوە‬ ‫س ا ا ا ا ااۆزداریە ااان بۆ اە ا ا ا ا اای بە ئااامااان ە ااان راااە & ‪Gratz‬‬ ‫‪Romer, 2004, 45‬ی و پێناس ااەی رێ‪:‬اری‪:‬بنی ەە لەو نم ە ەی‬ ‫ا‬ ‫کە ئەندامان نمنونەی ریتیینەوە لە وەڵمدانەوە اندا لەس ا ا ا ا ااەر‬ ‫باازاە ا ا ااان ئا ا ااامازازی راایتااییاانەوەکە کە را ا اااۆا ەرە بە ەاچاانانەکا دن‬ ‫کۆنی دن ە چنون وەریدەانت ‪.‬‬ ‫ی‬ ‫گونجان پیشەن‬ ‫ریتیینەوە ەج ا نگە بۆ دەو ەمەندک دن و زیادک دن‬ ‫‪-3‬‬ ‫س ا ااەرچاوەی زانس ا ااو‪ ،‬وەڕ ریتیینەوەی ە ال ا اانو لە ریتیینەوەی‬ ‫ئە اد مەدا بە ارب ا چیی ‪.‬‬ ‫ر اایت اایی اانەوە ەج ا ا ن ااگە ب ااۆ م ەن پەۆ اانەنا ا ااد ا ا اادار لە‬ ‫‪-4‬‬ ‫وەزارەن پەروەردە‪ ،‬بە دانااان کۆمە ە ەچنەرتااک بۆ د اااری ک دن‬ ‫الەن بەرتنەبەران‬ ‫ئاسو کۆنی دن ە چنون و انن ان ە ر‬ ‫نا ا اااوەنا ا اادە پەروەردەۆ ااەە ا ا ااان و ەروە ا ا ااا ک ا دنەوەوی ک ااۆمە ە‬ ‫خنلێ ااک‪ ،‬بۆ زی اااد ک دن ئ اااس ا ا ا ا ااو ئ ااا ا راان بەرتنەبەران لە س ا ا ا ا ااەر‬ ‫الەن‪.‬‬ ‫چۆنەەن کۆنی دن ە چنون و انن ان ە ر‬ ‫ی‬ ‫ئامانجەکان توێژینەوەکە‪:‬‬ ‫ا‬ ‫ئە ریتیینەوە ە لە ەو بەدۆ چنان ئە ئامان انەدا ە‪:‬‬ ‫ئ ااام ااانک ەکە ‪ :‬زانپو ئ اااس ا ا ا ا ااو کۆنی دن ە چنون می‬ ‫ا‬ ‫بەرتنەبەران باخچەی منداڵن اری سلێمان‪.‬‬ ‫ئامانک دووە ‪ :‬زانپو جەاوازی ئاسو کۆنی دن ە چنون‬ ‫ا‬ ‫اداڵن ا ا ا ا ا اااری س ا ا ا ا االێمااان ە‬ ‫بەەو‬ ‫می بەرتنەبەران باااخچەی منا‬ ‫اۆڕاوی‪:‬‬ ‫ا‪ .‬ڕەاەز ن ەیو ە‬ ‫ری ‪.‬‬ ‫ب‪ .‬جۆری باخچە وکنر و ناوکنری‪.‬‬ ‫الەن می بەرتنەبەران‬ ‫ئامانک سیەە ا ‪ :‬زانپو ئاسو انن ان ە ر‬ ‫باخچەی منداڵن اری سلێمان‪.‬‬ ‫اەن می‬ ‫ئامانک چنارە ‪ :‬زانپو‬ ‫جەاوازی ئاس ا ا ااو انن ان ە ال ا ا ا ر‬ ‫ا‬ ‫بەەو ە‬ ‫منداڵن ا ا ا ا اااری س ا ا ا ا االێمان ە‬ ‫بەەو‬ ‫بەرتنەبەران باخچەی‬ ‫اۆڕاوی‪:‬‬ ‫ە‬ ‫ە‬ ‫ا‪ .‬ڕەاەز نیو ری ‪.‬‬ ‫ب‪ .‬جۆری باخچە وکنر و ناوکنری‪.‬‬ ‫ئااامااانک ەین ە ‪ :‬د زینەوەی پەۆنەناادی لە نچنان کۆنی دن‬ ‫اەن می بەرتنەبەران با ا اااخچەی‬ ‫ە اچنون وانن ا ا ااان ە ال ا ا ا ا ا ر‬ ‫منداڵن اری سلێمان‪.‬‬ ‫سنووری توێژینەوەکە‪:‬‬ ‫س ا ا ا ا ااننوری ب ا ااابەت‪ :‬کۆنی دن ە چنون و پەۆنەن ا اادی بە‬ ‫ا‬ ‫انن ااان ە ال ا ا ا ا ااەۆەەوە می بەرتنەبەران ب اااخچەی من ااداڵن لە‬ ‫اری سلێمانەدا‪.‬‬ ‫ا‬ ‫سننوری م رن‪ :‬بەرتنەبەران باخچەی منداڵن اری سلێمان‬ ‫لە ەر دوو ڕەاەزی ن ا ا ا ەایو ە‬ ‫ر‪ ،‬لە ەردوو بەرت ا ا اانەبەرا ەن‬ ‫ا‬ ‫ر ژ ەڵت و ر ژئاوا وکنر و ناوکنری‪.‬‬ ‫سننوری ات‪ :‬سا ا خیتندن )‪.(2021-2022‬‬ ‫ا‬ ‫س ا ا ا ااننوری ا ا ا اایتن‪ :‬باخچە ان منداڵن ا ا ا اااری س ا ا ا االێمان‪ ،‬لە‬ ‫ا‬ ‫سننری ەر دوو بەرتنەبەرا ەن پەروەردەی ر ژ ەڵت و ر ژئاوا‬ ‫وکنر و ناوکنری‪.‬‬ ‫ی‬ ‫پێناسەی چەمک و زاراوەکان توێژینەوەکە‬ ‫پێناس ا ا ا ااەی ندی و ال کر‪ :(2017،‬ب ادی دە ێ کە بنی ەە لە‬ ‫ە س ااە ەج داۆنامە و بەردەوا کە راە ەەو ە دەس ا ێ ‪ ،‬بە‬ ‫ی‬ ‫ئ ااام ااانک اە ا ا ا ا اای بە انن ااان و اااوئ ااا ەن لە اە ژینگەی‬ ‫ە الا ا ا ا ا اەو اوپ ال ا ا ا ا ااە اندا و پارتز اری ک دن ەلو ندی و ال ک ‪،‬‬ ‫‪15:2017‬ی و بنج‪2001 ،‬ی‪ :‬انن ا ااان را ااا ە لەاە دونەا ااای‬ ‫اردا‪ .‬انن ان راە لەاە ئەو ۆ ارە ژینگەۆەە جۆراوجۆرانەی‬ ‫کە لە ا ا ا ا اایتو ااارەکە اادا دەوری دەدەن‪ ،‬انن ااان لەاە ئەو‬ ‫اۆڕان اریانەی کە لە ۆ ارانەدا ڕوودەدەن بەدرت ران ماوە ەڕ‪،‬‬ ‫و انن اویەەکەی لەاە راۆ ەرمەندۆەە ان خایدا دەانتتەوە‪،‬‬ ‫خندی ااارەکە‪ ،‬انن ااانەکەی لەاە اۆڕان ب ااارود ن ب ااازاڕدا‬ ‫ر اااۆ ەت بە ااارەکە‪ ،‬انن اااوی لەاە رنان ااا ااان خۆی‪ ،‬لەاە‬ ‫مە لە ااان و لەاە میاجەکەی بنج‪74:2001،‬ی پێناااسا ا ا ا ا ااەی‬ ‫رێ‪:‬اااری‪ :‬بنی ەە لەو نم ە ەی کە ئەناادامااان نمنونەی ریتیینەوە‬ ‫ا‬ ‫لە وەڵمدانەوە اندا لەس ا ا ا ا ااەر بزاە ان ئامزازی ریتیینەوەکە کە‬ ‫الەن وەریدەانت ‪.‬‬ ‫راۆ ەرە بە ەچنانەک دن انن ان ە ر‬ ‫ا‬ ‫بەرێوەبەری باخچەی منداڵن‬ ‫پێناسەی محمد‪:)2000،‬‬ ‫ا‬ ‫بەرپ سێکە کە دەستەی بەرتنەبەری باخچەی منداڵن داۆناوە‬ ‫ا‬ ‫ەر ئەو دەستە ە دەسەڵرە ان د اری دەکەن و دەدرت‬ ‫ارەز ران ەب لە بنارە ان بەرتنەب دن‬ ‫ەەو‪ ،‬ەروە ا دەب‬ ‫پەروەردەن و‬ ‫سەرەران مندا ەدا لە ڕووی‬ ‫پەروەردەن و‬ ‫ر‬ ‫ر‬ ‫ر‬ ‫محمد‪90:2000،‬ی‪.‬‬ ‫زانس پەەوە ڕ نبی ب‬ ‫ا‬ ‫باخچەی منداڵن‬ ‫ا‬ ‫ی‬ ‫بۆکان ‪ :(2020‬باخچەی منداڵن دەز ا ەج پەروەردەی‬ ‫ا‬ ‫کۆمەڵ ەریەە ئامانک اە ە ەج رەواوو اوسەنگە لە ەمنو‬ ‫ا‬ ‫جەستەن و دەروون و کۆمەڵ ەن و‬ ‫م ەنە جۆراوجۆرە ان‬ ‫ر‬ ‫ەروە ا ئامانک بەرزک دنەوەی رناناو و بە ە جەاوازە انە لە‬ ‫رتگەی اری و چام ئازادانەوە‪ .‬بۆ ان‪56:2020،‬ی‬ ‫ی‬ ‫ی‬ ‫ر‬ ‫هەڵچوون‬ ‫ۆڵکردن‬ ‫کۆنت‬ ‫پێنا ا اااسا ا ا ا ا ااەی ‪ :)2004،Gratz & Romer‬بنی ەە لە رننا ا ااای‬ ‫ڕووبەڕوو بننەوەی ە چنونە ا ااان ‪ ،‬و کۆنی ک دن ەوا ە‬ ‫ی‬ ‫توێژینەوەکان پێشوو‬ ‫چوارچێوەی تیۆری توێژینەوەکە و‬ ‫‪155‬‬ ‫‪Journal of the University of Garmian 10 (1), 2023‬‬ ‫ی‬ ‫ی‬ ‫ر‬ ‫هەڵچوون‬ ‫ۆڵکردن‬ ‫کۆنت‬ ‫ە چنونە ان لە بارود خە جەاوازەكاندا بناسینەوە و دەریانبین‬ ‫ارنون ۆنی ك دن ە چنونە ان‬ ‫و ۆنی ەان بكەۆن‪ .‬لچ‬ ‫ر‬ ‫كاریگەری لەسەر م ەنە جۆربەجۆرەكان ژیان م ڤ ە ە و‬ ‫دەبیتە ۆی جێگیی وسەیامگیی و اە ە دن ەسا ەن‬ ‫ا‬ ‫م ڤ ‪ .)Li L,other:2015:51‬لە ە سەی کۆنی‬ ‫ە چنوندا ەمنو وارا مەعنیفەە ان وەڕ اداە‪ ،‬ە اری‬ ‫رەدەخنل ب ات و ئەاەری ە ە‬ ‫بابەت‪ ،‬و تد‪ ،‬دەرنان‬ ‫ا‬ ‫ە چنون خۆنەویس وکۆنی ک او ا ئا ا‬ ‫ە سەی کۆنی‬ ‫ان نائا ا ب ‪ .‬لەبەرئەوەی ە چنونە ان ە سەاەلێ چەند‬ ‫ا‬ ‫ە چنون‬ ‫م ەنن کە لە ەر ارێکدا ئا ار دەبن‪ .‬کۆنی‬ ‫دەبیتە ۆی ڕودان کۆمە ە اۆڕان اریەڕ لە داۆنامە‬ ‫ە چنون ان دەبیتە ۆی اۆران لە اردا‪ ،‬بز و ماوە و چا سازی‬ ‫ا‬ ‫وەڵمدانەوە لە بنارە ان رەبتاری‪،‬ئەزمنن ان ب سپۆلۆژی‬ ‫ا‬ ‫ە چنون لەوانە ە لە سەر بنەمای ئامانک‬ ‫دەبیتەوە‪ .‬کۆنی‬ ‫را ‪ ،‬رتکخسی لە ە چنونەکەدا ب ات‪ ،‬ازو رپو ە‬ ‫بان ان بە‬ ‫ا‬ ‫ە چنون لەوانە ە‬ ‫ئاسان ب رستتەوە‪ .‬ەروە ا کۆنی‬ ‫ا‬ ‫ئاسو راۆ ەرمەندی پەۆنەند دار لە س ستەر وەڵمدەرەوەی‬ ‫ە چنون کە دەبیتە ۆی ئا ار بنون ە چنون بگۆرت‬ ‫)‪(Thompson,2009:879‬‬ ‫چنون كان ئەزمنون ان د خێکە کە سەما ەج وی دان‬ ‫ە ەو لە بنچپنەدا دەروونیەە و لە رەبتارو ئەرکە‬ ‫بسپۆلۆجپەە اندا دەردەکەوت ‪ .‬وو ەی وی دان ئاماژە ە بۆ‬ ‫ا‬ ‫چی و ی‬ ‫م ەن ە ی‬ ‫خۆش و ئازارو ژان‪ ،‬بەڵ دەستەواژەی لە‬ ‫بنچپنەدا دەروونیەە وارا لە دەروونەوە سەرچاوە دەانت و‬ ‫م ەن ب سپۆلۆن ناگنتتەوە وەڕ ب سچوی‪ .‬ئاستەمە لە‬ ‫ە چنوندا اۆڕان اری لطفلە ڕەبتارو لە ئەرکە بسپۆلۆجپەە ان‬ ‫ەناسەدان و لێدان د و سنڕان خیتن و بەرزبنونەوەی‬ ‫پلەی اەرما‪ ..‬تدی جەابکنتنەوەو ەریەکە ان لە سننری‬ ‫خایاندا دابیتن چننکە جەستە وەڕ ەکە ەج سەربەخۆو‬ ‫ا‬ ‫االتگی وەڵ بە ە چنونە ان دەدارەوەو رەن ا لە ڕوی‬ ‫ی‬ ‫بۆکان‪)366:2020،‬‬ ‫رپۆریەوە ئەو جەا اریەە ئەن ا دەدرت‬ ‫بنچپنەن ژیان م ە‪،‬‬ ‫ە چنونە ان بە ێ ا نگ و‬ ‫ر‬ ‫بەجۆرتک زەومەرە ژیان ە‬ ‫بەن ئەوان خە ا بکە ‪.‬‬ ‫راۆ ەرمەندی و اۆڕان ە چنونە ان‪ ،‬چۆنەەن پەۆنەندی‬ ‫سۆزداری و رێگەاالی و لێکدانەوەی ەسو ئەوان ر ڕ ێ‬ ‫ا نگەان ە ە لە اە ەک دن و ڕتکخستو کەسا ەن‪،‬‬ ‫ا‬ ‫پەرەسەندن ئەخنە پەۆنەندۆەە کۆمەڵ ەریەە ان‪،‬‬ ‫دروس بنون نناس و چەم خند لطف‬ ‫ابادی‪)651:2001‬‬ ‫ی‬ ‫پرۆسەی ر‬ ‫ۆڵکردن هەڵچوون‬ ‫کۆنت‬ ‫کۆنی دن ە چنون ئاماژە ە بۆ ئەو سی ی‬ ‫ارییانەی کە‬ ‫بە اردە چیتن بۆکەمک دنەوە‪ ،‬زیادک دن ان پاراستو ئەزمنونە‬ ‫ی‬ ‫ڕەج سی ی‬ ‫سۆزداریەە ان‪.‬‬ ‫ارییەە ان کۆنی ل ک دن‬ ‫ا‬ ‫ە چنونە ان اۆڕان سۆزداری و مەعنیب و کۆمەڵ ەریەە‪.‬‬ ‫ک دەوەی خ اپ لە سی ی‬ ‫اریانەدا دەرنان ڕ ێ ە ن ەرەوە‬ ‫ە‬ ‫بگیت لە دروستک دن و پاراستو رێکچنون دەروون‪ .‬لە‬ ‫وایە دا چەم کۆنی دن ە چنون ز ر ب اوان و االتگیە‬ ‫و کۆمە ێک ە سەی بییۆلۆژی و ڕەبتار و دەروون ۆ ەار و‬ ‫نائا ا لەخۆدەانت ‪ )191:2002،Gross‬مۆدێیل ە سەی‬ ‫کۆنی دن ە چنون رتگە ەج نیتەە بۆ رێگەاالی لە‬ ‫ا‬ ‫سی ی‬ ‫ارییە ان رتکخستو ە چنون‪ ،‬لە ساڵن ڕاب دوو لە‬ ‫لەم ەن زانا ا س ناسیندرا‪ ،‬کە لە نمنونە ەدا چەند‬ ‫ە ە‪،‬ئەوان یک‪:‬‬ ‫بنون‬ ‫سەرەج‬ ‫انیمانە ەج‬ ‫‪)551:2002،Gross‬‬ ‫ل ڕاس ەدا پچ اودەنگە ك نیە ل نچنان زانا اندا دەربارەی‬ ‫ا‬ ‫ی‪ ،‬ب ڵ ب چنە ی‬ ‫چنون وناوەڕ‬ ‫االتنون‬ ‫ەێگ‬ ‫ر‬ ‫االو دەرنانی ل س ر ۆم ێك ەێك ار ڕتك ك وین ئ ا ر‬ ‫االتنون چنون دێ‬ ‫ابنون وە ئ وكات ب وارای ەێگ‬ ‫ر‬ ‫‪:‬‬ ‫چنونو ش ەێگ االتنو‬ ‫بنیتی ل ‪ -:‬ەکە ‪ :‬انن اوی‬ ‫ا‬ ‫ەچییست وەڵمدان وەی ب بۆ ئ و بارود خان ی دێن‬ ‫ە یش ب چنە ج انن او وارا ن زیادەڕەوی بكات ن‬ ‫وارە‬ ‫چنون كان‪،‬‬ ‫ل‬ ‫من نرر یک بنیتنێ‬ ‫ا‬ ‫بارود خ ۆم ڵ رە كان‬ ‫چنون كان ەچییست ل ا‬ ‫ی بارەی وروژتن رەكان ئ‬ ‫بین وە‪ ،‬و انن اوبن ل ا‬ ‫چنون‪ :‬رناناكان راك‬ ‫چنون ‪ .‬دووە ‪ :‬کۆنی دن‬ ‫چنون كان وبایاردان‬ ‫دەردەخات ل س ر ڕتكخستو‬ ‫ل س ریان‪ ،‬وە رنانای م یک ری ك ل ئاوار كان ان‬ ‫ل ئ ستادا‬ ‫دواخستو ەری دن ەچییس ە كان دەردە وت‬ ‫دەورنت بایاردان ل‬ ‫س ەان ەێدەكات‪ ،‬وە ەلیەدا‬ ‫کۆنی دندا التحكم ە الض طی ب وارای بایاردان ل س ر‬ ‫ی ان‬ ‫ئ ندازەی چنون ونزر چنون ل ڕووی رنند‬ ‫سننرە ی سمور‪ .)73 :2012 ،‬مەبەس لە ۆنی ك دن‬ ‫و سۆز و ە چنونە ان ئەوە ە ە بتنانی‬ ‫ەس‬ ‫آ‪.‬بابەن کۆنی دن ە چنونە ان باسێ زیار ە لە زا بنون‬ ‫بەسەر ە چنونە نەرتنپەە ان ان رەن ا اۆڕیو ئەزمنونە‬ ‫سۆزداریەە ان‪ ،‬بۆ نمنونە با یک دن باری دەروون خ اپ‬ ‫ب‪ .‬کۆنی دن ە چنونە ان ئاماژە ە بۆ ئەو ە سانەی کە‬ ‫بە ایانەوە د اری دەکنت م ە ان ا ەس و سۆزیان لە‬ ‫‪156‬‬ ‫‪Journal of the University of Garmian 10 (1), 2023‬‬ ‫ارێک دەربین و چۆن ئە‬ ‫دەریان بین ‪.‬‬ ‫‪ ،‬ەروە ا بەڕتنەبەری ینرابخانە اردە ات بۆ دابینک دن‬ ‫ەمنو ەێداویسو و کەرەستە ان بەمەبەسو ئاسان اری لە‬ ‫پننە ان وانەورنەوە‪ ،‬پاراستو ڕتکیپێ ەۆل بۆ جچ ەجێک دن‬ ‫ە‬ ‫بیک دنێ ارا و چامە‪.‬سیەە ‪ :‬با یک دن ئەدای مامۆستا ان‬ ‫ا‬ ‫و اە ەەێدان رنانا انەان‪ :‬ا نگەدان ار ە‬ ‫ایی باخچەی منداڵن‬ ‫الەن مامۆستا و بەرزک دنەوەی ئاستەکەی‬ ‫بۆ اە ەەێدان ە ر‬ ‫ەکێکە لە ئەرکە سەرەکپەە ان بەڕتنەبەری ینرابخانە‪،‬‬ ‫لەڕتگەی کابننەوە و سەمپنار و خنلە انەوە دەرناننت ئەدای‬ ‫مامۆستا پەرەەی درت و رنانا انەان پەرەەی درت ‪ ،‬کە لە‬ ‫بەرام ەردا لە بەڕتنەب دن ەۆل و ڕتکخس ندا ڕەنگ دەدارەوە‪.‬‬ ‫چنارە ‪ :‬دەستەبەرک دن و بە ار چنان ئام از و کەرەستە‬ ‫ارمەرەدەرە ان‪:‬بە ار چنان ئام از و کەرەستە ارمەرەدەرە ان‬ ‫ەکێکە لەو ۆ ارە ا ن انەی کە اریگەری لەسەر بەڕتنەب دن‬ ‫ەۆلە ان ە ە‪ ،‬سەرن دان بەوەی کە مامۆستا ەندێک جار‬ ‫ڕووبەڕووی ک الەی چاوەڕواننەک او دەبیتەوە لە بەدەست چنان‬ ‫ە‬ ‫بەڕتنەبەر ار ب ات بۆ‬ ‫ئامیە ان‪ ،‬بایە دەب‬ ‫ئام از و‬ ‫ئامادەک دن ئام ازە ان بۆ مامۆستا لە ان خایدا و بە یەبارەی‬ ‫مامۆستا ان بە‬ ‫ەچییس بۆ ارە پەروەردەۆەە ان‪ ،‬و دەب‬ ‫راۆ ەت نیت ان ئاراستە بکنتن بۆ چۆنەەن بە ار چنان ئام ازە ان‬ ‫و سنودمەندبنون لچەان‪.‬ەین ە ‪ :‬سەردان ەۆل‪ ،‬ە سەن اندن‬ ‫ا‬ ‫مامۆستا و بەدواداچنون بۆ منداڵن لە ەۆلدا‪ :‬سەردان ەۆل ڕتگە‬ ‫بە بەڕتنەبەر دەدات وتنە ەج ڕوون ەریەڕ لە مامۆستا ان‬ ‫ا‬ ‫سەبارەت بە منداڵن باخچەکەی و ارلێک و‬ ‫ەب‬ ‫مامە ە ان لە ەۆلە انەان و دەربەت دەدات بە با یک دن‬ ‫ە یێستە ە‬ ‫بی اری و پەروەردەۆەە ان بە ارمەرەدان مامۆستا ان‬ ‫ە‬ ‫بۆ با ی ک دن م ەن جچ ەجێک دنەان لە ڕتکخستو بیبنون‬ ‫ا‬ ‫خیتند اران‪ .‬ە ە ‪ :‬ا نگەدان بە اروباری منداڵن‪ :‬بەڕتنەبەر‬ ‫ا‬ ‫بەرپ سەارتو لە ئەستۆدا ە بەرام ەر بە ئا اداربنون لە منداڵن‬ ‫و ئا اداربنونەان‪ ،‬ەروە ا ا نگیین ئەرکە ان بەڕتنەبەر لە‬ ‫ا‬ ‫ڕووەوە ەچییسو رێگەاال نە لە منداڵن لە ڕووی دەروون و‬ ‫ا‬ ‫کۆمەڵ ەریەەوە‪ ،‬و ارلێ ک دن لەاە ەاندا بە چنە ەڕ کە وا‬ ‫دە ات ەس ەان ەەو ب ات و ەەیانپو ەب و ار ب ات بۆ‬ ‫تنمان و ڕاوت اری بە چنە ەج االتگی‬ ‫ڕتکخستو بەرنامەی ڕ ر‬ ‫ا‬ ‫بۆ م ەنە دەروون و کۆمەڵ ەن و ئە اد ا و ئا اریەە ان ان‬ ‫بەدواداچنون ب ات بۆ ئەو ک الانەی کە دێینە سەر رت ای بە‬ ‫چامە و اریگەر‪ .‬وەورە ‪ :‬اە ەەێدان ە الەی‬ ‫چنە‬ ‫مامۆستا‪ :‬اە ەەێدان ە الەی مامۆستا و بەدواداچنون‬ ‫اە ەەێدان و بەرزک دنەوەی ئاسو کە دوو چنە لە خۆی‬ ‫دەانت ‪ :‬ا‪ .‬اە ەەێدان مامۆستا لە بابەن پسەۆڕیەەکە دا‪ :‬ان‬ ‫بەردەوامبنون لە بابەن پسەۆڕی ئە اد ا وەڕ مامۆستای‬ ‫پەروەردەن مامۆستا‪:‬‬ ‫بابەرێ د اریک او‪.‬ب‪ .‬اە ەەێدان م ەن‬ ‫ر‬ ‫ە چنونانە ئەزمنون بکەن و‬ ‫ج‪ .‬سی ی‬ ‫ارییەە ان کۆنی دن ە چنون ە بە چنەی‬ ‫ۆ ەاری و کۆنی او‪ ،‬و ە بە چنەی نا ۆ ەاری نائا ای‬ ‫و لەخۆوە بە اردە چیتن ‪.)551:2002،Gross‬‬ ‫ی‬ ‫گونجان پیشەن‬ ‫انن ان ئەو د خە ە کە ڕەچاوی ەێداویسو و داوا اریەە ان‬ ‫راە دە ات و بە ەەو ئەو ژینگە ەی کە ەریبنون رەواوەن خۆی‬ ‫قبقوب‪ ،‬زوقة‪ )67:2017،‬ەروە ا‬ ‫بەدەس دە چنێ‬ ‫ی‬ ‫اپلیی ەەو وا ە انن ان پەۆنەندۆەەج اوئا ەن م ە لە‬ ‫ا‬ ‫وکۆمەڵ ەرەەی ریەدا دەژت‬ ‫اە ئەو ژینگە بیی‬ ‫الداهری‪ .)15:2008،‬و زانا سیپەر چەم انن ان بە‬ ‫چنە ە کنرت دە ارەوە‪ :‬دەستکەورێ االو ان رێکە دن‬ ‫چەند جۆرتک لە م ەنە راۆ ەرە ان انن اندنە سازانەی‪ ،‬کە‬ ‫دوو چنە لە خۆی دەانت ‪ ،‬ئەوان یک بنیتی لە خۆانن اندن‬ ‫انن ان خندیی کە پەۆنەندی بە خۆڕتکخستو دەروونو‬ ‫ا‬ ‫را ەوە ە ە و ئەوی د کە انن ان کۆمەڵ ەرەە بە وارای‬ ‫پەۆنەندۆەە ان نچنان خند و ئەوان د کەوە دێ‬ ‫الخالدی‪. )99:2009،‬‬ ‫ی‬ ‫ی‬ ‫ی‬ ‫می‪:‬انتیم بەدەستهێنان گونجان پیشەن بۆ بەڕێوەبەری‬ ‫ا‬ ‫باخچەی منداڵن و دیسیپلینەکەی‬ ‫پەروەردەن جچ ەجێ ار لە‬ ‫بەڕتنەبەر وەڕ سەرپەر ەاری‬ ‫ر‬ ‫بەڕتنەب دندا اردە ات و ڕتکیپێ دەپارتزت بە ئەن امدان ئە‬ ‫ئەر انەی خنارەوە‪ :‬ەکە ‪ :‬پنندانان و ڕتکخستو اری‬ ‫ا‬ ‫باخچەی منداڵن‪ :‬اری ەۆل اریگەری لەسەر اری باخچەی‬ ‫ا‬ ‫و بە ەێچەوانە ەوە ەر راستە‪ ،‬پاراستو‬ ‫منداڵن دەب‬ ‫ا‬ ‫ڕتکیپێ االو باخچەی منداڵن اریگەری لەسەر ڕەون اری‬ ‫ەۆل و ئە پاراس نە ەچنسو بە ڕتکخسی و ‪ ،‬ەچییسو بە‬ ‫پنندانان ە ە ەروە ا‪ ،‬ەچییستە بەڕتنەبەر پەر یش ئەوە ب‬ ‫کە ە یک دەس ەێک دن دەوا زوو ئامادە ب بۆ ئەوەی بتنان‬ ‫پەروەردەن‬ ‫اری ڕ ژانەی دەوا ڕتک خات و ژینگە ەج‬ ‫ر‬ ‫رەندروس دروس ب ات ببێ بە نمننە ەڕ بۆ ارەکە را‬ ‫ئەوانپی چاوی ە‬ ‫ل بکەن‪ .‬دووە ‪ :‬ئاڕاستەک دن و بەدواداچنون‬ ‫ا‬ ‫بۆ اری باخچەی منداڵن‪ :‬بەز ری ئاراستەک دن دروس بە‬ ‫ڕ وێ م د ستانە ئەن ا دەدرت کە کە ێ دەروون و‬ ‫ا‬ ‫کۆمەڵ ەن ب خسینێ و ان ەمنوان دەدات بۆ ارک دن و‬ ‫د نەابنون لەوەی کە ارەکە چنە ەج رەواو ئەن ا دەدرت‬ ‫بەڕتنەبەر ەێداچنونەوە بە پنن وانە ان مامۆستا ان دە ات و‬ ‫وانە انەان ئامادە دە ات‪ ،‬د نەا دەبیتەوە لەوەی کە مامۆستا ان‬ ‫پنن با ەان بۆ ئەدارە ک دن ەۆل و پاراستو ڕتکیپێکەەکەی‬ ‫‪157‬‬ ‫‪Journal of the University of Garmian 10 (1), 2023‬‬ ‫بەو ارو چام ەانە دەورنت کە ەچییسی بۆ بەرتنەبەرە ان لە‬ ‫بنارە جەاجەا ان ارک دندا‪ ،‬ارەزای و زانەاری ەچییس ەان بۆ‬ ‫دابی ب ات بۆئەوەی ئاسو رنانا انەان و ئەدا ان بەرزب ارەوە بۆ‬ ‫بەدۆ چنان ئامان ە ان دەز ا ە‪ .‬ا نگیین ئەو بنارانە وەڕ‪:‬‬ ‫ەکە ‪ :‬بناری پنندانان‪ :‬پنن دانان ەکێکە لە ڕەاەزە‬ ‫ا نگە ان ە سەی ار ە‬ ‫ایی ە یک ە سە ان ر دەکەوت ‪،‬‬ ‫چننکە سەرکەورو ەر ارتک بە چنە ەج سەرەج لەسەر‬ ‫پنن با دارت راوە‪ ،‬پنن دانان یک بەمانای دانان ئامانە و‬ ‫سەاسەن نەخالەک الان و دانان مە انزر انن او و بایان و‬ ‫چۆنەەن ان ەچییس بۆ جچ ەجەک دنەان‪ .‬وەڕ سەرکەورو‬ ‫پننەکە لە د اریک دن جۆروچۆنەەن بەر ەمە اندا‪ .‬ڕادەی‬ ‫اوئا ەنگەان لەاە ەێداویس ەە ان کۆمە گە بەژداری دە ات‬ ‫ا‬ ‫لە ڕەخساندن بابەت و بارود ن زیار لە وەڵر ڕتکخ اوەکە‬ ‫بۆ ەێداویسو ژینگە و رنانای بەدۆ چنان ئامان ە دان اوە ان‬ ‫وەڕ ەچییس ‪.‬دووە ‪ :‬بناری ڕتکخسی‪ :‬ڕتکخسی ەکێکە لە‬ ‫رنخمە بنەڕەرەە ان ە سەی ار ە‬ ‫ایی‪ ،‬ئەوییک ەێک ارەی‬ ‫ڕتکخ اوەی ئەداریە ان ئەو چنارچچنە ە ە کە ەو ە ان رێدا بۆ‬ ‫بەدۆ چنان ئامان ە دان اوە انە‪ ،‬ەروەڕ دابە اریە ان اری‬ ‫ا‬ ‫ئەداری ڕوون دە ارەوە وئاماژە بەو دەسەڵرانە دە ات کە ەیەان‬ ‫دراوە ‪.‬ز رجارییک دەز ای ار ە‬ ‫ایی لە ەر دەز ا ەڕ لە ە‬ ‫ش‬ ‫ا‬ ‫ئاسو ار ە‬ ‫ئاسو ار ە‬ ‫ایی باڵ‪ ،‬ناوەند‪ ،‬خناروو‬ ‫ایی ەێک دێ‬ ‫ان جچ ەجێ اری ەریەڕ لەو ئاستانە ئەرڕ و بەرپ سەارتو‬ ‫خۆی ە ە کە جەاوازی ە ە لە ئاستە ان ر وسن‪،‬‬ ‫تنمان‪ :‬ئاڕاستەکە پەۆنەندی بە‬ ‫‪53:2015‬ی سچ ە ‪ :‬بناری ر ر‬ ‫رتنمان وئاڕاستەک دن ژتنبەژت ە ان ە یک و لە ان‬ ‫ر‬ ‫جچ ەجێک دن ارە انەدا ە ە لەڕووی ەێدان رتنمای ەچییس‬ ‫بۆ جچ ەجێک دن و چۆنەەن مامە ەک دن لەاە ئەوزەومەن و‬ ‫ک الانەی کە ڕووبەڕوویان دەبیتەوە‪ ،‬چننکە ەرکەس‬ ‫اردە ات لە ەر دەز او ڕووبەرت ژیاندا ەچییس ەان بە کەسێکە‬ ‫بۆ ئاڕاستەک دن و رتنماۆەک دنەان‪.‬چنارە ‪ :‬بناری کۆنی ‪:‬‬ ‫ە‬ ‫بەەو‬ ‫ارەکە‬ ‫کۆنی دن زامنک دنە بۆئەوەی‬ ‫ە‬ ‫و د نەابی لەوەی‬ ‫ەن بکنت‬ ‫نەخالەک الک اوەکە جچ‬ ‫کە ارمەندان ار بە اسا ان ارو ڕەبتارە و س سیتەر‬ ‫رتکخ اوەکە پەۆنەسی ەکێک لە ارە بنەڕەرەە ان کۆنی‬ ‫ەچنانەک دن ئاسو ئەدا و ڕاستک دنەوەی ئەو مدانانە ە کە لە‬ ‫ان ارک دندا ڕوودەدەن‪ .‬ەین ە ‪ :‬بناری رەنسپق‪ :‬ەکێکە لە‬ ‫ئامانک بە ەکەو ئەدارەکە‪ .‬رەنسەب اریگەر لە نچنان‬ ‫چام ەە ان دامەزراوەکەدا لە ئەن ار ئەدای اریگەرە لە‬ ‫ەمنو ارو چام ەە اندا وسن‪56:2015،‬ی ە ە ‪ :‬بناری‬ ‫ە سەن اندن‪ :‬ە سەن اندن ئەوە دەردەخات کە ئا ا ئەدارەی‬ ‫باخچە سەرکەورنو بنوە لە بەدۆ چنان ئامان ە پەرووەردەۆەە‬ ‫پەۆزەوک دن کۆمە ێک چناز بۆ ڕا چنان مامۆستا لە م ەنە‬ ‫جۆراوجۆرە ان پەروەردەدا‪ ،‬وەڕ چنازە ان وانەورنەوە‪،‬‬ ‫بە ار چنان ەندێک ئام از و پەرەەێدان رنانا ان لە بناری‬ ‫ە سەن اندن و چام پەە ان ەۆل ئازادنە‪ ،‬د نەا بنونەوە لە‬ ‫بنون رنانای ڕتکخستو رۆماری نم ە ان و رنانای ەک دنەوە‬ ‫و ڕەخنەا رن لە مەن ەج و کتچو خیتندن و دەربایو بۆچنون‬ ‫لەسەر خا ە بە ەی و موازە ان رنانا ان و پەرەەێدانەان بۆ‬ ‫مامە ەک دن لەاە ەمنو کەسێک کە بە دارە لە ارە انەدا‪،‬‬ ‫وەڕ خیتند ار و ار ە‬ ‫ای و دا ک و باوڕ و کەسان ر ‪.‬‬ ‫ال جیم‪)220-21:2000،‬‬ ‫ی‬ ‫گونجان پیشەن‪:‬‬ ‫پەیوەندی بەرێوەبەر بە‬ ‫ا‬ ‫ڕ بەڕتنەبەر ەکێکە لە ئەرکە ەرە رەوەدا و ڕازیکەرە ان‬ ‫اریگەری لەسەر‬ ‫جپ ان ار‪ ،‬چننکە بەڕتنەبەر دەرنان‬ ‫کەسان ر ب و ببچتە ما ەی ڕەزامەندی لە ار‪ ،‬لە بەرام ەردا‬ ‫دەرنان ببچتە ما ەی ەبیاری و نائنمێدی لە یتو اردا ئەاەر‬ ‫ب نەرنان مامە ە لەاە ک الە ان ئەو را انەدا ب ات کە بە‬ ‫اوەن د ‪ ،‬داوا‬ ‫ژی ی لەاە ەدا اردەکەن‪ ،‬ی ئ ستدمل دن و ک ر‬ ‫دە ات کە بەڕتنەبەر ا ن بە ەسو ئەو را انە بدات کە‬ ‫سەرک دا ەریەان دەکەن و ئەو بارود خانە با ی بکەن کە ارەکە‬ ‫ا‬ ‫لەژت دا بە ئەن ا دە ات و بەرزک دنەوەی‪ ...‬مۆڕا ئەو را انەی‬ ‫کە لەاە ەدا اردەکەن‪ ،‬و وەزیان لە ڕازیک دنەان ب لە یتو‬ ‫ی‬ ‫دەدات بە ارەکە‪.‬‬ ‫ەمان ا ن‬ ‫ارەکەدا‪ ،‬و ئەمە‬ ‫عداد‪)91:2017،‬‬ ‫الەن‪ ،‬چننکە‬ ‫ە ش سەرک دا ەن ز ر ا نگە بۆ انن ان ە ر‬ ‫ژینگەی ارک دن انن او لەم ەن بەڕتنەبەرەوە دابی دەکنت ‪،‬‬ ‫وبەرتنەبەر ەر چنازتک ان رت ا ەج بەرتنەبەردن کە لەاە‬ ‫ئەو را انەی کە ەێکەوە ار دەکەن پەۆزەوی دە ات ئەوە‬ ‫اریگەری لەسەر ڕادەی بالارە ان دەب کە ڕووبەڕوویان‬ ‫دەبیتەوە ان ئاسو انن ان ە الەۆەان لەاە ارە انەاندا‪،‬‬ ‫ی‬ ‫بەرینەبەر بۆ کەسانەی می ار دەکەن ەسو ا ن‬ ‫دەب‬ ‫ی‬ ‫روخۆش و زەردەخەنە ان ەیب ەخال ‪ ،‬ڕوخساری ا ژ‬ ‫ەەدان ‪،‬‬ ‫و ەی ک دن‪ ،‬نەب و ار ک دن ەمنو ێک نەب بە ن‬ ‫ا‬ ‫کەنا انن اوی پەۆنەندی نچنان خۆی و ئەوانەان بۆ دابی‬ ‫ب ات‪ ،‬و لە مامە ە ان لەاە ەاندا بەدوای دادپەروەریدا‬ ‫دەاەڕت ‪ ،‬و ب ەیاری و خەمۆج لە کە ن ەوای ار دوور‬ ‫دەخارەوە عداد‪)91:2017،‬‬ ‫ی‬ ‫ی‬ ‫گەشەپێدان پیشەن الی بەرێوەبەر یان باخچەی‬ ‫بوارەکان‬ ‫‪158‬‬ ‫‪Journal of the University of Garmian 10 (1), 2023‬‬ ‫)‪(OBQ‬‬ ‫‪Obsessive‬‬ ‫وەسناسەە ان ‪belief‬‬ ‫‪ questionnaire‬و رتکخستو مەعنیفەە ارنۆ س و‬ ‫او اران ی بە ار چیاون‪ .‬دەرئەن امە ان بە چییە بنون‬ ‫کە پەۆنەند ەج بە ەی ە ە لە نچنان لۆمە ک دن خند لە‬ ‫اە باورە وەسناسەە ان و ەکێک بنو لە ا نگیین‬ ‫ۆ ارە ان بۆ ە البپنەک دن بیوباوەڕە وەسنەسەۆەە ان‪،‬‬ ‫لە نچنان دوو اۆڕاوە ە البپنەکەرەکەی ر دا‪ ،‬یبن دن‪،‬‬ ‫دووبارە ە سەن اندن و ڕا چنان ە البپنەکەررنین‬ ‫اۆڕاوە ان بنون کە پەۆنەندۆەان بە بیوباوەڕە‬ ‫وەسنەسەۆەە انەوە ەبنو‪.‬‬ ‫‪2016‬ی‪ :‬پالکنپو‬ ‫‪ .３‬ریتیینەوەی ع دلملیک وئەوانپی‬ ‫پەۆنەندی نچنان ژینگەی سۆزداری ە‬ ‫خیان و کۆنی دن‬ ‫ە چنون ە الان ئالنودەبنون‪ .‬ا نگیین ئامانک‬ ‫ریتیینەوە زانپو پەۆنەندی نچنان ژینگەی سۆزداری ە‬ ‫خیان‬ ‫وکۆنی دن ە چنون لەاە ە الان ئالنودەبنون‬ ‫ە‬ ‫التەن یەزای یل ە سەر بە‬ ‫)‪323‬ی کەش دان التنان ن‬ ‫ار چکەی ی وەی ەئیانە‪،‬کە بە نمننەی ریتیینەوەکە بە‬ ‫ە ییت درابنو‪ .‬ەچنەری‬ ‫ەڕەمەج سادە‬ ‫چنەی‬ ‫ئامادەک اویان بۆ ەریەکە لە‪ ،‬ژینگەی سۆزداری ە‬ ‫خیان‬ ‫نۆدر اە بنتد یو ەچنەری کۆنی دن ە چنون‬ ‫ە‬ ‫سەلڤی‪ ،‬ڕ ف‪،‬ئپنرسۆن‪ ،‬بارس وب و ک و ا اران یو‬ ‫منسەوی‪،‬‬ ‫ئالندەبنون‬ ‫ە الان‬ ‫ەچنەری‬ ‫دوستقنین‪،‬رو نفک و دزبنل ی‪ .‬ریت ەران ئەن امەکە ان‬ ‫ی‬ ‫بۆ دەرکەوت بە بنون پەۆنەندی ا ن ئاماری بە گەدار لە‬ ‫ە‬ ‫خیان لە اە وەزی‬ ‫نچنان ژینگەی سۆزداری‬ ‫ئالنودەبنون‪ .‬و ەروە ا بنون پەۆنەندی بە گەداری‬ ‫ئاماری لە نچنان کۆنی دن ە چنون و ە الان‬ ‫ئالنودەبنون بە جۆرتک ئە پەۆنەند ە بە گەدارە بۆ‬ ‫ەمنو بنارە ان ەچنەری کۆنی دن ە چنونەە بێ گە‬ ‫لە رنڕەبنونی‪ .‬ەروە ا ئەن امە ان ن الان دەدەن‬ ‫ژینگەی سۆزداری ە‬ ‫ە البپو ە الان‬ ‫خیان دەرنان‬ ‫ا‬ ‫ئالنودەبنون ب ات‪ ،‬بەڵ نارنان ە البپو کۆنی دن‬ ‫ە چنون ب ات‪.‬‬ ‫ی‬ ‫ی‬ ‫توێژینەوەکان تایبەت بە گونجان پیشەن‬ ‫بەرنامە بۆ دارت راوە ان ان ئەاەر کسو چناوە نەۆتنانپنە‬ ‫ا ی و‬ ‫ئەن ا و ئامان ە خناسیاوانە بەدۆب چنێ‬ ‫صیاف‪16،‬ی‬ ‫ی‬ ‫توێژینەوەکان پێشوو‬ ‫ی‬ ‫ی‬ ‫ی‬ ‫توێژینەوەکان تایبەت بە ر‬ ‫هەڵچوون‬ ‫ۆڵکردن‬ ‫کۆنت‬ ‫‪ .１‬ریتیینەوەی ومدەللا ‪2020‬ی‪-‬ئنردن‪ :‬کۆنی دن‬ ‫ە چنون می بەرتنەبەران ینرابخانە ئە لپەە ان لە‬ ‫عەممان و پەۆنەندی بەئاسو رەبتاری ار ە‬ ‫ایی نەخنازراو‬ ‫لە ڕوانگەی مامۆستا انەوە‪ .‬ا نگیین ئامانک ریتیینەوە‪،‬‬ ‫زانپو ئاسو جچ ەجێک دن کۆنی دن ە چنون‬ ‫بەرتنەبەری ینرابخانە ئە لپەە ان لە روانگەی‬ ‫مامۆستا انەوە‪ .‬نمننەی ریتیینەوە بە رت ای ەڕەمەج‬ ‫سادە ە ییت درابنو‪ ،‬ەێک اربنو لە ‪ (204‬مامۆستا لە ەر‬ ‫دوو رەاەزی ەنیو ە‬ ‫ر‪ ،‬لە ینرابخانە ئە لەە ان عمان‪/‬اردن‬ ‫ا‬ ‫بۆ سا خیتندن) ‪2019-2020‬ی‪ .‬ریت ەر بۆرر ڕاە ش‬ ‫بە ار چناوە‪ ،‬ودەرئەن ار ریتیینەوەکە بە چنە ە بنو‬ ‫کە ئاسو کۆنی دن ە چنون لە ینرابخانە‬ ‫ئە لپەە ان اری عەممان اەاالتە پلە ەج بەرز و‬ ‫بەاالو و لە ەمنو بنارە اندا‪،‬و ەروە ا نم ەی ئاسو‬ ‫رەبتاری ار ە‬ ‫ایی نەخنازراو بە نم ە ەج نز ات‪ ،‬و‬ ‫ئەن امە ان یک بەوە ات ‪،‬بە بنون پەۆنەند ەج نەرتو‬ ‫بەلگەداری ئاماری لە نچنان کۆنی دن ە چنون و‬ ‫رەبتاری ار ە‬ ‫ایی نەخنازراو‪.‬‬ ‫ە‬ ‫‪ .２‬ریتیینەوەی واودی و ئەوانپی ‪2013‬ی‪-‬رە ‪-‬ئیان‪:‬‬ ‫پالکنپو پەۆنەندی لە نچنان کۆنی دن ە چنون و‬ ‫باورە‬ ‫رتکخستو مەعنیب‪ -‬ە چنون لە اە‬ ‫کک یۆنام ناوەندی‬ ‫وەسناسەە ان لە می خیتند اران ر‬ ‫اری رە ‪ -‬ەئیان‪ .‬ا نگیین ئامانک ریتیینەوە زانپو‬ ‫پەۆنەندی لە نچنان کۆنی دن ە چنون و رتکخستو‬ ‫مەعنیب‪ -‬ە چنون لە اە باورە وەسناسەە ان لە می‬ ‫کک یۆنام ناوەندی اری رە ‪ -‬ەئیان‪.‬‬ ‫خیتند اران ر‬ ‫کک یۆنام‬ ‫نمننەی ریتیینەوەکە )‪302‬ی خیتند اری ر‬ ‫ناوەندی خیتندن ا ون مەە ان اری ڕە تە‪ ،‬کە رت ای‬ ‫ە ییاردن نمننەکە بە رتگەی ەڕەمەج ب ە یۆناغ ان‬ ‫النون بنو‪ .‬ئام ازی ریتیینەوە ە سەارنامەی‬ ‫ر‬ ‫کۆنی دن ە چنون راج و ن اریان ی و باوڕە‬ ‫‪ .１‬ریتیینەوەی ع نة و ابن طی ‪2020‬ی‪ :‬بیک دنەوەی ئەرتو و‬ ‫پەروەردەن لە‬ ‫الەن می رتنماۆە اران‬ ‫ر‬ ‫پەۆنەندی بە انن ان ە ر‬ ‫ینرابخانە ان سەر بە بەرتنەبەرا ەن پەروەردەی دەوروبەری‬ ‫یندس‪ .‬ا نگیین ئامانە زانپو بیک دنەوەی ئەرتو و‬ ‫پەروەردەن لە‬ ‫الەن می رتنماۆە اران‬ ‫ر‬ ‫پەۆنەندی بە انن ان ە ر‬ ‫ینرابخانە ان سەر بە بەرتنەبەرا ەن پەروەردەی دەوروبەری‬ ‫پەروەردەن لە ینرابخانە ان‬ ‫یندس‪ ،‬سەرجە رتنماۆە اران‬ ‫ر‬ ‫سەر بە بەرتنەبەرا ەن پەروەردەی دەوروبەری یندس کە‬ ‫ا‬ ‫ژمارە ان‪ 66‬رتنماۆە ارە لە ەردوو رەاەزەکە بۆ سا‬ ‫‪159‬‬ ‫‪Journal of the University of Garmian 10 (1), 2023‬‬ ‫خیتندن)‪ .(2019-2020‬ریت ەر ەردوو ەچنەری بیک دنەوەی‬ ‫پەروەردەن‬ ‫الەن بۆ سەرجە رتنماۆە اران‬ ‫ر‬ ‫ئەرتو ‪ ،‬انن ان ە ر‬ ‫بە ار چناوە‪ .‬ئە ریتیینەوە ە اە تە ئە ئەن امانە بنون‬ ‫پەۆنەندی راستەوانەی بە گەداری ئاماری لە نچنان‬ ‫الەن‪ ،‬ەروە ا لە ئەن امدا‬ ‫بیک دنەوەی ئەرتو و انن ان ە ر‬ ‫دەرکەورنە کە ئاسو بیک دنەوەی ئەرتو می رتنماۆە اران‬ ‫پەروەردەن لە ینرابخانە ان سەر بە بەرتنەبەرا ەن‬ ‫ر‬ ‫ا‬ ‫پەروەردەی دەوروبەری یندس لە ئاس ێ باڵدا بنوە‪ .‬و پچ‬ ‫جەاوازیەج بە گەداری ئاماری بنون نەبنوە لە نچنان ناوەندی‬ ‫ە‬ ‫ژمی رەن لە ئاسو بیک دنەوەی ئەرتو می رتنماۆە اران‬ ‫پەروەردەن لە ینرابخانە ان سەر بە بەرتنەبەرا ەن‬ ‫ر‬ ‫پەروەردەی دەوروبەری یندس بە ەەو اۆڕاوی رەاەز‪.‬‬ ‫الەن‬ ‫ەروە ا لە ئەن امدا دەرکەورنە کە ئاسو انن ان ە ر‬ ‫پەروەردەن لە ینرابخانە ان سەر بە‬ ‫می رتنماۆە اران‬ ‫ر‬ ‫بەرتنەبەرا ەن پەروەردەی دەوروبەریب یندس لە ئاس ێ‬ ‫ا‬ ‫باڵدا بنوە‪.‬‬ ‫‪ .２‬ریتیینەوەی ما و ونة )‪ :(2018‬خۆ ار ران و پەۆنەندی بە‬ ‫انن ان ە الەن می مامۆستا ان ە‬ ‫بیک دن یۆنام ناوەندی"‬ ‫ر‬ ‫ریتیینەوە ەج مە دانیەە لە خیتند ای ناوەندی ە ەد‬ ‫س دی صالح لە سەدی عام ‪-‬المسەلة‪ ،‬جزائ ‪.‬ئامانک‬ ‫ریتیینەوەکە زانپو ئاسو خۆ ار ران‪ ،‬ئاسو انن ان ە الەۆەە‬ ‫می مامۆستا ان ە‬ ‫بیک دن یۆنام ناوەندی‪ ،‬ەروە ا ئامانک‬ ‫د اریک دن و و پەۆنەندی لە نچنان نم ەی االو‪ ،‬و‬ ‫الەن‪ .‬نمننەی‬ ‫ڕە ەندە ان بۆ ەچنەری خۆ ار ران‪ ،‬و انن ان ە ر‬ ‫ریتیینەوەکە ەێک اربنو لە )‪30‬ی مامۆستا لە ینرابخانەی‬ ‫ناوەندی ە ەد سەعدی سا ح لە سەدی عام ‪ ،‬لە ماوەی‬ ‫ا‬ ‫سا خیتندن ‪ .(2017-(2018‬ریت ەر لە ریتیینەوەکە دا بۆ‬ ‫الەن ەچنەری‬ ‫ەردووڕ اۆڕاوی خۆ ار ران و انن ان ە ر‬ ‫کۆران ریتیینەوەکەدا ئەن امە ان اە نە‬ ‫ئامادەک د‪ .‬لە‬ ‫ر‬ ‫پالەن اەورە ە‪ ،‬و‬ ‫ئەوەی کە ئاسو خۆ ار ران و انن ان‬ ‫ر‬ ‫ەروە ا پەۆنەند ەج بە گەداری ئاماری اەورە بنون ە ە لە‬ ‫نچنان نم ەی االو و رە ەندە ان ەچنەری خۆ ار ران‪ ،‬و نم ەی‬ ‫الەن‪ ،‬و نم ەی االو‪ ،‬و ەچنەری‬ ‫االو‪ ،‬و ەچنەری انن ان ە ر‬ ‫الەن‪،‬ونم ەی االو‪ ،‬و بنون جەاوازی بە گەداری‬ ‫انن ان ە ر‬ ‫ئاماری لە نچنان ناوەندە ان نم ەی مامۆستا ان ە‬ ‫بیک دن‬ ‫یۆنام ناوەندی‪.‬‬ ‫‪ .３‬ریتیینەوەی ولەمە و امیة ‪ :(2020‬بالاری دەروون و‬ ‫الەن لەمی ڕا چنەران باخچەی‬ ‫پەۆنەندی بە انن ان ە ر‬ ‫ا‬ ‫منداڵن‪ ،‬ریتیینەوە ەج مە دانەە لە اری المسەلة ‪-‬‬ ‫جزائ ‪.‬ئامانک ریتیینەوەکە بنیو بنو لە زانپو ئاسو بالاری‬ ‫الەن لە می ڕا چنەران باخچەی‬ ‫دەروون و ئاسو انن ان ە ر‬ ‫ا‬ ‫منداڵن‪ ،‬نمنونەی ریتیینەوەکە ‪40‬ی ڕا چنەر بنون ‪ ،‬لە‬ ‫ا‬ ‫ماوەی سا خیتندن )‪2019-2020‬ی‪ .‬سەبارەت بە ئامزازی‬ ‫ریتیینەوە‪ ،‬ریت ەر ەستاوە بە بننەاد نان ەچنەر بۆ بالاری‬ ‫الەن ەچنەری ئامادەک اوی‬ ‫دەرون و ەروە ا بۆ انن ان ە ر‬ ‫ا مان محمند عبەدی یبە ار چناوە‪ .‬ئەن امە ان بە چییە‬ ‫بنون‪ ،‬بنون پەۆنەند ەج بە گەداری ئاماری لە نچنان بالاری‬ ‫الەن لە می ڕا چنەران باخچەی‬ ‫دەرون و انن ان ە ر‬ ‫ا‬ ‫منداڵن‪.‬‬ ‫‪ .４‬ریتیینەوەی خلن ‪2019‬ی‪ :‬بالاری دەروون و پەۆنەندی بە‬ ‫سەرەران‪ ،‬ریتیینەوە ەج‬ ‫الەن لەمی مامۆستا ان‬ ‫ر‬ ‫انن ان ە ر‬ ‫مە دانەە لە ەندێک لە ینرابخانە سەرەراۆەە ان اری‬ ‫رق ب‪-‬جزائ ‪.‬ئامانک ریتیینەوە دەس ن الانک دن ئاسو‬ ‫بالاری دەروون بنوە لە نچنان مامۆستا ان ینرابخانە‬ ‫الەن لە نچنان‬ ‫سەرەراۆەە ان‪ ،‬ەروە ا ئاسو انن اوی ە ر‬ ‫سەرەران لە ەندێک لە ینرابخانە‬ ‫مامۆستا ان خیتندن‬ ‫ر‬ ‫سەرەراۆەە ان رق ت‪ ،‬ەروە ا زانپو پەۆنەندۆەانە‪ ،‬نمننەی‬ ‫ریتیینەوەکە بنیو بنون لە ‪120‬ی مامۆستا‪ ،‬ریت ەر بۆ اۆڕاوی‬ ‫الەن‬ ‫بالاری دەرون ەچنەری بنەات ناو و بۆاۆڕاوی انن ان ە ر‬ ‫ەچنەری ئامادەک اوەی منری امەلەا ‪ 2017‬ی بە ار چنا‪.‬‬ ‫وئەن امە ان بە چنە ە بنون‪ :‬ئاسو بالاری دەروون می‬ ‫سەرەران بەرزە‪ ،‬ئاسو انن اوی‬ ‫مامۆستا ان خیتندن‬ ‫ر‬ ‫الەن لە نچنان مامۆستا ان خیتندن سەرەراۆەدا بەرزە‪.‬‬ ‫ە ر‬ ‫پەۆنەندن لە نچنان بالاری دەروون و‬ ‫ەروە ا بنون‬ ‫ر‬ ‫الەن لە نچنان مامۆستا ان ینرابخانە‬ ‫خۆانن اندن ە ر‬ ‫‪.‬‬ ‫جەاوازن ئاماری لە نچنان بالاری‬ ‫ە‬ ‫سەرەراۆەە اندا ە‬ ‫ر‬ ‫الەن لە نچنان مامۆستا ان ینرابخانە‬ ‫دەروون و انن اندن ە ر‬ ‫ە‬ ‫‪.‬‬ ‫سەرەراۆەە اندا بنون نیەە بە ەو اۆڕاوی ڕەاەزیەەوە جەاوازی‬ ‫ئاماری بنون نیەە لە نچنان بالاری دەروون و انن اندن‬ ‫سەرەران بە ەەو‬ ‫الەن لە نچنان مامۆستا ان خیتندن‬ ‫ر‬ ‫ە ر‬ ‫اۆڕاوی پلەو ئاس ‪.‬‬ ‫ی‬ ‫ی‬ ‫توێژینەوەکان پێشوو‬ ‫گفتوگۆکردن‬ ‫ا‬ ‫لە ڕووی ئامانک ریتیینەوەوە ز ربەی ریتیینەوە ان لە ەو‬ ‫زانپو ئاسو جچ ەجێک دن ەریەکە لە اۆراوە انن لە سەر‬ ‫کۆمە گای ریتیینەوە‪ ،‬ەروە ا زانپو پەۆنەندی نچنان‬ ‫اۆراوە ان‪.‬ئامانک ریتیینەوەی ریت ەران یک بە ەمان چنە‬ ‫بنی پەە لە زانپو ئاسو کۆنی دن ە چنون و پەۆنەندی بە‬ ‫ا‬ ‫انن ان ە الەۆەەوە لە می بەرتنەبەران باخچەی منداڵن‬ ‫اری سلێمانەدا‪ .‬سەرجە ریتیینەوە ان و او ات ریت ەران یک‬ ‫مپتۆدی وەسفپەان بە ار چناوە‪ .‬لە ڕووی سامدڵەوە ەری کە لە‬ ‫ریتیینەوە ان بە ەەو کۆمە گای ریتیینەوەکەو جۆری‬ ‫ریتیینەوەکە ە ی‬ ‫ەانیاردووە ەروەڕ لە سەرەوە ئاماژە دراوە بە‬ ‫ا‬ ‫رت ەکەی‪ ،‬و سامدڵ ریتیینەوەی ئێمە بنیتو بنو لە ەمنو‬ ‫کۆمە گای ریتیینەوەکە‪ ،‬وارە ‪78‬ی بەرتنەبەر‪ .‬سەرجە‬ ‫ریتیینەوە ان بۆ بەدۆ چنان ئامان ە انەان ئامزازی انن اویان‬ ‫ا‬ ‫ی‬ ‫اوبەش نچنان ەمنویان ئەوە ە ان‬ ‫بە ار چناوە و خا‬ ‫ەچنەریان بنەات ناوە ان یه ئامادەک اویان بە ار چناوە‪ ،‬او ات‬ ‫ریت ەرییک لە ریتیینەوەکە دا بۆ بەدۆ چنان ئامانک‬ ‫‪160‬‬ ‫‪Journal of the University of Garmian 10 (1), 2023‬‬ ‫جەا اری ‪ -‬اوکۆلكەی پەۆنەندی نم ەی ەر بزاە ەڕ بە کۆی‬ ‫استگۆن روا ەت – جێگیی ی بۆ بزاە ان‬ ‫االو نم ە ان‪ -‬ڕ‬ ‫ر‬ ‫استگۆن وجێگیی‬ ‫ەچنانەکە ئەن امدا‪ .‬ریت ەران دوای ئەوەی ڕ‬ ‫ر‬ ‫بۆ ەچنانەی کۆنی دن ە چنونی د اریک د‪ ،‬ەچنانەکەی بە‬ ‫کۆران ‪ (42‬بزاە ما ەوە‪ ،‬بە چنە ەج االو بزاە ان‬ ‫چنەی‬ ‫ر‬ ‫ا‬ ‫ڕوون بنون لەمی بەرتنەبەران باخچەی منداڵن و ان‬ ‫ا‬ ‫ەچییس بۆ وەڵمدانەوە ‪6‬ی خنلەڕ بنو‪ ،‬ەروە ا ب اردە ان‬ ‫ا‬ ‫وەڵمدانەوە لە ناوەڕ ج بزاە ان بە جۆری ەینە ب اردە‬ ‫دارت رابنو‪ ،‬وارە)‪5‬یب اردە لەبەرام ەر بزاە ان ە ە‪ ،‬کە بنیتی‬ ‫لە بەبەردەوا ‪،‬ز رجار‪ ،‬ەندێک ار‪،‬کەم ار‪ ،‬ەرایی وبە ای‬ ‫ب اردە ان بنی ەە لە ‪1،2،3،4،5‬یبە ەمان ڕیزبەندی بەرام ەر‬ ‫بزاەنەرتنپەە ان نم ە ان ەێچەوانە‪ ،‬دەبنەوە‪ ،‬وارە بەرزرنین‬ ‫نم ەی ەچنەرەکە ‪210‬ی نم ە ە و نزمیین ‪42‬ینم ە ە‪.‬‬ ‫الەن‪ :‬لەبەر نەبنون‬ ‫بۆرر ڕاوەرا ی بۆ ەچنانەی انن ان ە ر‬ ‫الەن‬ ‫ەچنانە ەج انن او بەزمان کنردی بۆ ەچنان انن ان ە ر‬ ‫ا‬ ‫می بەرتنەبەران باخچەی منداڵن‪،‬ریت ەران پەنا ان ب دە بەر‬ ‫الەنی بە زمان‬ ‫بە ار چنان ەچنانە ک دن اۆڕاوی انن ان ە ر‬ ‫ەستا بە [ دار نەوەی بزاە انی‬ ‫عەرەن بۆ ئە مەبەستە‬ ‫الەن‪-‬‬ ‫ەک دنەوەی ئاماری بۆ بزاە ان ەچنانەی انن ان ە ر‬ ‫ەیی جەا اری ‪ -‬اوکۆلكەی پەۆنەندی نم ەی ەر بزاە ەڕ‬ ‫استگۆن روا ەت – جێگیی یه‬ ‫بە کۆی االو نم ە ان‪ -‬ڕ‬ ‫ر‬ ‫استگۆن وجێگیی بۆ‬ ‫ریت ەران دوای ئەوەی ڕ‬ ‫ر‬ ‫ەچنانەی کۆنی دن ە چنونی د اریک د‪ ،‬ەچنانەکەی بە‬ ‫کۆران ‪ (48‬بزاە ما ەوە‪ ،‬بە چنە ەج االو بزاە ان‬ ‫چنەی‬ ‫ر‬ ‫ا‬ ‫ڕوون بنون لەمی بەرتنەبەران باخچەی منداڵن و ان‬ ‫ا‬ ‫ەچییس بۆ وەڵمدانەوە ‪7‬ی خنلەڕ بنو‪ ،‬ەروە ا ب اردە ان‬ ‫ا‬ ‫ە‬ ‫ش ب اردە‬ ‫وەڵمدانەوە لە ناوەڕ ج بزاە ان بە جۆری‬ ‫دارت رابنو‪ ،‬وارە)‪3‬ی ب اردە لەبەرام ەر بزاە ان ە ە‪ ،‬کە بنیتی‬ ‫لە ەم الە‪ ،‬ەندێک ار‪ ،‬ەرایی وبە ای ب اردە ان بنی ەە‬ ‫لە ‪1،2،3‬یبە ەمان ڕیزبەندی بەرام ەر بزاەنەرتنپەە ان‬ ‫نم ە ان ەێچەوانە‪ ،‬دەبنەوە‪ ،‬وارە بەرزرنین نم ەی ەچنەرەکە‬ ‫‪144‬ی نم ە ە و نزمیین ‪48‬ینم ە ە‪ .‬دوای د نەابنون ریت ەر لە‬ ‫ڕاسو و جێگیی ئامزازی ریتیینەوەکەی‪ ،‬ریت ەران لە ڕتکەون‬ ‫‪ 27-4-2022‬ی بە چنەی دەسو و ئەلەکی ن دەس پەان ک د‬ ‫بە جپ ەجێک دن ەچنانە ان ریتیینەوەکەی بەسەر کۆمە گەی‬ ‫ریتیینەوەکە دا‪ ،‬کە ەێک اربنو لە ‪78‬ی بەرتنەبەران باخچەی‬ ‫ا‬ ‫منداڵن اری سلێمان لە ەر دوو ڕەاەزی ەنیو ە‬ ‫ر‪ ،‬لە ەردوو‬ ‫ا‬ ‫بەرتنەبەرا ەن ر ژ ەڵت و ر ژئاوا وکنر و ناون ای‪،‬دوای‬ ‫ەێدان ڕوونک دنەوە لەم ەن ریت ەرانەوە دەربارەی ریتیینەوەکە‬ ‫ا‬ ‫داوا ان لە بەرتنەبەران ک د بە وردی وڕوون وەڵر اال‬ ‫بزاە ان بدەنەوە‪ ،‬دوار سەرجە بۆرر ڕاە سەە ان کۆک دەوە‪،‬‬ ‫ریتیینەوەکەی و اەاالی بە ئەن ا ‪ ،‬بۆڕر ڕاوەرا ی راۆ ەت بە‬ ‫الەن بە ار چناوە‪.‬‬ ‫کۆنی دن ە چنون و انن ان ە ر‬ ‫سەبارەت بە ئەن ار ریتیینەوە ان‪ ،‬ەریەکە ان ئەن امێک‬ ‫دەرچنو‪،‬بە جۆرتک لە ەمنویاندا ئاسو کۆنی دن‬ ‫الەن بە ەەو اۆڕاوە ان خایان بەرز بنو‬ ‫ە چنون وانن ان ە ر‬ ‫و لە ەمان اردا لە ز ربە اندا پەۆنەندی بە گەداری ئاماری‬ ‫ەبنو ان بە ئەرتو ان نەرتو ەروەڕ لە ی‬ ‫بەش سەرەوە بە‬ ‫وردی ئاماژەی ەێدراوە‬ ‫ی‬ ‫ڕێ‪:‬ارییەکان توێژینەوەکە‬ ‫میتۆدی توێژینەوە‪:‬ریتیینەوەکە ریتیینەوە ەج وەسبی‬ ‫ە‪،‬باسەکە ەو دەدات پەۆنەندی نچنان کۆنی لك دن‬ ‫ە چنون و انن ان ە الەۆەدا لە می بەرتنەبەران باخچەی‬ ‫ا‬ ‫منداڵن بد زتتەوە‪.‬‬ ‫کۆمەڵگەی توێژینەوە‪:‬کۆمە گەی ریتیینەوەکە ەێک اربنو‬ ‫ا‬ ‫لەسەرجە بەرتنەبەران باخچەی منداڵن لە سەرجە‬ ‫باخچە ان ون ا و ناون ای سەر بە ەردووبەرتنەبەرا ەن‬ ‫ا‬ ‫ا‬ ‫پەروەردەی ر ژئاوا و ر ژ ەڵت اری سلێمان‪ .‬بۆ سا‬ ‫خیتندن ‪2021-2022‬ی و ژمارە ان ‪78‬ی بەرتنەبەرە‪10 ،‬ی‬ ‫بەرتنەبەر لە ڕەاەزی ەنی و ‪68‬ی بەرتنەبەر لە ڕەاەزی ە‬ ‫ر‪،‬‬ ‫وەڕ لە خالتەی ‪1‬ی دا خ اوەرەڕوو‪.‬‬ ‫خشتەی ژمارە ‪)1‬کۆمەڵگەی توێژینەوە‬ ‫ی‬ ‫ازەکان توێژینەوە‬ ‫ئامر‬ ‫ژمارەی بەرتنەبەر‬ ‫ب ڕتنەب را ن‬ ‫باخچ ی‬ ‫وكنر‬ ‫بهرێوەبهرایه رن‬ ‫پهروەردەی‬ ‫ڕۆژئاوا‬ ‫‪37‬‬ ‫ڕەا ز‬ ‫ەنی‬ ‫ە‬ ‫ر‬ ‫‪6‬‬ ‫‪31‬‬ ‫باخچ ی‬ ‫ناوكنر‬ ‫‪21‬‬ ‫ڕەا ز‬ ‫ەنی‬ ‫ە‬ ‫ر‬ ‫‪0‬‬ ‫‪21‬‬ ‫بهرێوەبهرایه رن‬ ‫پهروەردەی‬ ‫ا‬ ‫ڕۆژههڵت‬ ‫‪14‬‬ ‫‪3‬‬ ‫‪11‬‬ ‫‪6‬‬ ‫‪1‬‬ ‫‪5‬‬ ‫ر‬ ‫گشت‬ ‫كۆی‬ ‫‪51‬‬ ‫‪9‬‬ ‫‪42‬‬ ‫‪27‬‬ ‫‪1‬‬ ‫‪26‬‬ ‫ۆی‬ ‫االو‬ ‫ژمارەی‬ ‫بەرتنەبەر‬ ‫‪78‬‬ ‫بۆڕر ڕاوەرا ی بۆ ەچنانەی کۆنی دن ە چنون‪ :‬ریت ەران‬ ‫پەنای ب دە بەر بە ار چنان ەچنانەی کۆنی ک دن ە چنون‬ ‫زبی ی یفی کە بە زمان کنردی ئامادەک ابنو‪ ،‬و ژمارەی بزاە ان‬ ‫‪42‬ی بزاە بنون‪ .‬پا ان بۆ ئەوەی ەچنانەکە بگنن ێ لە اە‬ ‫ەیی‬ ‫کۆمە گەی ریتیینەوەکە ئەوە ەک دنەوەی ئاماری‬ ‫‪161‬‬ ‫‪Journal of the University of Garmian 10 (1), 2023‬‬ ‫کۆران ەچ ات‪.‬‬ ‫ئەن امدانەکە لەبەرواری )‪ 26-5-2022‬ی‬ ‫ر‬ ‫ریت ەران ارە ئاماریەە انیەان لە ڕتگەی بە ار چنان ە ا ار‬ ‫ا‬ ‫‪SPSS‬ی ەا ەی ئاماری بۆ زانستە کۆمەڵ ەرەە ان ەیژن‬ ‫‪Cronbach’s Alpha‬ی‬ ‫‪25‬ی بۆ بە ار چنان ئە ئامزازانە‬ ‫ئەلفا ک ن اخ بۆ د زینەوەی جێگیی ەچنانە‪One sample ،‬‬ ‫‪T-test‬ی بۆ دەرخستو ئاسو کۆنی دن ە چنون و‬ ‫الەن بە چنە ەج االو‪Independent ،‬‬ ‫انن ان ە ر‬ ‫‪sample T-test‬ی بۆ مەبەسو زانپو جەاوازی بە گەداری‬ ‫الەن بە ەەو‬ ‫ئاماری کۆنی دن ە چنون و انن ان ە ر‬ ‫اۆڕاوی ڕەاەز و جۆری باخچە‪ (Pearson Correlation)،‬بۆ‬ ‫زانپو پەۆنەندی نچنان کۆنی دن ە چنون و انن ان‬ ‫الەن‪.‬‬ ‫ە ر‬ ‫‪78‬‬ ‫ەوارە ە‬ ‫بەەو ئەو ئەن امەی لە س ا ااەرەوە دەرچنوە دەریدەخات‬ ‫ا‬ ‫بەڕتنەبەران باخچەی منداڵن ا ا اااری س ا ا االێمان کۆنی دن‬ ‫ە چنونە ا ااان ە ە بە ئ ا اااسا ا ا ا ا ا ێ بەرز‪ ،‬ریت ەر ۆ ا اااری ئە‬ ‫اتنون‬ ‫ئەن امە دەاەڕتنچتەوە بۆ ئاس ا ا ااو ر ا ا اانبیی و رێگەاال ا ا ا ر‬ ‫بەرتنەبەران‪ ،‬ەروە ا ااا ئەو کۆمە ە خنل و دورار ا ااانەی کە لە‬ ‫م ەن وەزارەن پەروەردەوە دەکنتتەوە اااریگەری اەورەی ە ە‬ ‫لە س ا ا ا ا ااەر رااا بەرتنەبەر‪ ،‬بەو مااانااا ەی ڕەچاااوی ڕتکخس ا ا ا ا اای و‬ ‫کۆنی دن ەسا ا ا و ە چنونەان خایان دەکەن و دەزانن‬ ‫بە ەا ەاو ە اایێسا ا ا ا ا ااتە جاەا ا اااوازە ا ا ااان چااۆن رەباتا ا ااار بااکەن‪ ،‬و بە‬ ‫افتناایەج ئاارا و ڕتزدارانە لەاە کەسا ا ا ا ا ااان د کەدا و ڕەخنە‬ ‫ی‬ ‫یبن ک دن و لە ڕتگەی دا ااالۆج ب ااابەریەەوە ب اااس لە ە س ا ا ا ا ااە‬ ‫راۆ ەت و اال ا ا ا پەە ان بکەن و بۆچنونە انەان بەڕتزەوە دەربین‬ ‫و ااان لە بااۆچاانون و باایوڕای بەرام ا ەر ب اگ ا ن‪.‬ئەن ا ا ا ااار ئەو‬ ‫راایتااییاانەوە ە ەکا ا اادەااانتااتەوە لەاە راایتااییاانەوە و اما ا اادەللا و‬ ‫الرصاۆ ة‪2020 ،‬ی‪.‬‬ ‫ی‬ ‫ی‬ ‫ر‬ ‫ر‬ ‫ی‬ ‫کۆنتۆڵکردن هەڵچوون الی‬ ‫ئامانج دووەم‪ :‬ا‪ .‬زانیت ئاسات‬ ‫ی‬ ‫ڕ‬ ‫ا‬ ‫ی‬ ‫ی‬ ‫ی‬ ‫بەڕێوەبەران باخچەی منداڵن شاری سلێمان بەپت گۆراوی‬ ‫ڕەگەزی نت ویم)‪.‬‬ ‫ە‬ ‫بۆ اەاال ا ا ا ا اای بە ئ ا ااام ا ااان ە ریت ەر ن ا اااوەن ا اادی ژمی رەن و مدان‬ ‫ا‬ ‫ەچنەرن نم ە ا ااان بەرتنەبەران ب ا اااخچەی من ا ااداڵن نمنونەی‬ ‫ر‬ ‫را ا اایتا ا ااییا ا اانەوەکەی بە ەا ا ا ەاو اا ا ااۆڕاوی ڕەاەز لەس ا ا ا ا ااەر ە ا ا اچا ا اانەری‬ ‫کۆنی دن ە چنون دەر چنا‪ ،‬بە بەكار چنان رایەک دنەوەی‬ ‫ی‬ ‫ران بۆ دوو نمنونەی س ا ااەربەخۆ ‪independent sample T-‬‬ ‫)‪test‬ی بە پال بەسی بە بەرنامەی ئاماری ‪SPSS‬ی دەرکەوت‬ ‫ە ن ن ‪t‬ی ئەژمارک او ‪0.61‬ی نم ە ە بچنکیە لە ن ن ‪ t‬ی‬ ‫یران‬ ‫اتەن ‪1.69‬ی بە نم ەی ئاازادی ‪76‬ی‪ ،‬لە س ا ا ا ا ااەر ئااس ا ا ا ا ااو‬ ‫خال ا ا ا ا ا ر‬ ‫بە گەداری ‪0.05‬ی‪ ،‬وەڕ لە خالتەی ژمارە ‪8‬ی د ارە‪.‬‬ ‫خستنەڕووی ئەنجامەكان و شیكردنەوەیان ‪:‬‬ ‫ئە بە ا ا ا ااە راۆ ەرە بە ا ا ا ااەک دنەوەی دارا ان و خسا ا ا ا ا نەڕووی‬ ‫ئەن امە ان بە ایت ەی ڕیزبەندی ئامان ە ان ریتیینەوە کە لە‬ ‫ی‬ ‫بەش ەکەر ریتیینەوەکەدا د اااری ک اوە و ڕا ەک دنە ااان و لە‬ ‫کۆراۆەدا راسداردەو پالنەار خ اونەرەڕوو‪.‬‬ ‫ی‬ ‫ی‬ ‫انیت ئاسا ا ا ا ا ا رات ر‬ ‫ئامانج یەكەم ‪:‬ز ی‬ ‫کۆنتۆڵکردن هەڵچوون الی‬ ‫ی‬ ‫ڕ‬ ‫ا‬ ‫ی‬ ‫ی‬ ‫ی‬ ‫بەڕێوەبەران باخچەی منداڵن شاری سلێمان‪.‬‬ ‫دوای کۆک دنەوەی دار ااا ااان و بە ااار چن ااان بەرن ااامەی ‪SPSS‬ی‬ ‫ئ ا ااامزازی ئ ا ااام ا اااری ‪One Sample T-test‬ی بابەراورد ک دن‬ ‫ناااوەناادی ە‬ ‫ژمی رەن نمنونەی ریتیینەوەکە بەناااوەناادی انیمااانە رن‪.‬‬ ‫ە‬ ‫لە ئەن امدا دەرکەوت‪ ،‬کەناوەندی ژمی رەن ‪162.46‬یبنو‪ .‬ئە‬ ‫ن خە اەورەر ە لە ناااوەناادی انیمااانەی ەچنانەکە کە بنیو بنولە‬ ‫‪126‬ی بە مدان ەچنەری ‪ . (1.57‬ەروە ااا دەرکەوت بە ااای‬ ‫‪t‬ی ەژمارک او بنی ەە لە ‪20.82‬ی لە ئاسا ااو بە گەداری ‪0.05‬‬ ‫ی ەروەڕ لە خالا ا ا ا ا ااتەی ‪7‬ی ڕووناکا اوەرەوە وارە جاەا ا اااوازی‬ ‫بە گەداری ئاماری بەد دەکنت وناوەندە ە‬ ‫ژمی رەن اەورەر ە لە‬ ‫انیمانەن ئەمە ئەوە دەردەخات ئاسو کۆنی دن‬ ‫ناوەندە‬ ‫ر‬ ‫ا‬ ‫ە چنون بەرینەبەران باخچەی منداڵن لە ئاس ێ بەرزدا ە‪.‬‬ ‫خشتەی ژمارە ‪)7‬‬ ‫ی‬ ‫ی‬ ‫ی‬ ‫ئەنجامەکان تاقیکردنەوەی تان یەک نموونە) بۆ نمرەکان‬ ‫ا‬ ‫بەرێوەبەر یان باخچەی منداڵن لە ناوەندی شاری سلێمانیبە‬ ‫ی‬ ‫ی‬ ‫گشت لە پێوەری ر‬ ‫ر‬ ‫هەڵچوون‬ ‫ۆڵکردن‬ ‫کۆنت‬ ‫شێوەیەیک‬ ‫نموونە‬ ‫ناوەندی‬ ‫ژمتەن‬ ‫ناوەندی‬ ‫گریمانەن‬ ‫ی‬ ‫الدان پێوەری‬ ‫هەژمارکراو‬ ‫خشتەن‬ ‫خشتەی ژمارەی ‪)8‬‬ ‫تان ‪ )t‬دوو نموونە) بۆ نمرەکانی‬ ‫ئەنجایم تاقیکردنەوەی ی‬ ‫ی‬ ‫بەرێوەبەر یان باخچە لەسەر پێوەری ر‬ ‫ۆڵکردن‬ ‫کۆنت‬ ‫ی‬ ‫هەڵچوون بەپت گۆڕاوی ڕەگەزی نت و یم)‬ ‫ی‬ ‫بەهای تان‬ ‫ئ‬ ‫ا‬ ‫س‬ ‫ر‬ ‫ن‬ ‫ب‬ ‫ە‬ ‫ڵ‬ ‫نم‬ ‫گ‬ ‫رە‬ ‫ەد‬ ‫ی‬ ‫ال‬ ‫ار‬ ‫ئ‬ ‫دا‬ ‫ی‬ ‫ا یز‬ ‫نا‬ ‫‪0.‬‬ ‫دن‬ ‫پ‬ ‫‪0‬‬ ‫او‬ ‫ی‬ ‫ێو‬ ‫ە‪5‬‬ ‫ەر‬ ‫ند‬ ‫ن‬ ‫ژم‬ ‫ی‬ ‫ار‬ ‫ژم‬ ‫ە‬ ‫ڕە‬ ‫ێر‬ ‫گ‬ ‫ە‬ ‫ەز‬ ‫ن‬ ‫نمرەی ئازادی‬ ‫بەهای ی‬ ‫تان‬ ‫ر‬ ‫ئاست‬ ‫بەڵگەداری‬ ‫‪0.05‬‬ ‫‪162.46‬‬ ‫‪126‬‬ ‫‪1.57‬‬ ‫‪77‬‬ ‫‪20.82‬‬ ‫‪1.69‬‬ ‫بەڵگەدارە‬ ‫‪162‬‬ ‫‪Journal of the University of Garmian 10 (1), 2023‬‬ ‫هەژمارکرا‬ ‫و‬ ‫خشتەن‬ ‫نت‬ ‫‪10‬‬ ‫‪165.‬‬ ‫‪30‬‬ ‫‪5.81‬‬ ‫‪76‬‬ ‫‪0.61‬‬ ‫بەڵگەدار نیە‬ ‫‪)0.538‬‬ ‫‪1.69‬‬ ‫یم‬ ‫‪68‬‬ ‫‪162.04‬‬ ‫‪16.40‬‬ ‫بە بەكااار چنااان ‪One Sample T-test‬ی ریت ەر ەسا ا ا ا ا اتااا بە‬ ‫بەراورک دن ناوەندی ە‬ ‫ژمی رەن نمنونەی ریتیینەوەکە بە ناوەندی‬ ‫انیمااانەی‪ .‬لە ئەن اااماادا دەرکەوت‪ ،‬کەناااوەناادی ە‬ ‫ژمی رەن بنی ەە‬ ‫لە ‪110.47‬ی‪ .‬ئە نا خە اەورەرا ە لە نا ا اااوەنا ا اادی ااانیاما ا ااانەی‬ ‫ە اچاانانەکە کە باانیااو باانو لە ‪96‬ی بە مدان ە اچاانەری ‪. (9.43‬‬ ‫ەروە ااا دەرکەوت بە ااای ‪t‬ی ەژمااارک او بنی ەە لە ‪13.55‬ی‬ ‫لە ئاااس ا ا ا ا ااو بە گەداری ‪ 0.05‬ی ەروەڕ لە خال ا ا ا ا ااتەی ‪10‬ی‬ ‫ڕوونک اوەرەوە وارە جەاااوازی بە گەداری ئاااماااری بەد اادەکنت ا‬ ‫ە‬ ‫اانەن ئەمە ئەوە‬ ‫وناااوەناادە ژمی رەن اەورەر ە لە ناااوەناادە انیما ر‬ ‫اەن می بەرینەبەران باخچەی‬ ‫دەردەخات ئاس ااو انن ان ە ال ا ر‬ ‫ا‬ ‫منداڵن لە ئاس ێ‬ ‫لەبەر ئەوەی بە ا ا ااای را ا ا یاان ئەژما ا ااارک ا او ب اچ اانک اایە لە بە ا ا ااای‬ ‫اتەن ئەمە ئەوە دەردەخا ا ااات کە جاەا ا اااوازی لە ناچاانان‬ ‫خالا ا ا ا ا ا ر‬ ‫ە‬ ‫ە‬ ‫ن اااوەن اادی ژمی رەن نم ە ااان ڕەاەزی نی و نم ە ااان ڕەاەزی ە‬ ‫ر‬ ‫لەس ا ا ا ا ااەر ەاچانەری کاۆنای ا ا دن ە اچانون ناەە‪ .‬بە باۆچنون‬ ‫ریت ەر ۆ ااارەکەی دەاەڕتنچتەوە بۆ ئەوەی کە ەردوو ڕەاەز‬ ‫لە ژینگەی اااو ا ا ا ا ااچنەدا ژیااان دەانزەرتی‪ .‬ئە دەرئەن ااامە‬ ‫ە انگی نەە لەاە پچ ا لە ریتیینەوە ان ە الن‪.‬‬ ‫ی‬ ‫ی‬ ‫انیت ئاسا ا ا ا ا ا رات ر‬ ‫ئامانج دووەم‪ :‬ب‪ .‬ز ی‬ ‫هەڵچوون‬ ‫ۆڵکردن‬ ‫کۆنت‬ ‫ی‬ ‫ڕ‬ ‫ا‬ ‫ی‬ ‫ی‬ ‫الی بەڕێوەبەر یان باخچەی منداڵن ش ا ا ا ا اااری س ا ا ا ا االێمان بەپت‬ ‫جۆری باخچە حکویم و ناحکویم)‬ ‫ە‬ ‫ەچنەرن‬ ‫بۆ زانپو ئە جەااوازیە ریت ەر نااوەنادی ژمی رەن و مدان‬ ‫ر‬ ‫ا‬ ‫بۆ نم ە ا ااان بەڕتنەبەران با اااخچەی منا ااداڵن لەس ا ا ا ا ااەر ەچنەری‬ ‫کۆنی دن ە چنون دەر چنا ااا پا ااا ا ا ا ا ا ا ااان بە بەكا ااار چنا ااان‬ ‫ر ا ا ااای ا ا اە ا ا اک ا ا ا دنەوەی ر ا ا ا یان ب ا ا ااۆ دوو ن ا ا ام ا ا اانونەی سا ا ا ا ا ااەربەخ ا ا ااۆ‬ ‫)‪independent sample T-test‬ی بە پال ا ا ا ا ا ا بەس ا ا ا ا اای بە‬ ‫بەرن ااامەی ئ ااام اااری ‪SPSS‬ی دەرکەوت ە ن ن ‪t‬ی ئەژم ااارک او‬ ‫‪1.08‬ی بە نم ەی ئازادی ‪76‬ی‪ ،‬لە س ا ا ا ا ااەر ئاس ا ا ا ا ااو بە گەداری‬ ‫‪0.05‬ی‪ ،‬وەڕ لە خالتەی ژمارە ‪9‬ی خ اوەرە ڕوو‪.‬‬ ‫بەرزدا ە‪.‬‬ ‫خشتەی ژمارە ‪)10‬‬ ‫تان یەک نموونە) بۆ نمرەکانی‬ ‫ئەنجامەکان تاقیکردنەوەی ی‬ ‫ی‬ ‫ا‬ ‫ی‬ ‫سلێمان‬ ‫بەرێوەبەر یان باخچەی منداڵن لە ناوەندی شاری‬ ‫ی‬ ‫ر‬ ‫گونجان پیشەن‬ ‫گشت لە پێوەری‬ ‫بە شێوەیەیک‬ ‫ەوارە ە‬ ‫بەەو ئەو ئەن امەی لە س ا ااەرەوە دەرچنوە دەریدەخات‬ ‫نموونە‬ ‫ناوەندی ژمتەی‬ ‫ناوەندی‬ ‫گریمانەن‬ ‫ی‬ ‫الدان پێوەری‬ ‫نمرەی ئازادی‬ ‫هەژمارکراو‬ ‫بەهای ی‬ ‫تان‬ ‫خشتەن‬ ‫بەهای ی‬ ‫تان‬ ‫‪9.4‬‬ ‫‪3‬‬ ‫ر‬ ‫ئاست بەڵگەداری‬ ‫‪0.05‬‬ ‫‪78‬‬ ‫‪110.4‬‬ ‫‪7‬‬ ‫‪96‬‬ ‫‪77‬‬ ‫‪13.5‬‬ ‫‪5‬‬ ‫‪1.6‬‬ ‫‪9‬‬ ‫بەڵگەدار‬ ‫ە‬ ‫ا‬ ‫بەڕتنەبەران ب اااخچەی من ااداڵن ا ا ا ا ا اااری س ا ا ا ا االێم ااان انن ااان‬ ‫ەا ال ا ا ا ا ااەۆاەا ا ااان ە ە بە ئا ا اااس ا ا ا ا ا ا اێا بەرز‪ .‬بە بااۆچاانون راایتا ەر‬ ‫ۆ ارەکەی لێکتێگەاالا ا ا اای و او اری و خۆ ا ا ا ااەویسا ا ا ااو نچنان‬ ‫مامۆستا ان و ستاە ار ە‬ ‫ایی و خیتند ارانەوە ب ‪ ،‬وە ەبنون‬ ‫اەان او اری و دەسا ا ا ا ا ە الا ا ا ا ااخەری لە ا ا ا ا اایتو اردا و زا بنون‬ ‫بەس ا ااەر بارود خە بال ا اااراویە ان و ڕووبەڕووبننەوە ان‪ ،‬وە خۆ‬ ‫انن اندن لەاە ژینگەی ئەرتو و نەرتو لە ناوەوە و دەرەوەی‬ ‫ژینگەن باخچە و ەبنون ئارەزووی ارک دنەان ئەمە پا نەرت‬ ‫ر‬ ‫اەورە ە بۆ ئەوان‪ ،‬ەروە ا ااا بنون لێکتێگەاال ا ا ا ا اای لە نچنان‬ ‫ئەن اادام ااان ە‬ ‫خیان و ئەمە ڕەنگە انن ااان اادنێکە ااان ەی اادات لە‬ ‫ە ال ا ا ا ا ااەکەدا‪ .‬ئە ئەن ااامە ەك دەانتتەوە لەاە ئەن ااار‬ ‫ریتیینەوەی ع نة و ابن طی‪2020 ،‬ی‪ ،‬ریتیینەوەی ما ا ااا و‬ ‫ونة‪2018 ،‬ی‪ ،‬ریتیینەوەی الال ا ا ا ا اااع و س ا ا ا ا اابەحة‪2019 ،‬ی ئە‬ ‫اەن بەرزە می‬ ‫ریتیینەوانە دەریانخس ا ا ا ئاسا ا ااو انن ان ە الا ا ا ر‬ ‫نمنونەی ریتیینەوەکە ان‪.‬‬ ‫ئامانج چوارەم‪:‬‬ ‫ی‬ ‫لەبەر ئەوەی بە ا ا ااای را ا ا یاان ئەژما ا ااارک ا او ب اچ اانک اایە لە بە ا ا ااای‬ ‫اتەن بە جۆرە دەردەکەوتا کە جەاااوازی بە گەداری‬ ‫خال ا ا ا ا ا ر‬ ‫ە‬ ‫نەە لە نچنان ناااوەناادی ژمی رەن نم ە ااان بەڕتنەبەران باااخچەی‬ ‫ا‬ ‫منداڵن لەس ااەر ەچنەری کۆنی دن ە چنون بە ەەو جۆری‬ ‫باخچە‪ .‬لە دوای سەردانەک دن خندی ریت ەر بۆ باخچە ان بە‬ ‫ەر دوو جۆرەکە ەوە ۆ اااری ئە ئەن ااامە دەاەڕتنچتەوە بۆ‬ ‫ەبنون ئا اااما ااانە و پە ا ااار ڕوون وئا ااا ا ا ا ا ااک ای با اااخچە ا ااان بە‬ ‫جۆرتک کە ەریەکە ان بە ە‬ ‫ن جەاوازی ەو بۆ بەرەوپ الیدن‬ ‫باخچەکە ان دەدەن‪ ،‬وارە ز ر بە ڕوون بەر ئەو ەو و ماندوو‬ ‫بنونەی بەرتنەبەر و ەمنو س ا ا ا ا ااتابە ان باخچە ان کەورم‪ ،‬کە‬ ‫چەند بەپەر و د س ا ااۆزانە ار دەکەن بە ە‬ ‫ن پچ جەاوازیەڕ‬ ‫لە ەر دوو جۆر وکنر و ناوکنمەەوە‪.‬‬ ‫ئامانک سیەە ‪:‬‬ ‫ی‬ ‫ی‬ ‫ز ی‬ ‫انیت ئاس ا ا ا ا رات گونجان پیش ا ا ا ااەن الی بەڕێوەبەران باخچەی‬ ‫ی‬ ‫ا ی‬ ‫منداڵن شاری سلێمان)‬ ‫‪163‬‬ ‫‪Journal of the University of Garmian 10 (1), 2023‬‬ ‫ی‬ ‫ز ی‬ ‫گونجان پیشا ا ا ااەن الی بەڕێوەبەر یان‬ ‫انیت ئاسا ا ا ا رات‬ ‫أ‪.‬‬ ‫ا‬ ‫ی‬ ‫ی‬ ‫باخچەی منداڵن شاری سلێمان بەپت گۆراوی ڕەگەز)‬ ‫بۆ اەاال ا ا ا ا اای بە ئ ا ااام ا ااان ە ریت ەر ن ا اااوەن ا اادی ە‬ ‫ژمی رەن و مدان‬ ‫ا‬ ‫ەچنەرن نم ە ان بەرتنەبەران باخچەی منداڵن بە ەەو اۆڕاوی‬ ‫ر‬ ‫اەن دەر چنا‪ ،‬پا ا ا ا ا ااان بە‬ ‫ڕەاەز لەسا ا ا ا ااەر ەچنەری انن ان ە الا ا ا ا ا ر‬ ‫بەك ااار چن ااان ر ااایەک دنەوەی ر ا یاان بۆ دوو نمنونەی س ا ا ا ا ااەربەخۆ‬ ‫)‪independent sample T-test‬ی بە پال ا ا ا ا ا ا بەس ا ا ا ا اای بە‬ ‫بەرن ااامەی ئ ااام اااری ‪SPSS‬ی دەرکەوت ە ن ن ‪t‬ی ئەژم ااارک او‬ ‫بنی ەە لە ‪1.58‬ی بە نم ەی ئ ااازادی ‪76‬ی‪ ،‬لە س ا ا ا ا ااەر ئ اااس ا ا ا ا ااو‬ ‫بە گەداری ‪0.05‬ی‪ ،‬وەڕ لە خالتەی ژمارە ‪11‬ی د ارە‪.‬‬ ‫لەبەر ئەوەی بە ا ا ااای را ا ا یاان ئەژما ا ااارک ا او ب اچ اانک اایە لە بە ا ا ااای‬ ‫اتەن بە جۆرە دەردەکەوتا کە جەاااوازی بە گەداری‬ ‫خال ا ا ا ا ا ر‬ ‫نەە لە نچنان ناااوەناادی ە‬ ‫ژمی رەن نم ە ااان بەڕتنەبەران باااخچەی‬ ‫ا‬ ‫من ااداڵن لەس ا ا ا ا ااەر ەچنەری انن ااان ە ال ا ا ا ا ااەۆو بە ەەو اۆڕاوی‬ ‫ڕەاەز‪ ،‬ریت ەر ۆ ا ااارەکەی دەاەڕتنچتەوە بۆ ئەوەی ە ال ا ا ا ا ااەی‬ ‫ی‬ ‫ە ە لە ەس ا ا ا ا ااتک دن بە‬ ‫بەڕتنەبەری با ا اااخچە ڕ ێ ا ن‬ ‫اندەن و سا ااەممەن و د نەا رن می ەردوو ڕەاەز‪ ،‬ەروە ا‬ ‫ئاسا ا ر‬ ‫ەبنون ەمان ئاس ااو رمیح و کۆ نەدان و جددی لە ارک دندا‬ ‫و پەۆنەنادۆەان بە ە ال ا ا ا ا ااەکە اانەوە و پااپەنادبنونەان ەیەەوە می‬ ‫ەردوو ڕەاەز وەک اان ەکە‪ ،‬وە دابا ایا انا اکا ا دن ەا اێا ا ااداویسا ا ا ا ا ا ا ااەە‬ ‫مادد ە ان و بنون اەان او اری و متمانە و خۆ ەویس ەان بۆ‬ ‫ارەکە ان می ەردوو ڕەاەز وا لێک دن وەڕ رەمێک اربکەن‬ ‫او چنەی‬ ‫و بگنن ەی لە ڕووی ە الەۆەەوە‪ .‬ئە دەرئەن امە‬ ‫دەرئەنا ا ا ا ااار ر اایت اایی اانەوەی خا ال اان‪2019،‬ی ئە ر اایت اایی اانەوە‬ ‫الەن جەاوازی نەە بە ەەو اۆڕاوی‬ ‫دەریخس ئاسو انن ان ە‬ ‫ر‬ ‫ڕەاەز‪.‬‬ ‫گونجان پیشا ا ا ااەن الی بەڕێوەبەرانی‬ ‫ی‬ ‫ز ی‬ ‫انیت ئاسا ا ا ا رات‬ ‫ب‪.‬‬ ‫ا‬ ‫ی‬ ‫ی‬ ‫باااخچەی منااداڵن شا ا ا ا ا ا اااری سا ا ا ا ا االێمااان بەپت جۆری باااخچە‬ ‫حکویم و ناحکویم)‬ ‫ە‬ ‫بۆ اەاال ا ا ا ا اای بە ئ ا ااام ا ااان ە ریت ەر ن ا اااوەن ا اادی ژمی رەن و مدان‬ ‫ا‬ ‫ەچنەرن نم ە ااان بەرتنەبەران باااخچەی منااداڵن بە ەەو جۆری‬ ‫ر‬ ‫اەن دەر چنا‪ ،‬پا ا ا ا ااان بە‬ ‫باخچە لەس ا ا ا ااەر ەچنەری انن ان ە ال ا ا ا ا ر‬ ‫بەك ااار چن ااان ر ااایەک دنەوەی ر ا یاان بۆ دوو نمنونەی س ا ا ا ا ااەربەخۆ‬ ‫)‪independent sample T-test‬ی بە پال ا ا ا ا ا ا بەس ا ا ا ا اای بە‬ ‫بەرن ااامەی ئ ااام اااری ‪SPSS‬ی دەرکەوت ە ن ن ‪t‬ی ئەژم ااارک او‬ ‫بنی ەە لە ‪1.036‬ی بە نم ەی ئااازادی ‪76‬ی‪ ،‬لە س ا ا ا ا ااەر ئاااس ا ا ا ا ااو‬ ‫بە گەداری ‪0.05‬ی‪ ،‬وەڕ لە خالتەی ژمارە ‪12‬ی د ارە‪.‬‬ ‫خشتەی ژمارەی ‪)12‬‬ ‫تان بۆ دوو نموونە بۆ نمرەکانی‬ ‫ئەنجامەکان تاقیکردنەوەی ی‬ ‫ی‬ ‫ا‬ ‫ی‬ ‫گونجان‬ ‫بەڕێوەبەر یان باخچەی منداڵن لەسەر پێوەری‬ ‫پیشەن بەپت جۆری باخچە‬ ‫لەبەر ئەوەی بە ا ا ااای را ا ا یاان ئەژما ا ااارک ا او ب اچ اانک اایە لە بە ا ا ااای‬ ‫اتەن بە جۆرە دەردەکەوتا کە جەاااوازی بە گەداری‬ ‫خال ا ا ا ا ا ر‬ ‫ە‬ ‫نەە لە نچنان ناااوەناادی ژمی رەن نم ە ااان بەڕتنەبەران باااخچەی‬ ‫ا‬ ‫من ااداڵن لەس ا ا ا ا ااەر ەچنەری انن ااان ە ال ا ا ا ا ااەۆو بە ەەو جۆری‬ ‫باخچە‪.‬بە بۆچنون ریت ەر ۆ ارەکەی دەاەڕتنچتەوە بۆ ئەوەی‬ ‫کە بەڕتنەبەران لە ەردوو جۆری بااخچە وکنر و نااوکنری‬ ‫ی‬ ‫ئاسندەن و سەممەن‬ ‫نەان و‬ ‫ر‬ ‫وەڕ ەڕ ەس بەخۆش و د ر‬ ‫و جێگیو ڕتزا رن دەکەن لە ژینگەی اردا‪ ،‬ەروە ا دابینک دن‬ ‫ا ا ااانا ا اادانە ما ا اااددی و ئەخنیپەە ا ا ااان و ریدەڕانا ا اادن با ا ااارود ن‬ ‫دەوروبەری ا ااان کە ئەوانە بەر ەر س ا ا ا ا ااەرکەورن و انن ا ااان لە‬ ‫اەن بە دەس ا ا دە چنێ لە ەردوو جۆری باخچە‬ ‫چام ە الا ا ر‬ ‫وەکن ەکە‪،‬ئە دەرئەن امە ە انگی نەە لەاە پچ ا لە‬ ‫ریتیینەوە ان ە النو‪.‬‬ ‫ئامانج پێنجەم ‪:‬‬ ‫ی‬ ‫ی‬ ‫ی‬ ‫ر‬ ‫ی‬ ‫اتۆڵ اک ااردن هەڵ اچ ااوون و‬ ‫زان ای اات پەی ااوەنا ا اادی لە ن اێ ااوان ک ااۆن ا ڕ‬ ‫ا‬ ‫ی‬ ‫گونجان پیش ااەن الی بەڕێوەبەر یان باخچەی منداڵ ین ش اااری‬ ‫ی‬ ‫سلێمان‬ ‫بەمەبەس ا ا ا ا ااو زانپو و ا ا ا ا ااو پەۆنەناادی نچنان کۆنی دن‬ ‫اەن می بەڕتنەبەران ب ا اااخچەی‬ ‫ە چنون و انن ا ااان ە ال ا ا ا ا ا ر‬ ‫ا‬ ‫منداڵن ریت ەر اوکۆل ەی پەۆنەندی ەەیسا ااۆن م امل ارر اط‬ ‫بیسا ا ا ا ااننی بە ار چنا بە پال ا ا ا ا ا بەسا ا ا ا اای بە بەرنامەی ئاماری‬ ‫‪SPSS‬ی‪ .‬لە ئەن ا ا اااما ا اادا دەرکەوت ن ن پەۆنەنا ا اادی لە نچنان‬ ‫ەردوو اۆڕاوەکە ەکس ا ا ا ا ا ا ااانە بە ‪0.34‬ی‪ .‬وارە پەۆنەن ا ااد ەج‬ ‫ە ااۆزەر اە ااب مواز ە ە لە ن اچ اانان ک ااۆن اای ا ا دن ە اچ اانون و‬ ‫اەن ‪.‬کە ئەن امەکەی لە خال ا تە ەی ژمارە ‪13‬ی‬ ‫انن ان ە ال ا ر‬ ‫خ اوەرەڕوو‪.‬‬ ‫خشتەی ژمارە ‪(13‬‬ ‫ی‬ ‫ئەنجامەکان دۆزینەوەی هاوکۆلکەی پەیوەندی لە نێوان‬ ‫ی‬ ‫ی‬ ‫ی‬ ‫ر‬ ‫گونجان پیشەن‬ ‫هەڵچوون و‬ ‫ۆڵکردن‬ ‫کۆنت‬ ‫ڕ‬ ‫گۆڕاوەکان‬ ‫ی‬ ‫ر‬ ‫ۆڵکردن‬ ‫کۆنت‬ ‫ڕ‬ ‫ی‬ ‫هەڵچوون‬ ‫ی‬ ‫گونجان پیشەن‬ ‫نموونەی‬ ‫توێژینەوە‬ ‫ی‬ ‫نرخ هاوکۆلکەی‬ ‫پەیوەندی‬ ‫پتسۆن‬ ‫ر‬ ‫ئاست بەڵگەداری‬ ‫ئاماری ‪0.05‬‬ ‫‪78‬‬ ‫‪0.34‬‬ ‫‪o. o1‬بەڵگەدارە‬ ‫ئە خالا ا ا ا ا ااتە ەی س ا ا ا ا ااەرەوە پەۆاانەنا ا اادی ناچاانان کااۆناای ا ا دن‬ ‫اەن می بەڕتنەبەران ب ا اااخچەی‬ ‫ە چنون و انن ا ااان ە ال ا ا ا ا ا ر‬ ‫ا‬ ‫ما انا ا ااداڵن روون دە ا ا ااارەوە وارە پەۆ اانەنا ا ااد ەکە پەۆ اانەنا ا ااد ەج‬ ‫ڕاس ا ااتەوانە ە ئەاەر کۆنی دن ە چنون بەرز ب ئاس ا ااو‬ ‫انن ان ە ال ا ا ااەایک بەرز دەب وە بە ەێچەوانە ا ا ااەوە‪ ،‬ریت ەر‬ ‫ااۆ ا ا اااری ئە ئەن ا ا ا ااامە بااۆ ئەوە دەاەڕت ان اچااتەوە کە ئا ا اااسا ا ا ا ا ااو‬ ‫کۆنی دن ە چنون دەبیتە ۆی بەرزبنونەوەی انن ان‬ ‫‪164‬‬ ‫‪Journal of the University of Garmian 10 (1), 2023‬‬ ‫اەن لەناااو بەڕتنەبەران باااخچە وە بە ەێچەوانەوە ا ا ا ا ااەوە‬ ‫ە ال ا ا ا ا ا ر‬ ‫انن ان ە ال ا ا ا ااەۆ یک دەبیتە ۆی بەرزبنونەوەی کۆنی دن‬ ‫ە اچ اانون‪ ،‬چ اانن ااکە ک ااۆن اای ا ا دن ە اچ اانون ب اانی ا اپ ااەە لە‬ ‫ڕتکخساتو خند کە ارمەن راە دەدات بە ئا اۆەەوە ڕووبەووی‬ ‫بارود خە ان و بال ا ا ا ا ااارو س ا ا ا ا ااەختەە ان ژیان ببچتەوە وە بایاری‬ ‫دروس ا بدات‪ ،‬ئەمە ئەاەری سااەرکەورو راە زیاد دە ات‬ ‫اەن‬ ‫لە ئەن ااام اادان ااارە ااان وا لە ر اااە دە ااات انن ااان ە ال ا ا ا ا ا ر‬ ‫ە‬ ‫کەورنن لە‬ ‫ەب لەاە اوڕ و اوپ الا ا ااە ان داو وە سا ا ااەر‬ ‫ما ا ااامە ا ا ا ا دن و پەۆ ا اانەنا ا ااد ا اک ا ا دن لەاە کەسا ا ا ا ا ا ا ااان ر ا ا دا‪...‬‬ ‫اەن رااۆ ەرمەنادی ئەو‬ ‫کۆنی دن ە چنن و انن اان ە ال ا ا ا ا ا ر‬ ‫کەس ا ا ا ا ااانە ە کە کەس ا ا ا ا ااا ەریەەج بە ەیو رەندروس ا ا ا ا ااو دەروون‬ ‫با ا ااا ا ا ا ا ا اەا ا ااان ە ە‪ .‬ئە دەرئەن ا ا ا ااامە لەاە اپااچ ا ا ااا لە‬ ‫ریتیینەوە ان ە النو ە انگی نەە‪.‬‬ ‫ڕاسپاردەکان‬ ‫بە ەا ا ەاو دەرئەن ا ا ا ا ااامە ا ا ااان ئە را اایتا ااییا اانەوە ە را اایت ا ا ەر چەنا ا ااد‬ ‫ڕاسا ا ا ا ا ادا اااردە ەڕ بۆ دەز ا ااا پەۆنەنا ااد اادارە ا ااان وکنمەن ەرتم‬ ‫دەخارەڕوو‪ ،‬بە چنە ەی خنارەوە‪:‬‬ ‫ەرچەنا ا ااد کە بە ە ا ەاو دەرئەنا ا ا ا ااامە ا ا ااان ئا ا اااسا ا ا ا ا ا ااو‬ ‫‪.1‬‬ ‫ا‬ ‫اەن بەرزە‬ ‫بەرینەبەران بۆ کۆنی‬ ‫ەل نون و انن ان ە ال ا ا ا ا ا ر‬ ‫ا‬ ‫بەڵ دەکنت ا بۆ زیااار پەرەەێاادان رنانااای بەڕتنەبەران باااخچە‬ ‫بە او اری س ا ا ا ا ااەرپەر ا ا ا ا ا ا ەاران بەرپ س بەڕتگەی ەێدانەان بۆ‬ ‫زانپو ئەن ار ریتیینەوەی زانسا ا ا پەانە ان و بانگ ال ا ااتک دنەان‬ ‫بۆ کۆنف انس و س ا ا ا ا ااەمپنااارە ااان بۆ ئااا ااادارک دنەوە ااان لە نیتیین‬ ‫ڕەورە ان‪.‬‬ ‫ەروە ااا ئەن اااماادان خنل بۆ پەرەەێاادان رنانااا اان‬ ‫‪.2‬‬ ‫بەرتنەبەران لە س ا ا ا ا ااەر پنناادانااان و جچ ەجێک دن ااارامەۆەە ااان‬ ‫ە‬ ‫بیک دن و بەرتنەب دن بە ەەو سپ ستەر بەرتنەب دن و سا نامە‪.‬‬ ‫ا‬ ‫ەێدان پادا ا ا ا ا ااو س ا ا ا ا اااڵنە بە بەرتنەبەران باخچە بە‬ ‫‪.3‬‬ ‫مەبەس ا ا ا ا ااو ا ااان ا اادانە ا ااان بۆ م ا ااانەوە لە ئ ا اااس ا ا ا ا ااتە ب ا ااا ا ا ا ا ااەی‬ ‫لچ ارنویەە انەان لە ارک دن بۆ کەررە پەروەردەۆەە ان‪.‬‬ ‫زیا ااادک دن ا اااو ا اااری لە نچنان بە ا ا ا ا ااە ا ااان وەزارەن‬ ‫‪.4‬‬ ‫ا‬ ‫بەش چاودێ ی‪ ،‬ی‬ ‫باڵی کنردسا ا ااتان‪ ،‬ی‬ ‫بەش‬ ‫پەروەردە و خیتندن‬ ‫ار ە‬ ‫ایی باخچە ان‪ ،‬لەاە سا ا ا ا ااەرپەر ا ا ا ا ااو رەکنە و ڕا چنان‬ ‫بەڕتنەبەران باخچە و مامۆستا ان‪.‬‬ ‫رەرخانک دن ا ا اایتن لەناو دامەزراوە پەروەردەۆەە ان‬ ‫‪.5‬‬ ‫ا‬ ‫باخچەی منداڵنی بۆ نە ال ا ااتو ا ژی و رە ا ااەنیج و بال ا ااار‪،‬‬ ‫وەڕ ژووری راۆ ەت بۆ ئ رساوەن دەروون‪.‬‬ ‫پێشنیارەکان‪:‬‬ ‫لەبەر ر ا ا ا ا اانەا ا ا راان ئا ا اااما ا ااان ە ا ا ااان ریتیینەوەکە ریت ەر چەن ا ااد‬ ‫پەروەردەن خستە ڕوو ئەوان یک بنیتی لە‪:‬‬ ‫ە النەارت‬ ‫ر‬ ‫دەرئەنجامەکان‬ ‫دوای خس ا نەڕووی ئەن امە ان ریتیینەوەکە و ااەک دنەوە ان‬ ‫ریت ەر بە ئەن امانە اەاال ‪:‬‬ ‫‪ -1‬ئاس ا ا ا ااو کۆنی دن ە چنون لە ئاس ا ا ا ا ێ بەرزدا ە می‬ ‫ا‬ ‫بەڕتنەبەران باخچەی منداڵن ا اااری س ا االێمان بە ا ااچنە ەج‬ ‫االو‪.‬‬ ‫‪ -2‬أ‪.‬جەاوازی بە گەداری ئاماری نەبنو بۆ ئاسا ااو کۆنی دن‬ ‫ا‬ ‫ە چنون می بەڕتنەبەران باخچەی منداڵن اااری س االێمان‬ ‫بە ەەو اۆڕاوی ڕەاەزی ەنی و ە‬ ‫ری ‪.‬‬ ‫ب‪ .‬جەاوازی بە گەداری ئاماری نەبنو بۆ ئاس ا ا ااو کۆنی دن‬ ‫ا‬ ‫ە چنون می بەڕتنەبەران باخچەی منداڵن اااری س االێمان‬ ‫بە ەەو جۆری باخچە وکنر و ناوکنری ‪.‬‬ ‫اەن لە ئ ا اااسا ا ا ا ا ا ێ بەرزدا ە می‬ ‫‪ -3‬ئ ا اااس ا ا ا ا ااو انن ا ااان ە ال ا ا ا ا ا ر‬ ‫ا‬ ‫بەڕتنەبەران باخچەی منداڵن ا ا اااری س ا ا االێمان بە ا ا ااچنە ەج‬ ‫االو‪.‬‬ ‫‪ -4‬أ‪.‬جە اااوازی بە گەداری ئ ااام اااری نەبنو لە ئ اااس ا ا ا ا ااو انن ااان‬ ‫ا‬ ‫اەن می بەڕتنەبەران باخچەی منداڵن اااری س االێمان بە‬ ‫ە ال ا ر‬ ‫ە‬ ‫ەەو اۆڕاوی ڕەاەزی نی و ە‬ ‫ری ‪.‬‬ ‫ب‪ .‬جەا اااوازی بە گەداری ئا اااما اااری نەبنو لە ئا اااس ا ا ا ا ااو انن ااان‬ ‫ا‬ ‫اەن می بەڕتنەبەران باخچەی منداڵن اااری س االێمان بە‬ ‫ە ال ا ر‬ ‫ەەو جۆری باخچە وکنر و ناوکنری‬ ‫‪ -5‬پەۆنەنا ا ااد ەج ڕاس ا ا ا ا ااتەوانەی ە ە لە نچنان کۆنی دن‬ ‫اەن می بەڕتنەبەران ب ا اااخچەی‬ ‫ە چنون و انن ا ااان ە ال ا ا ا ا ا ر‬ ‫ا‬ ‫منداڵن اری سلێمان‪.‬‬ ‫ئەنا ا ا ا ا ااار ر ا اایت ا اایی ا اانەوە ەڕ لەس ا ا ا ا ااەر پەرەەا ا اێا ا اادان‬ ‫‪.１‬‬ ‫ا‬ ‫بەرن ااامە ااان بۆ ئ ااام ااادەک دن بەڕتنەبەری ب اااخچەی من ااداڵن لە‬ ‫دوان باخچە اندا‪.‬‬ ‫بەر ر‬ ‫نان ە ال ارە ان ر‬ ‫ر‬ ‫ئەم ا ا ا ا اااما ا اادان را اایتا ااییا اانەوەی ا ا اااو ا ا ا ا ا ا اچا اانە لەاە‬ ‫‪.２‬‬ ‫ب اوانک دن نمنونەی ریتیینەوەکە وارە ن ا اااوچەو پ ا ااارتز ا ااا ا ااانپی‬ ‫بگنتتەوە‪.‬‬ ‫ئەنا ا ا ا اااما ا اادان ر اایت ااییا انەوە لەس ا ا ا ا ااەر ک ااۆن اای ا ا ا ا دن‬ ‫‪.３‬‬ ‫ە چنون و پەۆنەندی بە انن ان ە الەۆەەوە می مامۆستا ان‬ ‫ا‬ ‫باخچەی منداڵن‪.‬‬ ‫ئەن ااام اادان ریتیینەوە لەس ا ا ا ا ااەر ئەو ک ال ا ا ا ا ا ااانەی کە‬ ‫‪.４‬‬ ‫ڕووبەڕوی بەرتنەبەرە ان باخچە دەبیتەوە‪.‬‬ ‫‪165‬‬ ‫‪Journal of the University of Garmian 10 (1), 2023‬‬ ‫ئەن ا ا ا اااما ا اادان ر اایت اایی اانەوە ەڕ لەس ا ا ا ا ااەر ا ا اااری ااگەری‬ ‫‪.５‬‬ ‫بەرنااامە ااان ئااامااادەک دن و ڕا چنااان مااامۆسا ا ا ا ا اتااا ااان لە باااخچەی‬ ‫ا‬ ‫نان ڕەون سەردەمدا‪.‬‬ ‫منداڵن لە ژت ڕ‬ ‫ر‬ ‫سەرچاوە ان‬ ‫‪.１‬‬ ‫‪.1‬‬ ‫‪.2‬‬ ‫‪.3‬‬ ‫‪.4‬‬ ‫‪.１‬‬ ‫‪.２‬‬ ‫‪.３‬‬ ‫‪.４‬‬ ‫‪.５‬‬ ‫‪.６‬‬ ‫ەکە ‪ :‬سەرچاوە کنرد ە ان‬ ‫بۆ ان‪ ،‬صاب مصطب ‪2020‬ی‪ :‬ئپنس لاپەد ای زانستە دەروونەە ان‪،‬‬ ‫‪1‬ی‪ ،‬چاپخانەی چنارچ ا‪ ،‬سلێمان‪ ،‬ەرتا کنردستان‪.‬‬ ‫دووە ‪ :‬سەرچاوە عەرەبەە ان‬ ‫کتی ە ان‬ ‫ا‪ ،‬محمد وسنی ‪2000‬ی‪ :‬اإلدارة المدرسەة ‪،‬دار الفك ال رن‪،‬‬ ‫ال‬ ‫ر‬ ‫القا ة‪.‬‬ ‫الالا ی و ع دل زیز و باطمە محمد رصاف ‪-‬ی‪ ،‬الدورة التدریبەة‬ ‫ی‬ ‫للمرسوات للنظائف ام ی ابەةوظەفة مدۆ و مساعد‪ ،‬ط‪.1‬‬ ‫الدا ی ‪،‬وسن ‪2005‬ی‪ :‬سەکنلنجەة التنجی الم و و نظنیارە‪،‬ط‪،1‬‬ ‫دار وائل ‪،‬عمان‪ .‬نقل عن ضغنط ال مل و عنیت ا بالتنابق الم و‬ ‫لدی المم ضی‪ ،‬ننارة رەنۆپە‪ ،‬ن ەمەمخلنە ‪ ،٢٠١٧،‬جام ة ال ن‬ ‫بن م ەدی ‪ ،‬ا البناە ‪ ،‬لەة ال لن امجتماعەة و امنسانەة‪ ،‬یسم‬ ‫ال لن امجتماعەةی‪.‬‬ ‫‪:‬‬ ‫‪:‬‬ ‫الخالدی‪ ،‬اد ب ‪2002‬ی الم جع ە الصحة النفسەة ط‪ ،2‬الدار‬ ‫ال نبەة ی‬ ‫للنرس و التنزیا اع‪ ،‬المکت ة ال ام ەة‪ ،‬نیان‪ :‬لپبەا نقل عن‪:‬‬ ‫ریان‪ ،‬محمنس اسماعەل محمد ‪2006‬ی‪ :‬امرزان امنف ال و عنیتە‬ ‫ب ل من الرسعة امدرا ەة و التفکی امبت اری لدی طل ة الصف الحادی‬ ‫ی‬ ‫عرس بمحابظات زةی‬ ‫ریتیینەوە ان‪:‬‬ ‫ال واوية‪ ،‬بدرية محمد ينسف ‪ 2016‬ی‪ :‬التنابق الم رو وعنیت‬ ‫بالفاعلية الذارية المدر ة لدى عينة من المنظفی رە المدينية ال امة‬ ‫للیبية والت ليم بمحابظة الداخلية ‪.‬رسالة مستكمال متطل ات‬ ‫عیل درجة الماجيسی رە اال اد والتنجي ‪ .‬كلية ال لن‬ ‫الحصنل ر‬ ‫واآلداب‪ .‬جام ة نزوى‪.‬‬ ‫زرویة‪ ،‬الا و ی قنب‪ ،‬ع ش الثقابة التنظيمية وعالقت ا بالتنابق‬ ‫منظب مؤسسة ارصاال ال زائ – بنع‬ ‫الم رو دراسة ميدانية عیل‬ ‫ر‬ ‫بسك ة ‪.‬‬ ‫سمنر‪،‬اون ‪2012‬ی‪:‬المساۆ ة‪-‬المغاۆ ة و عنیت ا بالتنکەد ة و‬ ‫امرزان امنف ال لدی طل ة الصف الحادی ی‬ ‫عرس‪،‬رساالة ماجستی ی‬ ‫منالنرة‪ ،‬لەة الیبەة‪،‬ال ام ة امسنمەة بغزة‪.‬‬ ‫عداد‪،‬وسا ‪2017‬ی‪ :‬امم اض الم و و عنیت ا بالتنابق الم و لدی‬ ‫مدراء الت لەم المتنسط و الثاننی‪ ،‬دراسة مەدانەة ە ومۆن ا البناە‪-‬‬ ‫ال زائ ‪.‬‬ ‫وسن‪ ،‬وسا محمد ‪2015‬ی‪ :‬نمنذج مقیح لتطیی وایع التنمی‬ ‫الم نی لدی مدۆ ریاض امطفال ە ال م نریة ال نبی السنرین ە‬ ‫ضنء مدخل ال ندة المة و الت نبتی اممنیکپنە و الیطانی و الدارة‬ ‫الیبی‪ ،‬امط ووة الدکتنراة‪ ،‬جام ة دمالق‪ ،‬لەة الیبەة‪ ،‬یسم الیبەە‬ ‫المقارنن‪ .‬دمالق‪.‬‬ ‫زرویة‪ ،‬الا و ی قنب‪ ،‬ع ش ‪ (2017:‬الثقابة التنظيمية وعالقت ا‬ ‫منظب مؤسسة ارصاال ال زائ‬ ‫بالتنابق الم رو دراسة ميدانية عیل‬ ‫ر‬ ‫– بنع بسك ة‬ ‫سەرچاوە بارسەە ان‪:‬‬ ‫‪ .１‬لطف ابادی ‪2015‬ی‪ :‬ر د ارب دی ننجنان و جنان سازمان میل‬ ‫جنانان‪ ،‬ر ان‪ .‬وەرایاوە لە‪:‬‬ ‫سامر ع دلملیک و ئەوان‬ ‫عاطب خاننادە و نیل عناطف با‬ ‫رش رابط جن‬ ‫ر‬ ‫ر ‪2015‬ی‪:‬ب ر‬ ‫ا ايیک ب اعتياد‪ ،‬م لە خاننادە ە و یک‪،65.2016،‬ژ‪48.‬ی‪.‬‬ ‫سیەە ‪ :‬زمان ئین لیی‬ ‫‪Gratz, K. L., & Roemer, L. (2004). Multidimensional‬‬ ‫‪assessment of emotion regulation and‬‬ ‫‪dysregulation: Development, factor structure, and‬‬ ‫‪initial validation of the difficulties in emotion‬‬ ‫‪regulation scale. Journal of Psychopathology and‬‬ ‫‪Behavioural Assessment, 26(1), 41–54‬‬ ‫‪Gross JJ (2002): . Emotion regulation: Affective,‬‬ ‫‪cognitive, and social consequences.‬‬ ‫‪Psychophysiology; 39: 191-281‬‬ ‫‪Thompson, R.J., Dizén, m, Berenbaum, H (2009): The‬‬ ‫‪unique relations between emotional awareness and‬‬ ‫‪facets of affective instability. Journal of Research in.‬‬ ‫‪Personality. 43: 875–9‬‬ ‫‪Yiğit, A., Özpolat, A., & Kandemir, M (2014): Emotion‬‬ ‫‪regulation strategies as a predictor of life. satisfaction‬‬ ‫‪in university students. Psychology, 5, 523-532‬‬ ‫‪Li L, Zhu X, Yang Y, He J, Yi J, Wang Y(2015): Cognitive‬‬ ‫‪Emotion Regulation: Characteristics and Effect on‬‬ ‫‪Quality of Life in Women with Breast Cancer. Health‬‬ ‫‪Qual Life Outcomes; 13(1): 51.‬‬ ‫‪Dunham Y, Baron AS, Banaji MR((2008): the‬‬ ‫‪Development of Implicit Intergroup Cognition.‬‬ ‫‪Trends Cong Sci 12(7): 248 -53‬‬ ‫وەرایاوە لە سمەە جان نثاری‪،‬عیل ا دولق ‪ :٢٠١٨:‬نقیک کنیل‬ ‫عناطف‪ ،‬ب زیسو روان و ربتار ای خندم ایبو در ە البپو کەفە‬ ‫ی‬ ‫زندج بەماران یلو ع وە‪ ،‬م لە علا ەزو یش دانال اە علن‬ ‫ەد صدوە ۆزد‪،‬‬ ‫‪27).‬ەز‬ ‫‪166‬‬ ‫‪１.‬‬ ‫‪２.‬‬ ‫‪３.‬‬ ‫‪４.‬‬ ‫‪５.‬‬ ‫‪６.‬‬ ‫‪Journal of the University of Garmian 10 (1), 2023‬‬ ‫خشتەی ژمارەی ‪)9‬‬ ‫ا‬ ‫ۆڵکردن هەڵچوونی‬ ‫ی‬ ‫ی‬ ‫ئەنجامەکان تاقیکردنەوەی ی‬ ‫ی‬ ‫ر‬ ‫کۆنت‬ ‫نمرەکان بەڕێوەبەر یان باخچەی منداڵن لەسەر پێوەری‬ ‫تان بۆ دوو نموونە بۆ‬ ‫ڕ‬ ‫بە پت جۆری باخچە‬ ‫جۆری‬ ‫باخچە‬ ‫نموونەی‬ ‫توێژینەوە‬ ‫ناوەندی‬ ‫ژمتی‬ ‫ی‬ ‫الدان‬ ‫پێوەری‬ ‫حکویم‬ ‫‪52‬‬ ‫‪163.80‬‬ ‫‪15.19‬‬ ‫ناحکویم‬ ‫‪26‬‬ ‫‪159.76‬‬ ‫‪15.95‬‬ ‫بەهای ی‬ ‫تان‬ ‫هەژمارکراو‬ ‫نمرەی‬ ‫ئازادی‬ ‫ی‬ ‫تان بەهای‬ ‫خشتەن‬ ‫بەڵگەداری ئاماری‬ ‫‪0.05‬‬ ‫‪76‬‬ ‫بەڵگەدار نیە‬ ‫‪1.08‬‬ ‫‪1.69‬‬ ‫‪0.280‬‬ ‫خشتەی ژمارەی ‪)11‬‬ ‫ا‬ ‫ی‬ ‫ی‬ ‫ئەنجامەکان تاقیکردنەوەی ی‬ ‫ی‬ ‫گونجان پیشەن بەپت‬ ‫نمرەکان بەڕێوەبەر یان باخچەی منداڵن لەسەر پێوەری‬ ‫تان بۆ دوو نموونە بۆ‬ ‫گۆڕاوی ڕەگەز‬ ‫جۆری باخچە‬ ‫نموونەی‬ ‫توێژینەوە‬ ‫ناوەندی‬ ‫ژمتی‬ ‫ی‬ ‫الدان‬ ‫پێوەری‬ ‫حکویم‬ ‫‪52‬‬ ‫‪109.69‬‬ ‫‪9.42‬‬ ‫ناحکویم‬ ‫‪26‬‬ ‫‪112.03‬‬ ‫‪9.41‬‬ ‫نمرەی‬ ‫ئازادی‬ ‫ئەژمارکراو‬ ‫بەڵگەدار نیە‬ ‫‪76‬‬ ‫‪167‬‬ ‫بەهای ی‬ ‫تان‬ ‫بەهای‬ ‫ی‬ ‫تان‬ ‫خشتەن‬ ‫بەڵگەداری‬ ‫ئاماری ‪0.05‬‬ ‫‪1.036‬‬ ‫‪1.69‬‬ ‫‪0.303‬‬
https://openalex.org/W2102373577	https://aacr.figshare.com/articles/journal_contribution/Supplementary_Table_2_from_Interferon_Regulatory_Factor-1_Signaling_Regulates_the_Switch_between_Autophagy_and_Apoptosis_to_Determine_Breast_Cancer_Cell_Fate/22406499/1/files/39852369.pdf	unk	null	Interferon Regulatory Factor-1 Signaling Regulates the Switch between Autophagy and Apoptosis to Determine Breast Cancer Cell Fate	Cancer research	2,015	cc-by	25	Supplemental Table 2 ER status Negative Positive IRF1-nuc positive 13/35 (37%) 1/48 (2%) IRF1-cyto positive 26/35 (74%) 34/48 (71%) ATG7 positive 3/28 (11%) 1/38 (3%)
https://openalex.org/W586865457	https://link.springer.com/content/pdf/10.1007/s10951-015-0432-2.pdf	English	null	Decentralized subcontractor scheduling with divisible jobs	Journal of scheduling	2,015	cc-by	13,764	1 Introduction Abstract Subcontracting allows manufacturer agents to reduce completion times of their jobs and thus obtain sav- ings. This paper addresses the coordination of decentralized scheduling systems with a single subcontractor and several agents having divisible jobs. Assuming complete informa- tion, we design parametric pricing schemes that strongly coordinate this decentralized system, i.e., the agents’ choices of subcontracting intervals always result in efﬁcient sched- ules. The subcontractor’s revenue under the pricing schemes depends on a single parameter which can be chosen to make the revenue as close to the total savings as required. Also, we give a lower bound on the subcontractor’s revenue for any coordinating pricing scheme. Allowing private information about processing times, we prove that the pivotal mechanism is coordinating, i.e., agents are better off by reporting their true processing times, and by participating in the subcon- tracting. We show that the subcontractor’s maximum revenue with any coordinating mechanism under private information equals the lower bound of that with coordinating pricing schemes under complete information. Finally, we address the asymmetric case where agents obtain savings at different rates per unit reduction in completion times. We show that coordinating pricing schemes do not always exist in this case. As a common supply chain management practice, manufac- turers take advantage of external resources to alleviate the burden of internal operations through subcontracting and outsourcing. While outsourcing externalizes some internal operations, subcontracting allows manufacturers to carry out jobs both internally and externally (Van Mieghem 1999). In this manner, subcontracting enables a manufacturer to speed up the completion times of his jobs. Instances of subcontracting practices can be found in quick-response industries characterized by volatile demand and inﬂexi- ble capacities, e.g., metal fabrication industry (Parmigiani 2003), electronics assembly (Webster et al. 1997), high-tech manufacturing (Aydinliyim and Vairaktarakis 2011), textile production, and engineering services (Taymaz and Kiliçaslan 2005). Although a considerable number of papers in the literature analyzes the subcontracting strategies in production planning and scheduling problems of manufacturers (Kamien and Li 1990; Tan and Gershwin 2004; Chen and Li 2008; Lee and Sung 2008), the subcontractors’ scheduling problems have received less attention. In reality, a subcontractor by itself faces a limited capacity while providing service to several manufacturers. Given the time-sensitive nature of subcon- tracting operations, the subcontractor’s schedule has critical impact on the performance of manufacturers as well as the supply chain. Decentralized subcontractor scheduling with divisible jobs Behzad Hezarkhani1,2 · Wiesław Kubiak2 Published online: 2 June 2015 Published online: 2 June 2015 Published online: 2 June 2015 © The Author(s) 2015. This article is published with open access at Springerlink.com J Sched (2015) 18:497–511 DOI 10.1007/s10951-015-0432-2 J Sched (2015) 18:497–511 DOI 10.1007/s10951-015-0432-2 1 Department of Industrial Engineering, Eindhoven Technical University, Eindhoven, The Netherlands 2 Faculty of Business Administration, Memorial University, St. John’s, NL, Canada B Behzad Hezarkhani b.hezarkhani@tue.nl Wiesław Kubiak wkubiak@mun.ca 1 Introduction The lack of due attention to the subcontrac- tors’ operations can cause signiﬁcant complications in the extended supply chain. A well-documented real-life exam- ple of this issue has been reported in Boeing’s Dreamliner supply chain where the overloaded schedules of subcontrac- tors, each working with multiple suppliers, resulted in long delays in the overall production due dates (see Vairaktarakis (2013) and the references therein). Keywords Scheduling · Divisible jobs · Subcontracting · Coordination · Mechanism design B Behzad Hezarkhani b.hezarkhani@tue.nl Wiesław Kubiak wkubiak@mun.ca 12 3 3 J Sched (2015) 18:497–511 498 for every agent. In particular, Sect. 3 characterizes efﬁcient allocations of the centralized solution. Section 4 provides sufﬁcient conditions for the existence of coordinating pric- ing schemes and it introduces a family of pricing schemes that are strongly coordinating. With a strongly coordinating pricing scheme, the efﬁcient allocations in the centralized solutions are uniquely optimal for all agents. Moreover, we show a lower bound on the total payments for any coor- dinating pricing scheme. Finally we show that with the appropriate choice of a single parameter, our proposed pric- ingschemesenablethesubcontractortoobtainatotalrevenue anywhere between the lower bound and the maximum total savings. An important feature of subcontractor’s scheduling prob- lem is that the jobs belong to different manufacturers who are concerned with the processing of their own jobs only. Unlike centralized systems where a single decision maker controls all the relevant decision variables and possesses all the necessary information, the agents in a decentralized system have some control over their individual decisions and/or are privately informed about some aspects of the system. Therefore, in order for the system to achieve a particular objective a mechanism is needed that motivates the agents to make their decisions and reveal their private information in a way that the system’s ultimate objective is attained indirectly. In this paper, we focus on the efﬁciency objective, that is, we are seeking mechanisms that result in subcontracting schedules that maximize the total savings obtained by all manufacturers. In other words, we address the problem of coordination in decentralized subcontracting systems. In Sect. 5, we allow the true processing time of each job to be a private information of its agent and we address the intricacies resulting from agents possibly lying by report- ing false processing times in order to obtain their preferred subcontracting intervals. 1 Introduction In order to achieve efﬁciency, how- ever, the subcontractor elicits the true processing times of jobs using certain mechanism. We draw upon the class of efﬁcient and incentive compatible mechanisms and on the pivotal mechanism (Vickrey 1961; Clarke 1971; Groves 1973) in particular. The underlying reason for this choice is that the pivotal mechanism is the only efﬁcient and incentive compatible mechanism that always results in non- negative payments from agents to the subcontractor. This is a desirable property as agents must not be paid if their jobs are processed by the subcontractor in the subcontrac- tor scheduling problem. We obtain a simple closed-form formula for the payments in the pivotal mechanism and prove that the pivotal mechanism results in truth-telling being the unique optimal choice of all agents except the one scheduled last on the subcontractor’s machine who can possibly exaggerate his processing time without affect- ing anyone’s utility. This result is particularly interesting as “uniqueness of equilibrium might be thought of as the exception rather than the rule” (Jackson 2000). Since we also show that the mechanism guarantees that all agents are better off by subcontracting, we actually prove that the pivotal mechanism coordinates the decentralized subcon- tracting problem under private information. Finally, we show that under any coordinating mechanism, the subcontractor’s revenue is equivalent to the lower bound of that with the coordinating pricing schemes. Therefore, coordinated sys- tem under private information never generates higher total revenue for the subcontractor than that under complete infor- mation. The mechanisms considered in this paper are pricing and payment schemes that the subcontractor announces before the manufacturers choose their most desirable subcontract- ing intervals. Such mechanisms are in fact common in practice. An example is the online reservation system imple- mented by the Semiconductor Product Analysis and Design Enhancement (SPADE) center of the Hong Kong Univer- sity of Science and Technology1 wherein the semiconductor companies choose service time intervals in a ﬁrst-come-ﬁrst- book manner and in consideration of an announced price list for services in different time intervals. Naturally, the design of mechanisms in the decentral- ized subcontracting systems must consider the utility of agents, i.e., savings due to subcontracting minus payments, as well as the subcontractor’s revenue to ensure that all par- ties are sufﬁciently motivated to participate and operate in the system. 1 http://www.ust.hk/spade 2 Related work Aydinliyim and Vairaktarakis (2010) study a cooperative game where coalitions of agents reschedule their reserved time intervals to obtain savings. They prove non-emptiness of the core and the convexity of the corresponding game. Cai and Vairaktarakis (2012) investigate a problem with over- time and tardiness costs where the subcontractor announces the prices of time intervals before agents book their most preferred time intervals in a ﬁrst-come-ﬁrst-book manner. They show the balancedness of the corresponding coopera- tive game and implement the VCG mechanisms to elicit the private information of agents. Bukchin and Hanany (2007) compare the costs of a scheduling system comprised of mul- tiple capacitated agents and an uncapacitated subcontractor in decentralized and centralized systems. Qi (2012) investi- gates a subcontractor’s pricing problem with a single agent having multiple jobs which can be subcontracted (though not partially) to reduce the tardiness costs. Thedecentralizedschedulingproblemshavebeenconsidered in cooperative and non-cooperative settings. In the former, agents (jobs) are able to communicate and coordinate strategies. The early work of Curiel et al. (1989) studies the cost allocation problem in a cooperative game based on the perennial scheduling problem of Smith (1956). A survey of related research is given by Curiel et al. (2004). In the non-cooperative settings agents choose their strate- gies individually and in competition with other agents. This paper falls in this category. In the former, agents (jobs) are able to communicate and coordinate strategies. The early work of Curiel et al. (1989) studies the cost allocation problem in a cooperative game based on the perennial scheduling problem of Smith (1956). A survey of related research is given by Curiel et al. (2004). In the non-cooperative settings agents choose their strate- gies individually and in competition with other agents. This paper falls in this category. Heydenreich et al. (2007) provide an introduction to and a literature review of problems arising in the non-cooperative decentralized scheduling. One important problem pertains to determining the equilibria of individual decisions and the quality of the corresponding solution compared with that of the centralized optimal solution. The pioneering work of KoutsoupiasandPapadimitriou(1999),whichgaverisetothe literature on price of anarchy, analyzes the worst-case per- formance of a decentralized scheduling system with parallel machines in comparison with that in the centralized system. The subcontracting problem related to the one addressed in this paper was ﬁrst studied by Vairaktarakis and Aydin- liyim (2007) where they compare the performance in decen- tralized and centralized settings. 2 Related work Building upon the same model, Vairaktarakis (2013) analyzes the outcomes of a decentralized subcontracting system under different proto- cols announced by the subcontractor. However, both papers assume complete information and neither provides coordi- nating pricing schemes for the problem. The model consid- ered in this paper generalizes the model of Vairaktarakis and Aydinliyim (2007) by allowing an agent to use more than one interval on the subcontractor’s machine. This generalization is critical for a coordinating pricing schemes which need to give the agents freedom in choosing how many intervals on the subcontractor’s machine to buy—the Vairaktarakis and Aydinliyim (2007) model would a priori limit this choice to at most a single interval which does not make their model adequate for the study of pricing schemes. As the outcomes of a decentralized system depend on the “policies” employed to handle the agents, another impor- tant problem addresses a better design of such policies. The coordination mechanisms discussed by Christodoulou et al. (2004) seek policies which improve the performance of a decentralized parallel sequencing system—Immorlica et al. (2005) review and extend this line of research. Ideally, such policies could make the decentralized system as efﬁcient as the centralized system. Wellman et al. (2001) study pricing schemesforaschedulingproblemthatcouldachievethisgoal and show that such pricing schemes might not exist in gen- eral. Kutanoglu and Wu (1999) report similar non-existence results for another scheduling problem. Even if the existence of such pricing schemes could be proven, the problem of ﬁnd- ing the pricing scheme might be NP-hard (Chen et al. 2004). It is worth noticing that the models in Vairaktarakis and Aydinliyim (2007) and Vairaktarakis (2013) borrow from the concept of divisible jobs introduced in the context of job shops by Anderson (1981), and in the context of distributed computer systems scheduling by Bharadwaj et al. (1996), see also Drozdowski (2009) for a more recent review. g p g g Private information further complicates the coordination problem. The well-known incentive compatible mechanisms draw upon payment schemes to truthfully elicit the pri- vate information of the agents. The Vickery–Clarke–Groves (VCG) mechanisms (Vickrey 1961; Clarke 1971; Groves 1973) characterize all efﬁcient mechanisms that make truth- telling an undominated strategy of all agents. For single machine sequencing, Suijs (1996) shows that there exists no incentive compatible and individually rational mecha- nism that results in the total payment of zero. 1 Introduction Inthispaper,westudyasubcontractorschedulingproblem with several manufacturer agents and a single subcontractor that carries out the agents’ jobs on a single machine. Of par- ticular interest to this paper is the scenario where a divisible job can be processed simultaneously on the manufacturer’s private machine as well as on the subcontractor’s machine. The reduction in completion time of an agent’s job obtained by such parallel processing provides monetary saving for that agent. The paper is organized as follows. Section 2 contains a review of related work. In Sects. 3 and 4, we assume complete information and give pricing schemes that coor- dinate the decentralized system. That is the pricing schemes enforce a choice of subcontracting time intervals that coin- cides with efﬁcient allocations of the centralized solution In Sect. 6, we address the asymmetric case where the agents obtain savings at different rates. The efﬁcient central- ized allocations in this case are more difﬁcult to characterize. Moreover, we show that it is impossible to devise a coordinat- ing pricing scheme in general for this case. Section 7 contains the concluding remarks. 3 3 J Sched (2015) 18:497–511 499 2 Related work Nisan and Ronen (2007) propose payment schemes that guarantee a certain performance for a decentralized scheduling problem for which the payment schemes of VCG mechanisms are NP-hard to compute. The distributed computer systems provide another impor- tant application area for the results obtained in this paper, where agents carry out their computations on their private machines as well as buy computational time on shared CPUs with available capacity. 2 We refer to i as a job or an agent depending on the context. 3 The problem Consider a set of agents N = {1, . . . , n}. At time t = 0, agent i ∈N has a divisible job with processing time pi > 0.2 Let The decentralized subcontracting and outsourcing prob- lems have also been addressed in a number of papers. 3 500 J Sched (2015) 18:497–511 Fig. 1 Valuation in subcontracting p = (p1, . . . , pn) be the vector of processing times. We assume, except in Sect. 5, that p is given. An agent i has its own private machine to do its job in time pi, besides private machines a subcontractor is available that can process any portionof jobi onits machinethat canbesharedbyall agents. Thus, by subcontracting, agent i can reduce the completion time of its job to less than pi. Let Ti = ∪mi k=1T k i be the total time allocated to agent i consisting of mi non-overlapping subintervals T k i = [tk i , tk i + ¯tk i ) where tk i ≥0 and ¯tk i > 0 are the start time and the duration of the kth subinterval, respec- tively. The subintervals of agent i are indexed by the order of their start times. An allocation T = {Ti\|i ∈N} is a set of allocations Ti on subcontractor’s machine. Allocation T is feasible if and only if for any i, j, k, and k′ the subintervals [tk i , tk i + ¯tk i ) and [tk′ j , tk′ j + ¯tk′ j ) do not overlap. Let T denote the set of all feasible allocations T for the agents in N. Fig. 1 Valuation in subcontracting Fig. 1 Valuation in subcontracting Our model allows preemptions on subcontractor’s machine, that is the portion of a job allocated to the sub- contractor’s machine (or the subcontracted part of a job) is allowed to be executed in more than one disjoint time interval on that machine. This renders our model to be more general and arguably closer to real-life than the one in Vairaktarakis and Aydinliyim (2007) where only at most one time interval on subcontractor’s machine is allowed for any subcontracted part. 3 The problem The total saving If Ti = {[ti, ¯ti)} is a single interval, the Eq. (2) simpliﬁes to υi(Ti) = max min (pi −ti) /2, ¯ti , 0 . The total saving of a feasible allocation T is the sum of savings of all agents, that is υ(T ) = i∈N υi(Ti). 3 The problem Consequently two main decisions need to be made for each job in the model: one is the size of the subcontracted part of a job (this part can be executed on the subcontractor’s machine simultaneously with the remaining part of the job executed on private machine—thus the term divisible jobs), the other as to how to execute the subcontracted part (this part can possibly be executed in several disjoint time intervals— thus the term preemptions on the subcontractor’s machine). processing time of job i is pi −υi(T 1 i ) on i’s private machine. Thus the saving due to the kth subinterval T k i to agent i is obtained recursively by υi(T k i ) = max min pi −k−1 l=1 υi T l i −tk i 2 , ¯tk i , 0 (1) (1) total saving due to the allocation Ti is calculated by summing up the savings obtained by all of its subintervals: total saving due to the allocation Ti is calculated by summing up the savings obtained by all of its subintervals: υi(Ti) = mi k=1 υi T k i . (2) (2) The saving obtained by agent i from an allocation Ti is calculated recursively as follows. Assume that initially, agent i uses its private machine only in the interval [0, pi], i ∈N. Take the earliest interval allocated to i, T 1 i . If the start time t1 i < pi, then a portion of the remainder of job i done after t1 i , i.e., pi −t1 i , on i’s private machine can be transferred to the subcontractor. The most efﬁcient way to do this is for agent i to split the remainder equally between its private and subcontractor’s machines, unless the duration of the interval is too short (see Fig. 1). If the duration of the interval is too short, i.e., ¯t1 i < (pi −t1 i )/2, then T 1 i is fully utilized by i on the subcontractor’s machine. Therefore, by utilizing the allocated interval T 1 i , agent i can reduce the ﬁnish time of its job by an amount equal to If Ti = {[ti, ¯ti)} is a single interval, the Eq. (2) simpliﬁes to υi(Ti) = max min (pi −ti) /2, ¯ti , 0 . 3.1 Characterization of efﬁcient (centralized) allocations The objective of the centralized problem is to ﬁnd efﬁcient allocations on the subcontractor machine for N. An efﬁcient allocation T is a feasible allocation that maximizes the total saving, that is T ∈arg max T ∈T υ(T ). (3) (3) T ∈arg max T ∈T υ(T ). υi(T 1 i ) = max min pi −t1 i 2 , ¯t1 i , 0 . Let T = {Ti\|i ∈N} be an efﬁcient allocation. We have the following two simple observations that hold for any efﬁcient allocation. Clearly, the allocation Ti cannot be utilized at all by agent i if t1 i ≥pi. The saving obtained by the next interval can be calculated in the same manner considering that the new Observation 1 For i ∈N, job i does not ﬁnish on the sub- contractor’s machine later than on agent’s i private machine. 123 J Sched (2015) 18:497–511 501 Observation 2 The subcontractor’s machine is never idle when some agent’s private machine is busy. Aydinliyim (2007) observe that, for any efﬁcient allocation T with mi = 1, i ∈N, each job ﬁnishes simultaneously on its own private and subcontractor’s machines, that is We now prove that all efﬁcient allocations are non- preemptive. pi −¯ti = ti + ¯ti for i ∈N. (4) (4) Lemma 1 No efﬁcient allocation on subcontractor’s machine is preemptive. Lemma 1 No efﬁcient allocation on subcontractor’s machine is preemptive. They also show that for mi = 1, i ∈N, the agents’ efﬁ- cient allocations Ti are ordered in non-decreasing order of pi. Thus, we assume hereafter in this section and in Sect. 4 that N is arranged in the non-decreasing order of processing times. In this manner, job i would be sequenced in ith posi- tion on subcontractor’s machine. Finally, by Observation 2, there is no idle time on the subcontractor machine in efﬁcient allocations, thus we have They also show that for mi = 1, i ∈N, the agents’ efﬁ- cient allocations Ti are ordered in non-decreasing order of pi. Thus, we assume hereafter in this section and in Sect. 4 that N is arranged in the non-decreasing order of processing times. In this manner, job i would be sequenced in ith posi- tion on subcontractor’s machine. Finally, by Observation 2, there is no idle time on the subcontractor machine in efﬁcient allocations, thus we have Proof By contradiction. Let T be an efﬁcient and preemp- tive allocation and let n′ ≤n be the number of agents with allocations on the subcontractor’s machine. We show that then there exists another feasible allocation with higher total saving. Suppose that mi > 1 for some i ∈N. T ∈arg max T ∈T υ(T ). Without loss of generality we take the largest such i. Let T m j j = [t m j j , t m j j + ¯t m j j ) be the allocation immediately preceding T mi i . We have i ̸= j, and t m j j + ¯t m j j = tmi i by Observa- tion 2. Consider the following modiﬁcation: [Step 1] delay T mi−1 i by tmi i −(tmi−1 i + ¯tmi−1 i ); speed up all the alloca- tions between T mi−1 i and T mi i by ¯tmi−1 i . Thus job i would be preempted one less time. By Observation 1 this mod- iﬁcation does not change the completion time of any job. Therefore, total saving remains unchanged. [Step 2] Increase the duration of the last allocation of j by ¯tmi−1 i /2, so that by Observation 1 the private machine of agent j ﬁnishes ear- lier by ¯tmi−1 i /2. For k = i, . . . , n′, i.e., jobs including and after i, start the last allocation of k by ¯tmi−1 i /2k−i+1 later and ﬁnish it min ¯tmi−1 i /2k−i+2, ¯tk later on the subcontrac- tor’s machine (observe that if ¯tmi−1 i /2k−i+2 ≥¯tk then k will no longer be executed on the subcontractor’s machine) and min ¯tmi−1 i /2k−i+2, ¯tk later on agent k private machine. t1 = 0 and ti = ti−1 + ¯ti−1 for i > 1. (5) (5) The unique solution of the recursive equations (4) and (5) is given in the following theorem. Theorem 1 (Efﬁcient allocation) (Vairaktarakis and Aydin- liyim 2007) For efﬁcient allocations with mi = 1 for i ∈N, we have ¯ti = pi 2 − i−1 k=1 ¯tk 2 = pi 2 − i−1 k=1 pk 2i+1−k . (6) (6) By this theorem υi(T ) = υi(Ti) = ¯ti (7) υi(T ) = υi(Ti) = ¯ti (7) The resulting allocation is feasible. Furthermore, jobs in N \ { j, i, . . . , n′} complete as before Step 2, and for the jobs in { j, i, . . . , n′} the total saving increases by and thus, υ(T ) = i∈N ¯ti. Equations (6) and (5) deﬁne vectors of efﬁcient durations ¯t = (¯t1, . . . , ¯tn) and start times t = (t1, . . . T ∈arg max T ∈T υ(T ). , tn) on subcontractor’s machine. Finally, we let tn+1 = tn + ¯tn denote the completion time of the efﬁcient allocations, i.e., the makespan of efﬁcient allocations. ¯tmi−1 i 2 − ¯tmi−1 i 4 −· · · −min ¯tmi−1 i 2n′−i+2 , ¯tn′ ≥ ¯tmi−1 i 2 − ¯tmi−1 i 4 −· · · − ¯tmi−1 i 2n′−i+2 > 0. Though multiple efﬁcient allocations exist as long as there are jobs with equal processing times, in each one of them the jobs in the same position on subcontractor’s machine start at the same time and have the same efﬁcient durations. This observation is key to the coordinating pricing scheme developed in the next section. Hence, the alternative allocation has a higher total saving which contradicts the efﬁciency of T . ⊓⊔ The lemma excludes allocations with preemptions on sub- contractor’s machine from the set of efﬁcient allocations. This result is key for our coordinating pricing scheme in Sect. 4 since the efﬁcient allocations with preemptions on subcontractor’s machine could make the existence of coordi- nating pricing scheme questionable or possibly more difﬁcult to prove. 4 Strongly coordinating pricing schemes The fact that ti + 2¯ti = pi and q is non-increasing, implies that agent i cannot choose another interval to increase its valuation and, at the same time, reduce its payment. In order to improve its utility, the agent i may be able to choose another interval to: (a) increase its valuation as well as payment such that the added valuation is greater than the additional payment, or (b) decrease its valuation as well as payment such that the saving in payment is greater than the reduction in valuation. We enforce conditions on q such that neither (a) nor (b) could possibly happen for any job. Deﬁnition 1 (Coordinating pricing scheme) The pricing scheme q is coordinating if for any T = (T1, . . . , Tn), T ∈E, and any T = (T1, . . . , Tn), T ∈T \ E, πq(Ti) −πq(Ti) ≤υi(Ti) −υi(Ti) (10) (10) foreachi ∈N.Thepricingschemeq isstrongly coordinating if (10) holds strictly for each i ∈N. A coordinating pricing scheme results in the situation where no agent would be worse off by choosing an efﬁcient alloca- tion. However, the implementation of a coordinating pricing scheme may not necessarily result in the efﬁciency of the system. This is due to the possibility that an agent chooses an interval which is not a part of any efﬁcient allocation. Such a choice could hinder forthcoming agents to select their efﬁ- cient allocations. Therefore, the strong coordination requires all agents to exclusively choose efﬁcient allocations. (a) Suppose that the agent i chooses Ti in a way that υi(Ti) −υi(Ti) = ϵ, for some ϵ > 0. Note that this is not an option for i = 1. As q is non-increasing, Ti has the cheapest payment if it starts as late as possible. Therefore, Ti = [ti −2ϵ, ti+1 −ϵ) is the cheapest alternative interval for agent i which results in ϵ improvement in its valuation. Note that i cannot have negative start time, thus ϵ ≤ti/2. From Deﬁnition 1 it follows that for the pricing scheme q to be coordinating, it must hold for any i = 2, . . . , n and for every 0 < ϵ ≤ti/2 that A natural class of pricing schemes to consider for schedul- ing consists of those with non-increasingq where agents have to pay a higher price for earlier intervals. 4 Strongly coordinating pricing schemes . , n −1 and every ϵ, 0 < ϵ ≤¯ti/2, and for i = n and every ϵ, 0 < ϵ ≤¯tn: πq(ti, ti + 2ϵ) −πq(ti+1, ti+1 + ϵ) ≤ϵ. (12) C1. For any i = 2, . . . , n and every ϵ, 0 < ϵ ≤¯ti−1/2: πq(ti −2ϵ, ti) −πq(ti+1 −ϵ, ti+1) ≥ϵ; (11) (11) The subcontractor announces its pricing scheme, and subse- quentlyagenti buys Ti inaﬁrst-come-ﬁrst-servemanner.The agent makes its decision so as to maximize its utility, which for a given q, and assuming quasilinear utilities, equals C2. For any i = 1, . . . , n −1 and every ϵ, 0 < ϵ ≤¯ti/2, and for i = n and every ϵ, 0 < ϵ ≤¯tn: πq(ti, ti + 2ϵ) −πq(ti+1, ti+1 + ϵ) ≤ϵ. (12) (12) uq i (Ti) = υi(Ti) −πq(Ti), (8) (8) where where Proof All T ∈E deﬁne the same start times t = (t1, . . . , tn) and durations ¯t = (¯t1, . . . , ¯tn) as shown in the previous sec- tion. πq(Ti) = t∈Ti q(t)dt (9) (9) The individual rationality requires that for 0 ≤t < tn+1, q(t) must be less than 1, otherwise jobs would be either better off by not selecting the time with subcontracting price exceeding one or indifferent if the price equals one. Since q is non-increasing, the individual rationality holds if q(0) < 1. is the agent i’s payment for Ti made to the subcontractor. We require q to be a locally integrable function so that (9) always exists. Though each agent maximizes its utility with respect to q, the choice of q must ensure coordination in the decentral- ized system. That is q must guarantee that the agents choose their subcontracting intervals in the exact same manner as in some efﬁcient allocation T . We call such pricing schemes coordinating. Let E be the set of all efﬁcient allocations. We formally deﬁne. For q to be a coordinating pricing scheme, it must be that no agent can deviate from an efﬁcient allocation and improve its utility. The ﬁrst-come-ﬁrst-booked order breaks ties between jobs with equal processing times, if any, and picks a unique efﬁcient allocation T from E. In T agent i has the utility υi(Ti) −q(Ti). 4 Strongly coordinating pricing schemes When the choice of agent i consists of single interval only, i.e., Ti = [ti, ti + ¯ti), we let πq(Ti) = πq(ti, ti +¯ti). We now show that the class of non-increasing pricing schemes contain coordinating pricing schemes. πq (ti −2ϵ, ti+1 −ϵ) −πq (ti, ti+1) ≥ϵ. By the deﬁnition of πq in (9), the last inequality can be rewritten as (11) (see Fig. 2). We use induction and show that the latter would be the case if for any i = 2, . . . , n, (11) 4 Strongly coordinating pricing schemes In contrast to a centralized system where allocations are cho- sen for the agents so as to maximize the total saving, in a decentralized system the decisions are made by the agents in a distributed fashion. Though the agents are self-interested they need, by deﬁnition of coordinating mechanism, to col- lectively converge to an efﬁcient allocation. The convergence By Lemma 1, mi = 1 for i ∈N in any efﬁcient allocation T that is Ti = [ti, ti + ¯ti) for i ∈N. Vairaktarakis and 12 3 502 J Sched (2015) 18:497–511 depends on a mechanism used in the collective decision mak- ing. In this section we design such a mechanism based on a pricing scheme. A pricing scheme is a function q, deﬁned on t ≥0, which determines a price q(t) ≥0 for acquiring the time t on the subcontractor’s machine by any agent. The mechanism based on a pricing scheme q works as follows. The subcontractor announces its pricing scheme, and subse- quentlyagenti buys Ti inaﬁrst-come-ﬁrst-servemanner.The agent makes its decision so as to maximize its utility, which for a given q, and assuming quasilinear utilities, equals depends on a mechanism used in the collective decision mak- ing. In this section we design such a mechanism based on a pricing scheme. A pricing scheme is a function q, deﬁned on t ≥0, which determines a price q(t) ≥0 for acquiring the time t on the subcontractor’s machine by any agent. The mechanism based on a pricing scheme q works as follows. The subcontractor announces its pricing scheme, and subse- quentlyagenti buys Ti inaﬁrst-come-ﬁrst-servemanner.The agent makes its decision so as to maximize its utility, which for a given q, and assuming quasilinear utilities, equals Theorem 2 (Sufﬁcientconditions)Anon-increasingpricing scheme q is coordinating if q(0) < 1 and the following two conditions are met: depends on a mechanism used in the collective decision mak- ing. In this section we design such a mechanism based on a pricing scheme. A pricing scheme is a function q, deﬁned on t ≥0, which determines a price q(t) ≥0 for acquiring the time t on the subcontractor’s machine by any agent. The mechanism based on a pricing scheme q works as follows. C1. For any i = 2, . . . , n and every ϵ, 0 < ϵ ≤¯ti−1/2: πq(ti −2ϵ, ti) −πq(ti+1 −ϵ, ti+1) ≥ϵ; (11) C2. For any i = 1, . . 3 3 123 J Sched (2015) 18:497–511 503 Fig. 2 Non-increasing pricing schemes Thus, (13) would be the case if for every ϵ′ such that 0 < ϵ′ ≤¯ti−j−1/2 we have, πq ti−j −2ϵ′, ti−j −πq ti−j+1 −ϵ′, ti−j+1 ≥ϵ′. Condition C1 for the job i −j ensures that the latter holds. Therefore, if for any i = 2, . . . , n, (11) holds (strictly) for every 0 < ϵ ≤¯ti−1/2, then (11) holds (strictly) for every 0 < ϵ ≤ti/2. (b) It is straightforward to see that the interval which reducesthevaluationoftheagenti byϵ > 0,whilehavingthe largest decrease in its payment, is Ti = (ti + 2ϵ, ti+1 + ϵ), as long as ϵ ≤¯ti. Therefore, for q to be coordinating it must hold for every i = 1, . . . , n and for every ϵ such that 0 < ϵ ≤¯ti that, πq (ti, ti+1) −πq (ti + 2ϵ, ti+1 + ϵ) ≤ϵ. By the deﬁnition of πq in (9), the last inequality can be rewritten as (12). We use induction to show that for i = 1, . . . , n −1 the latter would be the case if (11) holds for every 0 < ϵ ≤¯ti/2, i.e., the condition stated in C2. Fix i = 1, . . . , n −1 and suppose that for j, 1 ≤j < i, (12) holds for every ϵ such that, Fig. 2 Non-increasing pricing schemes holds for every 0 < ϵ ≤¯ti−1/2, i.e., the condition stated in C1. This holds trivially for i = 2 as t2 = ¯t1. Fix i = 3, . . . , n and suppose that for j, 1 ≤j < i, (11) holds for every ϵ such that holds for every 0 < ϵ ≤¯ti−1/2, i.e., the condition stated in C1. This holds trivially for i = 2 as t2 = ¯t1. Fix i = 3, . . . , n and suppose that for j, 1 ≤j < i, (11) holds for every ϵ such that 0 < ϵ ≤min ⎧ ⎨ ⎩ j k=1 ¯ti+k−1/2, ¯ti ⎫ ⎬ ⎭. 0 < ϵ ≤ j k=1 ¯ti−k/2. 3 qO(t)= ⎧ ⎪⎨ ⎪⎩ 1 −2i−1δ +κi 1 −2(t −ti)/¯ti if ti ≤t < ti + ¯ti, i ∈N 1 −2n−1δ −κn if t ≥tn + ¯tn Condition C2 for the job i + j ensures that the latter holds. Therefore, if for i = 1, . . . , n −1, (12) holds (strictly) for every 0 < ϵ ≤¯ti/2, then (12) holds (strictly) for every 0 < ϵ ≤¯ti. ⊓⊔ (15) (15) ⊓⊔ The conditions in Theorem 2 are designed to make the deviations from t = (t1, . . . , tn) undesirable for all agents in the sense that the agent’s utilities would be reduced if they choose any intervals other than those deﬁned by t. Clearly with quasilinear utilities any agent can increase its utility either by choosing an interval that either increases its valua- tion (saving due to reduction in completion time) or reduces its payment (see Fig. 2). With a non-increasing pricing scheme attaining both of these at the same time is impos- sible. Therefore, a deviation which increases (decreases) an agent’s valuation, simultaneously increases (decreases) its payment. Proof The assumptions on κ ensure that: (a) qO is non- increasing, (b) qO(0) < 1, and (c) qO(t) ≥0 for t ≥0. To verify (a), it sufﬁces to consider the points of (possible) discontinuity, i.e., t = ti+1 for i = 1, . . . , n −1. There, it must hold that 1 −2i−1δ −κi ≥1 −2iδ + κi+1 or κi + κi+1 ≤2i−1δ. (16) (16) κi + κi+1 ≤2i−1δ. By extending the assumption κi < κi+1/2 we get Only strongly coordinating pricing schemes can guarantee efﬁcient schedules in the decentralized system. In order to introduce a family of strongly coordinating pricing schemes, we need the following technical lemma which states that the efﬁcientdurationofanyjobonsubcontractor’smachineisnot longer than twice the efﬁcient duration of the job proceeding it. κi < κn/2n−i (17) (17) κi < κn/2n−i for i = 1, . . . , n −1. Thus, κi +κi+1 < 3κn/2n−i. It follows that for (16) to hold, we need Lemma 2 For i = 1, . . . , n −1, ¯ti ≤2¯ti+1. Lemma 2 For i = 1, . . . , n −1, ¯ti ≤2¯ti+1. κn ≤2n−1δ/3. (18) κn ≤2n−1δ/3. 3 We show that (12) holds as well for every ϵ such that, We show that (11) holds as well for every ϵ such that, 0 < ϵ ≤min ⎧ ⎨ ⎩ j+1 k=1 ¯ti+k−1/2, ¯ti ⎫ ⎬ ⎭. 0 < ϵ ≤ j+1 k=1 ¯ti−k/2. In case j k=1 ¯ti+k−1/2 < ¯ti, observe that the latter would hold if for every 0 < ϵ′ ≤min ¯ti+ j/2, ¯ti we have, Observe that this would be the case if for every ϵ′ such that 0 < ϵ′ ≤¯ti−j−1/2 we have, πq ti+ j, ti+ j + 2ϵ′ −πq ⎛ ⎝ti+1+ j k=1 ¯ti+k−1/2, ti+1 + j k=1 ¯ti+k−1/2 + ϵ′ ⎞ ⎠ ≤ϵ′. (14) πq ti−j −2ϵ′, ti−j −πq ⎛ ⎝ti+1 − j k=1 ¯ti−k/2 −ϵ′, ti+1 − j k=1 ¯ti−k/2 ⎞ ⎠≥ϵ′. (13) ≤ϵ′. (14) (13) As q is non-increasing, we have As q is non-increasing, we have As q is non-increasing, we have As q is non-increasing, we have As q is non-increasing, we have As q is non-increasing, we have As q is non-increasing, we have As q is non-increasing, we have πq ⎛ ⎝ti+1 − j k=1 ¯ti−k/2 −ϵ′, ti+1 − j k=1 ¯ti−k/2 ⎞ ⎠ ≤πq ti−j+1 −ϵ′, ti−j+1 πq ⎛ ⎝ti+1 + j k=1 ¯ti+k−1/2, ti+1 + j k=1 ¯ti+k−1/2 + ϵ′ ⎞ ⎠ ≥πq ti+ j+1, ti+ j+1 + ϵ′ πq ⎛ ⎝ti+1 − j k=1 ¯ti−k/2 −ϵ′, ti+1 − j k=1 ¯ti−k/2 ⎞ ⎠ ≤πq ti−j+1 −ϵ′, ti−j+1 πq ⎛ ⎝ti+1 + j k=1 ¯ti+k−1/2, ti+1 + j k=1 ¯ti+k−1/2 + ϵ′ ⎞ ⎠ ≥πq ti+ j+1, ti+ j+1 + ϵ′ 3 504 504 J Sched (2015) 18:497–511 κn ≤2n−1δ/3 where 0 < δ ≤3/2n+1. The following pricing scheme is strongly coordinating: κn ≤2n−1δ/3 where 0 < δ ≤3/2n+1. The following pricing scheme is strongly coordinating: Thus, (14) would be the case if for every ϵ′ such that 0 < ϵ′ ≤min ¯ti+ j/2, ¯ti we have πq ti+ j, ti+ j + 2ϵ′ −πq ti+ j+1, ti+ j+1 + ϵ′ ≤ϵ′. 3 (18) Proof From (6) we get ¯ti −2¯ti+1 = pi 2 − i−1 k=1 ¯tk 2 −pi+1 + i−1 k=1 ¯tk + pi 2 − i−1 k=1 ¯tk 2 = pi −pi+1. In order for (b) to hold we must have 1 −δ + κ1 < 1 or κ1 < δ. Note that (17) and (18) yield κi < 2i−1δ/3 for i = 1, . . . , n −1 which implies that κ1 < δ/3. Thus (b) holds. Given that qO is non-increasing, to verify (c) we must check the non-negativity of qO(t) at t ≥tn + ¯tn. Note that from (17) we get The agents in N are sequence according to non-decreasing order of processing times, thus we have pi ≤pi+1 which obtains ¯ti ≤2¯ti+1. ⊓⊔ 1 −2n−1δ −κn ≥1 −2n−1δ −2n−1δ/3. Note that in Lemma 2, the equality holds for jobs with equal processing times. More precisely, pi = pi+1 if and only if ¯ti = 2¯ti+1. Hence, for (c) to hold we need δ ≤3/2n+1. Hence, for (c) to hold we need δ ≤3/2n+1. We now check the conditions in Theorem 2. Observe that qO is piecewise linear and has a constant slope in between any consecutive points of possible discontinuity. We are now ready to give a family of non-increasing pricing schemes that are strongly coordinating, that is, any deviation from efﬁcient allocation by any agent results in utility loss for that agent. With regard to condition C1, note that for 0 < ϵ ≤ ¯ti−1/2 both πqO (ti −2ϵ, ti) and πqO (ti+1 −ϵ, ti+1) can be obtained by calculating the areas of the corresponding trapezoids. Let t−= limϵ→0+(t −ϵ). For any i = 2, . . . , n Theorem 3 Consider the coefﬁcient set κ = {κ1, . . . , κn} such that for i = 1, . . . , n −1, 0 < κi < κi+1/2 and 0 < 123 123 J Sched (2015) 18:497–511 505 we have (20) πqO (ti, ti + 2ϵ) −πqO (ti+1, ti+1 + ϵ) < ϵ πqO (ti −2ϵ, ti) −πqO (ti+1 −ϵ, ti+1) = 1 22ϵ qO (ti −2ϵ) + qO t− i −1 2ϵ qO (ti+1 −ϵ) + qO t− i+1 = 1 22ϵ 1−2i−2δ−κi−1 1−4ϵ ¯ti−1 +1−2i−2δ−κi−1 ! −1 2ϵ 1 −2i−1δ −κi 1 −2ϵ ¯ti + 1 −2i−1δ −κi ! 3 = ϵ 1 −2κi−1 1 −2ϵ ¯ti−1 + κi 1 −ϵ ¯ti ! . If ¯ti/2 ̸= ¯ti+1 then the latter also holds for ϵ = ¯ti/2. In case of ¯ti/2 = ¯ti+1, (20) would hold as equality for ϵ = ¯ti/2. However, this requires pi = pi+1 which implies the existence of an alternative efﬁcient allocation. Hence, the choice of any later intervals for a given agent i which is not part of an efﬁcient allocation results in the loss of utility for that agent. Also, for jobs with equal processing times the choice of a later alternative efﬁcient intervals does not alter the utility to the corresponding agents. Consideringtheabove,weconcludethatqO isacoordinat- ing pricing scheme where for any T ∈E and every T ∈T\E, it holds for all i ∈N that πq(Ti)−πq(Ti) < υi(Ti)−υi(Ti). ⊓⊔ ⊓⊔ From Lemma 2 we know that ¯ti−1 ≤2¯ti and consequently 1 −2ϵ/¯ti−1 ≤1 −ϵ/¯ti. By assumption we have 2κi−1 < κi which leads to the observation that for every 0 < ϵ < ¯ti−1/2, To see how the pricing scheme introduced in Theorem 3 can coordinate the subcontractor scheduling problem ﬁrst note that for all agents with unequal processing times, corre- sponding efﬁcient allocations are exclusively the best choices of subcontracting intervals. For agents with equal processing time jobs, however, the best choices are multiple. Neverthe- less, qO would result in the situation where the number of best choices for the jobs with equal processing times are exactly the same as the number of those jobs. Therefore, a ﬁrst-come-ﬁrst-serve rule would result in strict coordination of individual choices. (19) πqO (ti −2ϵ, ti) −πqO (ti+1 −ϵ, ti+1) > ϵ (19) with regard to ϵ = ¯ti−1/2 we consider two cases. If ¯ti−1/2 ̸= ¯ti then at ϵ = ¯ti−1/2, (19) holds strictly as well which implies that (19) holds strictly for every 0 < ϵ ≤ti. Otherwise, if ¯ti−1/2 = ¯ti, then at ϵ = ¯ti−1/2, (19) holds as equality. However, this requires that pi−1 = pi which corresponds to an alternative efﬁcient allocation wherein job i is allocated with the interval allocated to job i −1. Hence, the choice of any earlier intervals for a given agent i which is not part of an efﬁcient allocation results in loss of utility for that agent. 3 Also, for jobs with equal processing times the choice of an earlier alternative efﬁcient intervals does not alter the utility to the corresponding agents. 4.1 Subcontractor’s revenue with coordinating pricing schemes Let Πq = i∈N πq(Ti) be the total payment from agents to the subcontractor under the pricing schemes q. We call Πq the subcontractor’s revenue under q. In this section we characterize the range of Πq with q being a coordinating pricing scheme. With regard to condition C2, note that for 0 < ϵ ≤¯ti/2 both πqO (ti, ti + 2ϵ) and πqO (ti+1, ti+1 + ϵ) can also be obtained by calculating the areas of the corresponding trape- zoids. for any i = 1, . . . , n −1 we have The coordinating pricing schemes introduced in Theo- rem 3 can extract (almost) all of the total saving υ(T ) from the agents by selecting δ small enough which is shown in the following theorem. πqO (ti, ti + 2ϵ) −πqO (ti+1, ti+1 + ϵ) = 1 22ϵ [qO (ti) + qO (ti + 2ϵ)] −1 2ϵ qO (ti+1) + qO (ti+1 + ϵ) = 1 22ϵ 1 −2i−1δ + κi + 1 −2i−1δ + κi 1 −4ϵ ¯ti ! −1 2ϵ 1 −2iδ + κi+1 + 1 −2iδ + κi+1 1 −2ϵ ¯ti+1 ! = ϵ 1 + 2κi 1 −2ϵ ¯ti −κi+1 1 − ϵ ¯ti+1 ! . Theorem 4 ΠqO = υ(T ) −δ i∈N 2i−1¯ti. Theorem 4 ΠqO = υ(T ) −δ i∈N 2i−1¯ti. Proof For every i ∈N we have, πqO (Ti) = ¯ti 1 −2i−1δ + κi + 1 −2i−1δ + κi −2κi /2 = ¯ti 1 −2i−1δ . Therefore ΠqO = i∈N ¯ti −δ i∈N 2i−1¯ti. Therefore ΠqO = i∈N ¯ti −δ i∈N 2i−1¯ti. ⊓⊔ ⊓⊔ Hence, the total payments can be made arbitrary close to the maximum possible which is the total saving υ(T ). From Lemma 2 we know that ¯ti ≤2¯ti+1 and consequently 1−2ϵ/¯ti ≤1−ϵ/¯ti+1. By assumption we have κi < κi+1/2. Therefore, for 0 < ϵ < ¯ti−1/2 we would have We now provide a lower bound for the total subcon- tractor’s revenue attainable under any coordinating pricing 123 3 506 J Sched (2015) 18:497–511 By induction on i it is easily veriﬁable that under the pricing scheme that satisﬁes the condition (21) and has the lowest possible price, every agent i ∈N pays an amount equal to ¯ti(1 −1/2n−i) to the subcontractor. Thus for every coordinating pricing scheme q, it must be the case that Πq ≥ i∈N ¯ti 1 −1/2n−i . ⊓⊔ scheme. We show in Corollary 1 that this bound is an upper bound on the subcontractor’s revenue attainable by any coor- dinating mechanism with private information. scheme. We show in Corollary 1 that this bound is an upper bound on the subcontractor’s revenue attainable by any coor- dinating mechanism with private information. Theorem 5 (Lower bound) For any coordinating pricing scheme q, we have Πq ≥ i∈N ¯ti 1 −1/2n−i . Theorem 5 (Lower bound) For any coordinating pricing scheme q, we have Πq ≥ i∈N ¯ti 1 −1/2n−i . ⊓⊔ Proof Let T ∈E be an efﬁcient allocation. If q is coordinat- ing, then for all i ∈N there is no Ti which results in higher utility than Ti. In particular, for any agent i = 2, . . . , n, the following deviation from efﬁcient allocation should not be proﬁtable: construct Ti by removing the last ϵ > 0 interval at the end of Ti and add instead another interval with dura- tion 2ϵ starting at time ti −2ϵ (to have feasible allocations it must be that ϵ ≤ti/2). With the new allocation, i ﬁnishes ϵ units of time earlier, i.e., υi(Ti)−υi(Ti) = −ϵ. Theorem 4 ΠqO = υ(T ) −δ i∈N 2i−1¯ti. To make this deviation unproﬁtable, the condition in (10) requires that We close with the observation that the lower bound in Theorem 5 is attainable by the following pricing scheme qL(t) = 1 −1/2n−i if ti ≤t < ti+1, i ∈N; 0 if t ≥tn. (26) (26) Clearly qL is non-increasing, and coordinating. To see the latter we check the conditions in Theorem 2: C1 holds since we have 1 −1/2n−i+1 2ϵ − 1 −1/2n−i ϵ = ϵ. Also, we have 1 −1/2n−i 2ϵ − 1 −1/2n−i−1 ϵ = ϵ. Thus C2 holds as (0) 2ϵ −(0) ϵ ≤ϵ. Clearly, qL(0) < 1. Finally we get ΠqL (T ) = i∈N ¯ti 1 −1/2n−i . Clearly qL is non-increasing, and coordinating. To see the latter we check the conditions in Theorem 2: C1 holds since Clearly qL is non-increasing, and coordinating. To see the latter we check the conditions in Theorem 2: C1 holds since we have 1 −1/2n−i+1 2ϵ − 1 −1/2n−i ϵ = ϵ. Also, we have 1 −1/2n−i 2ϵ − 1 −1/2n−i−1 ϵ = ϵ. Thus C2 holds as (0) 2ϵ −(0) ϵ ≤ϵ. Clearly, qL(0) < 1. Finally we get ΠqL (T ) = i∈N ¯ti 1 −1/2n−i . we have 1 −1/2n−i+1 2ϵ − 1 −1/2n−i ϵ = ϵ. Also, we have 1 −1/2n−i 2ϵ − 1 −1/2n−i−1 ϵ = ϵ. Thus C2 holds as (0) 2ϵ −(0) ϵ ≤ϵ. Clearly, qL(0) < 1. ( ) ( ) y qL( ) Finally we get ΠqL (T ) = i∈N ¯ti 1 −1/2n−i . y Finally we get ΠqL (T ) = i∈N ¯ti 1 −1/2n−i . πq (ti −2ϵ, ti) −πq (ti+1 −ϵ, ti+1) ≥ϵ. (21) (21) We construct a pricing scheme qL which satisﬁes the above condition for every i = 2, . . . , n and every 0 < ϵ ≤ ti/2, and has the lowest possible total revenue. We construct a pricing scheme qL which satisﬁes the above condition for every i = 2, . . . , n and every 0 < ϵ ≤ ti/2, and has the lowest possible total revenue. 5 Private processing times Section 4 shows how to coordinate a set of self-interested agents through a coordinating pricing scheme under the assumption that the vector of true processing times p is given and known to the subcontractor. In this section, we relax this assumption by allowing true processing times to be private information of agents. Thus reporting an untruth- ful processing time by an agent can deceive the subcontractor who consequently moves the agent’s job in the sequence of jobs in efﬁcient allocations which can potentially increase the agent’s time saving or reduce his payment. Let qL(t) = 0 for t ≥tn. Observe that in the latter range qL has the lowest possible prices, so under qL the agent n pays 0 for its efﬁcient allocation. The condition in (21) for i = n and any 0 < ϵ ≤tn/2 requires that πqL (tn −2ϵ, tn) ≥ϵ. (22) (22) To obtain the cheapest pricing scheme we require qL to yield πqL (tn −2ϵ, tn) = ϵ for every 0 < ϵ ≤¯tn−1/2. In particular for ϵ = ¯tn−1/2 we get πqL (tn−1, tn) = ¯tn−1/2. This means that under qL the agent i = n −1 pays ¯tn−1/2. Next, the condition (21) for agent i = n −1 and every 0 < ϵ ≤tn−1/2 requires that To obtain the cheapest pricing scheme we require qL to yield πqL (tn −2ϵ, tn) = ϵ for every 0 < ϵ ≤¯tn−1/2. In particular for ϵ = ¯tn−1/2 we get πqL (tn−1, tn) = ¯tn−1/2. This means that under qL the agent i = n −1 pays ¯tn−1/2. This section presents a mechanism that guarantees that the agents are always better off by reporting their true process- ing times, and that they are also better off by participating in the subcontracting—such mechanism is refereed to as the coordinating mechanism for the subcontractor scheduling problem under private information. Finally, the section cal- culates the amount the subcontractor forfeits to the agents to extract the true processing times from them. Next, the condition (21) for agent i = n −1 and every 0 < ϵ ≤tn−1/2 requires that πqL (tn−1 −2ϵ, tn−1) −πqL (tn −ϵ, tn) ≥ϵ. 5 Private processing times (23) (23) To obtain the cheapest pricing scheme we can consider qL to be such that (23) holds as equality for every 0 < ϵ ≤ ¯ ¯ To obtain the cheapest pricing scheme we can consider qL to be such that (23) holds as equality for every 0 < ϵ ≤ We draw upon mechanism design theory (Nisan 2007; Jackson 2000) and introduce a payment scheme that moti- vates agents to report true processing times knowing the subcontractor’s intention to maximize the total savings. ¯tn−2/2. In this case, for ϵ = ¯tn−2/2 we get πqL (tn−2, tn−1) −πqL tn −¯tn−2/2, tn = ¯tn−2/2. (24) (24) A pricing scheme in Sect. 4 determines payments made by agents to the subcontractor for time intervals that they choose to utilize on the subcontractor’s machine. In this section the payments are made for reporting particular processing times by the agents to the subcontractor. Formally, each agent reports a processing time ri from its individual processing time space Pi ⊂R+. We use pi to denote the true process- ing time of agent i. Let P = P1 ×· · ·× Pn be the processing time space of all agents. By Lemma 2, we know that ¯tn−2/2 ≤¯tn−1 so tn − ¯tn−2/2 ≥tn−1. Hence we have πqL tn −¯tn−2/2, tn = ¯tn−2/4 and eventually πqL (tn−2, tn−1) = 3¯tn−2/4. (25) (25) The latter implies that agent i = n −2 under qL pays ¯tn−2(3/4) to the subcontractor. 123 123 J Sched (2015) 18:497–511 507 πVCG i (r) = hi r−i − j∈N\{i} υ j (T (r)) , Deﬁnition 2 A mechanism M is deﬁned by a (n + 1)-tuple ( f M, π M 1 , . . . , π M n ) consisting of an allocation function f M : P →T , and a payment scheme π M : P →Rn. where hi is a function independent of agent i’s reported processing time ri. Among the class of VCG mechanisms, the pivotal mechanism with Given a vector of reported processing times r and the sequence in which the agents report them (to break ties in the allocation), the payment scheme of a mechanism deter- mines the monetary amount π M i (r) that i ∈N pays in return for receiving the allocation prescribed for i by f M. Lemma 3 For all i ∈N, we have Although incentive compatibility implies that agents are not better off by lying about their true processing times, Although incentive compatibility implies that agents are not better off by lying about their true processing times, it does not generally imply that the agents are better off by reporting their true processing times—there could gen- erally exist untruthful agent reports that result in the same utility as the truthful ones. Therefore, unfortunately, agents may generally choose to be untruthful after all even with the incentive compatible mechanisms. This is generally the case for VCG mechanisms, in particular the pivotal mecha- nism, which characterize the class of efﬁcient and incentive compatible mechanisms for quasilinear utilities (Green and Laffont 1977). However, we show later in Theorem 6 that for the subcontractor scheduling problem studied in this paper the pivotal mechanism makes the agents always better off by reporting their true processing times. π PM i (r) = ¯ti(r) 1 −1/2n−[i] . (31) (31) Proof We start by showing that for any i, j ∈N, we have ¯t j(r−i) −¯t j(r) = 0 if [ j] < [i] and ¯t j(r−i) −¯t j(r) = ¯ti(r)/2[ j]−[i] if [ j] > [i]. By (6) ri does not appear in ¯t j(r) for [ j] < [i]. Therefore, ¯t j(r−i) −¯t j(r) = 0 for [ j] < [i]. By (6) ri does not appear in ¯t j(r) for [ j] < [i]. Therefore, ¯t j(r−i) −¯t j(r) = 0 for [ j] < [i]. If [ j] > [i], then the exclusion of i affects the duration of allocated time to jobs j without altering the relative position of other jobs. By (4) and (5), we have ¯t j(r) = 1 2 ⎛ ⎝r j − ⎡ ⎣ti(r) + ¯ti(r) + [i]<[k]<[ j] ¯tk(r) ⎤ ⎦ ⎞ ⎠ 5 Private processing times hi r−i = j∈N\{i} υ j T r−i (29) (29) With quasilinear utilities, a mechanism M would result in the utility guarantees that all payments from the agents to the mecha- nism are non-negative. Here T (r−i) is an efﬁcient allocation for the set of jobs excluding job i and for processing time vector r. Thus, the payment by pivotal mechanism for i ∈N equals uM i f M(r) = υi f M(r) −π M i (r) (27) (27) for i ∈N. π PM i (r) = j∈N\{i} " ¯t j r−i −¯t j(r) # , (30) (30) Thesubcontractorisseekingtoallocatethesubcontracting intervals efﬁciently, thus we focus on mechanisms whose allocation functions yield efﬁcient allocations, i.e., f M ≡T . We call such mechanisms efﬁcient. where ¯t j(r) is the duration of subcontracting time allocated to j by the efﬁcient (optimal) allocation for the reportedr and ¯t j(r−i) is the duration of subcontracting time allocated to j by the efﬁcient allocation for the reported r−i that excludes i. We concentrate on incentive compatible mechanism since they have the potential to motivate the agents to report their true processing times. By (30) the payment π PM i (r) charged by the pivotal mech- anism to agent i is the total subcontracting time that could have been allocated to all other agents had not i asked for subcontracting and reported ri. It follows from the charac- terization of efﬁcient solutions that the exclusion of an agent i reporting ri would only affect the agents that follow i in T (r). Let σ be the permutation of jobs in T (r) and let [i], for i ∈N, be the position of i in T (r). Deﬁnition 3 A mechanism M is incentive compatible if for every reported processing time vector r = (r1, . . . ,rn) and for every agent i ∈N uM i f M pi,r−i ≥uM i f M(r) , (28) (28) where r−i is the vector r without its ith coordinate. where r−i is the vector r without its ith coordinate. Lemma 3 For all i ∈N, we have Lemma 3 For all i ∈N, we have Lemma 3 For all i ∈N, we have 5.1 Payment scheme u PM i (T (r)) = max ( pi −ti (r) 2 , 0 ) −ri −ti (r) 2 1 − 1 2n−l , (33) / j Based on the above observation, the total payment in (30) becomes π PM i (r) = ¯ti(r)/2[k]−[i] u PM i (T (r)) = max ( pi −ti (r) 2 , 0 ) −ri −ti (r) 2 1 − 1 2n−l , Based on the above observation, the total payment in (30) becomes u PM i (T (r)) = max ( pi −ti (r) 2 t ( ) Based on the above observation, the total payment in (30) becomes (33) π PM i (r) = [k]>[i] ¯ti(r)/2[k]−[i] = ¯ti(r) [k]>[i] 1/2[k]−[i] = ¯ti(r) 1 −1/2n−[i] . where n > l ≥h and ti(r) is the start time of job i in T (r). Suppose l = h. In this case we have ti(r) = ti(p). Since h < n, it directly follows from (32) and (33) that u PM i (T (r)) < u PM i (T (p)). where n > l ≥h and ti(r) is the start time of job i in T (r). Suppose l = h. In this case we have ti(r) = ti(p). Since h < n, it directly follows from (32) and (33) that u PM i (T (r)) < u PM i (T (p)). where n > l ≥h and ti(r) is the start time of job i in T (r). Suppose l = h. In this case we have ti(r) = ti(p). Since h < n, it directly follows from (32) and (33) that u PM i (T (r)) < u PM i (T (p)). i i Next, suppose l > h. Let σ(k) be the job in position k in T (p). By (4) and (5) and induction on l −h we have, ⊓⊔ ti (r) = ti (p) + l k=h+1 rσ(k) −ti (p) 2l+1−k = rσ(l) 2 + · · · + rσ(h+1) 2l−h + ti (p) 2l−h 5.2 Coordinating meachanism Lemma 3 provides a closed-form formula for the pivotal mechanism payments. With these payments, each agent pays an amount directly proportional to the duration of its alloca- tion on subcontractor’s machine and inversely proportional to its order in the sequence of jobs on subcontractor’s machine. Therefore if an agent announces a shorter than true process- ing time, which could possibly precipitate its position in the sequence and thus result in the agent’s higher time saving, then the agent pays more. We show that as a result of this misrepresentation, the agent’s utility decreases. At the same time, we show that reporting longer than true processing time reduces agent’s utility as well, unless the agent comes in the last position in the sequence. This position is special since it neither incurs any payment by (31) nor it affects any other agent. Therefore, whether the agent in this position lies or not is irrelevant. Alternatively, one can assume that the longest job always belongs to the subcontractor—this dummy job could represent the continuation of subcontractor’s opera- tions. Consequently, the mechanism enforces agents to report their actual processing time which we now formally prove. Using the fact that rσ(k) ≥pi for k ≥h we get ti (r) ≥ l k=h+1 pi 2l+1−k + ti(p) 2l−h = pi − pi 2l−h + ti(p) 2l−h (34) For a contradiction assume u PM i (T (r)) ≥u PM i (T (p)), i.e., by (32) and (33) assume that max ( pi −ti (r) 2 , 0 ) −ri −ti (r) 2 1 − 1 2n−l ≥pi −ti (p) 2 1 2n−h . If pi −ti (r) ≤0, then the contradiction readily follows. Otherwise, suppose pi −ti (r) > 0. Then, we have pi −ti (r) 2 −ri −ti (r) 2 1 − 1 2n−l ≥pi −ti (p) 2 1 2n−h . Theorem 6 Let r = (r1, . . . ,rn) be the reported processing times and let pi be true processing time of agent i. Then u PM i (T (pi,r−i)) > u PM i (T (r)), provided that pi ̸= ri and i is not in the last position of T (r). The last inequality simpliﬁes to 2l−h [ri −ti (r)] −[pi −ti (p)] ≥2n−h [ri −pi] . Using (34), note that the last inequality remains valid if ti(r) is replaced with pi −pi/2l−h + ti(p)/2l−h. 5.1 Payment scheme and We draw upon the VCG mechanisms to obtain a desirable payment scheme for the subcontractor scheduling problem. The payment scheme for VCG mechanisms is deﬁned as follows ¯t j r−i = 1 2 ⎛ ⎝r j − ⎡ ⎣ti(r) + [i]<[k]<[ j] ¯tk r−i ⎤ ⎦ ⎞ ⎠ 3 3 J Sched (2015) 18:497–511 508 Thus By (27), (31), (7), and (4), for pi, the utility of agent i is By (27), (31), (7), and (4), for pi, the utility of agent i is Thus By (27), (31), (7), and (4), for pi, the utility of agent i is Thus ¯t j r−i −¯t j(r) = 1 2 ⎛ ⎝¯ti(r) − [i]<[k]<[ j] " ¯tk r−i −¯tk(r) # ⎞ ⎠ u PM i (T (p)) = pi −ti (p) 2 −pi −ti (p) 2 1 − 1 2n−h = pi −ti (p) 2 1 2n−h . (32) Th f ( ) d (b) u PM i (T (p)) = pi −ti (p) 2 −pi −ti (p) 2 1 − 1 2n−h = pi −ti (p) 2 1 2n−h . (32) ¯t j r−i −¯t j(r) = 1 2 ⎛ ⎝¯ti(r) − [i]<[k]<[ j] " ¯tk r−i −¯tk(r) # ⎞ ⎠ = 1 2 ⎛ ⎝¯ti(r) − [i]<[k]<[ j] " ¯tk r−i −¯tk(r) # ⎞ ⎠ (32) There are two cases for ri: (a) ri > pi, and (b) ri < pi. (a) With ri > pi we have There are two cases for ri: (a) ri > pi, and (b) ri < pi. (a) With ri > pi we have By induction on [ j] −[i] we obtain ¯t j(r−i) −¯t j(r) = ¯ti(r)/2[ j]−[i] if [ j] > [i]. By induction on [ j] −[i] we obtain ¯t j(r−i) −¯t j(r) = ¯ti(r)/2[ j]−[i] if [ j] > [i]. 5.3 Subcontractor’s revenue with coordinating mechanisms ti (p) = ti (r) + h−1 k=l rσ(k) −ti (r) 2h−k = rσ(h−1) 2 + · · · + rσ(l) 2h−l + ti (r) 2h−l . The payment scheme π PM provides the subcontractor with a positive revenue for any given r. Let us deﬁne Π PM(r) = i∈N π PM i (r) as the total payment from agents to the sub- contractor in pivotal mechanism. By Theorem 6 all agents except the last in T (r) report their true processing times while the last one pays nothing to the subcontractor regard- less of his report. Therefore, we focus on the subcontractor’s revenue for the true processing times p. Using the fact that rσ(k) ≤pi for k ≤h we get ti (p) ≤ h−1 k=1 pi 2h−k + ti(r) 2h−l = pi − pi 2h−l + ti(r) 2h−l (36) (36) Although the subcontractor’s services result in the total savings of υ(T (p)) for all agents, it follows from Lemma 3 that the mechanism (T , π PM) can only achieve revenue Π PM(p) for the subcontractor which is always less than v(T (p)). In fact, instances can be found for which the ratio of subcontractor’s revenue to total savings obtained via sub- contracting, i.e., Π PM(p)/υ(T (p)), is arbitrarily close to zero. For example, consider a set of two jobs with p1 = 2 and p2 = 101. Despite having a total saving of 51, we have π PM 1 = 0.5 and π PM 2 = 0 resulting in the ratio υ(T (p))/Π PM(p) < 0.01. For a contradiction assume u PM i (T (r)) ≥u PM i (T (p)) or, by (32) and (35), ri −ti (r) 2 1 2n−l ≥pi −ti (p) 2 1 2n−h . The last simpliﬁes to ri −ti (r) ≥2h−l[pi −ti (p)]. Nevertheless, Moulin (1986) shows that if for all agents it holds that minT υi(T ) = 0—which is the case in subcon- tractor’s subcontracting problem with divisible jobs—then among all efﬁcient, incentive compatible, and individually rational mechanisms, the pivotal mechanism generates the highest total payment. Therefore, the subcontractor cannot gain a higher revenue from any other efﬁcient, incentive com- patible, and individually rational mechanisms than it does from (T , π PM). Thus, by the juxtaposition of Lemma 3 and Theorem 5 we get the following observation. where 1 ≤l ≤h. Suppose h = l. Then we have ti(r) = ti(p), and it follows from (32) and (35) that u PM i (T (r)) < u PM i (T (p)). i i Next, suppose 1 ≤l < h. By (4) and (5) and induction on h −l 5.2 Coordinating meachanism The inequality then simpliﬁes to 2l−h [ri −pi] ≥2n−h [ri −pi]. Proof Assume pi ̸= ri ∈Pi for some agent i. Let p = (pi,r−i). Suppose job i is in position h and l in efﬁcient solutions T (p) and T (r), respectively, and assume l ̸= n. 12 123 509 J Sched (2015) 18:497–511 This is a contradiction since we have 2l−h < 2n−h. Thus u PM i (T (r)) < u PM i (T (p)) for ri > pi. (b) With ri < pi we have This is a contradiction since we have 2l−h < 2n−h. Thus u PM i (T (r)) < u PM i (T (p)) for ri > pi. (b) With ri < pi we have subcontracting. Therefore the mechanism coordinates the subcontractor scheduling problem under private information. One possible disadvantage of coordination based on mechanism design, the VCG mechanisms in particular, is that this coordination is implicitly centralized—the efﬁcient schedule is computed by the subcontractor and then reported to the agents. Although distributed implementations of the pivotal mechanism where the schedule is determined by the self-interested agents themselves are beyond the scope of this paper, we refer the reader to Parkes and Shneidman (2004) where guidelines for the distributed implementations of the VCG mechanisms are proposed. u PM i (T (r)) =ri −ti (r) 2 −ri −ti (r) 2 1 − 1 2n−l =ri −ti (r) 2 1 2n−l , (35) (35) where 1 ≤l ≤h. 5.3 Subcontractor’s revenue with coordinating mechanisms Using (36), the last inequality remains valid if ti(p) is replaced with pi −pi/2h−l + ti(r)/2h−l. The inequality then simpliﬁes to ri ≥pi which is a contradiction. Thus u PM i (T (r)) < u PM i (T (p)) for ri < pi. ⊓⊔ ⊓⊔ Finally, to ensure agents’ participation in subcontracting, the efﬁcient mechanism M must satisfy the strict indi- vidual rationality condition requiring that for all agents, truth-telling results in positive utility, irrespective of the reported processing times of other agents. That is, for every r = (r1, . . . ,ri = pi, . . . ,rn) and all i ∈N, uM i (T (r)) > 0. The efﬁcient mechanism (T , π PM) satisﬁes the strict indi- vidual rationality condition since, by (27), (31) and (7), we have u PM i (T (r)) = ¯ti(r)/2n−[i] > 0, for ri = pi. Corollary 1 Subcontractor’s revenue with any efﬁcient, incentive compatible, and individually rational mechanisms under private information never exceeds that with a coordi- nating pricing scheme under complete information. i Thus we showed the mechanism (T , π PM) guarantees, by Theorem 6, that the agents are always better off by reporting their true processing times, and that, by strict indi- vidual rationality, they are better off by participating in the However, the construction of a coordinating pricing scheme q requires the knowledge of true processing times p, thus the difference Πq(p) −Π PM(p) may be considered 123 12 3 J Sched (2015) 18:497–511 510 scheme, we must have q(t) > 2 for 0 ≤t < 18 so that job 2 would not be able to choose any interval on subcontractor’s machine. as the amount the subcontractor forfeit to the agents to extract the true processing times from them. scheme, we must have q(t) > 2 for 0 ≤t < 18 so that job 2 would not be able to choose any interval on subcontractor’s machine. The condition in (38) for job i = 4 and ϵ = 0.25 requires that, (37) υw i (T ) = wiυi(T ). The condition in (38) for job i = 3 and ϵ = 0.5 requires that, The total saving for all agents is deﬁned as the sum of indi- vidual savings, that is υw(T ) = i∈N υw i (T ). The efﬁcient allocation is T = arg maxT υw(T ). Unlike the symmetric case where each job in N is allocated a non-empty inter- val on subcontractor’s machine, in asymmetric case efﬁcient allocations may exclude some jobs from the subcontractor’s schedule. Let I = {i ∈N\|¯ti > 0} be the set of jobs that receive non-empty allocations, and O = {i ∈N\|¯ti = 0} the set of jobs that receive no such allocations. We denote the cardinality of I with l. q (2, 3) −q(10.5, 11) ≥6. (40) (40) q (2, 3) −q(10.5, 11) ≥6. We already know that q (10.5, 11) > 3.25 thus it must be that q (2, 3) > 9.25. This implies that in the cheapest coor- dinating pricing scheme, agent 1 must pay 9.25 for acquiring the time interval [2, 3) on subcontractor’s machine. However, since the valuation of agent 1 is only 9, this means that he would be better off by not accepting that interval. This contra- dicts the fact that q is coordinating. Therefore, in situations with asymmetric valuations, coordinating pricing schemes may be impossible to ﬁnd. ⊓⊔ In the remainder of this section, we focus on coordinating pricing schemes in situations with asymmetric valuations. In these situations, any coordinating pricing scheme q meets the following conditions: for ti ≤t < ti+1 it holds that q(t) < wi, that is, it is required that subcontracting price across the allocation of job i do not exceed its unit saving from subcontracting. ⊓⊔ 6 Asymmetric valuations q (10.5, 11) −q(17.75, 18) ≥2.75. (39) (39) We now relax the assumption of symmetric valuations and assume that the value of a unit time saving for agent i is wi. Let w = (wi\|i ∈N). The saving to agent i resulting from the allocation T thus equals assume that the value of a unit time saving for agent i is wi. Let w = (wi\|i ∈N). The saving to agent i resulting from the allocation T thus equals Considering that we need q(t) > 2 for 0 ≤t < 18, it must be that q(17.75, 18) > 0.5 and consequently q (10.5, 11) > 3.25. Considering that we need q(t) > 2 for 0 ≤t < 18, it must be that q(17.75, 18) > 0.5 and consequently q (10.5, 11) > 3.25. υw i (T ) = wiυi(T ). (37) (37) 7 Final remarks and conclusions We studied the decentralized subcontractor scheduling prob- lem under complete and private information. We designed the coordinating pricing schemes in case of complete infor- mation and studied the range of subcontractor’s revenue for these schemes. Our coordinating pricing schemes are piece- wise linear and non-increasing. We show by a counterexample that it is generally impos- sible to construct coordinating pricing schemes in the asym- metric case. Without loss of generality we assume I = {1, . . . ,l} and that the jobs are sequenced in efﬁcient solu- tions by their index order. We also devised a mechanism that is incentive compatible, individually rational, and efﬁcient in case of private informa- tion. The mechanism is based on the pivotal mechanism and it is moreover coordinating. Another desirable property of the mechanism, that immediately follows from Theorem 6, is Envy-Freeness (Foley 1967) which is deﬁned as the con- dition where no agent prefers another agent’s allocation to its own, that is ui(Ti(p)) ≥ui(T j(p)) for all j and all i. The mechanism produces the highest subcontractor revenue among efﬁcient, incentive compatible, and individually ratio- nal mechanisms. Theorem 7 There exists no coordinating pricing scheme in the asymmetric valuation case. Theorem 7 There exists no coordinating pricing scheme in the asymmetric valuation case. Proof To construct coordinating pricing schemes, we must ensure that agents would not beneﬁt by deviating from the efﬁcient allocations. In particular, deviations of the similar type as in Theorem 5 must be unproﬁtable for any agent who receives non-zero allocations on the subcontractor’s machine. That is for every i = 2, . . . ,l and every 0 < ϵ < ti it must hold that, (38) q (ti −2ϵ, ti) −q(ti+1 −ϵ, ti+1) ≥wiϵ. (38) For the asymmetric case, we showed that the coordination through pricing schemes is not always possible. Consider the following example. There are four jobs p = (p1 = 6, p2 = 18, p3 = 19, p4 = 25) with w = (w1 = 9, w2 = 2, w3 = 12, w4 = 11). The unique efﬁcient allocation is I = {1, 3, 4} with T1 = [0, 3), T3 = [3, 11), T4 = [11, 18), and O = {2}. For any coordinating pricing Consider the following example. There are four jobs p = (p1 = 6, p2 = 18, p3 = 19, p4 = 25) with w = (w1 = 9, w2 = 2, w3 = 12, w4 = 11). The unique efﬁcient allocation is I = {1, 3, 4} with T1 = [0, 3), T3 = [3, 11), T4 = [11, 18), and O = {2}. For any coordinating pricing An open problem is the complexity of ﬁnding the efﬁcient allocations for the asymmetric case. Several other extensions are possible for this problem: job’s release dates and due dates, setup times required for subcontractor to prepare for processing different jobs, and 123 123 511 J Sched (2015) 18:497–511 transportation costs for transportation from and to manu- facturers. Finally, the divisibility of jobs can be altered by requesting that only certain parts of jobs with ﬁxed dura- tions can be subcontracted. We leave these issues for future research. Groves, T. (1973). Incentives in teams. Econometrica, 41, 617–631. Heydenreich, B., Muller, R., & Uetz, M. (2007). Games and mech- anism design in machine schedulingan introduction. 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https://openalex.org/W3035072246	https://europepmc.org/articles/pmc7298710?pdf=render	English	null	Aspergillus fumigatus Transcription Factors Involved in the Caspofungin Paradoxical Effect	MBio	2,020	cc-by	15,411	Aspergillus fumigatus Transcription Factors Involved in the Caspofungin Paradoxical Effect Clara Valero,a Ana Cristina Colabardini,a Jéssica Chiaratto,a Lakhansing Pardeshi,b,c Patrícia Alves de Castro,a Jaire Alves Ferreira Filho,a Lilian Pereira Silva,a Marina Campos Rocha,d Iran Malavazi,d Jonas Henrique Costa,e Taícia Fill,e Mário Henrique Barros,f Sarah Sze Wah Wong,g Vishukumar Aimanianda,g Koon Ho Wong,b,h Gustavo H. Goldmana aFaculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo Ribeirão Preto, Ribeirão Preto, Brazil aFaculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo Ribeirão Preto, Ribeirão Preto, Brazil bFaculty of Health Sciences, University of Macau, Macau SAR, China cGenomics and Bioinformatics Core, Faculty of Health Sciences, University of Macau, Macau SAR, China dDepartamento de Genética e Evolução, Centro de Ciências Biológicas e da Saúde, Universidade Federal de São Carlos, São Carlos, São Paulo, B eInstituto de Química, Universidade de Campinas, Campinas, São Paulo, Brazil fDepartamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, Brazil gUnité Mycologie Moléculaire, Institut Pasteur, UMR2000, CNRS, Paris, France hInstitute of Translational Medicine, University of Macau, Macau SAR, China ABSTRACT Aspergillus fumigatus is the leading cause of pulmonary fungal diseases. Azoles have been used for many years as the main antifungal agents to treat and prevent invasive aspergillosis. However, in the last 10 years there have been several reports of azole resistance in A. fumigatus and new strategies are needed to combat invasive aspergillosis. Caspofungin is effective against other human-pathogenic fun- gal species, but it is fungistatic only against A. fumigatus. Resistance to caspofungin in A. fumigatus has been linked to mutations in the fksA gene that encodes the tar- get enzyme of the drug -1,3-glucan synthase. However, tolerance of high caspofun- gin concentrations, a phenomenon known as the caspofungin paradoxical effect (CPE), is also important for subsequent adaptation and drug resistance evolution. Here, we identiﬁed and characterized the transcription factors involved in the re- sponse to CPE by screening an A. fumigatus library of 484 null transcription factors (TFs) in CPE drug concentrations. We identiﬁed 11 TFs that had reduced CPE and that encoded proteins involved in the basal modulation of the RNA polymerase II initiation sites, calcium metabolism, and cell wall remodeling. One of these TFs, FhdA, was important for mitochondrial respiratory function and iron metabolism. The ΔfhdA mutant showed decreased growth when exposed to Congo red or to high temperature. RESEARCH ARTICLE Molecular Biology and Physiology crossm RESEARCH ARTICLE Molecular Biology and Physiology crossm RESEARCH ARTICLE Molecular Biology and Physiology RESEARCH ARTICLE Molecular Biology and Physiology May/June 2020 Volume 11 Issue 3 e00816-20 Citation Valero C, Colabardini AC, Chiaratto J, Pardeshi L, de Castro PA, Ferreira Filho JA, Silva LP, Rocha MC, Malavazi I, Costa JH, Fill T, Barros MH, Wong SSW, Aimanianda V, Wong KH, Goldman GH. 2020. Aspergillus fumigatus transcription factors involved in the caspofungin paradoxical effect. mBio 11:e00816-20. https://doi.org/10.1128/mBio .00816-20. Aspergillus fumigatus Transcription Factors Involved in the Caspofungin Paradoxical Effect These TFs encode proteins in- volved in the basal modulation of the RNA polymerase II initiation sites, calcium me- tabolism or cell wall remodeling, and mitochondrial respiratory function. The study of those genes regulated by TFs identiﬁed in this work will provide a better under- standing of the signaling pathways that are important for caspofungin tolerance and resistance. KEYWORDS Aspergillus fumigatus, calcium, caspofungin, cell wall, transcription factors, mitochondria I nfections by human-pathogenic fungal species are a major cause of morbidity and mortality in immunosuppressed patients. It is estimated that more people die from the 10 main invasive fungal diseases than from tuberculosis or malaria. Among them, invasive aspergillosis is the most common human systemic infection caused by ﬁla- mentous fungi, with approximately 200,000 infections by year worldwide and a high rate of mortality that can reach 95% (1). Despite of the continuous increase of populations susceptible to fungal infections, the availability of antifungal drugs is still very limited. Azoles have been used for many years as the main antifungal agents to treat and prevent invasive aspergillosis (2, 3). Their mode of action relies on inhibition of ergosterol biosynthesis, which, coupled with the accumulation of a toxic interme- diate, disrupts cell membrane integrity (4). However, in recent decades, a steady increase in worldwide reports of azole resistance in Aspergillus fumigatus, resulting in therapeutic failures, has been a matter of serious clinical concern (5). Resistant pheno- types have been found to be associated with exposure to azole formulations during both antifungal therapy in patients and fungicide application in the environment (6). g py p g pp Due to the limited availability of effective antifungal agents and the emergence of azole resistance, new strategies are needed to combat invasive aspergillosis (7). Since fungal cell wall is absent in humans and is essential to fungal viability, drugs disrupting cell wall biosynthesis have gained more attention. Echinocandins, such as caspofungin (CSP), are the newest class of antifungal drugs approved to treat invasive fungal infections. They target the fungal cell wall by inhibiting 1,3--D-glucan synthase, which is responsible for the assembly of -D-glucan, a major component of the fungal cell wall (8). As a result, treatment with echinocandins leads to hyphae with defects in growth and morphology (9). These antifungal agents are also effective against Candida species, exhibiting fungicidal activity. In contrast, they are fungistatic against A. Aspergillus fumigatus Transcription Factors Involved in the Caspofungin Paradoxical Effect Transcriptome sequencing (RNA-seq) analysis and further experi- mental validation indicated that the ΔfhdA mutant showed diminished respiratory capacity, probably affecting several pathways related to the caspofungin tolerance and resistance. Our results provide the foundation to understand signaling pathways that are important for caspofungin tolerance and resistance. Citation Valero C, Colabardini AC, Chiaratto J, Pardeshi L, de Castro PA, Ferreira Filho JA, Silva LP, Rocha MC, Malavazi I, Costa JH, Fill T, Barros MH, Wong SSW, Aimanianda V, Wong KH, Goldman GH. 2020. Aspergillus fumigatus transcription factors involved in the caspofungin paradoxical effect. mBio 11:e00816-20. https://doi.org/10.1128/mBio .00816-20. Editor J. Andrew Alspaugh, Duke University Medical Center Copyright © 2020 Valero et al. This is an open- access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Gustavo H. Goldman, ggoldman@usp.br. Received 6 April 2020 Accepted 15 May 2020 Published 16 June 2020 Citation Valero C, Colabardini AC, Chiaratto J, Pardeshi L, de Castro PA, Ferreira Filho JA, Silva LP, Rocha MC, Malavazi I, Costa JH, Fill T, Barros MH, Wong SSW, Aimanianda V, Wong KH, Goldman GH. 2020. Aspergillus fumigatus transcription factors involved in the caspofungin paradoxical effect. mBio 11:e00816-20. https://doi.org/10.1128/mBio .00816-20. IMPORTANCE Aspergillus fumigatus, one of the most important human-pathogenic fungal species, is able to cause aspergillosis, a heterogeneous group of diseases that presents a wide range of clinical manifestations. Invasive pulmonary aspergillosis is the most serious pathology in terms of patient outcome and treatment, with a high mortality rate ranging from 50% to 95% primarily affecting immunocompromised patients. Azoles have been used for many years as the main antifungal agents to treat and prevent invasive aspergillosis. However, there were several reports of evo- lution of clinical azole resistance in the last decade. Caspofungin, a noncompetitive -1,3-glucan synthase inhibitor, has been used against A. fumigatus, but it is fungi- static and is recommended as second-line therapy for invasive aspergillosis. More in- ® mbio.asm.org 1 ® mbio.asm.org 1 ® Valero et al. formation about caspofungin tolerance and resistance is necessary in order to reﬁne antifungal strategies that target the fungal cell wall. Here, we screened a transcrip- tion factor (TF) deletion library for TFs that can mediate caspofungin tolerance and resistance. We have identiﬁed 11 TFs that are important for caspofungin sensitivity and/or for the caspofungin paradoxical effect (CPE). May/June 2020 Volume 11 Issue 3 e00816-20 RESULTS Eleven transcriptional factors govern caspofungin paradoxical effect (CPE). To assess if any other TF plays a role in CPE, a library of 484 A. fumigatus TF null mutants (25) was screened for sensitivity to high concentrations of caspofungin (16 g/ml). At this concentration, the CPE is induced in the CEA17 parental strain (22, 23). Eleven null mutants, including the ΔzipD mutant (ΔAFUB_020350), exhibited increased sensitivity to caspofungin at a concentration of 16 g/ml (the other 10 mutants included ΔAFUB_009970 [cbfA], ΔAFUB_029870 [nctA], ΔAFUB_045980 [nctB], ΔAFUB_058240 [nctC], ΔAFUB_091020 [fhdA], ΔAFUB_064180 [znfA], ΔAFUB_037850 [atfA], ΔAFUB_ 040580 [rlmA], ΔAFUB_08249 [zfpA], and ΔAFUB_054000) (Fig. 1A). Except for the ΔnctA and ΔrlmA mutants, all also showed sensitivity to lower caspofungin concentrations, suggesting a high correlation between caspofungin sensitivity and reduced CPE (Fig. 1B). The ΔnctA and ΔnctB mutant strains were previously described (25) in a report from a study whose results indicated that loss of the negative cofactor 2 complex led to resistance to most clinically used antifungals, including azoles and terbinaﬁne. We have conﬁrmed these results for the ΔnctA mutant, but there were no differences in antifungal drug susceptibility seen for any other TF mutant in comparison with the wild-type strain (Table 1). CbfA is the putative homologue of Saccharomyces cerevisiae DPB4, a putative subunit of the DNA polymerase epsilon and the ISW2 chromatin accessibility complex (identity, 34%; similarity, 55%; E value, 4e09). NctA and NctB, together with CbfA and NtcC, are members of the evolutionarily conserved CBF/NF-Y family of TFs (Fig. 1C; Pfam PF00808, histone-like transcription factor CBF/NF-Y and archaeal histone). This family encompasses archaebacterial histones and histone-like TFs from eukaryotes, which include the CBC transcription regulator complex (26). NctC is a putative A. fumigatus paralogue of the NctA and HapE CCAAT-binding factor complex subunit (26). FhdA and ZnfA encode novel TFs with fork-head-associated domains and zinc-ﬁnger-associated domains, respectively. AtfA is important for viability of conidia, and several kinds of stress, including oxidative, osmotic, and cell wall damage stress, while RlmA is required for the regulation of the cell wall integrity (CWI) pathway; both proteins have already been shown to be important for CPE (23, 27–30). We also previously found ZfpA transcripts upregulated in the presence of calcium and voriconazole (31, 32). However, AFUB_054000 is not known to have identity with any other known TF. Aspergillus fumigatus Transcription Factors Involved in the Caspofungin Paradoxical Effect fumigatus, promoting the lysis of the apical tips of expanding hyphae, altering hyphal morphol- ogy, and modifying cell wall composition and organization (10). Regardless, they are recommended for second-line therapy against invasive aspergillosis due to excellent tolerability and synergistic potential in combination with other antifungals (11). Resis- tance to echinocandins in A. fumigatus has been linked to mutations in the fksA gene (coding for 1,3--D-glucan synthase, the drug-target), representing a well-documented mechanism in Candida species (12, 13). Nevertheless, FKS1-independent resistance mechanisms have also been reported to exist (14, 15). A phenomenon different from resistance is known to occur when A. fumigatus is exposed to high concentrations of caspofungin, called the caspofungin paradoxical effect (CPE), which relies on the ability of the fungus to restore growth at suprainhibitory drug concentrations (16). Several studies have been performed to elucidate the mechanisms underlying this process, showing links between CPE and components of signaling pathways responsible for the fungal response to environmental stresses such as those imposed by Hsp90, calcineu- rin, or mitogen-activated protein kinases (MAPKs) (17–19). In Candida albicans, aneu- ploidy also enables the CPE by altering the copy number of several genes involved in cell wall remodeling or calcium metabolism (20, 21). Recently, our group identiﬁed a May/June 2020 Volume 11 Issue 3 e00816-20 mbio.asm.org 2 ® Regulation of Caspofungin Tolerance in Aspergillus novel basic leucine zipper ZipD transcription factor (TF) that was found to play a role in the calcium-calcineurin pathway and to be involved in CPE (22, 23). Moreover, it has been suggested that reactivation of -1,3-glucan synthase is essential for CPE mainte- nance (24). Given the polygenic and complex nature of CPE, a better understanding of mech- anisms of echinocandin resistance and tolerance in A. fumigatus has become manda- tory. Accordingly, in order to identify possible regulatory mechanisms controlling CPE, we screened an A. fumigatus library of null TFs in various CPE concentrations. In this work, we identiﬁed and characterized several TFs involved in the response to CPE. We identiﬁed 11 TF mutants that have reduced CPE, and 9 of them were more sensitive to caspofungin than the wild-type strain. The TFs identiﬁed encode proteins involved in the basal modulation of the RNA polymerase (Pol) II initiation sites, in calcium metab- olism, or in cell wall remodeling. Additionally, one of these TFs (FhdA) encodes a novel protein important for mitochondrial respiratory function and iron metabolism. RESULTS To obtain insight into the TF signaling networks governing caspofungin tolerance, we generate functional gene networks using STRING (https://string-db.org/) analysis. The TF interaction network showed two distinct networks that are highly connected and composed as follows: (i) CbfA (AFUB_009970), NctA (AFUB_029870), NctB (AFUB_045980), and NctC (AFUB_058240) and (ii) AtfA (AFUB_037850) and RlmA (AFUB_040580) (data not shown). These results suggest that the different TF members of the CBF/NF-Y family interact under conditions of caspofungin tolerance. In addition, mbio.asm.org 3 May/June 2020 Volume 11 Issue 3 e00816-20 ® Valero et al. FIG 1 Identiﬁcation of A. fumigatus transcription factor null mutants that have reduced caspofungin paradoxical effect (CPE). A collection of A. fumigatus 484 null transcription factor mutants was screened for sensitivity to caspofungin by growing them on MM plus 16 g/ml of caspofungin. (A and B) Eleven mutants that displayed reduced CPE had their radial growth quantiﬁed on MM plus 16 g/ml of caspofungin (A) or MM plus 0.5 g/ml of caspofungin (B). Statistical analysis was performed using one-tailed, paired t tests for comparisons to the control condition (, P 0.05). (C) Protein domains in NctA, NctB, NctC, and CbfA determined by the use of SMART (http://smart.embl-heidelberg.de/). FIG 1 Identiﬁcation of A. fumigatus transcription factor null mutants that have reduced caspofungin paradoxical effect (CPE). A collection of A. fumigatus 484 null transcription factor mutants was screened for sensitivity to caspofungin by growing them on MM plus 16 g/ml of caspofungin. (A and B) Eleven mutants that displayed reduced CPE had their radial growth quantiﬁed on MM plus 16 g/ml of caspofungin (A) or MM plus 0.5 g/ml of caspofungin (B). Statistical analysis was performed using one-tailed, paired t tests for comparisons to the control condition (, P 0.05). (C) Protein domains in NctA, NctB, NctC, and CbfA determined by the use of SMART (http://smart.embl-heidelberg.de/). RlmA and AtfA interact for cell wall remodeling under conditions of caspofungin tolerance. Here, we characterized in more detail the ΔcbfA, ΔnctC, and ΔfhdA mutants. CbfA and NctC are required for optimal growth under conditions of stresses and suppress production of secondary metabolites (SM). The ΔcbfA mutant showed reduced radial growth on minimal medium (MM) (about 50% of the levels seen with the wild-type and complemented strains; Fig. 2A), and it was more sensitive to higher temper- ature (44°C), CaCl2, caspofungin, and increased osmotic pressure (Fig. 2A to E). mbio.asm.org 4 RESULTS CbfA:GFP (CbfA:green ﬂuorescent protein) was constitutively present in the nucleus (Fig. 2F) since there was no change in its location or in the level of ﬂuorescence intensity seen upon exposure to all stressing conditions described above (data not shown). Interestingly, growth of the ΔcbfA mutant in liquid MM for up to 48 h at 37°C resulted in the secretion of compounds that gave a reddish color to the culture medium (Fig. 3A). Mass spectrometry analysis revealed that the ΔcbfA mutant showed increased production of fumiquinazoline A, C, and F and of pyripyropene A (Fig. 3B to E). Reverse transcription-quantitative PCR (RT-qPCR) conﬁrmed that two genes in the fumaquinazoline pathway, fmqB (AFUB_078050) May/June 2020 Volume 11 Issue 3 e00816-20 mbio.asm.org 4 ® Regulation of Caspofungin Tolerance in Aspergillus TABLE 1 MIC values and ranges obtained by CLSI antifungal susceptibility testing method for the A. fumigatus mutant strains identiﬁed in the caspofungin screening and the corresponding wild-type strain Strain MIC (g/ml)a Amphotericin B Voriconazole Itraconazole Posaconazole Wild-type A. fumigatus 2 0.25 0.5–1 1 A. fumigatus ΔcbfA 2 0.5 0.5 1 A. fumigatus ΔzipD 2 0.25 0.5 1 A. fumigatus ΔnctA 2 2 16 4 A. fumigatus ΔnctB 2 0.5 0.5 1 A. fumigatus ΔnctC 2 0.25 0.25 1 A. fumigatus ΔfhdA 2 0.25 0.5 1 A. fumigatus ΔznfA 2 0.25–0.5 0.5 1 A. fumigatus ΔatfA 2 0.25 0.5 1 A. fumigatus ΔrlmA 2 0.5 1.0 1 A. fumigatus ΔzfpA 2 0.5 0.25–0.5 1 A. fumigatus ΔAFUB_054000 2 0.25–0.5 0.25 1 aReference MIC values for A. fumigatus ATCC 204305 (g/ml): amphotericin B, 0.5 to 2; voriconazole, 0.5 to 4; itraconazole, 0.12 to 1; posaconazole, 0.06 to 0.5. TABLE 1 MIC values and ranges obtained by CLSI antifungal susceptibility testing method for the A. fumigatus mutant strains identiﬁed in the caspofungin screening and the corresponding wild-type strain and fmqD (AFUB_047560), and one in the pyripyropene A biosynthesis pathway, pypC (AFUB_081560), were upregulated by about 2-fold to 10-fold in the ΔcbfA strain (Fig. 3F), suggesting that CbfA has a negative role in these SM gene clusters. The ΔnctC mutant showed (about 30%) reduced growth in MM compared to the wild-type and complemented strains (Fig. 4A). It also displayed reduced growth in high osmotic concentrations and at a high temperature (44°C) (Fig. 4B to D). May/June 2020 Volume 11 Issue 3 e00816-20 RESULTS Growth phenotypes of the wild-type, ΔcbfA, and ΔcbfA::cbfA strains grown for 5 days in MM at different temperatures (A), in MM plus CaCl2 500 mM at 37°C (B), in MM plus 16 g/ml of caspofungin at 37°C (C), in MM plus NaCl 500 mM at 37°C (D), and in MM plus sorbitol 1.2 M (E). Statistical analysis was performed using one-tailed, paired t tests for comparisons to the control condition (, P 0.05; , P 0.01; , P 0.001; *, P 0.0001). (F) CbfA:GFP germlings (16 h old) were constitutively present in the nuclei. FIG 2 Molecular characterization of CbfA. Growth phenotypes of the wild-type, ΔcbfA, and ΔcbfA::cbfA strains grown for 5 days in MM at different temperatures (A), in MM plus CaCl2 500 mM at 37°C (B), in MM plus 16 g/ml of caspofungin at 37°C (C), in MM plus NaCl 500 mM at 37°C (D), and in MM plus sorbitol 1.2 M (E). Statistical analysis was performed using one-tailed, paired t tests for comparisons to the control condition (, P 0.05; , P 0.01; , P 0.001; **, P 0.0001). (F) CbfA:GFP germlings (16 h old) were constitutively present in the nuclei. FIG 2 Molecular characterization of CbfA. Growth phenotypes of the wild-type, ΔcbfA, and ΔcbfA::cbfA strains grown for 5 days in MM at different temperatures (A), in MM plus CaCl2 500 mM at 37°C (B), in MM plus 16 g/ml of caspofungin at 37°C (C), in MM plus NaCl 500 mM at 37°C (D), and in MM plus sorbitol 1.2 M (E). Statistical analysis was performed using one-tailed, paired t tests for comparisons to the control condition (, P 0.05; , P 0.01; , P 0.001; **, P 0.0001). (F) CbfA:GFP germlings (16 h old) were constitutively present in the nuclei. FIG 2 Molecular characterization of CbfA. Growth phenotypes of the wild-type, ΔcbfA, and ΔcbfA::cbfA strains grown for 5 days in MM at different temperatures (A), in MM plus CaCl2 500 mM at 37°C (B), in MM plus 16 g/ml of caspofungin at 37°C (C), in MM plus NaCl 500 mM at 37°C (D), and in MM plus sorbitol 1.2 M (E). Statistical analysis was performed using one-tailed, paired t tests for comparisons to the control condition (, P 0.05; , P 0.01; , P 0.001; **, P 0.0001). RESULTS NctC:GFP was constitutively present in the nuclei, and its localization was not affected by switching the temperature or exposure to the aforementioned stressing conditions (Fig. 4E and data not shown). Interestingly, the ΔnctC mutant also secreted reddish compounds in the culture medium under conditions of growth at 37°C for 48 h (Fig. 5A). Mass spectrometry analysis showed that the ΔnctC mutant had increased production of fumitremorgin C and tryprostatin A (Fig. 5B and C). Surprisingly, three genes in the fumitremorgin biosynthesis pathway, ftmE (AFUB_086320), ftmD (AFUB_086340), and ftmA (AFUB_060400), did not shown mRNA accumulation in the ΔnctC mutant (Fig. 5D). There are differences in the cell wall composition of these strains (Fig. 6). The ΔcbfA and ΔnctA strains had higher levels of alkali-insoluble glucosamine (AI-glucosamine) (representing chitin) and lower levels of alkali-soluble glucose (AS-glucose) (indicating -1,3-glucan content) than the wild-type and complemented strains (Fig. 6). The ΔnctC strain had higher levels of AS-glucose whereas the ΔnctA strain had lower levels of AS-glucose and of AI-glucose (representative of -1,3-glucan) than the wild-type and complemented strains (Fig. 6). In a neutropenic mouse model, the ΔcbfA mutant was as virulent as the wild-type strain whereas the ΔnctC mutant was avirulent (see Fig. S1A and S1B at doi.org/ 10.6084/m9.ﬁgshare.12315230). Overall, these results strongly indicate that CbfA and NctC are important regulators of genes that have an impact not only on caspofungin sensitivity and cell wall composition but also on the responses that occur under several stress conditions. FhdA, containing a forkhead-associated domain, is involved in stress toler- ance. The fhdA gene encodes a putative transcription factor of 513 amino acids in length with a molecular weight of 57.1 kDa and a forkhead-associated domain (SM00240; https:// smart.embl.de/) (between amino acids 108 and 166). Phylogenetic analysis of FhdA across fungal species representing the 13 different taxonomic classes or subphyla within Dikarya (https://www.fungidb.org) revealed that orthologues were restricted to Pezizomycotina (Eurotiomycetes, Sordariomycetes, Leotiomycetes, and Dothideomyce- tes), Saccharomycotina (Saccharomycetes), and Taphrinomycotina (Schizosaccharomy- cetes) (Fig. 7A; see also Table S1 at doi.org/10.6084/m9.ﬁgshare.12315230). FhdA orthologues were identiﬁed in other important fungal pathogens, such as Coccidioides spp., Fusarium spp., and Ajellomyces capsulatus (see Table S1). May/June 2020 Volume 11 Issue 3 e00816-20 mbio.asm.org 5 ® Valero et al. FIG 2 Molecular characterization of CbfA. May/June 2020 Volume 11 Issue 3 e00816-20 May/June 2020 Volume 11 Issue 3 e00816-20 RESULTS Standard deviations represent averages Statistical analysis was performed using one-way ANOVA for comparisons to the wild-type condition FIG 3 The ΔcbfA mutant has increased production of several secondary metabolites. (A) Cultures (left column) and supernatants separated from the sam cultures (right column) were grown with agitation for 5 days at 37°C. (B to E) Secondary metabolites were extracted from the wild-type and ΔcbfA cultures from those supernatants, and mass spectrometry was performed, identifying pyripyropene A (B), fumiquinazoline A (C), fumiquinazoline F (D), and fumiquinazolin C (E). WT, wild type; EIC, extracted ion chromatogram. (F) Determination of the expression of fmqB, fmqD, pypE, and pypC by RT-qPCR. tub, -tubulin. Th wild-type and ΔcbfA strains were grown for 48 h at 37°C. Gene expression was normalized using tubA (Afu1g10910). Standard deviations represent average of results from three independent biological repetitions. Statistical analysis was performed using one-way ANOVA for comparisons to the wild-type conditio (, P 0.05). RESULTS (F) CbfA:GFP germlings (16 h old) were constitutively present in the nuclei. The FhdA:GFP was constantly present in the nucleus even after caspofungin expo- sure (about 100 germlings exposed or not to 0.006 g/ml of caspofungin for 30 min; data not shown). There were no differences in the radial growth among the ΔfhdA mutant and wild-type and complemented strains in both MM and YAG medium (2% [wt/vol] glucose, 0.5% [wt/vol] yeast extract, trace elements) (Fig. 7B). However, the ΔfhdA mutant showed some conidiation defects since it produced about 10-fold to 100-fold less conidia depending on the culture media (Fig. 7C). The ΔfhdA mutant was very sensitive to low caspofungin concentrations and had reduced CPE. Furthermore, it exhibited decreased growth when exposed to Congo red or at higher temperature (Fig. 1, 7D to G). In a neutropenic mouse model, the ΔfhdA mutant is as virulent as the mbio.asm.org 6 May/June 2020 Volume 11 Issue 3 e00816-20 ® FIG 3 The ΔcbfA mutant has increased production of several secondary metabolites. (A) Cultures (left column) and supernatants separated from the same cultures (right column) were grown with agitation for 5 days at 37°C. (B to E) Secondary metabolites were extracted from the wild-type and ΔcbfA cultures from those supernatants, and mass spectrometry was performed, identifying pyripyropene A (B), fumiquinazoline A (C), fumiquinazoline F (D), and fumiquinazoline C (E). WT, wild type; EIC, extracted ion chromatogram. (F) Determination of the expression of fmqB, fmqD, pypE, and pypC by RT-qPCR. tub, -tubulin. The wild-type and ΔcbfA strains were grown for 48 h at 37°C. Gene expression was normalized using tubA (Afu1g10910). Standard deviations represent averages of results from three independent biological repetitions. Statistical analysis was performed using one-way ANOVA for comparisons to the wild-type condition (, P 0.05). Regulation of Caspofungin Tolerance in Aspergillus ® Regulation of Caspofungin Tolerance in Aspergillus everal secondary metabolites. (A) Cultures (left column) and supernatants separated from the same ys at 37°C. (B to E) Secondary metabolites were extracted from the wild-type and ΔcbfA cultures from ed, identifying pyripyropene A (B), fumiquinazoline A (C), fumiquinazoline F (D), and fumiquinazoline ) Determination of the expression of fmqB, fmqD, pypE, and pypC by RT-qPCR. tub, -tubulin. The Gene expression was normalized using tubA (Afu1g10910). FIG 3 The ΔcbfA mutant has increased production of several secondary metabolites. (A) Cultures (left column) and supernatants separated from the same cultures (right column) were grown with agitation for 5 days at 37°C. (B to E) Secondary metabolites were extracted from the wild-type and ΔcbfA cultures from those supernatants, and mass spectrometry was performed, identifying pyripyropene A (B), fumiquinazoline A (C), fumiquinazoline F (D), and fumiquinazoline C (E). WT, wild type; EIC, extracted ion chromatogram. (F) Determination of the expression of fmqB, fmqD, pypE, and pypC by RT-qPCR. tub, -tubulin. The wild-type and ΔcbfA strains were grown for 48 h at 37°C. Gene expression was normalized using tubA (Afu1g10910). Standard deviations represent averages of results from three independent biological repetitions. Statistical analysis was performed using one-way ANOVA for comparisons to the wild-type condition (, P 0.05). FIG 3 The ΔcbfA mutant has increased production of several secondary metabolites. (A) Cultures (left column) and supernatants separated from the same cultures (right column) were grown with agitation for 5 days at 37°C. (B to E) Secondary metabolites were extracted from the wild-type and ΔcbfA cultures from those supernatants, and mass spectrometry was performed, identifying pyripyropene A (B), fumiquinazoline A (C), fumiquinazoline F (D), and fumiquinazoline C (E). WT, wild type; EIC, extracted ion chromatogram. (F) Determination of the expression of fmqB, fmqD, pypE, and pypC by RT-qPCR. tub, -tubulin. The wild-type and ΔcbfA strains were grown for 48 h at 37°C. Gene expression was normalized using tubA (Afu1g10910). Standard deviations represent averages of results from three independent biological repetitions. Statistical analysis was performed using one-way ANOVA for comparisons to the wild-type condition (, P 0.05). wild-type strain (see Fig. S1C at doi.org/10.6084/m9.ﬁgshare.12315230). Taken to- gether, these data strongly suggest that FhdA is a TF important for caspofungin, Congo red, and high-temperature tolerance. Transcriptional proﬁling of the fhdA mutant after caspofungin exposure. To identify FhdA-dependent gene expression under conditions of extensive exposure to mbio.asm.org 7 mbio.asm.org 7 May/June 2020 Volume 11 Issue 3 e00816-20 caspofungin, the wild-type and ΔfdhA transcriptomes were assessed postexposure to FIG 4 Molecular characterization of NctC. (A to D) Growth phenotypes of the wild-type, ΔnctC, and ΔnctC::nctC strains grown for 5 days in MM at 37°C (A), MM plus sorbitol 1.2 M (B), MM plus NaCl 500 mM (C), and MM at 44°C (D). Standard deviations represent averages of results from three independent biological repetitions Statistical analysis was performed using one-tailed, paired t tests for comparisons to the control condition (, P 0.05; , P 0.01; , P 0.001; **, P 0.0001). (E) NctC:GFP germlings (16 h old) were constitutively present in the nuclei at 30°C and 37°C. Valero et al. ® ® Valero et al. FIG 4 Molecular characterization of NctC. (A to D) Growth phenotypes of the wild-type, ΔnctC, and ΔnctC::nctC strains grown for 5 days in MM at 37°C (A), MM plus sorbitol 1.2 M (B), MM plus NaCl 500 mM (C), and MM at 44°C (D). Standard deviations represent averages of results from three independent biological repetitions Statistical analysis was performed using one-tailed, paired t tests for comparisons to the control condition (, P 0.05; , P 0.01; , P 0.001; **, P 0.0001). (E) NctC:GFP germlings (16 h old) were constitutively present in the nuclei at 30°C and 37°C. FIG 4 Molecular characterization of NctC. (A to D) Growth phenotypes of the wild-type, ΔnctC, and ΔnctC::nctC strains grown for 5 days in MM at 37°C (A), MM plus sorbitol 1.2 M (B), MM plus NaCl 500 mM (C), and MM at 44°C (D). Standard deviations represent averages of results from three independent biological repetitions Statistical analysis was performed using one-tailed, paired t tests for comparisons to the control condition (, P 0.05; , P 0.01; , P 0.001; **, P 0.0001). (E) NctC:GFP germlings (16 h old) were constitutively present in the nuclei at 30°C and 37°C. caspofungin, the wild-type and ΔfdhA transcriptomes were assessed postexposure to 2 g/ml caspofungin for 48 h (Fig. 7G). Differentially expressed genes were deﬁned as those with a minimum of 2-fold change (FC) in gene expression (log2FC greater than or equal to 1.0 and less than or equal to 1.0) with a false-discovery rate (FDR) of less than 0.05 (see Table S2 at doi.org/10.6084/m9.ﬁgshare.12315230) compared to the wild-type strain under the equivalent conditions. In the wild-type strain, 1,720 and 1,411 genes were upregulated and downregulated, respectively, after 48 h of growth in mbio.asm.org 8 May/June 2020 Volume 11 Issue 3 e00816-20 ® Regulation of Caspofungin Tolerance in Aspergillus FIG 5 The ΔnctC mutant has increased production of several secondary metabolites. (A) Cultures (left column) and supernatants separated from the same cultures (right column) were grown with agitation for 5 days at 37°C. Secondary metabolites were extracted from the wild-type and ΔnctC cultures from those supernatants, and mass spectrometry was performed, identifying tryprostatin A (B) and fumitremorgin C (C). (E) Determination of the expression of ftmE, ftmD, and ftmA by RT-qPCR. The wild-type and ΔnctC strains were grown for 48 h at 37°C. Gene expression was normalized using tubA (Afu1g10910). Standard deviations represent averages of results from three independent biological repetitions. Statistical analysis was performed using one-way ANOVA for comparisons to the wild-type condition (, P 0.05). FIG 5 The ΔnctC mutant has increased production of several secondary metabolites. (A) Cultures (left column) and supernatants separated from the same cultures (right column) were grown with agitation for 5 days at 37°C. Secondary metabolites were extracted from the wild-type and ΔnctC cultures from those supernatants, and mass spectrometry was performed, identifying tryprostatin A (B) and fumitremorgin C (C). (E) Determination of the expression of ftmE, ftmD, and ftmA by RT-qPCR. The wild-type and ΔnctC strains were grown for 48 h at 37°C. Gene expression was normalized using tubA (Afu1g10910). Standard deviations represent averages of results from three independent biological repetitions. Statistical analysis was performed using one-way ANOVA for comparisons to the wild-type condition (, P 0.05). the presence of caspofungin. FunCat (https://elbe.hki-jena.de/fungifun/fungifun.php) enrichment analyses performed for the wild-type strain demonstrated a transcriptional upregulation of genes coding for proteins involved in C-compound and carbohydrate metabolism; sugar, glucoside, polyol, and carboxylate catabolism; lipid, fatty acid, and isoprenoid metabolism; secondary metabolism; ﬂavin adenine dinucleotide/ﬂavin mononucleotide (FAD/FMN) and NAD/NADP binding; and electron transport (Fig. 8A). On the other hand, there was a downregulation of genes coding for proteins involved in rRNA processing, tRNA processing, RNA binding, ribosome biogenesis, secondary metabolism, and degradation of serine (Fig. 8A). This implies that under conditions of extensive exposure to CPE drug concentrations, A. fumigatus upregulates energy production, carbon blocks for carbohydrate and lipid biosynthesis, and secondary metabolism while downregulating RNA metabolism and part of the secondary metab- olism. the presence of caspofungin. May/June 2020 Volume 11 Issue 3 e00816-20 FunCat (https://elbe.hki-jena.de/fungifun/fungifun.php) enrichment analyses performed for the wild-type strain demonstrated a transcriptional upregulation of genes coding for proteins involved in C-compound and carbohydrate metabolism; sugar, glucoside, polyol, and carboxylate catabolism; lipid, fatty acid, and isoprenoid metabolism; secondary metabolism; ﬂavin adenine dinucleotide/ﬂavin mononucleotide (FAD/FMN) and NAD/NADP binding; and electron transport (Fig. 8A). On the other hand, there was a downregulation of genes coding for proteins involved in rRNA processing, tRNA processing, RNA binding, ribosome biogenesis, secondary metabolism, and degradation of serine (Fig. 8A). This implies that under conditions of extensive exposure to CPE drug concentrations, A. fumigatus upregulates energy production, carbon blocks for carbohydrate and lipid biosynthesis, and secondary metabolism while downregulating RNA metabolism and part of the secondary metab- olism. Comparisons of the ΔfhdA mutant to the wild-type strain under basal conditions (without exposure to caspofungin) revealed enrichment of the transcriptional upregulation of genes encoding proteins related to C-compound and carbohydrate metabolism, transport facilities, siderophore-iron transport, and secondary metab- mbio.asm.org 9 May/June 2020 Volume 11 Issue 3 e00816-20 ® Valero et al. FIG 6 Composition of monosaccharides in the cell walls of the wild-type and mutant strains. Standard deviations represent averages of results from three independent biological repetitions. Statistical analysis was performed using one-way ANOVA for comparisons to the wild-type condition (, P 0.05). FIG 6 Composition of monosaccharides in the cell walls of the wild-type and mutant strains. Standard deviations represent averages of results from three independent biological repetitions. Statistical analysis was performed using one-way ANOVA for comparisons to the wild-type condition (, P 0.05). FIG 6 Composition of monosaccharides in the cell walls of the wild-type and mutant strains. Standard deviations represent averages of results from three independent biological repetitions. Statistical analysis was performed using one-way ANOVA for comparisons to the wild-type condition (, P 0.05). olism (Fig. 8B). Under conditions of constant exposure to caspofungin, the ΔfhdA mutant showed enrichment of the upregulation of genes related to amino acid metabolism, aerobic respiration, heat shock response, and mitochondrial transport (Fig. 8C); in addition, enrichment was observed for downregulated genes related to the tricarboxylic acid (TCA) cycle; metabolism of pyruvate; electron transport; alcohol fermentation; lipid, fatty acid, and isoprenoid metabolism; secondary metabolism; and C-compound and carbohydrate metabolism. Taken together, the data suggest that the ΔfhdA mutant evidenced some defects in the mitochondrial function that probably affected several pathways related to caspofungin tolerance. May/June 2020 Volume 11 Issue 3 e00816-20 (A) Phylogenetic distribution of FhdA across fungal genomes. Sequences were aligned through ClustalW implemented in the MEGA7 software (77). Phylogenetic analyzes were performed using MEGA7 software and the neighbor-joining method (78) and 1,000 bootstrap replications (79) for each analysis. The phylogenetic tree was visualized using the Figtree program (http://tree.bio.ed.ac.uk/software/ﬁgtree/). The asterisk () indicates FhdA. (B) Growth phenotypes of the wild-type, ΔfhdA, and ΔfhdA::fhdA strains grown for 5 days at 37°C. (C) Number of conidia in the wild-type, ΔfhdA, and ΔfhdA::fhdA strains grown for 5 days at 37°C on MM and YAG medium. (D to F) Growth phenotypes on MM plus Congo red grown at 37°C (D) and at the higher temperature of 44°C (E) and on MM plus caspofungin (F). (G) Growth in MM plus caspofungin 2 g/ml for 48 h at 37°C. Standard deviations represent averages of results from three independent biological repetitions. Statistical analysis was performed using one-tailed, paired t tests for comparisons to the control condition (, P 0.05; , P 0.01; , P 0.001; **, P 0.0001). FIG 7 Molecular characterization of FhdA null mutant. (A) Phylogenetic distribution of FhdA across fungal genomes. Sequences were aligned through ClustalW implemented in the MEGA7 software (77). Phylogenetic analyzes were performed using MEGA7 software and the neighbor-joining method (78) and 1,000 bootstrap replications (79) for each analysis. The phylogenetic tree was visualized using the Figtree program (http://tree.bio.ed.ac.uk/software/ﬁgtree/). The asterisk () indicates FhdA. (B) Growth phenotypes of the wild-type, ΔfhdA, and ΔfhdA::fhdA strains grown for 5 days at 37°C. (C) Number of conidia in the wild-type, ΔfhdA, and ΔfhdA::fhdA strains grown for 5 days at 37°C on MM and YAG medium. (D to F) Growth phenotypes on MM plus Congo red grown at 37°C (D) and at the higher temperature of 44°C (E) and on MM plus caspofungin (F). (G) Growth in MM plus caspofungin 2 g/ml for 48 h at 37°C. Standard deviations represent averages of results from three independent biological repetitions. Statistical analysis was performed using one-tailed, paired t tests for comparisons to the control condition (, P 0.05; , P 0.01; , P 0.001; *, P 0.0001). FIG 7 Molecular characterization of FhdA null mutant. (A) Phylogenetic distribution of FhdA across fungal genomes. Sequences were aligned through ClustalW implemented in the MEGA7 software (77). Independent validation that iron depletion impacts the fhdA mutant upon CPE. RT-qPCR experiments validated the transcriptome sequencing (RNA-seq) results determined for all seven genes selected from the caspofungin stress response exper- iments (Fig. 9A to G). We selected genes involved in iron starvation, such as hapX (AFUB_052420), which encodes a bZIP transcription factor required for adaptation to both iron depletion and excess and for transcriptional activation of the siderophore system (33); sidA (AFUB_023720), encoding an L-ornithine N5-oxygenase, representing the ﬁrst commit- ted step in siderophore biosynthesis (34); sidI (AFUB_016580), encoding a putative long- chain-fatty-acid–coenzyme A (CoA) ligase, mevalonyl-CoA ligase (35); amcA (AFUB_ 083830), encoding a putative mitochondrial ornithine carrier protein whose expression was upregulated under low-iron conditions (36); mrsA (AFUB_078550), which encodes a mito- chondrial iron transporter (37); cycA (AFUB_028740), encoding a cytochrome c whose transcript was derepressed during iron starvation in a hapX mutant (37); and aoxA (AFUB_022090), encoding an alternative oxidase that mediates the activity of the cyanide- insensitive respiratory pathway that was transcriptionally induced in response to oxidative stress (38) (Fig. 9A to G). The actA A. fumigatus 6g04740 (Afu6g04740) gene, which codes for -actin, was used as a normalizer due to its consistent expression in all strains during caspofungin stress (see Table S3 at doi.org/10.6084/m9.ﬁgshare.12315230). The expression levels of these seven genes showed a high degree of correlation with the RNA-seq data (Pearson correlation values ranging from 0.7737 to 0.9878; Fig. 9H). Iron is important for resistance to caspofungin and CPE for the wild-type and ΔfhdA::fhdA strains since they grew proportionally less extensively in iron-depleted MM (AMM) than in non-iron-depleted MM (MM plus caspofungin [CSP]; Fig. 10A, compare the middle graph to the left graph). Comparisons of growth in AMM and MM showed that there were no growth differences for the ΔfhdA mutant (Fig. 10A, compare the middle graph to the left graph). In contrast, iron excess in MM (MM plus Fe) improved ΔfhdA growth at lower caspofungin and CPE concentrations, while there were no differences for the wild-type and complementing strains (Fig. 10A, compare the right graph to the left graph). May/June 2020 Volume 11 Issue 3 e00816-20 mbio.asm.org 10 ® Regulation of Caspofungin Tolerance in Aspergillus The ΔmrsA strain (mrsA encodes a mitochondrial iron transporter [39]) was more sensitive to 0.25 and 2.0 g/ml of caspofungin in MM and 0.25 g/ml of caspofungin FIG 7 Molecular characterization of FhdA null mutant. Phylogenetic analyzes were performed using MEGA7 software and the neighbor-joining method (78) and 1,000 bootstrap replications (79) for each analysis. The phylogenetic tree was visualized using the Figtree program (http://tree.bio.ed.ac.uk/software/ﬁgtree/). The asterisk () indicates FhdA. (B) Growth phenotypes of the wild-type, ΔfhdA, and ΔfhdA::fhdA strains grown for 5 days at 37°C. (C) Number of conidia in the wild-type, ΔfhdA, and ΔfhdA::fhdA strains grown for 5 days at 37°C on MM and YAG medium. (D to F) Growth phenotypes on MM plus Congo red grown at 37°C (D) and at the higher temperature of 44°C (E) and on MM plus caspofungin (F). (G) Growth in MM plus caspofungin 2 g/ml for 48 h at 37°C. Standard deviations represent averages of results from three independent biological repetitions. Statistical analysis was performed using one-tailed, paired t tests for comparisons to the control condition (, P 0.05; , P 0.01; , P 0.001; *, P 0.0001). The ΔmrsA strain (mrsA encodes a mitochondrial iron transporter [39]) was more sensitive to 0.25 and 2.0 g/ml of caspofungin in MM and 0.25 g/ml of caspofungin in AMM than the wild-type and complementing strains (Fig. 10B). It also had a lower CPE in both MM and AMM (Fig. 10B). Iron excess in MM did not improve the growth of mbio.asm.org 11 mbio.asm.org 11 May/June 2020 Volume 11 Issue 3 e00816-20 ® Valero et al. May/June 2020 Volume 11 Issue 3 e00816-20 FIG 8 A summary of the FunCat terms overrepresented and upregulated or downregulated (adjusted P value, 0.05) upon growth in MM and MM plus caspofungin 2 g/ml for 48 h at 37°C. (A) Wild-type strain treated with 2 g/ml caspofungin versus (x) wild-type control. (B) ΔfhdA control strain x wild-type control. (C) ΔfhdA mutant treated with 2 g/ml caspofungin x wild-type strain treated with 2 g/ml caspofungin. FIG 9 Independent validation of the RNA-seq results. The wild-type and ΔfhdA strains were grown for 48 h at 37°C in the presence or absence of caspofungin 2 g/ml. Gene expression data were obtained for hapX (A), sidA (B), sidI (C), aoxA (D), mrsA (E), cycA (F), and amcA (G) and were normalized by using actA (Afu6g04740). Standard deviations represent averages of results from three independent biological repetitions (each with 2 technical repetitions). Statistical analysis was performed using one-way ANOVA for comparisons to the wild-type condition (, P 0.05). (H) The expression levels of these seven genes measured by RT-qPCR showed a high degree of correlation with the RNA-seq data (Pearson correlation ranging from 0.77 to 0.98). FIG 8 A summary of the FunCat terms overrepresented and upregulated or downregulated (adjusted P value, 0.05) upon growth in MM and MM plus caspofungin 2 g/ml for 48 h at 37°C. (A) Wild-type strain treated with 2 g/ml caspofungin versus (x) wild-type control. (B) ΔfhdA control strain x wild-type control. (C) ΔfhdA mutant treated with 2 g/ml caspofungin x wild-type strain treated with 2 g/ml caspofungin. FIG 8 A summary of the FunCat terms overrepresented and upregulated or downregulated (adjusted P value, 0.05) upon growth in MM and MM plus caspofungin 2 g/ml for 48 h at 37°C. (A) Wild-type strain treated with 2 g/ml caspofungin versus (x) wild-type control. (B) ΔfhdA control strain x wild-type control. (C) ΔfhdA mutant treated with 2 g/ml caspofungin x wild-type strain treated with 2 g/ml caspofungin. the ΔmrsA mutant in medium containing 0.25 g/ml of caspofungin; in contrast, it did improve the growth in medium containing 2 and 8 g/ml of caspofungin (Fig. 10B). Taken together, these data indicate that FhdA connects iron repletion to caspofun- gin resistance and CPE. FhdA is important for the mitochondrial respiratory function. The RNA-seq analysis suggested that FhdA was also important for the mitochondrial function upon CPE (Fig. 8). Thus, we veriﬁed the state of mitochondria in living cells by using Mitotracker green as a mitochondrial ﬂuorescent probe. When A. fumigatus wild-type and ΔfhdA germlings were left unexposed to caspofungin, they displayed an intact mbio.asm.org 12 May/June 2020 Volume 11 Issue 3 e00816-20 ® Regulation of Caspofungin Tolerance in Aspergillus FIG 9 Independent validation of the RNA-seq results. The wild-type and ΔfhdA strains were grown for 48 h at 37°C in the presence or absence of caspofungin 2 g/ml. Gene expression data were obtained for hapX (A), sidA (B), sidI (C), aoxA (D), mrsA (E), cycA (F), and amcA (G) and were normalized by using actA (Afu6g04740). Standard deviations represent averages of results from three independent biological repetitions (each with 2 technical repetitions). Statistical analysis was performed using one-way ANOVA for comparisons to the wild-type condition (, P 0.05). (H) The expression levels of these seven genes measured by RT-qPCR showed a high degree of correlation with the RNA-seq data (Pearson correlation ranging from 0.77 to 0.98). FIG 9 Independent validation of the RNA-seq results. The wild-type and ΔfhdA strains were grown for 48 h at 37°C in the presence or absence of caspofungin 2 g/ml. Gene expression data were obtained for hapX (A), sidA (B), sidI (C), aoxA (D), mrsA (E), cycA (F), and amcA (G) and were normalized by using actA (Afu6g04740). Standard deviations represent averages of results from three independent biological repetitions (each with 2 technical repetitions). Statistical analysis was performed using one-way ANOVA for comparisons to the wild-type condition (, P 0.05). (H) The expression levels of these seven genes measured by RT-qPCR showed a high degree of correlation with the RNA-seq data (Pearson correlation ranging from 0.77 to 0.98). May/June 2020 Volume 11 Issue 3 e00816-20 mbio.asm.org 13 ® Valero et al. i h d i l k I h li f b h h i d FIG 10 Iron is important for caspofungin resistance and CPE. Growth phenotypes were determined for the wild-type, ΔfhdA (A), and ΔmrsA (B) strains and the corresponding complementing strains grown for 5 days in MM, AMM, and AMM plus FeSO4 200 M at 37°C with and without CSP. Standard deviations represent averages of results from three independent biological repetitions. Statistical analysis was performed using one-tailed, paired t tests for comparisons to the control condition, MM plus CSP (, P 0.05; , P 0.01; , P 0.001; *, P 0.0001). CSP, caspofungin; Fe, FeSO4. FIG 10 Iron is important for caspofungin resistance and CPE. Growth phenotypes were determined for the wild-type, ΔfhdA (A), and ΔmrsA (B) strains and the corresponding complementing strains grown for 5 days in MM, AMM, and AMM plus FeSO4 200 M at 37°C with and without CSP. Standard deviations represent averages of results from three independent biological repetitions. Statistical analysis was performed using one-tailed, paired t tests for comparisons to the control condition, MM plus CSP (, P 0.05; , P 0.01; , P 0.001; **, P 0.0001). CSP, caspofungin; Fe, FeSO4. FIG 10 Iron is important for caspofungin resistance and CPE. Growth phenotypes were determined for the wild-type, ΔfhdA (A), and ΔmrsA (B) strains and the corresponding complementing strains grown for 5 days in MM, AMM, and AMM plus FeSO4 200 M at 37°C with and without CSP. Standard deviations represent averages of results from three independent biological repetitions. Statistical analysis was performed using one-tailed, paired t tests for comparisons to the control condition, MM plus CSP (, P 0.05; , P 0.01; , P 0.001; **, P 0.0001). CSP, caspofungin; Fe, FeSO4. mitochondrial network. In contrast, when germlings of both the strains were exposed to caspofungin, they showed a mitochondrial network fragmentation process, revealed by an intense punctuated ﬂuorescent distribution in the cytoplasm, in 100% and 60% of the germlings, respectively. g g y Alternatively, we subjected cellular extracts from wild-type and ΔfhdA strains to mitochondrial fractionation and evaluated their respiratory competence by measuring the activities of mitochondrial complexes I to IV (Fig. 11; see also Fig. S2 at doi.org/ 10.6084/m9.ﬁgshare.12315230). NADH cytochrome c reductase (NCCR, representing complexes I and III), succinate-cytochrome c reductase (SCRR, representing complexes II and III), and cytochrome c oxidase (COX, representing complex IV) exhibited 4-fold, 2-fold, and 7-fold lower activities in the ΔfhdA mutant than in the wild-type strain, respectively (Fig. 11). The wild-type NCCR, SCCR, and COX activities were also reduced about 2-fold to 7-fold when the strain was exposed for 48 h to caspofungin 2 g/ml (Fig. 11). The decrement in the activity of the tested respiratory complexes of caspofungin-treated wild-type cells is in agreement with the observed organellar fragmentation. In contrast, the NCCR, SCCR, and COX activities were the same or were increased or reduced about 2-fold under conditions of exposure of the ΔfhdA mutant to caspofungin 2 g/ml for 48 h (Fig. 11). In this case, the fragmentation of the mitochondrial network did not result in an additive effect in the respiratory impairment of the ΔfhdA cells. Mitochondrial network fragmentation is frequently associated with May/June 2020 Volume 11 Issue 3 e00816-20 mbio.asm.org 14 ® Regulation of Caspofungin Tolerance in Aspergillus FIG 11 FhdA is important for mitochondrial respiratory function in A. fumigatus. Enzymatic activities were determined for NADH cytochrome c reductase (NCCR, representing complexes I and III) (A), succinate-cytochrome c reductase (SCRR, representing complexes II and III) (B), and cytochrome c oxidase (COX, representing complex IV) (C). Statistical analysis was performed using one-tailed, paired t tests for comparisons to the control condition (, P 0.05; , P 0.01; , P 0.001; **, P 0.0001). FIG 11 FhdA is important for mitochondrial respiratory function in A. fumigatus. NctA, NctB, NctC, and CbfA are members of the evolutionarily conserved CBF/NF-Y Enzymatic activities were determined for NADH cytochrome c reductase (NCCR, representing complexes I and III) (A), succinate-cytochrome c reductase (SCRR, representing complexes II and III) (B), and cytochrome c oxidase (COX, representing complex IV) (C). Statistical analysis was performed using one-tailed, paired t tests for comparisons to the control condition (, P 0.05; , P 0.01; , P 0.001; **, P 0.0001). the organellar quality control and mitophagy (40); therefore, the removal of damaged mitochondria can provide some compensation for the already prejudiced respiration observed in ΔfhdA cells. May/June 2020 Volume 11 Issue 3 e00816-20 DISCUSSION The echinocandin caspofungin, considered a second-line therapy for use in com- batting IA, is a -1,3-glucan synthase inhibitor and, when used in high concentrations, restores the anticipated A. fumigatus growth inhibition, a phenomenon called the “caspofungin paradoxical effect” (CPE). CPE has been widely associated with increased chitin content in the cell wall due to a compensatory upregulation of chitin synthase- encoding genes (16, 41). We have previously demonstrated that CPE depends on the cell wall integrity (CWI) mitogen-activated protein kinase MpkA and its associated TF RlmA, which regulates chitin synthase gene expression in response to different con- centrations of caspofungin (23, 29). Moreover, we found that the calcium- and calcineurin-dependent TF CrzA binds to and regulates the expression of speciﬁc chitin synthase genes in the absence of caspofungin and under CPE conditions (23). We also identiﬁed a novel basic leucine zipper TF, ZipD, that is also involved in CPE. Here, we have identiﬁed 11 TFs that are involved in the A. fumigatus CPE. Among these TFs, nine are important not only for caspofungin tolerance but also for resistance. Six of them (ΔcbfA, ΔzipD, ΔnctC, ΔznfA, ΔatfA, and ΔrlmA) have been previously identiﬁed as being associated with calcium sensitivity and caspofungin sensitivity and displaying reduced CPE (22, 23, 28, 42). ZipD and RlmA are important for osmotic and cell wall stresses, collaborating for cell wall construction and the activation of several pathways impor- tant for osmotic stability (22, 23). Here, we demonstrated that there were increased levels of chitin composition in the cell wall of the ΔcbfA and ΔnctA mutants but not in that of the ΔnctC mutant. We previously demonstrated that the ΔzipD and ΔrlmA mutants have altered chitin composition (22, 23). It remains to be determined if the chitin concentration is altered in the ΔnctB, ΔfhdA, ΔznfA, ΔatfA, ΔzfpA, and ΔAFUB_054000 mutants. The identiﬁcation of these TFs increases the number of genetic determinants known for caspofungin resistance and CPE and provides new opportunities for the identiﬁcation of signaling pathways important for caspofungin resistance and tolerance. NctA, NctB, NctC, and CbfA are members of the evolutionarily conserved CBF/NF- mbio.asm.org 15 May/June 2020 Volume 11 Issue 3 e00816-20 ® Valero et al. family of transcription factors (26). ΔcbfA not only was more sensitive to caspofungin and displayed reduced CPE but also was more sensitive to osmotic, cell wall, high temperature, and calcium stresses. DISCUSSION CbfA was constantly present in the nucleus, and its localization was not affected by different stimuli. Interestingly, derepression of several secondary metabolites was seen in the ΔcbfA mutant, suggesting that CbfA is impor- tant for the regulation of the biosynthesis of A. fumigatus secondary metabolites. CbfA is the homologue of Dpb4p, one of the subunits of the S. cerevisiae DNA polymerase epsilon (43). Loss of the DPB4 gene in S. cerevisiae is not lethal but disturbs DNA replication (43). On the other hand, a C. albicans Dpb4p homologue has been identiﬁed as one of the regulators of Goa1p, a protein required for respiratory growth and resistance to oxidative agents (44, 45). C. albicans Δdpb4 is more sensitive to cell wall inhibitors, including caspofungin, and shows defects in mitochondrial function (45). NctA (AFUB_029870) is the putative homologue of the S. cerevisiae negative cofactor 2 (NC2) complex -subunit Bur6p. NctA has two paralogues in A. fumigatus, NctC and HapE. In addition, NctB is a putative homologue of the S. cerevisiae NC2 complex -subunit Ncb2p. It has been shown that, as in the case of S. cerevisiae (Bur6p and Ncb2p), A. fumigatus NctA and NctB physically interact (25). These two TFs are subunits of the heterotrimeric NC2 complex that acts as a negative regulator of RNA polymerase II transcription by inhibiting formation of the preinitiation complex (46, 47). Genome- wide studies in S. cerevisiae, C. albicans, A. fumigatus, and Homo sapiens have shown that the N-chlorotaurine (NCT) complex interacts with 20% to 30% of all RNA Pol II gene promoters (25, 48–51). Similarly to the ΔcbfA mutant, the ΔnctC mutant not only was more sensitive to caspofungin and exhibited a reduced CPE but also was more sensitive to several different stresses, such as osmotic stress and higher temperature. NctC was also constantly present in the nucleus, and its localization was not affected by different stimuli. In this mutant, derepression of several secondary metabolites was also ob- served, suggesting that not only CbfA but also NctC is important for the regulation of the synthesis of secondary metabolites in A. fumigatus. Recently, A. fumigatus nctA and nctB null mutants were shown to phenocopy each other, being more resistant to itraconazole, voriconazole, posaconazole, terbinaﬁne, miltefosine, and amphotericin and more sensitive to cell wall-perturbing agents such as Congo red, calcoﬂuor white, caspofungin, and micafungin (25). May/June 2020 Volume 11 Issue 3 e00816-20 mbio.asm.org 16 mbio.asm.org 16 DISCUSSION We were not able to observe caspofungin sensitivity in the ΔnctA mutant or resistance to azoles in the ΔnctB mutant, which could have been due to the different conditions used for the MIC and growth assays. It was reported previously that, in azole-resistant isolates of C. albicans, the azole transporter CDR1, which is regulated by the Ncb2 homologue, was overexpressed (52). Conditional NCB2 null mutants displayed decreased susceptibility toward azole drugs and enhanced transcription of CDR1 (52). Four TFs, ZnfA, FhdA, ZfpA, and AFUB_054000, are completely novel and have not yet been characterized. In addition to its sensitivity to caspofungin and reduced CPE, the ΔznfA mutant is more sensitive to calcium than the wild-type strain (22). The ZfpA mutant has increased mRNA accumulation in the presence of voriconazole or calcium (31, 32, 53). We investigated in more detail the FhdA TF; the ΔfhdA mutant not only was sensitive to caspofungin and had reduced CPE but also was more sensitive to Congo red exposure and high temperature. RNA-seq experiments performed with the ΔfhdA mutant in the absence or presence of caspofungin suggested that this strain had some defects in the mitochondrial function. Mitochondrial pathways such as those associated with amino acid degradation, electron transport chains, some mitochondrial lipids, fatty acids, and isoprenoid metabolism were downregulated when the ΔfhdA mutant was exposed to caspofungin in comparison with the wild-type strain. The ΔfhdA mutant had reduced mitochondrial fragmentation and lower levels of mitochondrial enzymatic activities, indicating its involvement in the functioning of mitochondria. The connection between mitochondrial function and caspofungin tolerance has been widely reported in the literature (15, 39, 54–58). Addition of respiratory inhibitors results in signiﬁcant decreases of the caspofungin MICs for C. parapsilosis (39). Caspofungin exposure mbio.asm.org 16 May/June 2020 Volume 11 Issue 3 e00816-20 ® Regulation of Caspofungin Tolerance in Aspergillus induces expression of A. fumigatus mitochondrial respiratory chain genes, speciﬁcally NADH-ubiquinone oxidoreductases (complex I), while CPE is abolished by addition of rotenone, a complex I inhibitor (58). Interestingly, those authors also described a connection between rotenone inhibition and increases in intracellular calcium concen- trations. Recently, it has been reported that caspofungin induced mitochondrion- derived reactive oxygen species (ROS) and that inhibition of ROS formation eliminated caspofungin-induced resistance (15). Those authors proposed a novel mechanism of caspofungin resistance through ROS production, as well as integrated oxidative stress and sphingolipid alterations (15). DISCUSSION We have extended those studies by demonstrating that caspofungin induced mitochondrial fragmentation and inhibited all the tested respiratory complexes in the wild-type strain. A link between iron chelation and caspofungin susceptibility has also been observed (59, 60). Gallium is a metal compound that can be taken up by cellular iron transport systems, replacing it in iron-containing proteins. However, it cannot be reduced under physiological conditions, inhibiting the functionality of gallium-complexed proteins and arresting growth (59). We demonstrated previously that gallium nitrate III [Ga(NO3)3] has a synergistic effect with caspofungin but not with azoles and that it impairs CPE (59). We have observed several genes that encode proteins important for iron assimilation were more highly expressed in the ΔfhdA mutant, suggesting prob- lems involving iron deﬁciency in this mutant. Mitochondria employ the majority of cellular iron and therefore have a central role in its homeostasis. Actually, addition of iron together with caspofungin improved the CPE in the ΔfhdA mutant whereas iron depletion in the presence of caspofungin increased caspofungin sensitivity and de- creased CPE. Interestingly, the same effect was observed in the ΔmrsA mutant (mrsA encodes a mitochondrial iron transporter), suggesting that iron deﬁciency in mitochon- dria affects caspofungin sensitivity and CPE. Our work opens new possibilities for understanding caspofungin tolerance and resistance in fungi. Although the clinical signiﬁcance of CPE remains unclear (16, 61), those pathways that are modulated during caspofungin responses could be considered to be promising targets for drug development. The implementation of the use of drugs targeting these pathways together with caspofungin could help to improve caspofun- gin efﬁcacy and, ultimately, aspergillosis treatment. May/June 2020 Volume 11 Issue 3 e00816-20 MATERIALS AND METHODS analyses were used to verify homologous cassette integration into the A. fumigatus genome (see Fig. S3 at doi.org/10.6084/m9.ﬁgshare.12315230). The primers used in this work are listed in Table S5. Microscopy. Conidiospores were grown on coverslips in 4 ml of liquid MM for 16 h at 30°C. After incubation, coverslips with adherent germlings were left untreated or treated with Hoechst 33342 dye (Molecular Probes, Eugene, OR, USA) 20 g/ml or MitoTracker green FM dye (Molecular Probes) (250 nM) for 10 min. For caspofungin experiments, after overnight incubation, cells were exposed to caspofungin for 1 h (0.003 or 0.006 g/ml) for 30 min or left unexposed. Subsequently, the coverslips were rinsed with phosphate-buffered saline (PBS; 140 mM NaCl, 2 mM KCl, 10 mM NaHPO4, 1.8 mM KH2PO4, pH 7.4) and mounted for examination. Slides were visualized on an Observer Z1 ﬂuorescence microscope using a 100 objective oil immersion lens objective. For GFP and MitoTracker green conditions, ﬁlter set 38 (high efﬁciency [HE]) was used with an excitation wavelength of 450 to 490 nm and emission wavelength of 500 to 550 nm. For Hoechst/DAPI [4,6-diamidino-2-phenylindole] staining, ﬁlter set 49 was used with an excitation wavelength of 365 nm and emission wavelength of 420 nm to 470 nm. DIC (differential interference contrast) images and ﬂuorescent images were captured with an AxioCam camera (Carl Zeiss, Oberkochen, Germany) and processed using AxioVision software (version 4.8). RNA extraction and qRT-PCR. After the stated treatments were performed, mycelia were collected and ground to a ﬁne powder in liquid N2. RNA was extracted with using TRIzol reagent (Invitrogen, Life Technologies, Camarillo, CA, USA) following DNA digestion with RQ1 RNase-free DNase (Promega, Fitchburg, WI, USA) according to the manufacturer’s instructions. Total RNA was reverse-transcribed into cDNA by using an ImProm-II reverse transcription system (Promega) and oligo(dT). The ampliﬁcation assay was carried out in a model 7500 real-time PCR system (Applied Biosystems). qPCRs were performed in a 10-l ﬁnal volume containing Sybr green PCR master mix (Applied Biosystems) under the following conditions: an initial step of 2 s at 50°C, followed by 10 min at 95°C and 40 cycles at 95°C for 15 s and 60°C for 1 min. Three independent biological replicates were used, and mRNA quantity relative fold change data were calculated using standard curves (67) and normalized by -tubulin or -actin expression. All the primers used for RT-qPCR experiments are described in Table S5 at doi.org/10.6084/ m9.ﬁgshare.12315230. MATERIALS AND METHODS RNA sequencing. A. fumigatus conidia (5 107) from the parental CEA17 and ΔfhdA mutant strains were inoculated in triplicate into liquid MM and cultured for 8 h at 37°C prior to the addition or not of 2 g/ml caspofungin and left to grown for 48 h. Mycelia were harvested, frozen, and ground in liquid nitrogen. Total RNA was extracted using TRIzol (Invitrogen), treated with RQ1 RNase-free DNase I (Promega), and puriﬁed using an RNeasy kit (Qiagen) according to manufacturer’s instructions. RNA from each treatment was quantiﬁed using a Qubit ﬂuorometer and analyzed using an Agilent 2100 Bioanalyzer system to assess the integrity of the RNA. RNA integrity number (RIN) values were calculated and ranged from 7.0 to 9.5. An Illumina TruSeq stranded mRNA sample preparation kit was used to construct cDNA libraries following the manufacturer’s instructions. Libraries were sequenced (2 100 bp) at the next-generation sequencing (NGS) facility of the Brazilian Bioethanol Science and Technology Laboratory (CTBE) using a HiSeq 2500 instrument, generating approximately 11 106 fragments per sample. Obtained fastq ﬁles were quality checked with FastQC (http://www.bioinformatics.babraham.ac.uk/ projects/fastqc/) and cleaned (quality trim, adaptor removal, and minimum length ﬁltering) with Trim- momatic (68). rRNA was removed using SortMeRNA (69). High-quality RNA-seq reads were mapped against the A. fumigatus genome using CLC Genomics Workbench software (CLC bio—v4.0; Finlands- gade, Denmark) and the following parameters: for the mapping settings, minimum length fraction 0.7, minimum similarity fraction 0.8, and maximum number of hits for read 1; for the alignment settings, minimum distance 180 and maximum distance 1,000. All samples achieved saturation of known exon-exon junctions. Reproducibility among biological replicates was assessed by exploring a principal- component-analysis (PCA) plot of the top 500 genes that had the largest biological variation between the libraries and by pairwise measuring of the Pearson correlations among the replicates over the whole set of genes. In order to assess transcript abundance, exonic reads were counted in a strand-speciﬁc way using the featureCounts function from the Rsubread Bioconductor package (70). Calling of differentially expressed genes was carried out using DESeq2 (71) and, as the threshold, an adjusted P value of 0.01 (72) Extraction of secondary metabolites and high-resolution mass spectrometry (HRMS) analyses. For mass spectrometry analyses, extractions were performed following a previously described method- ology (73) with few modiﬁcations. May/June 2020 Volume 11 Issue 3 e00816-20 MATERIALS AND METHODS Strains and media. Strains were grown at 37°C in either complete medium (YAG medium) (2% [wt/vol] glucose, 0.5% [wt/vol] yeast extract, trace elements) or minimal medium (1% [wt/vol] glucose, nitrate salts, trace elements, pH 6.5). Solid YAG medium and MM were the same as described above except that 1.7% (wt/vol) or 2% (wt/vol) agar was added. Trace elements, vitamins, and nitrate salts compositions were as described previously (62). For iron limitation experiments, strains were growth in solid AMM (containing glucose 1% [wt/vol], trace elements without FeSO4, agar 1.7% [wt/vol]). Wherever required, MM was supplemented, at the stated concentrations, with calcium chloride (CaCl2), sorbitol, sodium chloride (NaCl), Congo red, iron sulfate (FeSO4), and caspofungin. For phenotype characterization, solid-medium plates were inoculated with 104 spores per strain and the spores were left to grow for 120 h at 37, 30, or 44°C. Values representing levels of radial growth were expressed as ratios, determined by dividing colony radial diameter of growth under the stress condition by colony radial diameter under the control (no stress) condition. All the strains used in this work are described in Table S4 at doi.org/10.6084/m9.ﬁgshare.12315230. Construction of A. fumigatus mutant strains. TF deletion mutants of A. fumigatus were described previously (25) and were obtained as described in reference 63. All GFP fusion constructions were performed by in vivo recombination in S. cerevisiae as previously reported (64), and all methods were adapted to A. fumigatus as previously described (65). Approximately 1 kb from the 5= untranscribed region (5=-UTR)- and 3=-UTR-ﬂanking sequences of the targeted genes were selected for primer design. The 5F and 3R primers contained a short sequence homologous to the multiple-cloning site of the pRS426 plasmid. For NctC:GFP constructions, the pyrG gene was used as a marker for prototrophy and was ampliﬁed from the pCDA21 plasmid (66). For CbfA:GFP and FhdA:GFP constructions, the GFP-pyrG- trpC fragment was ampliﬁed from the pOB435 plasmid. Cassettes used for A. fumigatus transformation were ampliﬁed directly from yeast genomic DNA using TaKaRa Ex Taq DNA polymerase (Clontech TaKaRa Bio). MM was used as selective media for positive transformants. All complemented strains were obtained by cotransformation of the open reading frame (ORF) and plasmid pPTRI containing the gene that confers resistance to pyrithiamine. MM supplemented with pyrithiamine (1 g/ml) was used as selective media for positive transformants. Southern blotting and PCR mbio.asm.org 17 ® Valero et al. MATERIALS AND METHODS Brieﬂy, 100 mg of the lyophilized fungal supernatant was extracted with 1 ml of MeOH during 1 h in an ultrasonic bath. The extracts were centrifuged at 13,000 rpm for 5 min, and the supernatants were collected and dried under N2. Crude extracts were resuspended in 1 ml MeOH and ﬁltered using a 0.22-m-pore-size ﬁlter. All the extractions were performed in triplicate. The liquid chromatography-HRMS (LC-HRMS) analyses were performed in a LC Agilent 1200 liquid chromatography mass spectrometer coupled to an Agilent iFunnel 6550 quadrupole-time of ﬂight (Q-ToF) LC-MS system with an electrospray ionization (ESI) source. All operations and spectrum analyses were conducted using Agilent Mass Hunter Workstation software. The parameters of MS analysis were set as follows: ESI source in positive mode; nebulizing gas temperature at 290°C; capillary voltage at 3,000 V; nozzle voltage at 320 V; drying gas ﬂow of 12 ml min1; nebulization gas pressure at 45 lb/in2; auxiliary gas temperature at 350°C; auxiliary gas ﬂow at 12 ml min1; m/z mass range of 50 to 1,700. The chromatography was performed on a Thermo Scientiﬁc Accucore C18 column (2.6-m pore size, 2.1 mm by 100 mm). For the gradient elution, 0.1% formic acid (solvent A) and acetonitrile (solvent B) were used, and the eluent proﬁle (A:B) was as follows: 0 to 10 min, gradient from 95:5 to 2:98; 10 to 15 min and ﬁnal mbio.asm.org 18 ® Regulation of Caspofungin Tolerance in Aspergillus isocratic elution at 2:98. The ﬂow rate was set at 0.2 ml min1, and a 6-l volume of sample was injected in each injection. isocratic elution at 2:98. The ﬂow rate was set at 0.2 ml min1, and a 6-l volume of sample was injected in each injection. j Mycelial cell wall analysis. Conidia (1 108) were inoculated into 30 ml liquid MM. After 24 h of growth at 37°C, mycelia were harvested by ﬁltration, washed extensively with and suspended in Milli-Q water, and disrupted using a FastPrep homogenizer (MP Biomedicals) at 4°C with 0.5-mm-diameter glass beads. MATERIALS AND METHODS The mycelial suspension obtained after disruption was centrifuged (5,000 rpm, 10 min at 4°C), and the pellet (cell wall) was washed with Milli-Q water (three times); boiled in a water bath upon suspension of the pellet in Tris-HCl buffer (50 mM; pH 7.5) supplemented with EDTA (50 mM), SDS (2%), and -mercaptoethanol (-ME, 40 mM) for 15 min (two times); and washed with Milli-Q water (ﬁve times; centrifugation at 5,000 rpm for 10 min). The pellet (cell wall) obtained was subjected to alkali fraction- ation by incubation of the pellet in NaOH (1 M) containing NaBH4 (0.5 M) at 65°C for 1 h (two times). The insoluble fraction (alkali insoluble; AI) obtained after centrifugation of the alkali-fractionated cell wall was washed with Milli-Q water (six times; centrifugation at 5,000 rpm for 10 min) and freeze-dried. Excess of NaBH4 in the supernatant (alkali-soluble fraction; AS) was neutralized with acetic acid (2%), dialyzed against water until neutrality was reached, and freeze-dried. The AI and AS fractions were then subjected to gas chromatography (GC)-liquid chromatography (Perichrom, France) using an instrument equipped with a ﬂame ionization detector and a fused silica capillary column of 30 m and 0.32 mm internal diameter ﬁlled with BP1. In brief, AI and AS suspensions (100 g; 10 mg/ml stock), containing 4 g myo-inositol (internal standard), were hydrolyzed with triﬂuoroacetic acid (4 N; to determine levels of hexoses) and hydrochloric acid (8 N; to determine levels of hexosamines) at 100°C for 4 h, dried under vacuum using NaOH pellets, reduced with NaBH4 (10 mg/hydrolyzed sample in 1 ml of 0.1 M NH4OH) for 8 to 10 h, dried under vacuum, and washed with methanol (two times). Acetic anhydride (200 l) and pyridine (50 l) were added to each of these samples followed by incubation for 10 to 12 h for derivatization of hydrolyzed monosaccharides into alditol acetates. They were then subjected to chloroform-water fractionation, and the derivatives extracted in the chloroform fractions were dried, suspended in methanol, and subjected to GC analyses. The monosaccharide composition was deter- mined from the GC peak area in comparison with the peak area of the internal standard (myo-inositol). The analysis was performed with three biological replicates. Cell fractionation and mitochondria isolation. Mitochondria were isolated from A. fumigatus mycelium (10 g moist mass) based on a previously described protocol (74). MATERIALS AND METHODS Wild-type and ΔfhdA conidia (1 108) were precultured on 100 ml of MM for 8 h at 37°C prior to addition of 2 g/ml caspofungin and left to grown for 48 h to induce cell wall stress. The control was left untreated. Mycelia were harvested by ﬁltration in Miracloth (Calbiochem), frozen in liquid nitrogen, and subjected to cell fractionation methods. Mitochondrial extracts were quantiﬁed by the use of a modiﬁed Lowry method (75) and loaded on 12% SDS-PAGE, electroblotted to PVDF (polyvinylidene diﬂuoride) membranes (GE Health Care), and subsequently analyzed by Western blotting. Monoclonal VDAC1/porin antibody (16G9E6BC4) from S. cerevisiae (Thermo Scientiﬁc) and polyclonal homemade anti-CytC antibody (a gift from Mário H. Barros) were used to conﬁrm mitochondrial membrane enrichment. Mitochondrial enzymatic activities. Mitochondrial succinate-cytochrome c reductase (SCCR), NADH-cytochrome c reductase (NCCR), and cytochrome c oxidase (COX) were all assayed spectropho- tometrically at 23°C, as previously described (76). Brieﬂy, NCCR and SCCR activities were measured by following the increase in absorbance at 550 nm due to the reduction of cytochrome c in the presence of 10 mM NADH (NCCR) or 10 mM succinate (SCCR). COX was assayed by following the oxidation of ferro-cytochrome c at 550 nM. The speciﬁc activity was calculated using an extinction coefﬁcient of 18 mM1 cm1 for cytochrome c. Murine model of pulmonary aspergillosis. Outbred female BALB/c mice (body weight, 20 to 22 g) were housed in vented cages containing 5 animals. Mice were immunosuppressed with cyclophosph- amide (150 mg/kg of body weight) administered intraperitoneally on days 4 and 1 prior to infection and day 2 postinfection. Hydrocortisone acetate (200 mg/kg of body weight) was injected subcutane- ously on day 3. A. fumigatus conidia grown in YAG plates for 2 days prior to infection were harvested in PBS and ﬁltered through a Miracloth (Calbiochem). Conidial suspensions were washed three times with PBS, counted using a hemocytometer, and resuspended at a concentration of 5 106 conidia/ml. The viability of the administered inoculum was determined by incubating a serial dilution of the conidia on YAG medium, at 37°C. Mice were anesthetized by halothane inhalation and infected by intranasal instillation of 105 conidia in 20 l of PBS. As a negative control, a group of ﬁve mice received PBS only. Mice were weighed every 24 h from the day of infection and visually inspected twice daily. May/June 2020 Volume 11 Issue 3 e00816-20 Valero et al. Valero et al. Valero et al. Antifungal susceptibility testing. The MIC of azole drugs (voriconazole, itraconazole, and po- saconazole; Sigma-Aldrich, St. Louis, MO, USA) and amphotericin B (Sigma-Aldrich) was determined using increasing concentrations of the aforementioned compounds in RPMI 1640 based on protocol M38 (2017) of the antifungal microdilution test proposed by the Clinical and Laboratory Standards Institute (CLSI) (https://clsi.org/media/1711/clsistandardsdevelopmentpoliciesandprocessesﬁnal.pdf). Statistical analysis. Grouped column plots with standard deviation error bars were used for representations of data. For comparisons with data from wild-type or control conditions, we performed one-tailed, paired t tests (Fig. 1, 2, 4, 7, 10, and 11) and one-way analysis of variance (ANOVA) (Fig. 3, 5, 6, and 9). P values of 0.05 were considered signiﬁcant. P values are indicated by asterisks as follows: , P 0.05; , P 0.01; , P 0.001; ***, P 0.0001. 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ACKNOWLEDGMENTS We thank São Paulo Research Foundation (FAPESP) (grants 2016/07870-9 [to G.H.G.], 2018/00715-3 [C.V.], 2016/12948-7 [P.A.D.C.], 2016/22062-6 [J.C.], 2016/21392-2 [L.P.S.], and 2017/07536-4 [A.C.C.]) and Conselho Nacional de Desenvolvimento Cientíﬁco e Tecnológico (CNPq) (G.H.G.), both in Brazil, for ﬁnancial support. We also thank Ling Lu for providing us with the A. fumigatus mrsA mutants and the two anonymous reviewers for their comments and suggestions. REFERENCES 10. Perlin DS. 2015. Mechanisms of echinocandin antifungal drug resistance. Ann N Y Acad Sci 1354:1–11. https://doi.org/10.1111/nyas.12831. 1. Brown GD, Denning DW, Gow NA, Levitz SM, Netea MG, White TC. 2012. Hidden killers: human fungal infections. Sci Transl Med 4:165rv13. https://doi.org/10.1126/scitranslmed.3004404. 1. Brown GD, Denning DW, Gow NA, Levitz SM, Netea MG, White TC. 2012. Hidden killers: human fungal infections. Sci Transl Med 4:165rv13. https://doi.org/10.1126/scitranslmed.3004404. 11. 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W4307817193.txt	null	en		Stable nanovesicles formed by intrinsically planar bilayers	Journal of colloid and interface science	2,023	cc-by	10,164	Journal of Colloid and Interface Science 631 (2023) 202–211 Contents lists available at ScienceDirect Journal of Colloid and Interface Science journal homepage: www.elsevier.com/locate/jcis Stable nanovesicles formed by intrinsically planar bilayers Mariana Köber b,a,1, Sílvia Illa-Tuset a,1, Lidia Ferrer-Tasies a,c,1, Evelyn Moreno-Calvo a, Witold I. Tatkiewicz a, Natascia Grimaldi a, David Piña a, Alejandro Pérez Pérez a, Vega Lloveras a,b, José Vidal-Gancedo a,b, Donatella Bulone d, Imma Ratera a,b, Jan Skov Pedersen e, Dganit Danino f, Jaume Veciana a,b, Jordi Faraudo a,⇑, Nora Ventosa a,b,⇑ a Institut de Ciència de Materials de Barcelona (ICMAB-CSIC), Campus UAB, Cerdanyola del Vallès, Spain Networking Biomedical Research Networking Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Spain Nanomol Technologies SL, Campus UAB, 08193 Cerdanyola del Vallès, Spain d Institute of Biophysics, Palermo, National Research Council, Palermo, Italy e Department of Chemistry and Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Gustav Wieds Vej, 14, 8000 Aarhus, Denmark f CryoEM Laboratory of Soft Matter, Faculty of Biotechnology and Food Engineering, Technion, Haifa 3200003, Israel b c g r a p h i c a l a b s t r a c t a r t i c l e i n f o Article history: Received 2 August 2022 Revised 16 October 2022 Accepted 20 October 2022 Available online 29 October 2022 Keywords: Nanovesicles Molecular self-assembly Vesicle stability Composition asymmetry Quatsomes a b s t r a c t Hypothesis: Quatsome nanovesicles, formed through the self-assembly of cholesterol (CHOL) and cetyltrimethylammonium bromide (CTAB) in water, have shown long-term stability in terms of size and morphology, while at the same time exhibiting high CHOL-CTAB intermolecular binding energies. We hypothesize that CHOL/CTAB quatsomes are indeed thermodynamically stable nanovesicles, and investigate the mechanism underlying their formation. Experiments: A systematic study was performed to determine whether CHOL/CTAB quatsomes satisfy the experimental requisites of thermodynamically stable vesicles. Coarse-grain molecular dynamics simulations were used to investigate the molecular organization in the vesicle membrane, and the characteristics of the simulated vesicle were corroborated with experimental data obtained by cryo–electron microscopy, small- and wide-angle X-ray scattering, and multi-angle static light scattering. Findings: CHOL/CTAB quatsomes fulfill the requisites of thermodynamically stable nanovesicles, but they do not exhibit the classical membrane curvature induced by a composition asymmetry between the bilayer leaflets, like catanionic nanovesicles. Instead, CHOL/CTAB quatsomes are formed through the association of intrinsically planar bilayers in a faceted vesicle with defects, indicating that distortions in the organization and orientation of molecules can play a major role in the formation of thermodynamically stable nanovesicles. Ó 2022 Published by Elsevier Inc. ⇑ Corresponding authors at: Institut de Ciència de Materials de Barcelona (ICMAB-CSIC), Campus UAB, Cerdanyola del Vallès, Spain (N. Ventosa). 1 E-mail addresses: jfaraudo@icmab.es (J. Faraudo), ventosa@icmab.es (N. Ventosa). These authors contributed equally to this work. https://doi.org/10.1016/j.jcis.2022.10.104 0021-9797/Ó 2022 Published by Elsevier Inc. Journal of Colloid and Interface Science 631 (2023) 202–211 M. Köber, Sílvia Illa-Tuset, L. Ferrer-Tasies et al. 1. Introduction 2.2. Methods for molecular dynamics simulations The potential of quatsomes has been demonstrated in recent years in a variety of medical applications, for example as scaffolds for creating bright and homogeneous bioimaging and biosensing probes [1–6], inducing the rupture of biofilms [7,8], delivering the recombinant human epidermal growth factor in the treatment of complex wounds [9,10], and delivering small RNAs to treat neuroblastoma [11]. Quatsomes can be functionalized easily by modifying the cholesterol moiety [6,12], and their applicability for intravenous administration has been demonstrated in mice [13]. Over years of use, quatsomes have exhibited remarkable stability in terms of size and morphology, unlike conventional lipid vesicles such as phospholipid liposomes, which led us to investigate its molecular origin. The organization of amphiphilic molecules in a vesicle-forming self-assembly process can yield thermodynamically stable structures [14–16]. The most prominent example of such stable structures is catanionic vesicles, which are formed by mixtures of cationic and anionic surfactants [17–20]. The theoretical requirements for such stability have been reported to include the presence of a mixed component system exhibiting composition asymmetry between the two bilayer leaflets, thereby yielding spontaneous membrane curvature [21–23]. Recently, theoretical works have described spontaneous membrane curvature arising from asymmetric lipid densities in the two leaflets, but such lipid distributions are thought to be metastable equilibrium states that eventually relax to symmetric lipid densities in both leaflets through flip-flops between the leaflets [24,25]. Also asymmetric ion concentrations across the membrane have been shown to provoke membrane curvature [26,27]. While quatsomes are a mixed component system, it is not known whether they possess such composition asymmetries and spontaneous membrane curvature, related to the mechanism underlying their formation. In this work, we pursue two interrelated objectives: i) shed light on the mechanism of formation of CHOL/CTAB quatsomes, and ii) determine whether quatsomes are a thermodynamically stable vesicle system. To gain insight into the molecular organization within the quatsome membrane, we performed coarse-grain molecular dynamics (MD) simulations of a full CHOL/CTAB vesicle with an equimolar composition of CHOL and CTAB. We contrasted the characteristics of the simulated vesicle, such as the vesicle diameter, bilayer thickness and disordered liquid-like membrane organization, with experimental observations obtained using cryo–electron microscopy (cryo-EM), small- and wide-angle Xray scattering (SAXS and WAXS), and multi-angle static light scattering. Furthermore, we checked whether quatsomes satisfy the experimental requisites to be classified as thermodynamically stable vesicles. Our MD simulations were performed using GROMACS software [28–31] version 2018 and the implicit solvent MARTINI coarse grain force field, known as Dry-MARTINI [32]. All the technical details and protocols of the simulations are described below. Models and Force Field. The cholesterol and CTAB molecules were modelled using the Dry-MARTINI [29] coarse-grain force field, as shown in Fig. S1. For cholesterol molecules several models compatible with the MARTINI force field are available in the literature. In this work, we employed the model developed by the MARTINI developers [32]. For CTAB we applied the model developed in our group and detailed in Ref. [33]. Building of the initial configuration for MD simulations. In our simulations, we consider as initial condition a pre-built vesicle, to be thermalized and equilibrated in an extended MD run. The initial coordinates for the vesicle were generated using the CHARMMGUI input generator Martini Maker tool [34,35]. Using this tool, we generated a vesicle with an initial radius of 20 nm and 1:1 cholesterol / surfactant composition. As the CTAB molecule is not available in the CHARMM-GUI tool, our strategy was to select another surfactant molecule with a similar number of Martini beads and then replace the topology of the surfactant molecules by that of CTA+ surfactant before energy minimization (we chose the dodecylpyridinium chloride (DPC) surfactant). The system initially generated with CHARMM-GUI was a spherical vesicle with 2054 cholesterol molecules and 2054 surfactant molecules. After manually substituting the topology of DPC surfactant molecules by CTA+ surfactant, we randomly added 2054 MARTINI anions to neutralize the system charge using the insert-molecules tool from GROMACS package. The system built in this way contained 28756 beads in a cubic simulation box of 31.8 nm length in the three axes. The initial configuration of the system is shown in Fig. S9. This initial structure was energy minimized and thermalized before performing the production runs following the default protocols from CHARMM-GUI. Protocols for the MD simulation. The equations of motion were solved with a time step of 20 fs. In all our simulations, the temperature was maintained constant (303 K) using the Bussi-DonadioParrinello thermostat [36]. We employed periodic boundary conditions in all directions. We observed that an adjustment of the vesicle geometry occurs during the first 100 ns, with the development of planar bilayers and defects form the initially spherical vesicle. In Fig. S10-right, it is clearly observed how the vesicle layers are deformed in comparison with the initial configuration. After this initial time, we performed a production run of 1000 ns and no further changes in the geometry or diameter of the vesicle were observed. The estimated equilibrium diameter is 18-19 nm (see Fig. 1). The analysis of the simulations was performed using the VMD software [37], and custom scripts to obtain the radial profiles reported in Fig. 1. 2. Materials and methods 2.3. Quatsome production 2.1. Materials Quatsome production using ultrasounds (US). Unless otherwise stated, quatsomes were prepared according to the following procedure: For all preparations, the CTAB concentration was kept constant 10 times above its critical micelle concentration (CMC), at a value of 10 mM. An equimolar ratio of cholesterol was used, i.e. 10 mM. Desired amounts of cholesterol and CTAB were weighted in a flask (38.7 mg and 36.4 mg, respectively), and subsequently suspended in 10 mL of Milli-Q water. Using a Vibracell Sonifier titanium probe working at 20 kHz and a power amplitude of 40% (max. 500 W) (Sonic and Materials Corporation, USA), the resulting dispersion was sonicated at 298 K for 4 minutes, until a homogenous dispersion was obtained. 5-Cholesten-3b-ol (CHOL, purity 95%) was obtained from Panreac AppliChem (Barcelona, Spain). Cetyltrimethylammonium bromide (CTAB, ultra for molecular biology, purity 99%) was purchased from Sigma-Aldrich (Madrid, Spain). All chemicals were used without further purification. The water used was pretreated with the Milli-Q Advantage A10 water purification system (Millipore Ibérica, Madrid, Spain). Ethanol (HPLC grade) was obtained from Teknokroma (Sant Cugat del Vallès, Spain), and carbon dioxide (purity 99.9%) was supplied by Carburos Metálicos S. A. (Barcelona, Spain). 3-b-DOXYL-5a-cholestane was purchased from Santa Cruz Biotechnology (USA). 203 M. Köber, Sílvia Illa-Tuset, L. Ferrer-Tasies et al. Journal of Colloid and Interface Science 631 (2023) 202–211 ple containing CTAB and cholesterol using the same experimental conditions. Aliquots were taken at EtOH volume fractions of 30 %, 20 % and 0 %, and all samples were characterized by means of dynamic light scattering (DLS) and cryogenic transmission electron microscopy (cryo-TEM) Fig. S6C. Note that in the spontaneous formation and DELOS-SUSP processes the presence of some EtOH helps in the vesicle formation process by avoiding the formation of cholesterol crystals, thus facilitating the formation of CHOL/CTAB synthons. However, quatsome vesicles can also be prepared in pure water, i.e. in the absence of any EtOH, as shown in the preparation by ultrasounds, where cholesterol crystals are broken apart through the application of mechanical energy, also facilitating the formation of CHOL/CTAB synthons. We therefore conclude that while the presence of EtOH may help to reach the equilibrium state, equilibrium can be reached likewise in the absence of EtOH. The temporal stability of quatsomes in pure water indicates that this system is stable for years and completely free of precipitates. These experimental observations are in agreement with free energy calculations [39], which have shown a slight preference of a cholesterol molecule to transfer into a 1:1 bilayer membrane (DG = 55 kcal/mol) instead of a cholesterol nanocrystal (DG = 50 kcal/mol). Coexistence in equilibrium with neighboring single-phase regions in phase diagrams, i.e. with thermodynamically stable CTAB micelles and cholesterol crystals. To demonstrate the coexistence of quatsomes with CTAB micelles and cholesterol crystals in equilibrium, different molar ratios of CTAB and CHOL were sonicated, following the protocol described above. The CTAB concentration was kept constant 10 times above its critical micelle concentration (CMC), at a value of 10 mM, and was either mixed with half the cholesterol concentration (5 mM) or with 3/2 of the cholesterol concentration (15 mM). Furthermore, Isothermal titration calorimetry experiments were performed (see below). 2.4. Experiments to demonstrate the thermodynamic stability Quatsome production by CO2-based processing. Quatsome production via CO2-based processing (DELOS-SUSP methodology) [12,38] consisted of loading a 7.5 mL high-pressure autoclave with a solution of 72.8 mg cholesterol in 2.88 mL ethanol. The solution was heated to the working temperature of 308 K (controlled by an external heating jacket) and then pressurized with compressed CO2 (using a thermostated syringe pump, model 260D, ISCO Inc., Lincoln, US) at a molar fraction of XCO2 = 0.6 until reaching a working pressure of 10 MPa, which produced a volumetric expanded organic solution of cholesterol (a co-solvent behavior of CO2 is observed until Xi = 0.76). The pressurized organic phase was left to equilibrate during one hour at constant pressure. Finally, the CO2-expanded organic phase was depressurized to atmospheric pressure over 24 mL of Milli-Q water containing 68.6 mg of CTAB. To maintain the pressure in the reactor constant during depressurization, N2, pressurized to the working pressure, was used as embolus to push down the expanded solution. The membrane component concentration, prior to process losses, was 5.3 mg/mL (7 mM cholesterol and 7 mM CTAB), and the dispersant contains a volume fraction of 10 % ethanol. To remove ethanol and excess CTAB, quatsome suspensions were diafiltered using a KrosFlo Research IIi TFF System (Spectrum Labs from Repligen Corporation; Waltham, Massachusetts, USA) equipped with a mPES MicroKros filter column (100 kDa molecular weight cut-off and a surface area of 20 cm2), at a feed flow rate of 15 mL/min and a transmembrane pressure of about 1.5 psi, unless otherwise noted. Samples were diafiltered applying six cycles of recircularization to ensure the full elimination of all hydrophilic components that were not incorporated in the vesicle membrane. Spontaneous formation of quatsomes. For the spontaneous formation of quatsomes, we started with CTAB micelles in water and gradually added cholesterol dissolved in ethanol (EtOH). After mixing both, the molarity of CTAB and cholesterol was 7 mM each and the final volume fraction of EtOH 10.7 %. As a first step, stock solutions of CTAB and cholesterol were prepared and equilibrated at 313 K, i.e. well above the Krafft temperature (TK = 298 K) to assure CTAB micelle formation and also to improve the solubility of cholesterol. In particular, an aqueous solution of CTAB was prepared at 7.8 mM ( 7.8 CMC), dissolving 16.25 mg CTAB in 5.7 mL H2O, and a cholesterol solution in ethanol was prepared at 66 mM by dissolving 17.25 mg cholesterol in 0.68 mL EtOH. Subsequently, the cholesterol stock solution was added gradually, in drops of 5 lL, to the aqueous solution containing CTAB micelles, under mild stirring (at the lowest speed that allowed constant stirring, 60 rpm) and while remaining in the 313 K bath. To allow complete equilibration, the sample was left at 313 K with mild stirring for 48 hours. Reversibility of quatsome formation. To conduct the reversibility experiments, we used quatsomes prepared by DELOS-SUSP. Samples were diafiltered to remove ethanol and excess CTAB. First, we determined the EtOH concentration that entails the complete disintegration of the quatsome membrane and the solubilization of the membrane components. EPR spectra (see below) of quatsomes tagged with the nitroxide spin label 3-b-DOXYL-5a-choles tane in their membranes indicated the complete membrane disintegration at EtOH volume fractions of 50 % (Fig. S5C). Consequently, we fixed the volume fraction of EtOH in the dispersant to 60 % and checked if the reformation of quatsomes occurred upon ethanol evaporation at low pressure. For this, first we determined the evaporation rate of an EtOH-H2O mixture at low pressure, monitoring the changes in the density of the liquid with a pycnometer. We have found that the volume fraction of EtOH was reduced by 6 % per minute at 55 mbar, 311 K and under a slow rotation of the flask. Next, we evaporated the EtOH from the sam- 2.5. Quatsome characterization For all characterizations, quatsome preparations were left to stabilize at 298 K for at least 1 week before characterizations were carried out. Cryo-transmission electron microscopy (cryo-TEM). The morphology of quatsomes was studied by cryo-TEM using two microscopes. Some samples were examined in a JEOL JEM-2011 TEM (JEOL LTD., Tokio, Japan) operating at 120 kV. The samples were vitrified by plunge-freezing in liquid ethane, and storage in liquid nitrogen until loaded onto a cryogenic sample holder (Gatan 626 CTH, Gatan, USA). The working temperature was kept below 98 K. Images were recorded on a Gatan 724 CCD camera under lowdose conditions using the Digital Micrograph 3.9.2. Software package (Gatan Inc.). Other samples were examined using a Tecnai 12 G2 TEM (FEI, USA). Images were recorded on an UltraScan 1000 2k x 2k high resolution CCD camera (Gatan, UK) at up to 50x nominal magnifications, under low dose conditions, following lab protocols [40]. Quantitative analysis of the vesicles’ diameters (Fig. 2A) was performed using the ImageJ freeware software. To obtain a statistically relevant size distribution, we have determined the diameter of 8700 spherical vesicles that originated from different experimental batches, all produced under identical experimental conditions, following the protocol for quatsome production by ultrasounds (detailed above). The resolution of the vesicle diameter in cryo-EM images was 2 nm. A Gaussian was fitted to the diameter histogram (bin size 3.5 nm), leaving the mean and standard deviation as fit parameters. The indicated errors of the mean and standard deviation are the standard error of the fit for each parameter. 204 Journal of Colloid and Interface Science 631 (2023) 202–211 M. Köber, Sílvia Illa-Tuset, L. Ferrer-Tasies et al. r2i ).[47] The instrumental smearing of the data due to finite colli- High-performance liquid chromatography (HPLC). For the determination of the molar ratio of membrane components we have used quatsomes prepared by US and CO2-based processing. Quatsomes were diafiltered in H2O to eliminate any excess CTAB not incorporated in the quatsome membrane. All quatsomes systems were disrupted completely before their injection in the HPLC column. With this objective, 1 mL of diafiltered samples (in duplicate or triplicate, as indicated) were lyophilized using a LyoQuest -85 freezedryer (Telstar), with a first freezing step at 193 K for four hours and a subsequent sublimation step at low pressure (0.05 mbar) and increasing temperature. After that, a known volume of organic solvent was added to the lyophilized samples. For the detection of cholesterol and CTAB in the quatsomes we have used an HPLC (1100 series, Agilent Technologies, Santa Clara, CA) equipped with an Evaporative Light Scattering Detector, ELSD (1260 infinity ELSD, Agilent Technologies, Santa Clara, CA). Due to the distinct chemical nature of CTAB and cholesterol we followed two different methods with different columns. Cholesterol separation was carried out using a C18 SymmetryÒ (5 lm; 4.6 150 mm) column (Waters Cromatografía S.A.) at room temperature with an ELSD nebulization temperature of 313 K and evaporative temperature of 353 K. The mobile phase for the cholesterol was a mixture of methanol with water (95:5) (mobile phase A) and formic acid in isopropanol (0.1% HCOOH) (mobile phase B) using gradient flow conditions. For the separation of CTAB we used an InfinityLab Poroshell 120 EC-C18 (4 lm; 4.6 100 mm) column (Agilent Technologies) at 308 K with an ELSD nebulization temperature of 313 K and evaporative temperature of 363 K. The mobile phase was a mixture of 50 mM ammonium acetate pH = 3.6 acetic acid (mobile phase A) and acetic acid in methanol (1% HAcO) (mobile phase B) using gradient flow conditions. In the accuracy test of matrix validation, recoveries between of 96–104 % were achieved. The precision, estimated through the relative standard deviation (RSD) in the measurements, was always < 5 %. For both quatsome production methods, the molar ratio between cholesterol and CTAB showed only small deviations from the equimolar ratio (Table S1). Small-angle X-ray scattering (SAXS). Small-angle X-ray scattering measurements of quatsomes prepared by ultrasounds were performed at the optimized Bruker AXS NanoStar instrument [41,42] at Aarhus University. It uses an Excillum Ga metal jet source operated at 200 W [43] and an optimized collimation section with an octagonal homebuilt scatterless pinhole [44,45] in front of the sample. A homebuilt flow-through cell with an automated sample handler was used for measurements of the samples in a reusable 2.0 mm Ø quartz capillary, which was thermostated using a Peltier element. Typical acquisition time was 2 hrs. The data are displayed as a function of the modulus of the scattering vector q = 4p sin(h)/k, where 2h is the scattering angle and k the wavelength of the Xrays. Note that the recorded SAXS data in most cases are quite noisy at low q as the vesicles are nearly overall contrast match in SAXS due to the positive-negative electron density contributions to the cross-section structure of nearly the same magnitude. Furthermore, the displayed data are obtained after subtraction of a relatively large background signal from the solvent. To obtain quantitative information on the vesicle structure from SAXS data, a model of bilayer vesicles with a core-shell structure including overall size polydispersity was fitted to the data using least-squares methods [46]. The cross-section structure was described by three layers, where the central one described the scattering from the electron-poor hydrocarbon part and the two others describe the electron-rich quaternary ammonium groups and counter ions on both sides of the core. To obtain good fits to the data, the sharp box-like cross-section profile was smeared by a Gaussian of width ri by multiplying the intensity by exp(-q2 mation and detector resolution was also estimated and included in the fit by smearing with a resolution function [48]. Fitting of the SAXS data with form factors for structures that deviate from the spherical shape was not considered, since the facet-related ‘slip of phase’ of the oscillations at high q will barely be observable, as polydispersity smears out the oscillations at higher q [49]. To obtain the outer diameter, the bilayer width (i.e. 5 nm) was added to the diameter for the central plane. Multi-angle static light scattering (MALS). The molecular weight (Mw) of CHOL/CTAB quatsomes was estimated from MALS measurements in aqueous solution at 298 ± 0.1 K. Samples were placed in a thermostatic cell compartment of a Brookhaven Instrument BI200-SM goniometer, and the temperature was controlled to within 0.1 K using a thermostatic recirculating bath. Upon irradiation with a 100 mW solid-state laser (Quantum-Ventus MPC 6000) tuned at k = 532 nm the scattered light intensity was measured using a Brookhaven BI-9000 correlator. Measurements were taken at various scattering vectors q = 4p n k1 0 sin (h/2), where n is the refractive index of the solution, k0 is the wavelength of the incident light, and h is the scattering angle. Static light scattering data were corrected for the background scattering of the solvent and normalized by using toluene as calibration liquid. All samples were filtered through 200 nm cellulose acetate (MiniSart) syringe filters to remove dust or contaminants. The refractive index increment dn/dc of quatsomes in aqueous solution was determined at 303 K and k = 620 nm with a (SEC 3010) Brookhaven Instrument differential refractometer. The MALS data (Fig. S4) were analyzed by means of the Zimm plot method and taking into account the measured dn/dc values [50]. Wide-angle X-ray scattering (WAXS). WAXS measurements of quatsomes prepared by ultrasounds were performed at the X33 beamline of the EMBL at 298 K. A sample to detector distance of 1.2 m was used (q range around 3.8–17.5 nm1), and the wavelength of the beamline was 1.5 Å. WAXS patterns were recorded using a Pilatus 300K-W 2D Photon counting detector. Eight patterns of 30 s each were acquired per sample. Two duplicate measurements of the same batch were performed. Dynamic light scattering (DLS). The hydrodynamic quatsome diameter was determined using a dynamic light scattering analyzer combined with non-invasive backscatter technology (Malvern Zetasizer Nanoseries, Malvern Instruments, UK). The optimal quatsome concentration for DLS measurements was determined by performing dilutions in Milli-Q water. 10 to 1000-fold dilutions of the as-prepared quatsome suspensions yielded overlapping size distributions, eliminating multiple scattering events observed for undiluted samples. Consequently, all reported data were acquired at 10-fold dilutions. Three consecutive measurements were performed on each sample. Averaged scattering-intensity weighted particle size distributions were determined using the software provided by Malvern, and then converted to number distributions by scaling with R4. The average hydrodynamic diameter dhyd was calculated from the discrete number distribution n(di), taking into account the variation of the step length on the logarithmic scale: dhyd ¼ P d nðdi Þ ðdiþ1 di1 Þ=2 Pi nðdi Þ ðdiþ1 di1 Þ=2 Electron paramagnetic resonance (EPR). For EPR experiments, CHOL/CTAB quatsomes containing the commercial spin probe 3bDOXYL-5a-cholestane radical at a mol fraction of 1% were produced by DELOS-SUSP, adding the spin probe to the organic phase in the high-pressure autoclave, together with the cholesterol. EPR spectra were obtained with an X-Band (9.7 GHz) Bruker ELEXSYS E-500 spectrometer equipped with a ST8911 microwave cavity, a Bruker variable temperature unit, a field frequency lock 205 M. Köber, Sílvia Illa-Tuset, L. Ferrer-Tasies et al. Journal of Colloid and Interface Science 631 (2023) 202–211 employs a simplified representation of the molecules, wherein on average every 3–4 heavy atoms are replaced by a coarse grain building block (a ‘‘bead”) with specific chemical interactions parametrized from thermodynamic data. This method enables full vesicle simulations to be conducted with molecular resolution up to the microsecond time scale, well beyond the possibilities of previous atomistic simulations performed on this system [3,39,52]. Our simulations of a 1:1 CHOL/CTAB mixture comprising 4108 molecules at 303 K during 1 ls predict a vesicular structure, which evidently deviates from a perfectly spherical object. Our results (Fig. 1A–C) clearly indicate that the vesicle is formed through the assembly of planar bilayers, joined together via small defects that provide curvature to the structure, eventually generating a closed vesicle. Cryo-TEM, the best method to directly examine the vesicle morphology at high resolution [40], confirms the presence of these facets and associated slight deviations from a perfectly spherical vesicle shape (Fig. 1D and Fig.S2 (SI)). These deviations become even more evident upon major irradiation, in contrast to phosphatidyl serine liposomes of similar size, which remain round even upon excessive irradiation of > 200 e/Å2 that turned the ice crystalline (see SI, Fig. S3). These results substantiate the presence of facets in quatsomes and show that these are different from planar faces present in any vesicle due to thermal fluctuations [53] or in the gel phase. Importantly, the simulations indicate a uniform distribution of the two components within the leaflets (Fig. 1A), i.e. the absence of any segregation or aggregation of the two components. Moreover, each leaflet contained exactly an equimolar ratio of cholesterol and CTAB (Fig. 1B), and no composition asymmetry existed between the inner and outer leaflet, suggesting that the bilayer curvature did not result from composition asymmetry between the two leaflets. The CHOL/CTAB synthon (bimolecular amphiphile) was found to be present throughout the whole vesicle, maintaining its stable arrangement in the planar regions [39] and exhibiting strong deformations in the defect regions. However, even in the defect regions no segregation of cholesterol or CTAB molecules was observed, and the equimolar ratio was still maintained. On the other hand, on the timescale of the simulation, we found an asymmetric lipid density in the two leaflets, containing 871 synthons in the inner leaflet and 1183 synthons in the outer leaflet. Overall, our results suggest that the bimolecular CHOL/CTAB synthon forming the membrane [39] has a tendency to form planar bilayers, which in turn form a closed vesicle due to the presence of defect regions that exhibit strong deformations in the molecular orientation of cholesterol and CTAB but no segregation of these two components. This mechanism of vesicle formation is different from that proposed thus far for stable vesicular systems, such as catanionic vesicles, which is based on compositional asymmetry leading to membrane curvature [21,22,54]. Experimentally, we found a 10 % deviation from an equimolar composition of the entire vesicular system using high-performance liquid chromatography (see SI, Table S1), which is small compared with the deviations used to produce stable catanionic vesicles, which are generally > 50 % [14,54,55]. In accordance with these results, the size distribution obtained through analyzing the vesicle diameter in cryo-TEM images of more than 8600 vesicles from different experimental batches, despite being roughly Gaussian, exhibited a non-Gaussian tail towards larger sizes (Fig. 2A), thereby pointing towards a vesicle formation mechanism that is not dominated by a spontaneous bilayer curvature resulting from composition asymmetries [54]. It is important to highlight that the characteristics of the simulated vesicle were consistent with several experimental observations: a) The vesicle diameter of 18–19 nm (Fig. 1C) corresponds to the smaller quatsomes observed using cryo-TEM (Fig. 2A). b) The bilayer thickness of 4 nm (including the surfac- system Bruker ER 033 M and a NMR Gaussmeter Bruker ER 035 M. The modulation amplitude was kept well below the line width, and the microwave power was well below saturation. We started with the sample of 10% vol. EtOH and then diluted step by step (30%, 40%, 45% vol.) until reaching the 50% in vol. All samples were previously degassed with Ar and measured in a flat quartz EPR cell. The same acquisition parameters were used: microwave power: 2.49 mW; modulation amplitude: 2.0 G; modulation frequency: 100 KHz; receiver gain: 60; time constant: 20.48 ms; conversion time: 40.96 ms; sweep width: 150 G; 15 scans. Isothermal titration calorimetry (ITC). ITC experiments were performed with a MicroCal VP-ITC System (Malvern Panalytical, UK). The sample cell contained a volume of 1.4 mL of quatsomes, prepared by ultrasounds, with a concentration of CTAB and cholesterol of 50 lM each, thus establishing an equimolar ratio of CHOL/CTAB at the beginning of the experiment. Subsequently, every 300 seconds 5 lL of a CTAB 0.7 mM solution were injected with a syringe, and the heat exchanged per injection was monitored. Blanks consisted in adding 5 lL of the 0.7 mM CTAB solution to H2O every 300 seconds, as well as adding H2O to the quatsomes at the above stated concentration. Note that the critical micellar concentration (CMC) of CTAB is approximately 0.9 mM. Conductivity. The electric conductivity was measured with a Hanna HI 5521 (Hanna Instruments, USA) using a four-ring platinum probe electrode HI76312 (Hanna Instruments, USA). For pure CTAB, a micellar suspension of 5 mM was prepared, and sequentially diluted up to 0.5 mM by adding Milli-Q water (resistivity of 18.2 MX*cm). For measurements on CHOL/CTAB quatsomes, sequential dilutions of two different quatsome batches were prepared by adding Milli-Q water. Measurements were performed at different time points up to three days after diluting. For all conductivity measurements the temperature was maintained within ± 0.3 K. 2.6. Calculation of the average number of molecules per vesicle Calculation from MALS data. The average molecular mass of quatsomes was determined by multi-angle light scattering, yielding mQ S ¼ 13.3 ± 0.8 106 g/mol. With a molecular mass of CHOL and CTAB of mCHOL ¼ 386.65 g/mol and mCTAB ¼ 364.45 g/mol, respectively, and assuming an equimolar composition, the mean mQS = 35 ± 2 103. number of molecules per vesicle is N ¼ ðmCHOL þm CTAB Þ=2 Scaling the simulated vesicle size to the experimentally observed average size. The average bilayer volume V exp bilayer was calculated for an outer vesicle radius R of RSAXS = 23.15 nm and a total bilayer thickness t of t SAXS = 5 nm, both mean values obtained from SAXS: 3 3 4 V exp : bilayer ¼ 3 p RSAXS ðRSAXS t SAXS Þ The bilayer volume of the simulated vesicle was equally calculated, with an outer vesicle radius RCGMD = 9 nm and a bilayer thick 3 3 4 ness of tCGMD = 4 nm: V sim : bilayer ¼ 3 p RCGMD ðRCGMD t CGMD Þ Scaling the number of molecules in the simulated vesicle N CGMD ¼ 4108 to the average volume obtained experimentally, we V exp = 44 ± 4 103 molecules. obtain N ¼ N CGMD V CGMD 3. Results and discussion To gain insight into the molecular organization within the quatsome membrane, we performed molecular dynamics (MD) simulations of a full CHOL/CTAB vesicle with an equimolar composition of cholesterol and CTAB (see SI, Fig. S1). We used the implicit solvent Martini coarse-grain force field for lipids [32] and surfactants [33] (nicknamed ‘‘Dry Martini”), which has been successfully employed in previous simulations to determine lipid properties, including vesicle formation and fusion [32,51]. The Dry Martini approach 206 Journal of Colloid and Interface Science 631 (2023) 202–211 M. Köber, Sílvia Illa-Tuset, L. Ferrer-Tasies et al. Fig. 1. Molecular composition of bilayer leaflets and supramolecular organization. (A–C) Coarse-grain MD simulation of a CHOL/CTAB quatsome vesicle, at a concentration of 0.1 mM of each component. (A) Snapshot of an equilibrated (303 K) full quatsome vesicle and a cross-section thereof. The head groups of the CTA+ and cholesterol are depicted as blue and orange balls, respectively (Br counterions are omitted for clarity), and their tails are depicted as cylinders. (B) Radial distribution of vesicle components. Left: radial density profile with .(r) calculated as the number of head group beads per nm3. Right: accumulated (integrated) number of molecules of each component as a function of the radial distance averaged over all equilibrium configurations. The peaks R1 and R2 in the CTA+ head group distribution are also indicated, which correspond to the inner and outer radii of the vesicle, respectively, and the minimum, indicated by an arrow, corresponds to the center of the bilayer. The diameter of the simulated vesicle is estimated as 18–19 nm, taking into account the size of the CTA+ head group bead (0.47 nm). (C) A detailed snapshot depicting the internal molecular organization of CTA+ and cholesterol of a 7 Å wide slice of the simulated quatsome vesicle, exposing regions with perfectly ordered planar bilayers (see the enlarged portion of the image) and disordered joining regions, with a 1:1 ratio of the two components. (D) Cryo-TEM image of quatsome vesicles depicting the faceting and deviations from a completely spherical shape (scale bar represents 20 nm). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) (I d4). Furthermore, the inter-technique variation in size was within the expected range [58]. ii) Quatsome size and polydispersity remain stable over time. Quatsome vesicles maintained their size distribution and unilamellar morphology for years, as observed using DLS and cryo-TEM (Fig. 3B). iii) Quatsome formation is reversible upon the change of an external factor. Quatsome rupture and reformation can be controlled through varying the volume fraction of ethanol in water. When adding EtOH to a quatsome suspension, vesicle rupture occurred at EtOH volume fractions > 30%, which is evident from the analysis of size distribution and cryo-TEM images (see SI, Fig. S6A–B). Complete membrane disintegration took place at volume fractions 50%, as demonstrated through the electron paramagnetic resonance spectra of quatsomes containing the 3b-DOXYL-5a-cho lestane radical as spin probe (see SI, Fig. S6C), exhibiting the typical signal corresponding to freely moving nitroxyl radicals (in contrast to the characteristic signature of restricted radical movement observed at low ethanol volume fractions). Upon subsequent ethanol evaporation, quatsome reformation occurred once the EtOH volume fraction was 30% again (Fig. S6A), and at 0% EtOH, we mainly observed small unilamellar vesicles (inset in Fig. 3C) that exhibited a size distribution that overlapped with the distribution obtained prior to the addition of EtOH (Fig. 3C). iv) Quatsomes are generated spontaneously. Quatsome vesicles are formed in the absence of any energy input, simply through the gradual addition of cholesterol dissolved in EtOH to an aqueous solution of CTAB micelles. This procedure yielded small unilamellar vesicles (inset in Fig. 3D) that were similar in size to those tant head groups) (Fig. 1C) is concordant with the value of 4.3 nm obtained in all atomic MD simulations [39] and 5 nm obtained experimentally using small-angle X-ray scattering (SAXS) (Fig. 2B). c) The ratio of molecules per bilayer volume is proportional to the experimentally observed value: Scaling the bilayer volume to the average volume obtained experimentally [56], the simulated vesicle would contain 44 ± 4 103 CTAB and cholesterol molecules, close to the mean value of ca. 35 ± 2 103, which is obtained experimentally from the molecular weight of 13.3 ± 0.8 MDa determined using multi-angle light scattering (see SI, Fig. S4). d) The molecular organization observed in Fig. 1C is consistent with disordered liquid-like packing, which is also observed experimentally using atomic force microscopy and wide-angle X-ray scattering (see SI, Fig. S5) [3,52]. Because CHOL/CTAB quatsomes exhibit a completely different mechanism of vesicle formation than previously proposed for thermodynamically stable vesicle systems [54,21–27], we aimed to confirm that quatsomes satisfy the experimental requisites of thermodynamically stable vesicles [14], as follows: i) The preparation route does not affect the vesicle size. Regardless of whether quatsomes were prepared through applying ultrasounds (US) or compressed CO2 (DELOS-SUSP methodology) [12,38], small unilamellar vesicles (insets in Fig. 2A and 3A) with similar size distributions were obtained (Fig. 3A). The somewhat larger polydispersity observed using DLS in comparison with the narrow vesicle size distribution obtained using SAXS (Fig. 2B) and cryo-TEM image analysis (Fig. 2A) was likely due to the large impact of few larger vesicles (visible in cryo-TEM images, Fig. 2A) on the scattered light intensity I 207 M. Köber, Sílvia Illa-Tuset, L. Ferrer-Tasies et al. Journal of Colloid and Interface Science 631 (2023) 202–211 Fig. 2. Relevant geometric characteristics of CHOL/CTAB quatsomes. (A) Vesicle diameter distribution, determined by analyzing cryo-TEM images of 8600 vesicles from Gauss various experimental batches, and the Gaussian fit yielding a mean diameter dTEM = 31.4 ± 0.2 nm and a standard deviation of rGauss TEM = 3.4 ± 0.2 nm. The arithmetic mean arith diameter of the entire ensemble is slightly larger, i.e. dTEM = 43.2 ± 0.2 nm (mean ± standard error of the mean), due to the presence of few larger vesicles. Inset: Representative cryo-TEM image (scale bar represents 50 nm). (B) SAXS data of CHOL/CTAB quatsomes at a concentration of 6 mM of each component, indicating the scattered intensity as a function of the modulus of the scattering vector q. The broad bump originates from the presence of a three-layer cross-section of the vesicle bilayer, and the oscillations [57] are attributed to the remarkably narrow vesicle size distribution of the overall size, and are generally not observed for conventional vesicles of this size owing to the polydispersity in their size. Fitting the data using a model of bilayer vesicles with an overall size polydispersity [46] yielded an average outer quatsome diameter of dSAXS = 46.3 ± 0.3 nm and a standard deviation of rSAXS = 1.56 ± 0.02 nm. From the fit to the SAXS data, the thickness of the bilayer membrane was found to be approximately 5 nm, which is concordant with the values obtained in previous atomistic simulations [39]. Note that instrumental smearing was included when fitting the data; the green curve represents the model without smearing and the red curve represents the model with smearing. The SAXS data did not display any signs of multi- or oligo-lamellarity, which would appear as relatively broad additional peaks on top of the main peak. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Fig. 3. Experimental evidence indicating that CHOL/CTAB quatsomes are thermodynamically stable. Hydrodynamic diameter distribution of quatsomes under different experimental conditions (with indicated arithmetic mean hydrodynamic diameters¯dhyd ± 1 SD from at least two separate measurements), obtained using dynamic light scattering (DLS) at a concentration of 0.6 mM of each component, and corresponding cryo-TEM images in the inset (scale bars represent 100 nm). (A) Quatsomes obtained using different preparation routes, i.e. through the application of ultrasound (black line) and the depressurization of a CO2-expanded liquid (red line). (B) Temporal stability of quatsomes over three years. Inset: Cryo-TEM image of quatsomes that have aged for three years, and photograph of the corresponding quatsome suspension, which was completely free of precipitate. (C) Quatsomes as-prepared through the depressurization of a CO2-expanded liquid (red line), and after ethanol-induced quatsome rupture and subsequent reformation upon ethanol removal (dark blue line). (D) Quatsomes produced through spontaneous formation (dark yellow), i.e. upon gradual addition of a cholesterol solution in EtOH to an aqueous solution containing CTAB micelles, compared to quatsomes formed through the application of ultrasound (black line). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 208 Journal of Colloid and Interface Science 631 (2023) 202–211 M. Köber, Sílvia Illa-Tuset, L. Ferrer-Tasies et al. Table 1 Average values for different structural features of CHOL/CTAB quatsomes in water at 298 K. Molecular weight (MDa) a Average outer diameter (nm) b Molar ratio CHOL/ CTAB c Lipid phase in the bilayer 13.3 ± 0.8 46.3 ± 0.3 1.11 ± 0.01 Disordered liquid-like packing d Equilibrium concentration with CTAB monomers (lM) e <5 a)Obtained from MALS measurements (Fig. S4). b)Obtained from SAXS measurements (Fig. 2B). c)Obtained from HPLC measurements (Table S1). d)Obtained from WAXS measurements (Fig. S5). e)Obtained from conductivity measurements (Fig. S8). bilayer regions joined through defect regions that exhibit strong deformations in the molecular orientation of the membrane components. Experimental observations obtained with cryo-TEM imaging, SAXS, HPLC, MALS, and WAXS, substantiate the simulated results, and further demonstrate a very high vesicle-to-vesicle homogeneity, distinguishing CHOL/CTAB quatsomes from other common nanovesicular structures [61–62]. At the same time, we demonstrate that quatsomes show all the experimental features of a thermodynamically stable vesicle system, with a particularly relevant preparation-route-independent size distribution that is stable over time (monitored along three years). For this demonstration, we have employed different experimental techniques for quatsome production (ultrasound application, depressurization of a CO2-expanded liquid, spontaneous formation) and characterization (DLS, ITC, EPR). With this data at hand, we propose a novel mechanism underlying the formation of thermodynamically stable nanovesicles, which is based on distortions in the orientation of bilayer molecules in the absence of any segregation of membrane components between the two bilayer leaflets. This mechanism of vesicle formation is different from that proposed thus far for stable vesicular systems (Table 2), based on spontaneous membrane curvature due to composition asymmetry between the two bilayer leaflets [54,21–23], and facilitates the long-term stability of asymmetric lipid densities in the two leaflets, in the absence of the relaxation to symmetric lipid densities through flip-flops between the leaflets described previously [24–25]. In the future, it will be of interest to study the ion distribution across the quatsome membrane, and determine its role in the vesicle stabilization [26–27]. This comprehensive study provides a conceptual framework demonstrating that CHOL/CTAB quatsomes form a stable phase, and it goes well beyond previously published data describing the obtained through the application of ultrasound (Fig. 3D). The small differences in size may indicate a partially different molecular organization in the vesicular membrane, related to the similar free energies of a cholesterol molecule transferred into a 1:1 bilayer membrane (DG = 55 kcal/mol) or a cholesterol nanocrystal (DG = –50 kcal/mol) [39], relevant at the later stages of the vesicle formation process [59]. These results suggest that the quatsome formation per se does not require any energy supply, but an additional energy input facilitates the formation of CTAB-cholesterol synthons that build up the vesicle membrane. v) Quatsomes coexist in equilibrium with neighboring single-phase regions in phase diagrams, i.e. with thermodynamically stable CTABrich colloidal assemblies and cholesterol crystals. The prolonged coexistence of the vesicular quatsome phase with thermodynamically stable CTAB-rich assemblies or cholesterol crystals was observed when cholesterol and CTAB were mixed in non-equimolar ratios; excess of CTAB (e.g. a molar ratio CHOL/CTAB of 1:2) led to the coexistence of CTAB-rich assemblies and quatsomes, as observed using cryo-TEM and isothermal titration calorimetry experiments (see SI, Fig. S7A–C), while an excess of cholesterol (e.g. a molar ratio CHOL/CTAB of 3:2) yielded both unilamellar vesicles and cholesterol crystals (see SI, Fig. S7D). Finally, to illustrate the resistance of quatsome nanovesicles towards dilutions, we have measured the concentration at which monomerically dissolved CTAB is in equilibrium with the CHOL/ CTAB quatsome, and found it to be < 5 lM (see SI, Fig. S8, and Table 1), which is more than two orders of magnitude lower than the critical micelle concentration of CTAB of 1 mM. Therefore, importantly, the quatsome vesicles are more resistant to dilution than micelles, an important requirement for their use as drug delivery systems [60]. This is consistent with the practically equimolar ratio of CHOL and CTAB even after multiple diafiltrations (Table 1), which implicitly shows that the monomerically dissolved CTAB in equilibrium with the quatsome is practically zero. Moreover, it suggests that the chemical potential of CTAB in the quatsome is much lower than that in the pure micelle, i.e., CTAB in the quatsome is thermodynamically more stable than in the micelle, likely due to the formation of the stable CHOL/CTAB synthon. Table 2 Formation mechanisms of thermodynamic stable nanovesicles. Mechanism of nanovesicle formation 4. Conclusions Spontaneous The data presented here points out that i) CHOL/CTAB quatsomes are thermodynamically stable nanovesicles, and ii) CHOL/ CTAB quatsomes are formed through the association of intrinsically planar bilayers in a faceted vesicle with defects. High intermolecular binding energies between CHOL and CTAB hinder the segregation of these two components in the vesicle membrane, and a stable nanovesicle is formed in the absence of major composition asymmetries between the two bilayer leaflets, with a stably persisting lipid density asymmetry. In particular, coarse-grain molecular dynamics simulations of a complete CHOL/CTAB quatsome demonstrated the absence of spontaneous membrane curvature related to composition asymmetries between the two bilayer leaflets, and instead the presence of planar [54,21–23] Spontaneous [24–25] Spontaneous [26–27] Association of This work 209 Experimental examples of thermodynamically stable nanovesicles reported References membrane curvature Composition asymmetry between leaflets Yes membrane curvature Asymmetry in lipid density between leaflets No membrane curvature Asymmetric ion concentrations across the membrane No intrinsically planar bilayers Defect regions with distortions in the orientation of bilayer molecules Yes Supramolecular organization of nanovesicle bilayer M. Köber, Sílvia Illa-Tuset, L. Ferrer-Tasies et al. Journal of Colloid and Interface Science 631 (2023) 202–211 124297NB-C33) and the FUNFUTURE-FIP-2020 Severo Ochoa project, from Generalitat de Catalunya through grant 2017-SGR-918, from CSIC through grant 2019AEP133, and from the European Commission through the H2020 PHOENIX project (contract no. 953110). We acknowledge the support of the Israel scienceIsrael science Foundation, grant 1117/2016, and thank Dr. Inbal Ionita for her professional assistance in the cryo-TEM analysis. We thank Jannik Nedergaard Pedersen and Beatrice Plazzotta for help with the SAXS measurements. The simulations reported here were performed using the Cori Supercomputing facility of the National Energy Research Scientific Computing Center (NERSC), a U.S. Department of Energy Office of Science User Facility operated under Contract No. DE-AC02-05CH11231. structure of these nanovesicles [39] and their use, for example for bioimaging [1–5] and wound healing [9,10]. Furthermore, the study addresses fundamental principles related to the formation of thermodynamically stable nanovesicles with well-defined physico-chemical characteristics and an infinite shelf life, of particular relevance in the development of nanovesicle based products, for example in the pharmaceutical and cosmetics fields. CRediT authorship contribution statement Mariana Köber: Conceptualization, Methodology, Formal analysis, Investigation, Data curation, Writing – original draft. Sílvia IllaTuset: Conceptualization, Formal analysis, Investigation, Data curation, Writing – original draft. Lidia Ferrer-Tasies: Conceptualization, Methodology, Formal analysis, Investigation, Data curation, Writing – original draft. Evelyn Moreno-Calvo: Methodology, Formal analysis, Investigation, Data curation. Witold I. Tatkiewicz: Methodology, Methodology, Formal analysis. Natascia Grimaldi: Methodology, Methodology, Formal analysis, Investigation. David Piña: Methodology, Methodology, Formal analysis. Alejandro Pérez Pérez: Methodology. Vega Lloveras: Methodology, Formal analysis. José Vidal-Gancedo: Methodology, Formal analysis, Resources. Donatella Bulone: Methodology, Formal analysis, Resources, Data curation, Funding acquisition. Imma Ratera: Methodology. Jan Skov Pedersen: Formal analysis, Data curation, Writing – review & editing, Project administration, Funding acquisition. Dganit Danino: Formal analysis, Resources, Data curation, Writing – review & editing, Project administration, Funding acquisition. Jaume Veciana: Conceptualization, Methodology, Investigation, Resources, Writing – review & editing, Funding acquisition. Jordi Faraudo: Conceptualization, Methodology, Investigation, Data curation, Writing – original draft, Supervision, Project administration, Funding acquisition. Nora Ventosa: Conceptualization, Methodology, Investigation, Resources, Data curation, Writing – original draft, Supervision, Project administration, Funding acquisition. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.jcis.2022.10.104. References [1] X. Liu, X. Liu, A. Ardizzone, B. Sui, M. Anzola, N. Ventosa, T. Liu, J. Veciana, K.D. Belfield, Fluorenyl-Loaded Quatsome Nanostructured Fluorescent Probes ACS Omega 2 (8) (2017), pp. 4112–4122, 10.1021/acsomega.7b00779. [2] D.A. Silbaugh, L. Ferrer-Tasies, J. Faraudo, J. Veciana, N. Ventosa, B.A. Korgel, Highly Fluorescent Silicon Nanocrystals Stabilized in Water Using Quatsomes, Langmuir 33 (50) (2017) 14366–14377, https://doi.org/10.1021/acs. langmuir.7b03539. [3] A. Ardizzone, S. Kurhuzenkau, S. Illa-Tuset, J. Faraudo, M. Bondar, D. Hagan, E. W. Van Stryland, A. Painelli, C. Sissa, N. Feiner, L. Albertazzi, J. Veciana, N. 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Melgarejo, D. Pulido, A. Córdoba, E. Moreno, U. Unzueta, E. Vazquez, I. Abasolo, S. Schwartz, A. Villaverde, F. Albericio, M. Royo, M.F. García-Parajo, N. Ventosa, J. Veciana, Multifunctional Nanovesicle-Bioactive Conjugates Prepared by a One-Step Scalable Method Using CO2-Expanded Solvents, Nano Lett. 13 (8) (2013) 3766–3774, https://doi.org/10.1021/nl4017072. Data availability Data will be made available on request. Simulation data is openly accessible at the ICMAB SoftMatter Theory GitHub repository https://github.com/soft-matter-theory-at-icmab-csic. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments This work was supported by funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 712949 (TECNIOspring PLUS) and from the Agency for Business Competitiveness of the Government of Catalonia. The production of quatsomes and part of their characterization has been performed by the ICTS ‘‘NANBIOSIS”, more specifically by the Biomaterial Processing and Nanostructuring Unit (U6), Unit of the CIBER in Bioengineering, Biomaterials & Nanomedicine (CIBER-BBN) located at the Institute of Materials Science of Barcelona (ICMAB-CSIC). ICMAB-CSIC acknowledges support from the MINECO through the Severo Ochoa Programme for Centres of Excellence in R&D (SEV-2015-0496 and CEX2019-000917-S). 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https://openalex.org/W1987580326	https://breast-cancer-research.biomedcentral.com/counter/pdf/10.1186/bcr1725	English	null	Algorithm for identification of individuals with hereditary predisposition to breast cancer and other breast cancer-associated forms of malignant neoplasms	Breast cancer research	2,007	cc-by	30,034	Available online http://breast-cancer-research.com/supplements/9/S1 Available online http://breast-cancer-research.com/supplements/9/S1 Breast Cancer Research Volume 9 Supplement 1, June 2007 Meeting abstracts VII Madrid Breast Cancer Conference: Changes in the treatment of breast cancer Madrid, Spain 20–22 June 2007 Received: 23 May 2007 Published: 19 June 2007 © 2007 BioMed Central Ltd Received: 23 May 2007 Published: 19 June 2007 © 2007 BioMed Central Ltd Speaker Abstracts (TailoRx). Whether these multigene predictors of prognosis will have greater utility than Adjuvant!Online remains to be determined. In the meantime, exploratory analyses are ongoing to identify reliable predictors of response to individual drugs and modern combination drug regimens. These are expected to lead to individualized selection of treatment, or personalized medicine. (TailoRx). Whether these multigene predictors of prognosis will have greater utility than Adjuvant!Online remains to be determined. In the meantime, exploratory analyses are ongoing to identify reliable predictors of response to individual drugs and modern combination drug regimens. These are expected to lead to individualized selection of treatment, or personalized medicine. Prognostic and Risk Factors (Part I) Prognostic and Risk Factors (Part I) Breast Cancer Research 2007, 9(Suppl 1):S1 (doi: 10.1186/bcr1684) Since the introduction of systemic adjuvant chemotherapy (ACT) and endocrine therapy in the early 1970s, the determination of risk of recurrence and death from breast cancer became a critical piece of information in the selection of the optimal postoperative treatment strategy. Classical histopathological prognostic factors included tumor size, regional lymph node metastases and number of axillary nodes involved, tumor grade, presence of lymphovascular invasion, and, more recently, estrogen receptor (ER) and progesterone receptor status, measurement of proliferative activity (S-phase fraction, mitotic index, Ki-67), and HER2 overexpression/amplification. As isolated factors, they have limited predictive ability in the case of individual patients. For that reason, prognostic indices were developed. The most successful is Adjuvant!Online, an online nomogram developed by Peter Ravdin. This nomogram incorporates tumor size, axillary nodal status, tumor grade, ER status, age and comorbidity. The nomogram will provide an assessment of recurrence and mortality rates at 10 years, including deaths due to comorbid conditions. In addition, the nomogram also calculates relative and absolute benefit from various adjuvant inter- ventions: tamoxifen, aromatase inhibitors, and first-generation, second- generation and third-generation ACT regimens. The prognostic and predictive value of this nomogram has been externally validated, with a margin of error ≤1%. Over the past decade, high-throughput technologies have been developed based on gene expression profiling. These include between two and a couple of hundred genes, and have the ability to separate patients with excellent outcomes from those with higher risk. One of these prognostic profiles has been externally validated and is currently undergoing testing for clinical utility in a large, multicenter, prospective randomized trial (MINDACT). Another approach was based on prospectively identifying a set of genes from the literature and from the results of gene expression profiling. Mathematical modeling then led to the selection of 16 genes related to cell proliferation, ER-driven genes, HER2 and proteases, as well as five ‘housekeeping’ genes (OncotypeDx). This assay is based on RT-PCR, is reproducible and applicable to archival, paraffin-embedded material, and has been shown to predict prognosis in patients with lymph-node- negative, ER-positive primary breast cancer. Further testing indicated that the assay might also predict sensitivity to tamoxifen, or first- generation adjuvant chemotherapy. This assay is also under evaluation for clinical utility in a large, multicenter, prospective randomized trial SYMPOSIUM I Department of Breast Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Breast Cancer Research Vol 9 Suppl 1 VII Madrid Breast Cancer Conference Breast Cancer Research Vol 9 Suppl 1 VII Madrid Breast Cancer Conference optimal biological dose of anti-angiogenic drugs, which are being used with increasing frequency in medical oncology. References status, tumor size, nodal status and histopathological grading, ITC was of independent prognostic significance for subsequent reduced breast cancer specific survival (RR 6.3, 95% CI 2.3–17.6, P < 0.0001), next to nodal status at time of primary diagnosis (RR 2.9, 95% CI 1.8–4.7). Given these inspiring results on ITC in bone marrow, several trials currently analyze the prognostic relevance of circulating tumor cells (CTCs) in peripheral blood in the adjuvant setting. In Germany, for example, CTCs in peripheral blood of breast cancer patients at primary diagnosis and during adjuvant chemotherapy as well as endocrine and bisphosphonate treatment within the SUCCESS Trial (n = 3,658 patients) are currently analyzed. In this study, the CellSearchSystem (Veridex, Warren, USA) is used for isolation and enumeration of CTCs from 23 ml peripheral blood. Briefly, after immunomagnetic enrichment with an anti-Epcam antibody, cells were labelled with anticytokeratin (8,18,19) and anti-CD45 antibodies to distinguish epithelial cells and leukocytes. In an interim analysis, 456 breast cancer patients at the time of primary diagnosis before the start of systemic adjuvant treatment were analyzed. In this patient group, 28% of patients (n = 129) presented with ≥1 CTC in peripheral blood. In patients with the detection of CTCs, the mean number of cells was four (range 1–166). In 19 healthy individuals we found one CTC in the blood of two patients. The presence of CTCs did not correlate with tumor size (P = 0.18), presence of lymph node metastases (P = 0.09), histopatho- logical grading (P = 0.56), hormone receptor status (P = 0.49) or Her2/neu status of the primary tumor (P = 0.33). optimal biological dose of anti-angiogenic drugs, which are being used with increasing frequency in medical oncology. References 1. Braun S, Pantel K, Müller P, et al.: Cytokeratin-positive cells in the bone marrow and survival of patients with stage I, II or III breast cancer. N Engl J Med 2000, 342:525-533. 1. Braun S, Pantel K, Müller P, et al.: Cytokeratin-positive cells in the bone marrow and survival of patients with stage I, II or III breast cancer. N Engl J Med 2000, 342:525-533. 2. Cristofanilli M, Budd GT, Ellis M, et al.: Circulating tumor cells predict progression free survival and overall survival in metastatic breast cancer. S3 10. Mancuso P, et al.: Circulating endothelial cell kinetics and via- bility predict survival in breast cancer patients receiving metronomic chemotherapy. Blood 2006, 108:452-459. Circulating tumor and endothelial cells as predictors of response in metastatic breast cancer M Cristofanilli 11. Smirnov DA, Foulk BW, Doyle GV, et al.: Global gene expres- sion profiling of circulating endothelial cells in patients with metastatic carcinomas. Cancer Res 2006, 66:2918-2922. Department of Breast Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Breast Cancer Research 2007, 9(Suppl 1):3 (doi: 10.1186/bcr1686) Breast Cancer Research 2007, 9(Suppl 1):3 (doi: 10.1186/bcr1686) The detection of microscopic disease is associated with prognostic implications in primary breast cancer [1]. In metastatic breast cancer (MBC), reliable detection of circulating tumor cells (CTCs) has been obtained using immunomagnetic separation and subsequent analysis by the CellSpotter™ analyzer (Veridex LLC, a Johnson & Johnson company, Warren, NJ, USA) [2,3]. This technology is becoming a standard tool for the ‘real-time’ assessment of prognosis and response to treatment [4,5]. The detection of CTCs in patients with MBC about to start a new line of treatment has been shown to predict progression- free survival and overall survival and treatment benefit. The prognostic value was independent of the line of therapy (for example, first line versus second line or more) [2,3]. Moreover, in multivariate analysis CTCs demonstrated superior value compared with the site of meta- stasis (for example, visceral versus soft tissue/bone), type of therapy, and length of time to recurrence after definitive primary surgery. Furthermore, the prognostic value of CTCs has been shown to be superior to standard tumor markers (for example, Ca27-29), tumor burden and phenotype of disease [6]. We have recently focused on determining the feasibility of the genotypic characterization of CTCs and correlating with the expression of similar genes in primary or metastatic lesions [7]. Prognostic and Risk Factors (Part II) Prognostic and Risk Factors (Part II) Prognostic and Risk Factors (Part II) Breast Cancer Research Vol 9 Suppl 1 VII Madrid Breast Cancer Conference N Engl J Med 2004, 351:781-791. 3. Cristofanilli M, Hayes DF, Budd GT, et al.: Circulating tumor cells: a novel prognostic factor for newly diagnosed metasta- tic breast cancer. J Clin Oncol 2005, 23:1420-1430. 4. Hayes DF, Cristofanilli M, Budd GT, et al.: Circulating tumor cells at each follow-up time point during therapy of metastatic breast cancer patients predict progression-free and overall survival. Clin Cancer Res 2006, 12:4218-4224. 5. Budd GT, Cristofanilli M, Ellis M, et al.: Circulating tumor cells versus imaging – predicting overall survival in metastatic breast cancer. Clin Cancer Res 2006, 12:6403-6409. 6. Cristofanilli M, Kristine Broglio KR, Guarneri V, et al.: Circulating tumor cells in metastatic breast cancer (MBC): biological staging beyond tumor burden. Clin Breast Cancer 2007, 7: 471-479. 7. Reuben JM, Lee B-N, Li C, et al.: Genomic of circulating tumor cells in metastatic breast cancer [abstract]. ASCO 2007, 26:in press. Persisting MRD after primary treatment may lead to an indication for extended adjuvant therapy. Until clinical consequences of MRD detection in solid tumors and particularly in breast cancer have been validated, however, the detection of isolated tumor cells in bone marrow should be performed mainly in clinical trials. 8. Cines DB, et al.: Endothelial cells in physiology and in the pathophysiology of vascular disorders. Blood 1998, 91:3527- 3561. 9. Mancuso P, et al.: Resting and activated endothelial cells are increased in the peripheral blood of cancer patients. Blood 2001, 97:3658-3661. W Janni I Frauenklinik Innenstadt, Klinikum der Ludwig-Maximilians-Universität, München, Germany Breast Cancer Research 2007, 9(Suppl 1):S2 (doi: 10.1186/bcr1685) Minimal residual disease (MRD) (that is, isolated tumor cells (ITC) in bone marrow) may be the source of potentially fatal overt distant metastases in solid tumors even years after primary treatment. MRD can be detected by immunohistochemical methods using antibodies directed against cytokeratins, cell-surface markers, or molecular, PCR- based techniques. Among solid tumors, the clinical relevance of MRD has been most extensively studied in breast cancer patients. Recently, the highest level of evidence for the prognostic impact of MRD in primary breast cancer was reached by a pooled analysis comprising more than 4,000 patients, showing poor outcome in patients with MRD at primary therapy. Yet, clinical application of MRD detection is hampered by the lack of a standardized detection assay. Moreover, clinical trial results demonstrating the benefit of a therapeutic interference derived from bone marrow status are still missing. Recent results suggest that, in addition to its prognostic impact, MRD can be used for therapy monitoring or as a potential therapeutic target after phenotyping of the tumor cells. Persisting MRD after primary treatment may lead to an indication for extended adjuvant therapy. y j py In a pooled analysis, bone marrow aspirates of 726 patients from academic breast cancer units in Oslo (n = 356), Munich (n = 228) and Tuebingen (n = 142) were analyzed during recurrence-free follow-up at a mean interval of 31.7 months after primary diagnosis of breast cancer pT1-4, pN0-3 pM0. Persistent ITC were detected in 15.4% of the patients (n = 112). The Kaplan–Meier estimate for mean distant relapse-free survival estimate was 163.6 months in patients with negative bone marrow status and 105.2 months in patients with positive bone marrow status. Patients without evidence of persistent ITC had a significantly longer overall survival (165.6 months), than patients with positive bone marrow status (103.3 months, P < 0.0001). In multivariate Cox regression analysis, allowing for bone marrow S1 Available online http://breast-cancer-research.com/supplements/9/S1 van ‘t Veer LJ, Dai H, van de Vijver MJ, et al.: Gene expression profiling predicts clinical outcome of breast cancer. Nature 2002, 415:530-536. 1. van ‘t Veer LJ, Dai H, van de Vijver MJ, et al.: Gene expression profiling predicts clinical outcome of breast cancer. Nature 2002, 415:530-536. 5. Gianni L, Zambetti M, Clark K, et al.: Gene expression profiles in paraffin-embedded core biopsy tissue predict response to chemotherapy in women with locally advanced breast cancer. J Clin Oncol 2005, 23:7265-7277. 2. van de Vijver M, He Y, Van ‘t Veer L, Dai H, Hart A, Voskuil D, Schreiber G, Peterse J, Roberts C, Marton M, et al.: A gene- expression signature as a predictor of survival in breast cancer. N Engl J Med 2002, 347:1999-2009. 6. Folgueira MA, Carraro DM, Brentani H, et al.: Gene expression profile associated with response to doxorubicin-based therapy in breast cancer. Clin Cancer Res 2005, 11:7434- 7443. g 3. Hannemann J, Oosterkamp HM, Bosch CAJ, Velds A, Wessels LFA, Loo C, Rutgers EJ, Rodenhuis S, van de Vijver MJ: Changes in gene expression associated with response to neoadjuvant chemotherapy in breast cancer. J Clin Oncol 2006, 23:3331- 3342. g 3. Hannemann J, Oosterkamp HM, Bosch CAJ, Velds A, Wessels LFA, Loo C, Rutgers EJ, Rodenhuis S, van de Vijver MJ: Changes in gene expression associated with response to neoadjuvant chemotherapy in breast cancer. J Clin Oncol 2006, 23:3331- 3342. 7. Dressman HK, Hans C, Bild A, Olson JA, et al.: Gene expression profiles of multiple breast cancer phenotypes and response to neoadjuvant chemotherapy. Clin Cancer Res 2006, 12(3 Pt 1): 819-826. 8. Thuerigen O, Schneeweiss A, Toedt G, et al.: Gene expression signature predicting pathologic complete response with gem- citabine, epirubicin, and docetaxel in primary breast cancer. J Clin Oncol 2006, 24:1839-1845. 9. Park S, Shimizu C, Shimoyama T, et al.: Gene expression profil- ing of ATP-binding cassette (ABC) transporters as a predictor of the pathologic response to neoadjuvant chemotherapy in breast cancer patients. Breast Cancer Res Treat 2006, 99:9-17. Available online http://breast-cancer-research.com/supplements/9/S1 signature (genomic profile) was obtained in node-negative patients with a representative tumor sample; node-positive patients were excluded. Between 2004 and 2006, 812 patients were enrolled and 427 (53%) genomic profiles were obtained. The remaining 385 (47%) patients were excluded mainly because of node-positive disease (22%). The logistics of obtaining fresh-frozen material from the tumor has gone very well in each of the participating hospitals. Approximately 50% of the tumors were shown to have a good prognosis signature. signature (genomic profile) was obtained in node-negative patients with a representative tumor sample; node-positive patients were excluded. pathways remain highly controversial as predictors of response or resistance to particular drugs. To date, the strongest (although still indirect) evidence supporting a molecular predictor of response to a particular regimen comes from a retrospective subset analysis of a variety of studies that showed a link between topoisomerase II amplification and increased sensitivity to anthracyclines. However, the best methodology for determining amplification of topoisomerase II and the appropriate cut-off value to distinguish between individuals with and without amplification have not been established. Between 2004 and 2006, 812 patients were enrolled and 427 (53%) genomic profiles were obtained. The remaining 385 (47%) patients were excluded mainly because of node-positive disease (22%). The logistics of obtaining fresh-frozen material from the tumor has gone very well in each of the participating hospitals. Approximately 50% of the tumors were shown to have a good prognosis signature. Another area of research is to identify gene expression signatures associated with the response to chemotherapy. Within a single- institution prospective phase II trial, patients with locally advanced breast cancer received six courses of either doxorubicin-cyclo- phosphamide (AC) (n = 25) or doxorubicin-docetaxel (AD) (n = 24) containing neoadjuvant chemotherapy. Gene expression profiles were generated from core needle biopsies obtained before treatment and correlated with the response of the primary tumor to the chemotherapy administered [3]. Additionally, pretreatment gene expression profiles were compared with those in tumors remaining after chemotherapy (n = 15). Several small studies provided ‘proof-of-principle’ that the gene expression profile of cancers that are highly sensitive to chemotherapy is different from that of tumors resistant to treatment. Table 1 (overleaf) presents a summary of the current literature. The most exciting possibility implicit to gene expression profiling-based diagnostic tests is that multiple predictors could be applied to a single data set. S5 Molecular and gene expression-based predictors of response to preoperative chemotherapy S5 10. Cleator S, Tsimelzon A, Ashworth A, et al.: Gene expression pat- terns for doxorubicin (Adriamycin) and cyclophosphamide (cytoxan) (AC) response and resistance. Breast Cancer Res Treat 2006, 95:229-233. Available online http://breast-cancer-research.com/supplements/9/S1 It is currently technically feasible to perform gene expression profiling on a single biopsy and assess prognosis using a 76-gene, or 70-gene signature, determine the ER and HER-2 status by measuring receptor mRNA levels, predict endocrine sensitivity among the ER-positive patients and estimate the probability of response chemotherapy. R f Eleven (22%) of the 49 patients showed a (near) pathological complete remission of the primary tumor after treatment. No gene expression pattern correlating with response could be identified for all patients as well as for the AC-treated or AD-treated group separately. The comparison of the pretreatment biopsy and the tumor excised after chemotherapy revealed differences in gene expression in those tumors that showed a partial remission but not in tumors that did not respond to chemotherapy. 1. Chang JC, Wooten EC, Tsimelzon A, et al.: Gene expression profiling for the prediction of therapeutic response to doc- etaxel in patients with breast cancer. Lancet 2003, 362:362- 369. 1. Chang JC, Wooten EC, Tsimelzon A, et al.: Gene expression profiling for the prediction of therapeutic response to doc- etaxel in patients with breast cancer. Lancet 2003, 362:362- 369. 2. Ayers M, Symmans WF, Stec J, et al.: Gene expression profiles predict complete pathologic response to neoadjuvant pacli- taxel and fluorouracil, doxorubicin, and cyclophosphamide chemotherapy in breast cancer. J Clin Oncol 2004, 22:2284- 2293. 2. Ayers M, Symmans WF, Stec J, et al.: Gene expression profiles predict complete pathologic response to neoadjuvant pacli- taxel and fluorouracil, doxorubicin, and cyclophosphamide chemotherapy in breast cancer. J Clin Oncol 2004, 22:2284- 2293. No gene expression profile predicting the response of primary breast carcinomas to AC-based or AD-based neoadjuvant chemotherapy could be detected in this study. We are currently expanding the series of patients in this neoadjuvant chemotherapy study. 3. Iwao-Koizumi K, Matoba R, Ueno N, et al.: Prediction of doc- etaxel response in human breast cancer by gene expression profiling. J Clin Oncol 2005, 23:422-431. We conclude that gene expression profiling is a method that has greatly accelerated the identification of prognostic and predictive factors in breast cancer and will lead to novel diagnostic tests that can be reliably implicated in clinical practice. References 4. Hannemann J, Oosterkamp HM, Bosch CA, et al.: Changes in gene expression associated with response to neoadjuvant chemotherapy in breast cancer. J Clin Oncol 2005, 23:3331- 3342. 1. M van de Vijver Department of Pathology, Netherlands Cancer Institute, Amsterdam, The Netherlands Breast Cancer Research 2007, 9(Suppl 1):S4 (doi: 10.1186/bcr1687) Tumor factors that can predict prognosis and response to specific therapies (for example, chemotherapy regimens) are of great potential benefit for tailored treatment of patients with invasive breast cancer. We have previously defined a gene expression profile of 70 genes that is predictive for a short interval to distant metastases (<5 years) in lymph node negative (LN0) patients [1]. We have subsequently validated the prognostic value of this 70-gene profile in a cohort of 295 stage I and II breast cancer patients younger than 53 years of age [2]. To test whether gene expression profiling can be used in clinical practice, we have performed a study in 16 hospitals in The Nether- lands. Female patients younger than 61 years presenting with primary operable cT1-2N0M0 breast carcinoma were eligible and entered into this prospective feasibility study. Fresh tumor samples in a standard- ized fashion were collected within 1–3 hours of primary surgery and sent in RNAlater® to the Netherlands Cancer Institute. The 70-gene Breast Cancer Research 2007, 9(Suppl 1):S4 (doi: 10.1186/bcr1687) Tumor factors that can predict prognosis and response to specific therapies (for example, chemotherapy regimens) are of great potential benefit for tailored treatment of patients with invasive breast cancer. Increases in the number of circulating endothelial cells (CECs) and progenitors (CEPs) have been reported in various pathological conditions including cancer [8,9]. At the clinical level, evidence is emerging that CEC kinetics and viability might correlate with clinical outcomes in cancer patients who undergo anti-angiogenic treatment [10]. The CellSpotter™ analyzer is also being investigated for measure- ment and characterization of CEC [11]. Therefore, CEC and CEP measurement has potential as a surrogate marker for monitoring anti- angiogenic treatment and drug activity, and could help to determine the S2 L Pusztai The University of Texas MD Anderson Cancer Center, Houston, TX, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 11. Hess KR, Anderson K, Symmans WF, et al.: Pharmacogenomic predictor of sensitivity to preoperative chemotherapy with paclitaxel and fluorouracil, doxorubicin, and cyclophos- phamide in breast cancer. J Clin Oncol 2006, 24:4236-4244. Breast Cancer Research 2007, 9(Suppl 1):S5 (doi: 10.1186/bcr1688) Many single-gene molecular markers have been evaluated as predictors of response to specific regimens. However, no reliable and routinely used molecular chemotherapy response predictors exist today. Molecular markers of proliferative activity remain nonspecific predictors of chemotherapy sensitivity in general. Multidrug-resistance transport proteins, p53 gene mutations, and defects in apoptotic 12. Mina L, Soule SE, Badve S, et al.: Predicting response to primary chemotherapy: gene expression profiling of paraffin- embedded core biopsy tissue. Breast Cancer Res Treat 2006, 103:197-208. S3 Breast Cancer Research Vol 9 Suppl 1 VII Madrid Breast Cancer Conference Table 1 (abstract S5) Patient Reference characteristics Treatment Chang et al. [1] 24 (discovery) and 4 cycles docetaxel 6 (validation) every 3 weeks patients. with LABC Ayers et al. [2] 24 (discovery) and 3 or 12 cycles paclitaxel and 12 (validation) 4 cycles of FAC (T/FAC) patients with breast cancer Iwao-Koizumi 44 (discovery) and 4 cycles of docetaxel et al. [3] 26 (validation) patients. with stage II/IV breast cancer Hannemann et al. [4] 48 patients with 6 cycles of doxorubicin/docetaxel LABC (AD, n = 24) or doxorubicin/ cyclophosphamide (AC, n = 24) Gianni et al. [5] 89 (INT-Milan) and 3 cycles of doxorubicin/paclitaxel, 82 (MDACC) 12 cycles of weekly paclitaxel patients with LABC Folgueira et al. [6] (1) 38 (discovery), 13 (validation) (2) 31 (discovery), 13 (validation) Dressman et al. [7] 37 patients with Liposomal doxorubicin x 4/paclitaxel stage IIB/III breast combined with local whole breast cancer hyperthermia Thuerigen et al. [8] 52 (discovery/ GEsDoc (gemcitabine, epirubicin GEsDoc), 48 every 2 weeks x 5 followed by (validation/GEDoc) docetaxel every 2 weeks x 2), patients with GEDoc (gemcitabine, epirubicin, T2-4N0-2M0 docetaxel every 3weeks) breast cancer Park et al. [9] 21 patients with 4 cycles of FEC every 3 weeks stage II/III breast followed by 12 cycles of weekly cancer paclitaxel Cleator et al. [10] 40 patients with 6 cycles doxorubicin/ primary breast cyclophosphamide (AC) cancer Hess et al. [11] 82 (discovery), 3 or 12 cycles paclitaxel and 51 (validation) 4 cycles of FAC (T/FAC) Mina et al. L Pusztai [12] 45 patients with 3 cycles doxorubicin every 22 of 274 candidate genes correlated with pCR (P < 0.05) forming three larg stage II/III breast 2 weeks and 6 cycles docetaxel clusters: angiogenesis-related genes, proliferation-related genes, invasion-rela cancer weekly x 6 genes, no correlation between RS and pCR, 24/274 genes correlated with inflammatory phenotype ABC transporter, ATP binding cassette transporter; discovery, development/training set; FAC, 5-fluorouracil, doxorubicine, cyclophosphamide; FEC, 5-fluorouracil, epirubicin, cyclophosphamide; IBC, inflammatory breast cancer; LABC, locally advanced breast cancer; LOOCV, leave-one-out cross-validation; NPV, negative predic value; pCR pathological complete response; PPV positive predictive value; RD residual disease; RS recurrence score (Oncotype DX©); validation validation set No differentially expressed genes identified, no possible multigene predictor developed Patient samples did not cluster distinctly for pCR/near pCR compared with RD in hierarchical clustering 86 genes correlating with pCR (P < 0.05) forming three clusters – Immune-related gene cluster RS correlated with pCR in INT-Milan patients 6-gene model developed on INT-Milan patients validated on MDAC 11 differentially expressed ABC transporters Multigene predictor model with the ABC transporters differentially expressed between the two classes (P < 0.003) with predictive of pCR with: – Predictive accuracy of 92.8% (95% CI, 88.0–97.4%), PPV of 93.2% (95% CI, 85.2–100%), NPV of 93.6% (95% CI, 87.8–99.4%), sensitivity of 88.1% (95% CI, 76.8–99.4%), specificity of 95.9% (95% CI, 87.8–100%) 253 differentially expressed genes; – 75 genes overexpressed in resistant tumors (that is, transcription, differentiation, signal transduction, amino acid metabolism) – 178 genes overexpressed in sensitive tumors (that is, cell cycle, survival, stress response, and estrogen-regulated genes) 30-gene predictor identified and validated by fivefold cross-validation, permutation testing and application to an independent data set: – Predictive accuracy of 76% (95% CI, 62–87%), PPV of 52% (95% CI, 31–73%), NPV of 96% (95% CI, 82–100%), sensitivity of 92% (95% CI 64–100%) specificity of 71% (95% CI 54–85%) – 75 genes overexpressed in resistant tumors (that is, transcription, differentiation, signal transduction, amino acid metabolism) – 178 genes overexpressed in sensitive tumors (that is, cell cycle, survival, stress response, and estrogen-regulated genes) 30-gene predictor identified and validated by fivefold cross-validation, permutation testing and application to an independent data set: 30-gene predictor identified and validated by fivefold cross-validation, permutation testing and application to an independent data set: g pp p – Predictive accuracy of 76% (95% CI, 62–87%), PPV of 52% (95% CI 31 73%) NPV of 96% (95% CI 82 100%) sensitivit – Predictive accuracy of 76% (95% CI, 62–87%), PPV of 52% – Predictive accuracy of 76% (95% CI, 62–87%), PPV of 52% (95% CI, 31–73%), NPV of 96% (95% CI, 82–100%), sensitivity of 92% (95% CI, 64–100%), specificity of 71% (95% CI, 54–85%) 22 of 274 candidate genes correlated with pCR (P < 0.05) forming three large clusters: angiogenesis-related genes, proliferation-related genes, invasion-related genes, no correlation between RS and pCR, 24/274 genes correlated with inflammatory phenotype ABC transporter, ATP binding cassette transporter; discovery, development/training set; FAC, 5-fluorouracil, doxorubicine, cyclophosphamide; FEC, 5-fluorouracil, epirubicin, cyclophosphamide; IBC, inflammatory breast cancer; LABC, locally advanced breast cancer; LOOCV, leave-one-out cross-validation; NPV, negative predictive value; pCR, pathological complete response; PPV, positive predictive value; RD, residual disease; RS, recurrence score (Oncotype DX©); validation, validation set. ABC transporter, ATP binding cassette transporter; discovery, development/training set; FAC, 5-fluorouracil, doxorubicine, cyclophosphamide; FEC, 5-fluorouracil, epirubicin, cyclophosphamide; IBC, inflammatory breast cancer; LABC, locally advanced breast cancer; LOOCV, leave-one-out cross-validation; NPV, negative predictive value; pCR, pathological complete response; PPV, positive predictive value; RD, residual disease; RS, recurrence score (Oncotype DX©); validation, validation set. L Pusztai [12] 45 patients with 3 cycles doxorubicin every stage II/III breast 2 weeks and 6 cycles docetaxel cancer weekly x 6 ABC transporter, ATP binding cassette transporter; discovery, development/training s epirubicin, cyclophosphamide; IBC, inflammatory breast cancer; LABC, locally advanc value; pCR, pathological complete response; PPV, positive predictive value; RD, resid Table 1 (abstract S5) Patient Reference characteristics Treatment Results Chang et al. [1] 24 (discovery) and 4 cycles docetaxel 92 differentially expressed genes (P < 0.001) 6 (validation) every 3 weeks LOOCV results in training: predictive accuracy of 88% (95% CI, 68–97%), P patients. with LABC of 92%, NPV of 83%, sensitivity of 85% (95% CI, 55–98%), specificity of 91 (95% CI, 59–100%) Independent validation: all six patients with sensitive tumors correctly identifie Ayers et al. [2] 24 (discovery) and 3 or 12 cycles paclitaxel and No single gene sufficiently associated with pCR to serve as single valid marke 12 (validation) 4 cycles of FAC (T/FAC) 74-gene predictor for response to chemotherapy patients with breast – Predictive accuracy of 78% (95% CI, 52–94%), PPV of 100% cancer (95% CI, 29–100%), NPV of 73% (95% CI, 45–92%), sensitivity of 43% (95% CI, 10–82%), specificity of 100% (95% CI, 72–100%) Iwao-Koizumi 44 (discovery) and 4 cycles of docetaxel 85-gene-predictor of pCR with: et al. [3] 26 (validation) – Predictive accuracy of 80.7% (95% CI, 63.5–92.5%), PPV of 73.3% patients. with (95% CI, 49.5–90.3%), NPV of 90.9% (95% CI, 65.9–99.4%), sensitivity of stage II/IV breast cancer 91.7% (95% CI, 68.1–99.5%), specificity of 71.4% (95% CI, 46.7–89.5%) Hannemann et al. [4] 48 patients with 6 cycles of doxorubicin/docetaxel No differentially expressed genes identified, no possible multigene predictor LABC (AD, n = 24) or doxorubicin/ developed cyclophosphamide (AC, n = 24) Patient samples did not cluster distinctly for pCR/near pCR compared with R hierarchical clustering Gianni et al. [5] 89 (INT-Milan) and 3 cycles of doxorubicin/paclitaxel, 86 genes correlating with pCR (P < 0.05) forming three clusters 82 (MDACC) 12 cycles of weekly paclitaxel – ER gene cluster patients with LABC – Proliferation gene cluster – Immune-related gene cluster RS correlated with pCR in INT-Milan patients 86-gene model developed on INT-Milan patients validated on MDACC sampl Folgueira et al. L Pusztai [6] (1) 38 (discovery), (1) 25 (3.8%) differentially expressed transcripts between responders and 13 (validation) non-responders, three-gene classifier could not be validated (2) 31 (discovery), (2) Three-gene classifier successfully developed and validated both by LOOC 13 (validation) and in an independent validation set Dressman et al. [7] 37 patients with Liposomal doxorubicin x 4/paclitaxel (a) 22-gene signature characterizing IBC identified and validated by LOOVC stage IIB/III breast combined with local whole breast (b) 18-gene signature characterizing IBC identified and validated by LOOVC cancer hyperthermia (c) No gene signature predicting clinical response could be identified Thuerigen et al. [8] 52 (discovery/ GEsDoc (gemcitabine, epirubicin 512-gene-signature predictive of pCR: predictive accuracy of 88% GEsDoc), 48 every 2 weeks x 5 followed by (95% CI, 75–95%), PPV of 64% (95% CI, 39–81%), NPV of 95% (validation/GEDoc) docetaxel every 2 weeks x 2), (95% CI, 82–99%), sensitivity of 78% (95% CI, 40–97%), specificity of 90% patients with GEDoc (gemcitabine, epirubicin, (95% CI, 76–97%) T2-4N0-2M0 docetaxel every 3weeks) breast cancer Park et al. [9] 21 patients with 4 cycles of FEC every 3 weeks 11 differentially expressed ABC transporters stage II/III breast followed by 12 cycles of weekly Multigene predictor model with the ABC transporters differentially expressed cancer paclitaxel between the two classes (P < 0.003) with predictive of pCR with: – Predictive accuracy of 92.8% (95% CI, 88.0–97.4%), PPV of 93.2% (95% CI, 85.2–100%), NPV of 93.6% (95% CI, 87.8–99.4%), sensitivity of 88.1% (95% CI, 76.8–99.4%), specificity of 95.9% (95% CI, 87.8–100%) Cleator et al. [10] 40 patients with 6 cycles doxorubicin/ 253 differentially expressed genes; primary breast cyclophosphamide (AC) – 75 genes overexpressed in resistant tumors (that is, transcription, differenti cancer signal transduction, amino acid metabolism) – 178 genes overexpressed in sensitive tumors (that is, cell cycle, survival, st response, and estrogen-regulated genes) Hess et al. [11] 82 (discovery), 3 or 12 cycles paclitaxel and 30-gene predictor identified and validated by fivefold cross-validation, permut 51 (validation) 4 cycles of FAC (T/FAC) testing and application to an independent data set: – Predictive accuracy of 76% (95% CI, 62–87%), PPV of 52% (95% CI, 31–73%), NPV of 96% (95% CI, 82–100%), sensitivity of 92% (95% CI, 64–100%), specificity of 71% (95% CI, 54–85%) Mina et al. Available online http://breast-cancer-research.com/supplements/9/S1 S6 determining the patterns of primary tumor shrinkage and the amount and location of any residual disease in the breast. Role of MR and digital mammography for screening DD Dershaw y Surgical treatment after neoadjuvant systemic therapy focuses on the management of the primary breast tumor and that of axillary lymph nodes. Regarding the primary breast tumor, several studies have shown that neoadjuvant systemic therapy converts a proportion of mastectomy candidates to candidates for breast-conserving surgery. Neoadjuvant systemic therapy can also decrease the amount of breast tissue that needs to be removed at lumpectomy even in patients who are lumpectomy candidates at presentation. Memorial Sloan-Kettering Cancer Center, New York, NY, USA Breast Cancer Research 2007, 9(Suppl 1):S6 (doi: 10.1186/bcr1689) New technologies have raised the issue of their application as a replacement for or in addition to screen-film mammography in screen- ing of women at normal risk and elevated risk of breast cancer. Digital mammography images the breast using the identical information obtained in screen-film mammography. The image is processed, stored and displayed electronically. This conveys several advantages over film techniques, but the approval of digital mammography by the US Food and Drug Administration has been based on comparable ability to detect cancer, not any diagnostic advantage. y Neoadjuvant systemic therapy (primarily neoadjuvant chemotherapy) downstages axillary lymph nodes in up to 30–40% of the patients. Although this observation was of little clinical significance when axillary node dissection was the sole method for staging the axilla, the development and validation of sentinel lymph node biopsy has provided an additional potential advantage for neoadjuvant chemotherapy; that is, the possibility of decreasing the extent and morbidity of axillary surgery. This approach is, naturally, predicated on the premise that sentinel node biopsy is feasible and accurate following neoadjuvant chemotherapy. Until recently, only small, single-institution studies have examined the efficacy of lymphatic mapping and the accuracy of sentinel node biopsy after neoadjuvant chemotherapy with significant variability in the rate of sentinel node identification and in the rate of false negative sentinel node [1]. When these studies are examined collectively [1,2] or when larger, multicenter data sets are analyzed [3], however, sentinel node biopsy after neoadjuvant chemotherapy appears to have similar performance characteristics to those of sentinel node biopsy before systemic therapy [4-6]. Four prospective studies comparing digital and film mammography on the same patients have shown that for population-based screening there is no advantage for digital over film. The last and largest of these studies [1] initially reported an advantage for several subgroups of women for digital screening. Reference 1. Pisano ED, Gatsonis C, Hendrick E, et al.: Diagnostic perfor- mance of digital versus film mammography for breast-cancer screening. N Engl J Med 2005, 353:1773-1783. SYMPOSIUM II 1. Mamounas EP: Sentinel lymph node biopsy after neoadjuvant systemic therapy. Surg Clin North Am 2003, 83:931-942. 1. Mamounas EP: Sentinel lymph node biopsy after neoadjuvant systemic therapy. Surg Clin North Am 2003, 83:931-942. 1. Mamounas EP: Sentinel lymph node biopsy after neoadjuvant systemic therapy. Surg Clin North Am 2003, 83:931-942. New Strategies for Primary Treatment (Part I) New Strategies for Primary Treatment (Part I) 2. Xing Y, Ding D, Cox D, et al.: Meta-analysis of sentinel lymph node biopsy following preoperative chemotherapy in patients with operable breast cancer [abstract 561]. Proc Am Soc Clin Oncol 2004, 23:17. L Pusztai S4 Available online http://breast-cancer-research.com/supplements/9/S1 Role of MR and digital mammography for screening DD Dershaw Later analysis of data from this study, however, concluded that only women with dense breasts may benefit and that screening of entire populations with digital mammography is excessively costly and not beneficial. Analysis of MR as a screening tool has been directed at women with greatly elevated lifetime risk of developing breast cancer. The ability of MR to detect a large percentage of cancers in these women earlier than mammography, sonography or physical examination and at a stage at which they should be curable has been clearly demonstrated. This has lead the American Cancer Society, along with others, to recommend the use of MR to annually screen women with at least a 20% lifetime risk of developing breast cancer starting at age 25 years. Those at less risk were not included due to lack of supporting data and concern over excessive biopsies in those women. Some have proposed that candidates for neoadjuvant systemic therapy should have a sentinel node biopsy before, rather than after, neoadjuvant systemic therapy so that information on the status of the axillary nodes is obtained without the potential confounding effects of systemic treatment, and sentinel node-negative patients can avoid axillary dissection [7-9]. Although this approach may be useful in patients who will not need systemic therapy (that is, chemotherapy) if the sentinel node is negative, it is not generally useful for the majority of candidates for neoadjuvant systemic therapy, for whom little – if anything – is to be gained by knowing the pathologic nodal status upfront. In addition, this approach commits patients to two surgical procedures and does not take advantage of the down-staging effect of neoadjuvant chemotherapy on the axillary nodes. Surgical issues in the breast and axillary nodes in patients treated with neoadjuvant systemic therapy EP Mamounas1,2 3. Mamounas EP, Brown A, Anderson S, et al.: Sentinel node biopsy after neoadjuvant chemotherapy in breast cancer: results from National Surgical Adjuvant Breast and Bowel Project Protocol B-27. J Clin Oncol 2005, 23:2694-2702. SA Khan1,2, H Hazard1, D Scholtens2,3 For many years in the past there was the misconception that breast reconstruction must be delayed for several years after mastectomy because reconstruction might prevent or delay detection of local recurrence. Hence, a large population of women has sought and presently still seeks to undergo delayed postmastectomy recon- struction months to years later. The outcomes of these reconstructions are good to excellent but the cosmetic outcome is always somewhat inferior to the results obtainable by immediate reconstruction because of the loss of the breast skin envelope after mastectomy and the need to replace so much of the chest wall skin. In regard to the all-important question of local recurrence, several longer term follow-up studies at the MD Anderson Cancer Center actually indicate a lower incidence of local recurrence after immediate reconstruction. Results A total of 16,024 patients with stage IV disease were identified in the NCDB over 4 years, of whom 42.8% received only diagnostic or palliative procedures, and 57.2% underwent partial or total mastectomy. A multivariate proportional hazards model identified the number of metastatic sites, the type of metastatic burden, and the extent of resection of the primary tumor as significant independent prognostic covariates. Women treated with surgical resection with free margins, when compared with those not surgically treated, had superior prognosis, with a hazard ratio of 0.61 (95% CI 0.58–0.65). In the NMH study, we identified 114 women; 48 (42%) underwent resection of the primary tumor. Local control of the chest wall was maintained in 36/48 (75%) of the surgical group, versus 31/66 (47%) patients without surgical therapy (P = 0.002). TTFP was prolonged in the surgical group, adjusted HR 0.639 (P = 0.03). The HR for overall survival in the surgical group was 0.724 (P = 0.160). Notably, in women with controlled chest walls we observed an overall survival benefit with a HR of 0.418 (P < 0.0002). Current comparative statistics for the numbers of patients undergoing immediate as well as delayed reconstruction from 2002–2006 are presented in Table 1. Since 2002, the MD Anderson Plastic Surgery Department has seen a progressively increasing demand of early breast cancer patients for implant-based reconstructions performed on an immediate basis. The opposite trend of a preference of autologous tissue reconstructions is apparent for delayed reconstructions. Conclusion Recent retrospective studies suggest that surgical resection of the primary tumor in women who also have distant disease may be beneficial [1-3]. SA Khan1,2, H Hazard1, D Scholtens2,3 Division of Plastic Surgery, Department of Surgery, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 1Department of Surgery, 2Robert H. Lurie Comprehensive Cancer Center and 3Department of Preventive Medicine, Feinberg School of Medicine of Northwestern University, Chicago, IL, USA 1Department of Surgery, 2Robert H. Lurie Comprehensive Cancer Center and 3Department of Preventive Medicine, Feinberg School of Medicine of Northwestern University, Chicago, IL, USA Breast Cancer Research 2007, 9(Suppl 1):S9 (doi: 10.1186/bcr1692) With numerous advances in the field of plastic surgery, breast recon- struction is available today to almost any woman undergoing surgery for breast cancer. Several methods can be used for restoration of the breast either at the same time as breast cancer surgery (immediate reconstruction) or months or even years later, at the patient’s discretion (delayed reconstruction). The skin sparing approach to mastectomy, originated in the early 1990s, has especially facilitated immediate reconstruction of the breast and resulted in a high standard of cosmetic outcomes. In general, the reconstructive options include using autologous soft tissues from the patient’s donor areas, such as the abdomen, hips, back, or buttock, or using a prosthetic implant to create a new breast mound. For well over a decade now, skin-sparing mastectomy and immediate reconstruction has offered the compelling advantages of both superior cosmetic appearance of the reconstructed Breast Cancer Research 2007, 9(Suppl 1):S8 (doi: 10.1186/bcr1691) Background Approximately 12,000 women with primary breast cancer present with stage IV disease annually in the United States. The present treatment paradigm is that systemic therapy is the standard of care and surgical therapy for the primary tumor is provided only to palliate symptoms. Methods We have examined the impact of local therapy on survival in patients presenting with stage IV breast cancer at initial diagnosis, reporting to the National Cancer Database (NCDB) between 1990 and 1993. In a subsequent study, we reviewed the medical records of women presenting with stage IV breast cancer to Northwestern Memorial Hospital (NMH) over the 10-year period 1995–2005. Cox regression models and logistic regression was used to estimate adjusted overall survival in both studies, and the time to first progression (TTFP) and chest wall status in the NMH population. g p pp breast as well as favorable psychological and economic benefits [1]. S8 S8 3. Babiera GV, Rao R, Feng L, Meric-Bernstam F, Kuerer HM, Sin- gletary SE, Hunt KK, Ross MI, Gwyn KM, Feig BW, Ames FC, Hortobagyi GN: Effect of primary tumor extirpation in breast cancer patients who present with stage IV disease and an intact primary tumor. Ann Surg Oncol 2006, 13:776-782. EP Mamounas1,2 1Aultman Cancer Center, Canton, OH, USA; 2Northeastern Ohio Universities College of Medicine, Rootstown, OH, USA 1Aultman Cancer Center, Canton, OH, USA; 2Northeastern Ohio Universities College of Medicine, Rootstown, OH, USA 4. Veronesi U, Paganelli G, Viale G, et al.: A randomized compari- son of sentinel-node biopsy with routine axillary dissection in breast cancer. N Engl J Med 2003, 349:546-553. Breast Cancer Research 2007, 9(Suppl 1):S7 (doi: 10.1186/bcr1690) Several unique surgical issues arise in the management of patients who are candidates for neoadjuvant systemic therapy. These involve the original diagnostic assessment of the extent of disease in the breast and axilla, the preoperative planning, and ultimately the surgical management of the primary breast tumor and that of axillary lymph nodes. Careful consideration of these issues is crucial in order to maximize local control of the disease, while minimizing the extent of the required surgical resection and the ensuing surgical morbidity. g 5. McMasters KM, Tuttle TM, Carlson DJ, et al.: Sentinel lymph node biopsy for breast cancer: a suitable alternative to routine axillary dissection in multi-institutional practice when optimal technique is used. J Clin Oncol 2000, 18:2560-2566. 6. Krag D, Weaver D, Ashikaga T, et al.: The sentinel node in breast cancer – a multicenter validation study. N Engl J Med 1998, 339:941-946. Adequate diagnostic assessment with core needle biopsy before the initiation of neoadjuvant systemic therapy ensures the presence of invasive carcinoma and provides adequate material for routine bio- marker evaluation (such as ER, PR and HER-2 neu), while minimally disturbing the primary breast tumor. Consideration should also be given in assessing the status of axillary nodes by minimally invasive techniques such as ultrasound of the axilla and fine needle aspiration of suspicious nodes. Optimal preoperative planning aims at accurately 7. Bedrosian I, Reynolds C, Mick R, et al.: Accuracy of sentinel lymph node biopsy in patients with large primary breast tumors. Cancer 2000, 88:2540-2545. 8. Schrenk P, Hochreiner G, Fridrik M, et al.: Sentinel node biopsy performed before preoperative chemotherapy for axillary lymph node staging in breast cancer. Breast J 2003, 9:282-287. 9. Sabel MS, Schott AF, Kleer CG, et al.: Sentinel node biopsy prior to neoadjuvant chemotherapy. Am J Surg 2003, 186:102-105. 9. Sabel MS, Schott AF, Kleer CG, et al.: Sentinel node biopsy prior to neoadjuvant chemotherapy. Am J Surg 2003, 186:102-105. S5 Breast Cancer Research Vol 9 Suppl 1 VII Madrid Breast Cancer Conference SA Khan1,2, H Hazard1, D Scholtens2,3 These are remarkably consistent in the magnitude of the survival advantage, with a hazard ratio of about 0.6 when surgical intervention is used. There is a paucity of data supporting the assumption that surgical resection will maintain a disease-free chest wall. A study of women with in-breast recurrence of breast cancer and synchronous distant metastases found that women undergoing resection were more likely to avoid uncontrolled chest-wall disease and to survive longer [4]. Our study at NMH (the largest reported to date examining this issue) finds that maintenance of a disease-free chest wall is associated with improved survival. This further emphasizes the need for prospective randomized trials to establish the role of local therapy in the setting of metastatic breast cancer. pp y The controversy today is that we continue to undergo an important evolution in our approach to breast reconstruction because of the identified advantage of the addition of postmastectomy radiation therapy to mastectomy and chemotherapy. The inability to determine which patients will require postmastectomy radiation therapy, especially which patients with early-stage breast cancer, has increased the complexity of planning for immediate breast reconstruction. There are two potential problems with performing an immediate breast reconstruction in a patient who will require postmastectomy radiation therapy. One problem is that postmastectomy radiation therapy can adversely affect the aesthetic outcome of an immediate breast reconstruction [1-3]. The other potential problem is that an immediate breast reconstruction can interfere with the delivery of postmastectomy radiation therapy. Because radiation therapy is one of the most important considerations affecting the timing and technique of breast reconstruction, plastic surgeons and radiation oncologists must work together to maintain an appropriate balance between minimizing the risk of recurrence and providing the best possible aesthetic outcome. Our experience at the MD Anderson Cancer Center and many of the other experiences reported in the literature indicate that breast reconstruction should probably be delayed in patients who are known preoperatively to require postmastectomy radiation therapy [2,4]. Considering, then, our evolving approach for early breast cancer patients who want immediate breast reconstruction but are at higher risk for postmastectomy radiation therapy and likely delayed recon- struction, our updated ‘delayed-immediate’ [5] reconstruction experience between May 2002 and November 2006 includes 42 References 1. Khan SA, Stewart AK, Morrow M: Does aggressive local therapy improve survival in metastatic breast cancer? Surgery 2002, 132:620-626. 2. 4. Dalberg K, Liedberg A, Johansson U, Rutqvist LE: Uncontrolled local disease after salvage treatment for ipsilateral breast tumour recurrence. Eur J Surg Oncol 2003, 29:143-154. S10 S10 The nipple sparing mastectomy: a 5-year experience at the European Institute of Oncology of Milan JY Petit1, U Veronesi2, R Orecchia3, A Luini4 1Department of Plastic Surgery, 2Scientific Director, 3Department of Radiotherapy and 4Department of Senology, European Institute of Oncology, Milan, Italy 1Department of Plastic Surgery, 2Scientific Director, 3Department of Radiotherapy and 4Department of Senology, European Institute of Oncology, Milan, Italy 1Department of Plastic Surgery, 2Scientific Director, 3Department of Radiotherapy and 4Department of Senology, European Institute of Oncology, Milan, Italy Breast Cancer Research 2007, 9(Suppl 1):S10 (doi: 10.1186/bcr1693) Background Under certain conditions, the nipple sparing mastectomy can be proposed for the treatment of breast cancer when the mastectomy remains indicated. The nipple areola complex (NAC) preservation improves the quality of life, reducing the feeling of mutilation. The main argument against the procedure is the lack of radical resection of the retroareolar ductal system. A novel radiosurgical treatment combining subcutaneous mastectomy with intraoperative radiotherapy is proposed. Breast reconstruction plays an important role in the multidisciplinary, comprehensive care of the breast cancer patient. Advances in techniques have allowed immediate reconstruction to minimize incisional scars on the breast and improve overall breast contour, shape, and appearance. The improved aesthetic outcomes over delayed reconstruction, achieved also by the skin-preserving ‘delayed- immediate’ approach for early breast cancer patients at risk for radiation therapy and similarly for selected patients actually undergoing PMRT, has convinced many breast cancer patients to view mastectomy with reconstruction as a viable and positive treatment choice. Those patients who are candidates for and who will undergo delayed reconstruction, either because of the planned need for adjuvant radiotherapy or because of poorly controlled comorbidities that must be stabilized before a reconstruction can be undertaken, can still anticipate an excellent cosmetic outcome with a record of high patient satisfaction. Methods Seven hundred and seventy-three nipple sparing mastec- tomies have been performed since March 2002, for invasive carcinoma in 63% and for in situ carcinoma in 37%. Clinical complications, aesthetic results, and oncological and psychological results were recorded. Results The NAC necrosed totally in 26 cases (3.3%) and partially in 49 cases (6.3%), and was removed in 36 cases (4.6%). Thirteen infections (1.6%) were observed and 32 (4.14%) prostheses removed. The median rating of the patients for global cosmetic results on a 0 (worst) to 10 (excellent) scale was 8. The surgeon in charge of the follow-up has given the same rating. References 1. Robb GL: Reconstructive surgery. In Breast Cancer. MD Ander- son Cancer Care Series. Edited by Hunt KK, Strom EA, Ueno NT. New York: Springer-Verlag, Inc.; 2001:223-253. 1. Robb GL: Reconstructive surgery. In Breast Cancer. MD Ander- son Cancer Care Series. Edited by Hunt KK, Strom EA, Ueno NT. New York: Springer-Verlag, Inc.; 2001:223-253. 2. Tran NV, Evans GRD, Kroll SS, Baldwin BJ, Miller MJ, Reece GP, Robb GL: Postoperative adjuvant irradiation: effects on trans- verse rectus abdominis muscle flap breast reconstruction. Plast Reconstr Surg 2000, 106:313-317. 3. Kronowitz SJ, Robb GL: Breast reconstruction and radiation therapy. In Advanced Therapy of Breast Disease. 2nd edition. Edited by Singletary SE, Robb GL, Hortobagyi GN. London: BC Decker, Inc.; 2003:427-438. SA Khan1,2, H Hazard1, D Scholtens2,3 Complication rates with delayed-immediate reconstruction are considered quite acceptable and included: stage I (three of 42 patients, 7%), stage II (four of 26 patients, 15%), skin- preserving delayed reconstruction (one of 13 patients, 7%), and expander loss (surgery-related, 2%; PMRT-related, 6%). Only 2% (one of 42 patients) of the patients developed a recurrence of breast cancer after an average follow-up of 24 months. patients who were considered preoperatively to be at an increased risk of requiring postmastectomy radiation therapy (PMRT) and underwent delayed-immediate breast reconstruction at the MD Anderson Cancer Center. After review of the permanent pathology, 62% (26 of 42 patients) of patients did not require PMRT and, because of the preserved breast envelope, could go ahead with essentially an immediate reconstruction. The remaining 38% (16 of 42 patients) did require PMRT, but selected patients are being managed on a similar IRB reconstruction protocol that will allow a ‘skin-preserving’ delayed reconstruction following completion of the PMRT. Fifteen of these 16 patients (94%) were evaluated as having uncompromised radiation delivery. Cosmetic results of this approach are very promising and compare favorably with immediate reconstruction in the short-term follow-up to date. Complication rates with delayed-immediate reconstruction are considered quite acceptable and included: stage I (three of 42 patients, 7%), stage II (four of 26 patients, 15%), skin- preserving delayed reconstruction (one of 13 patients, 7%), and expander loss (surgery-related, 2%; PMRT-related, 6%). Only 2% (one of 42 patients) of the patients developed a recurrence of breast cancer after an average follow-up of 24 months. New Strategies for Primary Treatment (Part II) S10 A radio dystrophy was observed on the areola in 5.1% of the patients. The sensitivity of the NAC recovered slowly and partially in only 30% of the cases. Twelve local recurrences (1.5%) occurred in an average follow-up period of 18 months (range 1–61 months). Most recurrences were located far from the nipple areola site, outside the radiated field. Two recurrences were Paget disease associated with an in situ carcinoma. Overall, we observed 23 metastases and three deaths. Sixty-eight per cent of patients were satisfied with their reconstructed breast and 97% were satisfied with having preserved the NAC. SA Khan1,2, H Hazard1, D Scholtens2,3 Rapiti E, Verkooijen HM, Vlastos G, Fioretta G, Neyroud-Caspar I, Sappino AP, Chappuis PO, Bouchardy C: Complete excision of primary breast tumor improves survival of patients with metastatic breast cancer at diagnosis. J Clin Oncol 2006, 24: 2743-2749. 2. Rapiti E, Verkooijen HM, Vlastos G, Fioretta G, Neyroud-Caspar I, Sappino AP, Chappuis PO, Bouchardy C: Complete excision of primary breast tumor improves survival of patients with metastatic breast cancer at diagnosis. J Clin Oncol 2006, 24: 2743-2749. 3. Babiera GV, Rao R, Feng L, Meric-Bernstam F, Kuerer HM, Sin- gletary SE, Hunt KK, Ross MI, Gwyn KM, Feig BW, Ames FC, Hortobagyi GN: Effect of primary tumor extirpation in breast cancer patients who present with stage IV disease and an intact primary tumor. Ann Surg Oncol 2006, 13:776-782. 4. Dalberg K, Liedberg A, Johansson U, Rutqvist LE: Uncontrolled local disease after salvage treatment for ipsilateral breast tumour recurrence. Eur J Surg Oncol 2003, 29:143-154. 4. Dalberg K, Liedberg A, Johansson U, Rutqvist LE: Uncontrolled local disease after salvage treatment for ipsilateral breast tumour recurrence. Eur J Surg Oncol 2003, 29:143-154. S6 Available online http://breast-cancer-research.com/supplements/9/S1 Table 1 (abstract S9) Immediate versus delayed breast reconstruction, 2002–2006, MD Anderson Cancer Center Number of immediate reconstructions Number of delayed reconstructions Total number of Number of Year Implant Patient’s own tissue Implant Patient’s own tissue reconstructions mastectomies 2002 167 153 43 55 418 418 2003 186 176 53 68 483 482 2004 256 158 45 84 543 541 2005 239 164 48 75 526 523 2006 313 234 39 98 684 683 Immediate versus delayed breast reconstruction, 2002–2006, MD Anderson Cancer Center SYMPOSIUM II patients who were considered preoperatively to be at an increased risk of requiring postmastectomy radiation therapy (PMRT) and underwent delayed-immediate breast reconstruction at the MD Anderson Cancer Center. After review of the permanent pathology, 62% (26 of 42 patients) of patients did not require PMRT and, because of the preserved breast envelope, could go ahead with essentially an immediate reconstruction. The remaining 38% (16 of 42 patients) did require PMRT, but selected patients are being managed on a similar IRB reconstruction protocol that will allow a ‘skin-preserving’ delayed reconstruction following completion of the PMRT. Fifteen of these 16 patients (94%) were evaluated as having uncompromised radiation delivery. Cosmetic results of this approach are very promising and compare favorably with immediate reconstruction in the short-term follow-up to date. Introduction C In our technique, 16 Gy were delivered on the NAC in one shot with lead and aluminium disk protection of the pectoralis muscle and thoracic wall (see Figure 1). However, in 145 cases, the same radiotherapy was postponed to the following day, due to technical problems with the machine in the operating room. (a) The sterile collimator of the LINAC. (b) The gauze over the areola allows a more homogeneous distribution of the dose to the nipple and the areola. (c) Protective lead and aluminum disks. (d) Pectoralis major muscle. Sensitivity, colour, radio dystrophy, position and symmetry, and global evaluation by the surgeon and the patient, were rated according to a scale (0–10), 10 indicating the best results. (The results were rated poor when rated from 0 to 3, fair from 5 to 6 and good or excellent from 7 to 10.) Local recurrences, distant metastases and death were also recorded. A psychological study was conducted concomitantly. A detailed questionnaire was sent to the patients 1 year after the operation to evaluate the degree of satisfaction. The statistic analysis of psychological results was performed with the chi-square test and exact Fisher test. Results appears an identity mark of the breast [12]. Several authors have evaluated the risk of nipple areola involvement and investigated the possibility of nipple areola preservation [13-18]. Focusing on such risk, and the possible cancer recurrence in the breast tissue preserved beneath the NAC for the blood supply, we proposed to combine the subcutaneous mastectomy technique with intraoperative radiotherapy with electrons (ELIOT), previously trialled in breast-conserving treatment [19]. The preliminary results of the ‘nipple sparing mastec- tomy’ (NSM) were published in 2003 [20]. Today, we report the results of 773 NSMs performed at the European Institute of Oncology. Materials and methods appears an identity mark of the breast [12]. Several authors have evaluated the risk of nipple areola involvement and investigated the possibility of nipple areola preservation [13-18]. Focusing on such risk, and the possible cancer recurrence in the breast tissue preserved beneath the NAC for the blood supply, we proposed to combine the subcutaneous mastectomy technique with intraoperative radiotherapy with electrons (ELIOT), previously trialled in breast-conserving treatment [19]. The preliminary results of the ‘nipple sparing mastec- tomy’ (NSM) were published in 2003 [20]. Today, we report the results of 773 NSMs performed at the European Institute of Oncology. p Materials and methods From March 2002 to March 2007, 898 patients were invited to undergo a NSM. The inclusion criteria were primary tumours located at least 1 cm outside the areola margins, an absence of nipple retraction or bloody discharge, and an absence of retro areola micro- calcifications. Multifocality was not a cause for exclusion, provided that all the tumour sites were distant from the areola. Invasive as well as in situ carcinomas were included. Patients were excluded at the time of the operation when the frozen examination of the retro areola tissue had been positive for carcinoma: in these cases, ELIOT was not delivered and the NAC was removed. Among the 898 cases, 98 were excluded, mainly because of the positive extemporaneous examination Cosmetic results and sequelea were evaluated by the surgeon in charge of the follow-up according to the scale previously mentioned. The average evaluation of the sensitivity of the areola and the periareolar area was 2/10. Thirty per cent of the patients recovered some kind of sensitivity in several months. The colour of the areola was judged 8/10. The radio dystrophy was absent in most cases: 9/10. A radio dystrophy such as telangiectasia has been observed in 5.1% of the cases. The symmetry of the breasts was evaluated well in most Introduction C 3. Kronowitz SJ, Robb GL: Breast reconstruction and radiation therapy. In Advanced Therapy of Breast Disease. 2nd edition. Edited by Singletary SE, Robb GL, Hortobagyi GN. London: BC Decker, Inc.; 2003:427-438. Conservative treatment is now well accepted for a majority of breast cancers and is performed in around 70–80% of cases [1]. However, mastectomy remains indicated in at least 20% of patients. An important improvement resulted from the skin-sparing mastectomy technique, validated by several publications [2-11]. The preservation of the skin envelope has favoured the quality of the breast reconstruction, and breast reconstructive surgery has made important progress, providing more natural shape. However, despite the reconstruction, patients often deplore a feeling of mutilation. The nipple areola complex (NAC) 4. Kronowitz SJ, Robb GL: Breast reconstruction with postmas- tectomy radiation therapy: current issues. Plast Reconstr Surg 2004, 114:950-960. 5. Kronowitz SJ, Hunt KK, Kuerer HM, Babiera G, McNeese MD, Buchholz TA, Strom EA, Robb GL: Delayed-immediate breast reconstruction. Plast Reconstr Surg 2004, 113:1617-1628. 5. Kronowitz SJ, Hunt KK, Kuerer HM, Babiera G, McNeese MD, Buchholz TA, Strom EA, Robb GL: Delayed-immediate breast reconstruction. Plast Reconstr Surg 2004, 113:1617-1628. S7 Breast Cancer Research Vol 9 Suppl 1 VII Madrid Breast Cancer Conference Figure 1 (abstract S10) and in several cases because of the poor blood supply requiring the NAC removal. Fifty-one candidates were excluded because no radiotherapy was delivered, most often when the blood supply was poor and the risk of necrosis very high. Finally, 749 patients underwent a NSM, among whom 24 had a bilateral NSM. (a) The sterile collimator of the LINAC. (b) The gauze over the areola allows a more homogeneous distribution of the dose to the nipple and the areola. (c) Protective lead and aluminum disks. (d) Pectoralis major muscle. The mean patient age was 46 years (range 20–73 years). Seventy-five per cent of the patients have a mean follow-up of 18 months (range 1–61 months). The surgical technique has already been described [20]. Breast reconstruction was achieved with a definitive prosthesis in 689 cases (89%) and with an expander in 185 (24%). Only six patients underwent a reconstruction with an autologous transverse rectus abdominis myocutaneous and one with an autologous Latissimus dorsi. Once the glandular tissue has been removed and the frozen section of the retro areola tissue proved free of cancer, ELIOT was performed on the NAC area. The ELIOT technique has already been described [21-24]. Introduction C Materials and methods NAC total necrosis was observed in 26 cases out of 773 NSMs (3.3%). Partial necrosis was observed in 49 cases (6.3%). The NAC was removed in 36 cases (4.6%). The definitive histology of the 773 NSMs was invasive carcinoma in 63% and in situ carcinoma in 37%. In 53 cases (6%), the definitive histology of the retro areola tissue returned positive for carcinoma (invasive in 35 cases and in situ in 18 cases). Thirteen infections (1.6%) were observed in the immediate postoperative period; 32 prostheses were removed (4.1%). A capsulo- tomy has been required in 137 cases (17.7%). Figure 2 (abstract S10) Case 1. (a) Preoperative drawing and result. (b) and (c) Final result with contralateral augmentation. Case 1. (a) Preoperative drawing and result. (b) and (c) Final result with contralateral augmentation. S8 Available online http://breast-cancer-research.com/supplements/9/S1 Figure 3 (abstract S10) C 2 ( ) Ri h NSM (b) B i i h TRAM fl Figure 4 (abstract S10) Case 3. (a) Left nipple sparing mastectomy, right prophylactic mastectomy. (b) Bilateral reconstruction with prosthesis. Figure 4 (abstract S10) Figure 3 (abstract S10) Figure 4 (abstract S10) Case 3. (a) Left nipple sparing mastectomy, right prophylactic mastectomy. (b) Bilateral reconstruction with prosthesis. Case 2. (a) Right NSM. (b) Breast reconstruction with TRAM flap. Case 3. (a) Left nipple sparing mastectomy, right prophylactic mastectomy. (b) Bilateral reconstruction with prosthesis. Case 2. (a) Right NSM. (b) Breast reconstruction with TRAM flap. longer follow-up is required to prove the efficacy of the intraoperative radiotherapy. The dose of ELIOT chosen to reduce the recurrence risk could be questioned [20-24]. The single application of 16 Gy corresponds to the classic fractioned radiotherapy of 45 Gy for tumour cells and of 70–80 Gy for late responding normal tissue. This dose should be sufficient to sterilise more than 90% of the residual cancer cells, and to obtain an acceptable risk (less than 5%) of severe late complications (necrosis). However, the percentage of patients with positive persistent carcinoma behind the NAC in our series justifies ELIOT, although other authors advocate the possibility of NAC preservation without ELIOT [13,26-29]. cases: 7/10. The global result was rated by the patient as 8/10 and also as 8/10 by the surgeon (see cases 1–3, Figures 2–4). Cancer results cases: 7/10. The global result was rated by the patient as 8/10 and also as 8/10 by the surgeon (see cases 1–3, Figures 2–4). Cancer results cases: 7/10. The global result was rated by the patient as 8/10 and also as 8/10 by the surgeon (see cases 1–3, Figures 2–4). Cancer results Despite the frozen section performed underneath the NAC to eliminate the cases with positive results, the final histology of the retro areola tissue was positive in 53 (6.8%) cases, among which 35 were invasive carcinomas (66%). However, the NAC has been preserved in most cases when the final examination returned positive, taking into account the intraoperative radiotherapy to avoid recurrences. The secondary removal was due to local necrosis. Twelve local recurrences were observed. Discussion g g 5. Ho CM, Mak CK, Lau Y, et al.: Skin involvement in invasive breast carcinoma: safety of skin sparing-mastectomy. Ann Surg Oncol 2003, 10:102-107. 5. Ho CM, Mak CK, Lau Y, et al.: Skin involvement in invasive breast carcinoma: safety of skin sparing-mastectomy. Ann Surg Oncol 2003, 10:102-107. Our study on 773 skin-sparing mastectomies performed at the European Institute of Oncology confirms the feasibility of the procedure, with a majority of good results after the preservation of the NAC. However, a partial or total NAC necrosis due to insufficient blood supply was observed in 9.7% of the cases. Moreover, 6.6% of the final retro areola histology return positive while it was observed negative at the frozen section. Such results could be improved with a better selection of the NSM, if we cancel the cases requiring an extensive retro areola tissue removal and if we change the surgical indications of the reconstruction in cases of large breast, more often using autologous tissue reconstruction. Concerning the retro areola free margins, we should verify with more accuracy the clinical proximity between the tumour and the NAC. g 6. Carlson GW, Styblo TM, Lyles RH, et al.: Local recurrence after skin-sparing mastectomy: tumor biology or surgical conser- vatism? Ann Surg Oncol 2003, 10:108-112. 7. Simmons RM, Fish SK, Gayle L, et al.: Local and distant recur- rence rates in skin-sparing mastectomy compared with non- skin-sparing mastectomies. Ann Surg Oncol 1999, 6:676-678. 8. Slavin SA, Schnitt SJ, Duda RB, et al.: Skin-sparing mastectomy and immediate reconstruction: oncologic risks and aesthetic results in patients with early stage breast cancer. Plast Recon- str Surg 1998, 102:49-62. 9. Kroll SS, Khoo A, Singletary SE, et al.: Local recurrence risk after skin-sparing and conventional mastectomy: a 6-year follow-up. Plast Reconstr Surg 1999, 104:421-425. A partial or global return of the NAC sensitivity was present in only 30% of the patients. As expected, the return is incomplete and takes months to happen [25]. The risk of radio dystrophy is low with such intraoperative radiotherapy at the level of 16 Gy. Seven per cent of the patients had a severe or moderate radio dystrophy, but a mild pigmentation was observed in around one-third of the women with at least 1 year follow-up. 10. Medina-Franco H, Vasconez LO, Fix RJ, et al.: Factors associ- ated with local recurrence after skin-sparing mastectomy and immediate breast reconstruction for invasive breast cancer. References 1. Veronesi U, Volterrani F, Luini A, et al.: Quadrantectomy versus lumpectomy for small size breast cancer. Eur J Cancer 1990, 26:671-673. Twenty-three cases of distant metastases and three deaths were observed in our series. Twenty-three cases of distant metastases and three deaths were observed in our series. 2. Carlson GW, Losken A, Moore B, et al.: Results of immediate breast reconstruction after skin-sparing mastectomy. Ann Plast Surg 2001, 46:222-228. The psychological study was completed for 159 patients who had at least 1 year follow-up and who answered our questionnaire. Ninety- seven per cent of the patients interviewed declare being very satisfied with the appearance of the breast after the NSM. No patient regretted having undergone reconstructive surgery, and 91.5% agreed with the mutilation being decreased by having preserved the NAC. Similarly, 93% of women responded that conserving the nipple aided in facing illness, and only 1.6% expressed a total dissatisfaction. 3. Carlson GW, Bostwick J, Styblo TM, et al.: Skin-sparing mastec- tomy. Oncologic and reconstructive considerations. Ann Surg 1997, 225:570-575. 4. Peyser PM, Abel JA, Straker VF, et al.: Ultra conservative skin- sparing ‘keyhole’ mastectomy and immediate breast and areola reconstruction. Ann R Coll Surg Engl 2000, 82:227-235. Figure 2 (abstract S10) Two were located on the NAC and the 10 others at a distance from the radiated area, usually in the same area of the tumour location. Both recurrences on the NAC were a DCIS associated with a Paget disease on the nipple. No recurrence on the NAC was observed in the group of patients with persistent positive histology. Finally, the primary goal of the NSM is the psychological improvement of patients requiring a mastectomy. The preliminary results of the questionnaire report a high level of satisfaction and confirm the psychological importance of NAC conservation. R Orecchia1,2 1Department of Radio-Oncology, European Institute of Oncology, Milan, Italy; 2University of Milan School of Medicine, Milan, Italy g 14. Lambert PA, Kolm P, Perry RR: Parameters that predict nipple involvement in breast cancer. J Am Coll Surg 2000, 191:354- 359. Over the past few years an increasing number of papers have appeared in peer-reviewed journals detailing various partial breast irradiation (PBI) approaches, utilizing high-precision external beam radiation therapy, single-dose intraoperative radiation therapy or brachytherapy. The concept of PBI has been associated with the use of accelerated schedules of fractionation. More recently, several preliminary clinical reports of retrospective series and the 5-year results of the only randomized study from Budapest using brachytherapy have produced significant discussion and confirmed the widespread interest toward PBI. The comparison between the current standard with early data coming from PBI techniques poses a dilemma as to when preliminary results are sufficiently mature to consider a new treatment approach as safe. 15. Gajdos C, Tartter PI, Bleiweiss IJ: Subareolar breast cancers. Am J Surg 2000, 180:167-170. 16. Vyas JJ, Chinoy RF, Vaidya JS: Prediction of nipple and areola involvement in breast cancer. Eur J Surg Oncol 1998, 24:15- 16. 17. Laronga C, Kemp B, Johnston D, Robb GL, Singletary SE: The incidence of occult nipple-areola complex involvement in breast cancer patients receiving a skin sparing mastectomy. Ann Surg Oncol 1999, 6:609-613. 18. Gerber B, Krause A, Reimer T, et al.: Skin-sparing mastectomy with conservation of the nipple-areola complex and autolo- gous reconstruction is an oncologically safe procedure. Ann Surg 2003, 238:120-127. PBI allows reducing the radiation field to only the initially involved quadrant of the breast and significantly shortens the duration of radiation therapy. This may represent the possibility of overcoming constraints such as accessibility to the radiation therapy centres, and the socioeconomic impact on the working life and on the personal habits of the patient. PBI seems to have a positive impact on patients’ quality of life. Another important advantage is the avoidance of interactions with systemic therapy that may determine delays in the initiation or in the carrying out of the conventional treatment. g 19. Merrick HW, Battle JA, Padgett BJ, Dobelbower RR: ELIOT for early breast cancer: a report on long-term results. Front Radiat Ther Oncol 1997, 31:126-130. 20. S11 S11 12. Wellisch DK, Schain WS, Noone RB, Little JW 3rd: The psycho- logical contribution of nipple addition in breast reconstruction. Plast Reconstr Surg 1987, 80:699-704. Partial breast irradiation and intraoperative radiotherapy O 1 2 13. Cense HA, Rutgers EJ, Lopes-Cardozo M, Van Lanschot JJ: Nipple sparing mastectomy in breast cancer: a viable option? Eur J Surg Oncol 2001, 27:521-526. Discussion Ann Surg 2002, 235:814-819. 11. Beer GM, Varga Z, Budi S, et al.: Incidence of the superficial fascia and its relevance in skin-sparing mastectomy. Cancer 2002, 94:1619-1625. Two in situ local recurrences were observed on the preserved NAC, which is reassuring although the median follow-up is only 18 months. A Two in situ local recurrences were observed on the preserved NAC, which is reassuring although the median follow-up is only 18 months. A S9 Breast Cancer Research Vol 9 Suppl 1 VII Madrid Breast Cancer Conference R Orecchia1,2 Petit JY, Veronesi U, Orecchia R, et al.: The nipple-sparing mas- tectomy: early results of a feasibility study of a new applica- tion of perioperative radiotherapy (ELIOT) in the treatment of breast cancer when mastectomy is indicated. Tumori 2003, 89: 288-291. 21. DuBois J-B, Hay M, Gely S, Saint-Aubert B, Rouanet P, Pujol H: ELIOT in breast carcinomas. Front Radiat Ther Oncol 1997, 31: 131-137. These possible benefits must be balanced with the potential risk of recurrence within the untreated tissue in the breast receiving PBI as well as the unknown long-term cosmetic results with the accelerated techniques. Significant practical considerations also include the choice of technique, interstitial brachytherapy (high dose-rate versus low dose- rate), balloon-based brachytherapy (MammoSite®), external beam (3D- conformal versus intensity modulated radiation therapy), and intra- operative irradiation (electrons versus low-energy X-ray device). The choice of the technique has an impact on the schedules and dose-rate that can be used, on the volume that can be irradiated and on the dose homogeneity achievable. 22. Battle JA, DuBois J-B, Merrick HW, Dobelbower RR: ELIOT for breast cancer. In Current Clinical Oncology: Intraoperative Irradi- ation: Techniques and Results. Edited by Gunderson LL, et al. New York: Humana Press, Inc.: 1999:521-526. 23. Veronesi U, Orecchia R, Luini A, et al.: Focalised intraoperative irradiation after conservative surgery for early stage breast cancer. Breast 2001, 10(Suppl 3):84-89. 24. Veronesi U, Orecchia R, Luini A, et al.: A preliminary report of intraoperative radiotherapy (ELIOT) in limited-stage breast cancers that are conservatively treated. Eur J Cancer 2001, 37:2178-2183. Patient selection also remains uncertain, including questions regarding age exclusions, applicability with various primary tumour sizes, hystopathological features, the tumour-free margin and amount of tumour near the margins, and negative (including micrometastasis) or positive axillary lymph node status (up to three or more). 25. Benediktsson KP, Perbeck L, Geigant E, Solders G: Touch sen- sibility in the breast after subcutaneous mastectomy and immediate reconstruction with a prosthesis. Br J Plast Surg 1997, 50:443-449. For this reason, data coming from the multicentric or unicentric large phase III ongoing trials in the United States and Europe comparing standard irradiation with the different PBI schedules and techniques will hopefully support the movement of PBI into routine clinical practice. Issues of patient selection, target volume definition, total dose, fractionation, and the quality assurance programme should be addressed and defined from analysing the results of such randomized trials. q References References Available online http://breast-cancer-research.com/supplements/9/S1 SYMPOSIUM III Basal-like breast cancer has some unique characteristics. It is the type of breast cancer that BRCA1 mutation carriers generally develop, although most basal-like breast cancers are sporadic. It comprises approximately 15% of all breast cancers, but is overrepresented among young African- American women who develop breast cancer, in whom it comprises 39%. Although it does not occur at higher stages than other breast cancer subtypes, it is usually high grade and highly proliferative, which may explain the poor prognosis associated with this subtype in several series. Controversies in Clinical Practice (Part I) Controversies in Clinical Practice (Part I) PF Conte, V Guarneri, C Bengala 2. Colleoni M, Gelber S, Goldhirsch A, Aebi S, Castiglione-Gertsch M, Price KN, Coates AS, Gelber RSP: International Breast cancer Study Group: Tamoxifen after adjuvant chemotherapy for premenopausal women with lymph node positive breast cancer. J Clin Oncol 2006, 24:1332-1341. Division of Medical Oncology, Department of Oncology and Hematology, University Hospital, University of Modena and Reggio Emilia, Modena, Italy Breast Cancer Research 2007, 9(Suppl 1):S14 (doi: 10.1186/bcr1697) Metastatic breast cancer patients represent a very heterogeneous population and several factors are important for therapeutic decision: patient characteristics including age, comorbidities, and performance status, tumor biological characteristics, site and extension of metastatic spread, prior adjuvant therapies and disease-free interval. In the past years, adjuvant treatment has been rapidly changing as new cytotoxics, new endocrine agents and targeted therapies are becoming the mainstay of treatment [1]. For endocrine-sensitive breast cancer, several large randomized trials have shown that third-generation aromatase inhibitors, can reduce the risk of relapse of early breast cancer in comparison with tamoxifen. Nowadays, aromatase inhibitors administered up front, as a switch after 2–3 years of tamoxifen or as extended treatment after 5 years of tamoxifen represent the standard treatment in postmenopausal women. As anthracycline and taxanes are the most active cytotoxic agents in breast cancer, anthracycline/taxane- containing regimens are becoming the mainstay of adjuvant chemotherapy. Finally, the growing understanding of the biology of breast cancer cells led to identification of key molecular points that represent possible therapeutic targets. Trastuzumab, the monoclonal antibody against the HER-2 receptor, is approved for the treatment of high-risk early breast cancer overexpressing HER-2 and represents the standard treatment in these patients. With increasing use of very active drugs in the adjuvant setting, there is a greater need for effective 3. Goldstein LJ: Controversies in adjuvant endocrine treatment of premenopausal women. Clin Breast Cancer 2006, 6:S36-S40. 4. Kaufmann M, Jonat W, Blamey R, Cuzick J, Namer M, Fogelman I, de Haes JC, Schumacher M, Sauerbrei W, Zoladex Early Breast Cancer Research Association (ZEBRA) Trialists Group: Survival analyses from the ZEBRA study. Goserelin (Zoladex) versus CMF in premenopausal women with node positive breast cancer. Eur J Cancer 2003, 39:1711-1717. A Howell CRUK Department of Medical Oncology, University of Manchester, Christie Hospital NHS Foundation Trust, Manchester, UK CRUK Department of Medical Oncology, University of Manchester, Christie Hospital NHS Foundation Trust, Manchester, UK Breast Cancer Research 2007, 9(Suppl 1):S12 (doi: 10.1186/bcr1695) The increased risk of death from cancer in young women attests to its aggressive nature and a greater likelihood of being oestrogen receptor (ER)-negative. Approximately 60% of tumours are ER-positive, however, and thus endocrine therapy is an important consideration in this group. In some countries chemotherapy alone was used for many years until the importance of the additional therapeutic effect of amenorrhea became evident and later, after much scepticism, tamoxifen was shown to be effective in this group of women. Sadly this rather staccato history of treatment has left many questions concerning endocrine therapy in young women unanswered. For example, there have been at least nine studies that show ovarian suppression is equivalent for disease-free and overall survival to chemotherapy, showing equivalent therapeutic advantage. However, the correct design of their studies should have included a third arm of chemotherapy and ovarian ablation and some randomisation to investigate the role of additional tamoxifen. New trials such as SOFT, TEXT and PROMISE will help answer the question of optimal endocrine therapy ± chemotherapy in young women and will also help decide whether aromatase inhibitors are effective in the presence of ovarian suppression. Two further points are important to consider. One is that chemotherapy used alone in very young women with ER-positive disease is detrimental since they do not have chemotherapy-induced amenorrhoea. Secondly, the value of chemotherapy in strongly ER-positive disease is being increasingly questioned. Treatment of metastatic disease after current adjuvant approaches (taxanes, aromatase inhibitors, trastuzumab) 1. Brown RJ, Davidson NE: Adjuvant hormonal therapy for pre- menopausal women with breast cancer. Semin Oncol 2006, 33:657-663. 1. Brown RJ, Davidson NE: Adjuvant hormonal therapy for pre- menopausal women with breast cancer. Semin Oncol 2006, 33:657-663. PF Conte, V Guarneri, C Bengala Current status of adjuvant endocrine therapy for premenopausal patients with primary breast cancer A H ll p p p g yp Given the triple-negative status of basal-like breast cancer, it cannot be treated with ER-targeted or HER2-targeted therapies, so is primarily treated with chemotherapy. Fortunately, advances in adjuvant therapy appear to benefit ER-negative breast cancer even more than ER- positive breast cancer, and women with the basal-like subtype have similarly benefited. In addition, in cohorts of breast cancer treated with neoadjuvant chemotherapy, the pathologic complete response to anthracycline/taxane-based therapy was significantly higher among basal-like breast cancers than luminal breast cancers. The women with pathologic complete response have good outcome; the poor prognosis of basal-like breast cancer appears to relate to a particularly high risk of early distant relapse among those that had residual disease. Among the targeted agents for breast cancer, bevacizumab added to paclitaxel in a randomized phase III trial appeared to have similar benefit in the triple-negative subset as the other breast cancer phenotypes, suggest- ing effectiveness in basal-like breast cancer. Current investigations into other therapeutic options for this subtype include confirmation of effectiveness of VEGF targeting, examination of EGFR-targeted strategies, determining whether the association of basal-like breast cancer with BRCA1 mutations means that this DNA repair pathway is dysfunctional in all basal-like breast cancer, and determining whether specific chemotherapy agents have greater or lesser efficacy in this subtype. Future studies should also focus upon identifying whether there are particular genetic or environmental risk factors for basal-like breast cancer that differ from other subtypes, and whether there are metastatic or predictive signatures that can be identified within the basal-like subtype. R Orecchia1,2 Acknowledgements The authors thank the support of the American Italian Cancer Foundation and Associazione Italiana Ricerca sul Cancro to the research programme of the division. For this reason, data coming from the multicentric or unicentric large phase III ongoing trials in the United States and Europe comparing standard irradiation with the different PBI schedules and techniques will hopefully support the movement of PBI into routine clinical practice. Issues of patient selection, target volume definition, total dose, fractionation, and the quality assurance programme should be addressed and defined from analysing the results of such randomized trials. 26. Simmons RM, Brennan M, Christos P, et al.: Analysis of nipple/ areolar involvement with mastectomy: can areola be pre- served? Ann Surg Oncol 2002, 9:165-168. 27. Chagpar AB: Skin-sparing and nipple-sparing mastectomy: preoperative, intraoperative and postoperative considera- tions. Am Surg 2004, 70:425-432. Acknowledgements The authors thank the support of the American Italian Cancer Foundation and Associazione Italiana Ricerca sul Cancro to the research programme of the division. 28. Crowe JP Jr, Kim JA, Yetman R, Bambury J, Patrick RJ, Baynes D: Nipple-sparing mastectomy: technique and results of 54 pro- cedures. Arch Surg 2004, 139:148-150. g 29. Simmons Rm, Hollenbeck ST, Latrenta GS: Areola-sparing mas- tectomy with immediate breast reconstruction. Ann Plast Surg 2003, 51:547-551. S10 Breast Cancer Research Vol 9 Suppl 1 VII Madrid Breast Cancer Conference Breast Cancer Research Vol 9 Suppl 1 VII Madrid Breast Cancer Conference metastases. These observations have been made in a variety of tumor types. The optimal duration of bisphosphonate therapy has not been determined, and while the usual schedule of administration is monthly intravenous therapy for this indication, there are ongoing trials attempting to determine the most effective and safest schedule for patients with bone metastases. Similar studies are ongoing for patients with osteoporosis and osteopenia. Although bisphosphonates are very well tolerated, the last few years have brought a number of anecdotal reports of osteonecrosis of the jaw associated with bisphosphonate administration. More recently, systematic retrospective analyses of large databases have indicated a low but definite incidence of this complication, predominantly in patients receiving third-generation, potent bisphosphonates. These reports suggest the incidence to be in the range of 0.1–2.0%, varying with tumor type and research methodo- logy. There is insufficient information about prevention and manage- ment of this condition, although risk factors include poor oral hygiene, oral/dental interventions, increasing cumulative dose of bisphospho- nates and duration of therapy. therapy at the time of relapse [2]. In endocrine-sensitive breast cancer, sequencing data for anastrozole and tamoxifen have shown that tamoxifen is likely to be effective after progression on anastrozole. Moreover, in recent years Fulvestran, a selective ER downregulator acting as a pure antiestrogen agent, has been developed. Tamoxifen, Fulvestran or an aromatase inactivator are possible options after failure of adjuvant aromatase inhibitors. At present, it is not possible to define the optimal sequence of these endocrine agents. In endocrine-resistant disease, a treatment-free interval (TFI) >12 months after adjuvant chemotherapy has been shown to be an important factor for determining sensitivity to drug rechallenge. If the tumor has been exposed to an anthracycline and a taxane in adjuvant setting and the TFI is >12 months, the re-treatment with the same agents may be an option [3]. If the TFI is <12 months it is preferable to use a different agent. Capecitabine, gemcitabine and vinorelbine have demonstrated substantial activity in metastatic breast cancer. Finally, patients with HER2-positive tumors receiving adjuvant trastuzumab might be refractory (primary resistance) or might become resistant (secondary resistance) to trastuzumab. The mechanisms of primary and secondary trastuzumab resistance are not yet fully elucidated. Lapatinib, a new target agent that simultaneously inhibits both HER-2 and EGFR tyrosine kinases, has been shown to be active in trastuzumab-resistant metastatic breast cancer. References 1. Conte PF, Bengala C, Guarneri V: Controversies of chemother- apy for the treatment of metastatic breast cancer. Eur J Cancer 2007, Suppl 5:11-16. S16 3. Gennari A, Bruzzi P, Orlandini C, et al.: Activity of first line epiru- bicin and paclitaxel in metastatic breast cancer is indepen- dent of type of adjuvant therapy. Br J Cancer 2004, 90: 962-967. SYMPOSIUM IV Primary Systemic Therapy and Adjuvant Treatment Primary Systemic Therapy and Adjuvant Treatment 2. Conte PF, Guarneri V: The curability of breast cancer and the treatment of advanced disease. Eur J Nucl Med Mol Imaging 2004, 31(Suppl 1):S149-S161. S15 Memorial Sloan-Kettering Cancer Center, New York, NY, USA Breast Cancer Research 2007, 9(Suppl 1):S16 (doi: 10.1186/bcr1699) Breast cancer is an increasing global public health burden with more than 1 million new cases anticipated worldwide and more than 200,000 new cases anticipated in the United States in 2007. Screening and awareness have increased the detection of breast cancer at its early stages where it is most curable, but many such patients have microscopic metastatic disease even when diagnosed. The outcome for such patients can only be improved through the effective control of this undetected systemic disease, thus motivating adjuvant therapy. For patients with sufficient risk, based historically on nodal status, tumor size, specific histology, and hormone receptor status, chemotherapy can be the only option or a component of regimens including hormonal agents and anti-HER2 drugs. Genetic profiling is a recent addition to our risk stratification methods and can allow for more precise selection of chemotherapy. Historical chemotherapy regimens were largely empiric combinations of drugs with proven activity in advanced disease. More recent advances have been translational, with preclinical development of the taxanes leading to their testing in the adjuvant setting and mathematical modeling leading to improvements in the delivery of otherwise standard agents. Despite this mixture of empiricism and translational research, adjuvant chemotherapy is highly effective, particularly in selected subsets of patients, such as those with no expression of hormone receptors. Building on the demonstration of activity for the humanized monoclonal anti-HER2 antibody (trastuzumab) as adjuvant treatment, newer HER2 targeting drugs are in development and will be tested in this setting (lapatinib). For the majority of tumors lacking overexpression of HER2, planned research focuses on anti-angiogenic agents (bevacizumab). For many patients, an optimized chemotherapy plan (that is, dose- Breast Cancer Research Vol 9 Suppl 1 VII Madrid Breast Cancer Conference Moreover, several new agents targeting HER-2 are currently under clinical development. There are no data on rechallenge with trastuzumab in patients who had received this agent as adjuvant treatment and relapsed after a long TFI, and this issue is a new area of research. There is much interest and ongoing trials to define the role of bisphosphonates in the preventive treatment of primary breast cancer. Whether bisphosphonates can prevent osseous metastases or not, emerging information about their use in treatment-induced bone loss might lead to the incorporation of these agents into the combined modality management of primary breast cancer anyway. This makes the determination of therapeutic benefit, optimal dose and schedule of administration very important. Triple-negative (basal-like) breast cancer: a new entity LA C Hematology/Oncology Department, University of North Carolina, Chapel Hill, NC, USA Breast Cancer Research 2007, 9(Suppl 1):S13 (doi: 10.1186/bcr1696) Breast Cancer Research 2007, 9(Suppl 1):S13 (doi: 10.1186/bcr1696) , ( pp ) ( ) Unsupervised gene expression array profiling has provided biological evidence for the heterogeneity of breast cancer through the identification of intrinsic subtypes such as luminal A, luminal B, HER2+/ER– and the basal-like subtype. The basal-like subtype is characterized by low expression of both the ER and HER2 clusters of genes, so is typically ER-negative, PR-negative, and HER2-negative on clinical testing; for this reason, it is often referred to as ‘triple-negative’ breast cancer. , ( pp ) ( ) Unsupervised gene expression array profiling has provided biological evidence for the heterogeneity of breast cancer through the identification of intrinsic subtypes such as luminal A, luminal B, HER2+/ER– and the basal-like subtype. The basal-like subtype is characterized by low expression of both the ER and HER2 clusters of genes, so is typically ER-negative, PR-negative, and HER2-negative on clinical testing; for this reason, it is often referred to as ‘triple-negative’ breast cancer. S11 Breast Cancer Research Vol 9 Suppl 1 VII Madrid Breast Cancer Conference Preoperative systemic treatment for operable disease: biological correlates of response to neoadjuvant treatment GN Hortobagyi Methods The results of key randomised trials addressing these issues are reviewed. Results In the P24 trial for patients not suitable for conservative surgery, neoadjuvant letrozole was shown to achieve a significantly greater clinical overall response rate than tamoxifen (56% versus 36%, P < 0.001) and a significantly better incidence of breast-conserving surgery (45% versus 35%, P = 0.02). In the IMPACT trial, neoadjuvant anastrozole was compared with tamoxifen and the combination (three arms, equivalent to adjuvant ATAC). No significant difference in clinical overall response was seen between the three arms although breast- conserving surgery was considered feasibly significantly more often with anastrozole (46%) than with tamoxifen (22%) or with the combination (26%) (P = 0.03). In the PROACT trial, neoadjuvant anastrozole achieved a higher clinical overall response rate than tamoxifen (50% versus 40%) but this difference was not significant (in the endocrine therapy alone group – excluding patients also treated with chemotherapy). Breast-conserving surgery was achieved signifi- cantly more often with anastrozole (43% versus 31% P = 0 04) Results In the P24 trial for patients not suitable for conservative surgery, neoadjuvant letrozole was shown to achieve a significantly greater clinical overall response rate than tamoxifen (56% versus 36%, P < 0.001) and a significantly better incidence of breast-conserving surgery (45% versus 35%, P = 0.02). In the IMPACT trial, neoadjuvant anastrozole was compared with tamoxifen and the combination (three arms, equivalent to adjuvant ATAC). No significant difference in clinical overall response was seen between the three arms although breast- conserving surgery was considered feasibly significantly more often with anastrozole (46%) than with tamoxifen (22%) or with the combination (26%) (P = 0.03). In the PROACT trial, neoadjuvant anastrozole achieved a higher clinical overall response rate than tamoxifen (50% versus 40%) but this difference was not significant (in the endocrine therapy alone group – excluding patients also treated with chemotherapy). Breast-conserving surgery was achieved signifi- cantly more often with anastrozole (43% versus 31%, P = 0.04). The optimum duration of neoadjuvant endocrine therapy prior to surgery has not been established but circumstantial evidence suggests that it is at least 4 months, and probably longer. In the P24 trial and the IMPACT trial, patients with higher levels of ER expression had a significantly higher chance of clinical response. Likewise in both trials, aromatase inhibitors appeared strikingly more effective than tamoxifen in the subgroup of patients with HER2- positive, ER-positive tumours. EP Winer Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, USA Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, USA Breast Cancer Research 2007, 9(Suppl 1):S17 (doi: 10.1186/bcr1700) Approximately 75% of all breast cancer is hormone receptor positive and the majority arises in postmenopausal women. In the United States alone, there are over 100,000 women annually who are diagnosed with postmenopausal hormone-receptor-positive disease. Adjuvant hormonal therapy substantially decreases a woman’s risk of developing recurrent breast cancer and improves overall mortality. For almost two decades, a 5-year course of tamoxifen was the standard treatment offered to almost all women with hormone-receptor-positive disease. Over the past decade, the aromatase inhibitors have emerged as highly effective agents in the treatment of hormone-receptor-positive breast cancer. Multiple large trials including 25,000 women have assessed the role of the aromatase inhibitors in the adjuvant setting. These studies have demonstrated that the use of an aromatase inhibitor, either in place of tamoxifen or following a 2-year to 5-year course of tamoxifen, will reduce the risk of disease recurrence compared with the use of a 5-year course of tamoxifen alone. To date, these studies have not demonstrated a substantial improvement in overall survival, but the follow-up was relatively short. The optimal treatment approach has not been identified. Recently completed and ongoing trials are comparing a 5-year course of an aromatase inhibitor with the use of sequential therapy. Given the natural history of hormone-receptor-positive breast cancer, there is also great interest in treatment strategies that extend beyond 5 years. The toxicity profile of the aromatase inhibitors differs from tamoxifen, and these differences need to be considered in making clinical decisions. Ultimately, it is probable that different treatment approaches will be appropriate for different women based on tumor biology, pharmacogenetic variability, and susceptibility to side effects. Neoadjuvant endocrine therapy: clinical and biological issues IE Smith The Royal Marsden Hospital and Institute of Cancer Research, London, UK The Royal Marsden Hospital and Institute of Cancer Research, London, UK Breast Cancer Research 2007, 9(Suppl 1):S19 (doi: 10.1186/bcr1702) Background Neoadjuvant endocrine therapy is being increasingly used with two aims: to downstage cancers and reduce the need for mastectomy; and to establish short-term clinical and/or biological surrogate endpoints predicting for long-term outcome. Present and future role of bisphosphonates in treatment GN Hortobagyi Department of Breast Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Breast Cancer Research 2007, 9(Suppl 1):S15 (doi: 10.1186/bcr1698) Bone metastases are part of a spectrum of conditions based on dysregulated osseous metabolism. Metastatic cancer cells activate osteoclasts, both directly, by producing and releasing local humoral factors that result in osteoclast activation, and by activating osteo- blasts, which in turn further affect osteoclast activity. Activated osteo- clasts, when uncoupled and unbalanced from osteoblast activity, result in net excess bone resorption, resulting in bone loss and eventually lytic or mixed metastatic lesions. Therefore, the osteoclast, and the humoral activation system that leads to osteoclast recruitment and activation have become the major targets for developing therapies active in bone metastases. Bisphosphonates are potent osteoclast inhibitors and interfere with recruitment of osteoclast precursors, activation and release of bone resorbing substances. Bisphosphonates represent the treatment of choice for osteoporosis. They might also have weak, but direct, antitumor effects. As single agents, bisphosphonates relieve pain, and reduce the skeletal complication rate associated with bone metastases. Prospective randomized trials have clearly demonstrated that when added to standard antitumor therapy, bisphosphonates reduce skeletal morbidity, fractures, pain and analgesic requirements, as well as the need for radiotherapy or orthopedic interventions. Bisphosphonates also increase the time to first skeletal event, thus extending the complication-free survival of patients with bone S12 Available online http://breast-cancer-research.com/supplements/9/S1 Available online http://breast-cancer-research.com/supplements/9/S1 Available online http://breast-cancer-research.com/supplements/9/S1 dense administration of anthracyclines and taxanes) combined with appropriate targeted agents is likely to offer even larger improvements in outcomes than those already achieved. widely accepted that the magnitude of benefit from chemotherapy is proportionately more modest for patients with ER+ tumors than for ER– tumors. This is dramatically expressed in preoperative trials, where the pathological complete remission (pCR) rate is fourfold to sixfold higher for ER– BC than for ER+ BC. Similar variation in the pCR rate is observed by grade. More recent reports have indicated that the pCR rate was very low for invasive lobular cancers (almost always ER+) compared with invasive ductal cancers. Paradoxically, although pCR identifies a group of patients with improved survival compared with patients who do not achieve pCR, patient groups with lower pCR rates (those with ER+ tumors, those with low grade or lobular cancer) have better overall survival than those who tend to have higher pCR rates. This observation emphasizes the importance of understanding the biological heterogeneity of BC, and parallels the observations made in lymphoma over the past several decades: high-grade lymphomas respond more readily to chemotherapy but have lower survival rates in early follow-up than low-grade lymphomas. Studies based on gene expression profiling in BC have confirmed the existence of at least three distinct forms of BC: ER+/HER2–, HER2+, and ‘triple-negative’. These three groups differ by much more than the individual gene (ER or HER2) expression, and their clinical course and responsiveness to different treatments is quite different too. Thus, HER2+ and triple- negative tumors achieve a high pCR rate (40–50%) with standard combinations, while ER+/HER2– tumors do not (pCR rate <10%). There are other ramifications to drug sensitivity, and, most importantly, gene profiling leads to the identification of potential new targets for therapeutic intervention. Validation of such novel targets and development of specific therapeutics might be the best legacy of neoadjuvant treatment. H Cortés-Funes Hospital Universitario 12 de Octubre, Madrid, Spain Hospital Universitario 12 de Octubre, Madrid, Spain Breast Cancer Research 2007, 9(Suppl 1):S22 (doi: 10.1186/bcr1705) Vascular endothelial growth factor (VEGF), a potent angiogenic factor, has been reported to be associated with a poor prognosis in primary breast cancer and other solid tumours [1]. VEGF was observed to be one of the most important mediators of tumour angiogenesis in human breast cancer and was the only one linked to poor relapse-free survival [2]. VEGF affects tumour growth and metastasis in many forms of breast cancer, including invasive/noninvasive, node-negative/node-positive, inflammatory and metastatic disease. Also, VEGF expression has been observed across a number of disease-related variables, including hormonal and HER-2 status. Hospital Universitario 12 de Octubre, Madrid, Spain Breast Cancer Research 2007, 9(Suppl 1):S22 (doi: 10.1186/bcr1705) Vascular endothelial growth factor (VEGF), a potent angiogenic factor, has been reported to be associated with a poor prognosis in primary breast cancer and other solid tumours [1]. VEGF was observed to be one of the most important mediators of tumour angiogenesis in human breast cancer and was the only one linked to poor relapse-free survival [2]. VEGF affects tumour growth and metastasis in many forms of breast cancer, including invasive/noninvasive, node-negative/node-positive, inflammatory and metastatic disease. Also, VEGF expression has been observed across a number of disease-related variables, including hormonal and HER-2 status. Specific issues relating to chemotherapy for MBC include the following. There is evidence from older trials supporting the principle that endocrine therapy should be used before chemotherapy in patients with nonlife-threatening hormone-receptor-positive disease. Patients whose primary is HER2-unknown should have this checked at the time of relapse to determine whether trastuzumab should be given in addition to chemotherapy. Ideally, patients with primary HER2- negative disease should have metastatic disease rechecked since there is evidence of HER2-positive conversion in up to 25% of such patients. The choice of combination versus single-agent sequential chemotherapy is a complex one. The weight of current evidence suggests no significant survival difference with the combination, and this may be associated with more toxicity. There is no absolute answer here; choice depends on the nature of the drugs being used. A complex relationship between oestrogen, the oestrogen-receptor subtypes, and VEGF has been seen in preclinical breast cancer models. Oestrogen modulates VEGF expression at the gene transcriptional level [3]. Advances in Metastatic Disease Advances in Metastatic Disease Advances in Metastatic Disease SYMPOSIUM VI Targeted Therapy: Present and Future Targeted Therapy: Present and Future Conclusion These results suggest that neoadjuvant endocrine therapy is an effective clinical strategy to reduce the need for mastectomy in postmenopausal women with large hormone receptor-positive breast cancer. Short-term changes in molecular markers may be superior to clinical response in predicting long-term outcome, and this hypothesis is being further tested in a large prospective randomised trial. Targeted Therapy: Present and Future Preoperative systemic treatment for operable disease: biological correlates of response to neoadjuvant treatment GN Hortobagyi Department of Breast Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Breast Cancer Research 2007, 9(Suppl 1):S18 (doi: 10.1186/bcr1701) Experiments on preclinical models have suggested that preoperative introduction of chemotherapy of endocrine therapy can improve survival by abrogating a postsurgical growth spurt and by addressing micrometastatic disease at an earlier stage than when given post- operatively. Clinical trials have not confirmed this hypothesis, although they have demonstrated that preoperative systemic therapy reduced the overall tumor burden, expanded the indications for breast- conserving surgery to patients with more advanced disease, and provided an in vivo assessment of response that facilitated the safe and effective administration of systemic treatment. Preoperative administration of systemic therapy also provides an improved investigational model, since sequential monitoring with the primary tumor in situ offers the opportunity for multiple biopsies to monitor the biological effects of treatment. Large randomized trials have shown preoperative chemotherapy to be at least equivalent in disease-free and overall survival to the same chemotherapy administered postoperatively. Emerging data suggest, however, that the effects of systemic therapy in general, and preoperative therapy in particular, vary with different subclasses of breast cancer (BC). The first such observations were based on the estrogen receptor (ER). It is now S13 Breast Cancer Research Vol 9 Suppl 1 VII Madrid Breast Cancer Conference reast Cancer Research Vol 9 Suppl 1 VII Madrid Breast Cancer Conference Multiple lines of chemotherapy are now available for MBC. Current evidence suggests that it is only worth proceeding to third-line treatment if there has been a response to first-line and/or second-line therapy. New drug trials are appropriate for fit patients who have failed two lines of conventional therapy, and sometimes earlier than this in patients with nonlife-threatening disease. In the IMPACT trial, the change in proliferation rates after 2 weeks of treatment as measured by Ki67 was significantly greater for anastrozole than for tamoxifen or the combination, reflecting results in the adjuvant ATAC trial, and suggesting that Ki67 might be a better marker for long-term outcome than clinical response. Likewise, long- term follow-up of the IMPACT trial showed that the absolute level of Ki67 suppression after 2 weeks of neoadjuvant endocrine therapy significantly correlated with long-term disease-free survival outcome, and more so than baseline pre-treatment Ki67. Changes in expression of >2,800 genes were observed after 2 weeks of aromatase inhibitor therapy. Epigenetics of breast cancer M Esteller Cancer Epigenetics Laboratory, Spanish National Cancer Centre (CNIO), Madrid, Spain Cancer Epigenetics Laboratory, Spanish National Cancer Centre (CNIO), Madrid, Spain Breast Cancer Research 2007, 9(Suppl 1):S21 (doi: 10.1186/bcr1704) In breast cancer there is a global 40% loss of methylated cytosine, but DNA hypermethylation indicates which genes are turned off in tumors and tumor cell lines. Every tumor type has its own pattern of hyper- methylation. Genes important in familial breast cancer are also epigenetically silenced. In sporatic tumors, BRCA1 expression was wiped out by a combination of loss of heterozygosity (gene deletion) and epigenetic silencing (hypermethylation). In familial BRCA1 tumors, hypermethylation occasionally serves as an alternate mechanism for the ‘second hit’, when there was not loss of heterozygosity. Other known cancer genes are hypermethylated in breast cancers, such as those that affect cell cycle (p16 INK4a), and hormone receptors (ER, PR). New epigenomic approaches revealed the novel importance of potential tumor suppressor genes, such as the prolactin receptor and WRN, the Werner syndrome gene associated with premature aging and increased cancer risk, which is silenced in a subset of breast cancers. In addition to direct DNA hypermethylation, modification of histones is another epigenetic mechanism with implications in breast cancer. Overall, there is global loss of monomethylation and trimethy- lation of histone H4 in cancer. New epigenetic drugs targeting DNA methylation and histone deacetylation are in development for the treatment of breast cancer. Present and future roles of bevacizumab in breast cancer Symptom improvement has been shown to correlate with response rate in one trial but this important endpoint is not used often enough. Quality-of-life endpoints are appropriate in principle, but in reality many trials have shown a poor correlation with outcome, suggesting that this tool may be relatively insensitive in this context. Time to disease progression is a much used and important endpoint because it takes into account the fact that symptom relief and prolonged survival relate not just to patients achieving objective response, but also to those with minor response or stable disease. The Royal Marsden Hospital and Institute of Cancer Research, London, UK The Royal Marsden Hospital and Institute of Cancer Research, London, UK Many different cytotoxic agents are currently available for the treatment of metastatic breast cancer (MBC), and multiple factors determine the choice of treatment. These include previous adjuvant therapy, tumour characteristics (for example, molecular receptors, sites of relapse), patient characteristics (for example, performance status, age) and patient preference. p p The aims of treatment with chemotherapy for MBC are: to prolong survival; to relieve symptoms; to improve and maintain quality of life; and to delay disease progression. Only a small minority of MBC chemotherapy trials has shown a significant survival benefit, and to some extent this is explained by the multiple lines of potential further therapy following completion of the trial. Survival is therefore an uncertain endpoint in MBC trials. Available online http://breast-cancer-research.com/supplements/9/S1 Investigators will assign patients to a chemotherapy regimen, including specified regimens of anthracycline-based combina- tion chemotherapy, taxane (docetaxel or Abraxane™) every 3 weeks, or Xeloda. Patients are then randomized to receive bevacizumab (15 mg/kg every 2 weeks) or placebo. Another combination that is also being explored is bevacizumab plus trastuzumab. There is a strong rational for combining both antibodies as the different factors that bevacizumab and trastuzumab inhibit play important roles in tumour growth and progression. It has been shown that activation of the HER family of receptors induces VEGF expression and angiogenesis in cancer cell models [13]. These observations suggest that combined inhibition of VEGF and HER2 may enhance antitumour activity. y p p Bevacizumab has since been studied in several phase II trials with different chemotherapy regimens (docetaxel, vinorelbine), targeted agents (trastuzumab, erlotinib) and hormonal therapies (letrozol), showing encouraging results and acceptable toxicity profiles in these combinations. Nowadays, there is evidence for efficacy of bevacizumab from two phase III trials (AVF2119g and E2100). The first phase III trial published (AVF2119g) compared a combination of bevacizumab (15 mg/kg every 3 weeks plus capecitabine 2,500 mg/m2 daily for 2 weeks of a 3-week cycle) versus capecitabine alone. A total of 462 women with MBC previously treated with an anthracycline and a taxane were randomized to any of the study arms. The primary study endpoint was progression-free survival (PFS) and secondary endpoints included the OR rate, DR and OS. There was no significant difference in PFS between the two arms. The proportion of progression-free patients at 6 months was 33.8% for the Xeloda-alone arm and 33.0% for the Xeloda plus bevacizumab arm. Median PFS was 4.17 and 4.86 months, respectively (HR = 0.98, P = 0.857). Overall survival data for the Xeloda-alone arm (14.5 months) compared with the Xeloda plus bevacizumab arm (15.1 months) was also not significant. Although the addition of bevacizumab to capecitabine did improve PFS nor OS, there was a significant increase in the OR rate in the capecitabine plus bevacizumab arm compared with the capecitabine-only arm when the data were analysed both by investigators (30.2% versus 19.1%; P = 0.006) and by an independent review facility (19.8% versus 9.1%; P = 0.001). However, no significant differences in PFS or in OS were found between the two arms. Available online http://breast-cancer-research.com/supplements/9/S1 In multiple prospective and retrospective studies assessing blood and tumour samples in lung cancer patients, VEGF and angiogenesis have been associated with larger tumour size, increased rate of metastasis, recurrence of disease and poorer overall and disease-free survival [5-7]. Bevacizumab is a humanized recombinant antibody that prevents VEGF receptor binding, and inhibits angiogenesis and tumour growth. In patients receiving an irinotecan plus fluorouracil/leucovorin regimen for first-line treatment of metastatic colorectal cancer, the addition of bevacizumab significantly increased overall survival. In the second-line treatment of advanced colorectal cancer, patients who received bevacizumab in combination with a fluorouracil/leucovorin plus oxaliplatin (FOLFOX4) regimen had an overall survival time 2 months longer than that in patients receiving FOLFOX4. Another reason could be that patients were heavily pretreated, having received both an anthracycline and a taxane prior to entering the trial. received both an anthracycline and a taxane prior to entering the trial. The other phase III trial (E2100) is an ongoing open-label, phase III trial in which bevacizumab is being evaluated in combination with weekly paclitaxel as first-line therapy for MBC. Patients recruited to this trial are less heavily pretreated than patients in AVF2119g. A total of 722 patients have been randomized to one of two arms: paclitaxel 90 mg/m2 alone every week for 3 weeks followed by 1 week without treatment or paclitaxel plus bevacizumab 10 mg/kg every 2 weeks. No crossover is permitted in this trial. p The objectives are to compare PFS, the OR rate and OS between the arms. Preliminary results from the first preplanned interim analysis showed a significant increase in median PFS in patients receiving bevacizumab plus paclitaxel compared with paclitaxel alone (11.4 versus 6.11 months; P < 0.0001). This PFS result was obtained following 89% of the required events. The OR rate for all patients was 29.9% (bevacizumab/paclitaxel) versus 13.8% (paclitaxel) (P < 0.0001), and was 37.7% (bevacizumab/paclitaxel) versus 16% (paclitaxel) (P < 0.0001) for patients with measurable disease only. The survival data are still immature [12]. Bevacizumab has been tested in many tumours, proving efficacy when added to almost any chemotherapy regimen. It has been hypothesized that there are three main mechanisms by which bevacizumab exerts its antitumour activity: regression of existing tumour microvasculature, normalization of surviving mature tumour vasculature and inhibition of vessel regrowth and neovascularization as an effect with continuous treatment [8]. Available online http://breast-cancer-research.com/supplements/9/S1 Differences in the patient populations may help to explain the differences in results between the two phase III trials. Trial AVF2119 included a higher proportion of patients who had received chemo- therapy, including those who had received both an anthracycline and a taxane. A greater number of HER2-positive patients were also enrolled in AVF2119, most of whom had previously received trastuzumab. Based on preclinical findings that have shown the activity of bevacizumab in breast cancer, bevacizumab monotherapy was tested in a phase I/II dose escalation trial (AVF0776g) in metastatic breast cancer (MBC) with three doses (3, 10 and 20 mg/kg every 2 weeks) in 75 patients who had relapsed following at least one conventional chemotherapy regimen for metastatic disease. Combining data from the three arms, the overall response (OR) rate was 6.7%, the median duration of response (DR) was 5.6 months and overall survival (OS) was 10.2 months. Bevacizumab was well tolerated although four patients presented with headache with nausea and vomiting at the higher dose (dose limiting toxicity). Comparison of data from the three study arms indicated that a bevacizumab dose of 10 mg/kg every 2 weeks produced the best therapeutic ratio in this population of heavily pretreated patients [9]. Trial AVF2119 patients were generally more heavily pretreated than those enrolled in the E2100 trial. This may partly explain why the significant response rate improvement seen in this trial did not translate into a PFS benefit. Showing these results, it can be hypothesized that bevacizumab should be used as early as possible to obtain the maximum benefit. There are many other ongoing phase III trials that are testing the use of bevacizumab in combination with standard chemotherapy regimens. bevacizumab in combination with standard chemotherapy regimens. The AVADO trial is an ongoing randomized, double-blind, placebo- controlled, multicentre phase III trial that will randomize 705 patients to receive docetaxel at a dose of 100 mg/m2 every 3 weeks with either placebo, bevacizumab 7.5 mg/kg or bevacizumab 15 mg/kg. The primary trial endpoint will be PFS. Secondary endpoints will include the OR rate, duration of response, time to treatment failure, overall survival, safety and quality of life. Another trial exploring bevacizumab in combination with standard regimens is the RIBBON-1 study, a phase III trial to be conducted in countries worldwide that will investigate further combinations of bevacizumab and chemotherapy. The primary trial endpoint is PFS. H Cortés-Funes VEGF expression in breast cancer cells promotes oestrogen-independent tumour growth in ovariectomized mice and VEGF-stimulated neovascularization, and contributed to tumour growth in both oestrogen-treated and nonoestrogen-treated mice. Tumour growth was highest in VEGF-expressing mice treated with oestrogen [4]. S14 Available online http://breast-cancer-research.com/supplements/9/S1 The combination was found to be well tolerated, with no increase in the frequency or severity of capecitabine- related adverse events in the bevacizumab-containing arm [10]. In this study, although the addition of bevacizumab to capecitabine did not improve PFS (the primary endpoint), the OR rate doubled. y y The feasibility of combining bevacizumab with trastuzumab was evaluated in the phase I, open-label AVF2473s trial. A total of nine patients with HER2-positive advanced or MBC were treated in one of three cohorts. Data from this trial led to the recommendation of bevacizumab 10 mg/kg every 2 weeks for use in combination with trastuzumab 2 mg/kg weekly (after a loading dose of 4 mg/kg) in phase II trials. The maximum tolerated dose was not reached. Analyses of pharmacokinetic parameters showed that coadministration of bevacizumab and trastuzumab does not alter the pharmacokinetics of either agent, indicating no interaction between the two agents. The combination was well tolerated, with no grade 3/4 adverse events [14]. Recently presented at the San Antonio Breast Cancer Symposium 2006 have been the first efficacy data from an ongoing, There have been a number of reasons hypothesized that could explain these observations. First of all, it has been demonstrated that VEGF inhibition is more effective in earlier disease, since in advanced disease there are also other angiogenic factors that are overexpressed [11]. S15 Breast Cancer Research Vol 9 Suppl 1 VII Madrid Breast Cancer Conference in order to compare tumour angiogenesis in patients given bevacizumab and placebo. nonrandomized, open-label, uncontrolled, phase II study trial (TORI-B-03) that explores the combination of bevacizumab plus trastuzumab for the first-line treatment of Her2-positive locally recurrent, surgically unresectable, or metastatic breast cancer. The interim data of 37 patients showed that the combination of both targeted agents induces an overall response rate of 54% with an acceptable safety profile without inducing the typical chemotherapy- related side effects. Other trial objectives include investigation of the clinical benefit of adding bevacizumab to TAC in the neoadjuvant setting. Parameters to be evaluated are the clinical objective response rate (complete response plus partial response), pathologic complete response rate and rate of breast-conserving surgery. As wound-healing complications have previously been reported in bevacizumab clinical trials, the effect of bevacizumab on postsurgical wound-healing will also be investigated as well as the rate of cardiac heart failure. Available online http://breast-cancer-research.com/supplements/9/S1 There is currently running a phase III trial of bevacizumab, docetaxel and trastuzumab (AVEREL) in patients with previously untreated, HER2-positive MBC: 320 patients will be randomized to receive either docetaxel and trastuzumab or docetaxel, trastuzumab and bevacizumab until disease progression. Patients from the control arm will not be permitted to crossover to receive bevacizumab upon progression. The primary trial endpoint is PFS. Other endpoints include response rate, duration of response, OS and safety. The most successful use of antiangiogenic therapy has been predicted to be in the adjuvant treatment, and there is a clear biologic basis and rationale for exploring bevacizumab in this clinical setting. A large proportion of patients with breast cancer have been reported to have primary tumours that overexpress VEGF, and overexpression of VEGF is associated with increased rates of relapse [18]. The trials planned with bevacizumab in the adjuvant setting will confirm the efficacy of adding an antiangiogenic therapy to standard combinations. There is also evidence from phase II trials proving the efficacy of adding bevacizumab to other agents such as vinorelbine, erlotinib or letrozol. All this combinations have demonstrated encouraging results with acceptable toxicity profiles that warrant further exploration. The data from the trials currently ongoing will tell us the efficacy of combining bevacizumab with other drugs used in the metastatic setting, besides docetaxel or capecitabine, the exact role of bevacizumab in neoadjuvant and adjuvant settings, and perhaps it will be possible to identify which patients are most likely to benefit from VEGF-targeted therapies. Besides the use of bevacizumab in the metastatic setting, it has been hypothesized that the greater benefit could be obtained when used in earlier stages. The progression of breast cancer is accompanied by the production of a wide array of proangiogenic growth factors that promote and support tumour growth. When tumours are small they secrete VEGF, which acts as a paracrine factor to induce endothelial cell proliferation and blood vessel formation, mediating tumour progression. As the tumour develops further, additional factors are also secreted [15]. This evidence suggests that the inhibition of VEGF may be more beneficial in earlier stages, as neoadjuvant or adjuvant treatment. There are many trials, planned and ongoing, exploring bevacizumab in the neoadjuvant setting. An ongoing, phase II trial of bevacizumab in combination with docetaxel in the neoadjuvant setting (AVF2307s) is being conducted in patients with locally unresectable breast cancer with or without metastasis. References References 1. Foekens JA, et al.: Cancer Res 2001. 2. Relf M, et al.: Cancer Res 1997. 3. Buteau H, et al.: Cancer Res 2002. 4. Guo P, et al.: Cancer Res 2003. 5. Macchiarini P, et al.: Ann Thorac Surg 1994. 6. Lucchi M, et al.: Eur J Cardiothorac Surg 1997. 7. Yuan A, et al.: Int J Cancer Pred Oncol 2000. 8. Gerber H, et al.: Cancer Res 2005. 9. Cobleigh MA, et al.: Semin Oncol 2003. 10. Miller KD, et al.: J Clin Oncol 2005. 11. Relf M, et al.: Cancer Res 1997. 12. Miller KD, et al.: Breast Cancer Res Treat 2005. 13. Russell KS, et al.: Am J Physiol 1999. 14. Pegram M, et al.: Breast Cancer Res Treat 2004. 15. Relf M, et al.: Cancer Res 1997. 16. Lyons JA, et al.: J Clin Oncol 2006. 17. Lyons JA, et al.: J Clin Oncol 2006. 18. Linderholm BK, et al.: Clin Breast Cancer 2003. 1. Foekens JA, et al.: Cancer Res 2001. 5. Macchiarini P, et al.: Ann Thorac Surg 1994. 6. Lucchi M, et al.: Eur J Cardiothorac Surg 1997. 7. Yuan A, et al.: Int J Cancer Pred Oncol 2000. 8. Gerber H, et al.: Cancer Res 2005. 9. Cobleigh MA, et al.: Semin Oncol 2003. 10. Miller KD, et al.: J Clin Oncol 2005. 11. Relf M, et al.: Cancer Res 1997. 12. Miller KD, et al.: Breast Cancer Res Treat 2005. 13. Russell KS, et al.: Am J Physiol 1999. 14. Pegram M, et al.: Breast Cancer Res Treat 2004. 15. Relf M, et al.: Cancer Res 1997. 16. Lyons JA, et al.: J Clin Oncol 2006. 17. Lyons JA, et al.: J Clin Oncol 2006. y 18. Linderholm BK, et al.: Clin Breast Cancer 2003. Available online http://breast-cancer-research.com/supplements/9/S1 Patients receive bevacizumab 10 mg/kg every 2 weeks and docetaxel 35 mg/m2 weekly for the first 6 weeks of an 8-week cycle. After two cycles, patients with stable disease or response, undergo surgery and radiotherapy, followed by further chemotherapy (not containing bevacizumab). The study objectives are to evaluate the efficacy and safety of neoadjuvant bevacizumab with docetaxel in breast cancer patients. Preliminary efficacy data showed that bevacizumab plus docetaxel reduces tumour vascular permeability and microvessel density [16]. Combined response data for both study arms revealed seven complete responses (14.3%), 32 partial responses (65.3%), five patients with stable disease and a further five with progressive disease. This gave an OR rate of 79.6%, with no difference between the study arms. Both PFS and OS were 40 months in the docetaxel arm and neither had been reached in the docetaxel plus bevacizumab arm [17]. TORI-B-02 is a phase II neoadjuvant trial currently being conducted at the University of California, Los Angeles. A total of 90 patients are being enrolled into four different treatment arms, in order to compare two different bevacizumab doses with placebo: Arm 1: bevacizumab 7.5 mg/kg, followed by six cycles of bevacizumab 7.5 mg/kg in combination with TAC (docetaxel, doxorubicin and cyclophosphamide) every 3 weeks; Arm 2: placebo 7.5 mg/kg, followed by six cycles of placebo 7.5 mg/kg in combination with TAC every 3 weeks; Arm 3: bevacizumab 15 mg/kg, followed by six cycles of bevacizumab 15 mg/kg in combination with TAC every 3 weeks; and Arm 4: placebo 15 mg/kg, followed by six cycles of placebo 15 mg/kg in combination with TAC every 3 weeks. Then, 28–42 days after receiving chemotherapy, patients eligible for surgery will undergo resection. After either receiving surgery or completing chemotherapy, patients in Arms 1 and 3 will receive bevacizumab at their previous dose until disease progression. Patients in Arms 2 and 4 will not receive further therapy before disease progres- sion. The primary objectives of the TORI-B-02 trial are to evaluate the safety and toxicity of bevacizumab given as preoperative therapy to patients with stage II/III breast cancer in combination with the TAC regimen. Analysis of changes in HIF-1α levels will also be carried out, Prognostic factors for the node-negative breast cancers Prognostic factors for the node negative breast cancers B-W Park, J-H Kim, K-S Kim, KS Lee Yonsei University College of Medicine, Seoul, Korea Breast Cancer Research 2007, 9(Suppl 1):P1 (doi: 10.1186/bcr1707) B-W Park, J-H Kim, K-S Kim, KS Lee Yonsei University College of Medicine, Seoul, Korea Breast Cancer Research 2007, 9(Suppl 1):P1 (doi: 10.1186/bcr1707) B-W Park, J-H Kim, K-S Kim, KS Lee Yonsei University College of Medicine, Seoul, Korea Cancer Research 2007, 9(Suppl 1):P1 (doi: 10.1186/bcr1707) Objective The proportion of node-negative breast cancer patients has been increasing with improvement of diagnostic modalities and early detection. However, there is a 20–30% recurrence in node-negative breast cancer. To identify the prognostic factors for node-negative breast cancers, we studied the impact of many clinico-pathologic para- meters on the outcome of the node-negative breast cancer patients. Methods The data of 1,110 node-negative breast cancer patients who underwent curative surgery at the Severance Hospital, Yonsei University College of Medicine, were reviewed. The impact of many clinico-pathological parameters on the outcome was investigated. Univariate survival curves for disease-free survival and death were estimated using the Kaplan–Meier method: group differences in survival time were tested by the log-rank test. Multivariate Cox regres- sion analysis was performed to compare and identify independent prognostic factors. 1. Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL: Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science 1987, 235:177-182. 1. Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL: Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science 1987, 235:177-182. 2. Esteva FJ, Valero V, Booser D, Guerra LT, Murray JL, Pusztai L, Cristofanilli M, Arun B, Esmaeli B, Fritsche HA, et al.: Phase II study of weekly docetaxel and trastuzumab for patients with HER-2-overexpressing metastatic breast cancer. J Clin Oncol 2002, 20:1800-1808. Results The mean age was 47.2 years. The median follow-up was 88 months. Recurrence occurred in 161 patients; 64 patients with locoregional recurrences, 129 with systemic recurrences, and 32 with both. The 5-year overall survival rate was 93.3%. The rate of locoregional recurrence for a 10-year period was significantly lower in the mastectomy group compared with those in the breast conservation therapy group (94.7% versus 79.6%, P = 0.000). No other prognostic factors except age affected locoregional recurrence. FJ Esteva Novel therapies targeted against these aberrant molecular pathways are being studied in laboratory and clinical settings, and offer hope that the efficacy and duration of response to trastuzumab can be greatly improved. 10. 10. Geyer CE, Forster J, Lindquist D, Chan S, Romieu CG, Pienkowski T, Jagiello-Gruszfeld A, Crown J, Chan A, Kaufman B, et al.: Lapatinib plus capecitabine for HER2-positive advanced breast cancer. N Engl J Med 2006, 355:2733-2743. Lapatinib (Tykerb) is a small molecule tyrosine kinase inhibitor that targets EGFR and HER-2 [8]. Clinical responses were observed in patients with HER2-overexpressing metastatic breast cancer [9]. A recent phase III randomized trial showed that a combination of lapatinib and capecitabine (Xeloda) was superior to capecitabine alone [10], leading to the FDA approval of lapatinib in 2007. Clinical trials are ongoing to determine the role of lapatinib in the frontline setting for metastatic breast cancer in combination with trastuzumab and taxanes, as well as in the neoadjuvant and adjuvant settings. P1 Other novel strategies being tested in patients with HER2-over- expressing breast cancer include monoclonal antibodies targeting HER- 2 on different epitopes than trastuzumab (for example, pertuzumab), attaching toxins to trastuzumab (for example, trastuzumab-DM1), Hsp90 inhibitors that degrade the HER-2 protein (for example, 17-AAG), irreversible small molecule tyrosine kinase inhibitors (for example, HKI- 272), agents directed against IGF-IR and multitarget kinase inhibitors. Indirect approaches include immunotherapy and anti-angiogenic therapy. Clinical trials are evaluating the safety and efficacy of targeted biologic therapies, both as single agents and in combination with other biologics and in combination with standard chemotherapy and endocrine therapy. One of the main challenges is to match the right patient with the right drug(s) at the right time. Prognostic factors for the node-negative breast cancers There was less systemic recurrence in patients with age greater than 35, with histologic grade I, and with intraductal components greater than 20%. Thus, the 10-year distant relapse-free survival rates were 87.4% versus 79.8% (P = 0.039), 93.5% versus 85.5% (P = 0.024), and 94.4% versus 82.0% (P = 0.007), respectively. There was no statistical significance in the other prognostic factors that influence systemic recurrence. 3. Nahta R, Yu D, Hung MC, Hortobagyi GN, Esteva FJ: Mecha- nisms of disease: understanding resistance to HER2-targeted therapy in human breast cancer. Natl Clin Pract Oncol 2006, 3: 269-280. 4. Nahta R, Yuan LX, Zhang B, Kobayashi R, Esteva FJ: Insulin-like growth factor-I receptor/human epidermal growth factor receptor 2 heterodimerization contributes to trastuzumab resistance of breast cancer cells. Cancer Res 2005, 65:11118- 11128. 5. Nagata Y, Lan KH, Zhou X, Tan M, Esteva FJ, Sahin AA, Klos KS, Li P, Monia BP, Nguyen NT, et al.: PTEN activation contributes to tumor inhibition by trastuzumab, and loss of PTEN predicts trastuzumab resistance in patients. Cancer Cell 2004, 6:117- 127. Conclusion Patient age, histologic grade, and presence of intraductal component were identified as independent prognostic factors in node- negative breast cancer patients. 6. Nagy P, Friedlander E, Tanner M, Kapanen AI, Carraway KL, Isola J, Jovin TM: Decreased accessibility and lack of activation of ErbB2 in JIMT-1, a herceptin-resistant, MUC4-expressing breast cancer cell line. Cancer Res 2005, 65:473-482. FJ Esteva The University of Texas MD Anderson Cancer Center, Houston, TX, USA The University of Texas MD Anderson Cancer Center, Houston, TX, USA Breast Cancer Research 2007, 9(Suppl 1):S23 (doi: 10.1186/bcr1706) The HER2 tyrosine kinase receptor is overexpressed in approximately 25% of invasive breast cancers, and has been associated with a poor prognosis [1]. Trastuzumab (Herceptin) and lapatinib (Tykerb) are the two therapies currently approved by the FDA for the treatment of HER2- overexpressing metastatic breast cancer. The majority of patients that initially respond to trastuzumab monoclonal antibody-based therapy develop progressive disease within 1 year of treatment initiation [2]. Preclinical studies have indicated several molecular mechanisms that may contribute to the development of trastuzumab resistance [3]. Increased signaling via the phosphatidylinositol 3-kinase/protein kinase B (or Akt) pathway could contribute to trastuzumab resistance due to activation of multiple receptor pathways. These include HER2-related receptors or non-HER receptors such as the insulin-like growth factor-I receptor, which appears to cross-talk to HER2 in resistant cells [4]. Additionally, loss of function of the tumor suppressor PTEN, the negative regulator of Akt, results in heightened Akt signaling that leads S16 Available online http://breast-cancer-research.com/supplements/9/S1 Available online http://breast-cancer-research.com/supplements/9/S1 Phase I safety, pharmacokinetics, and clinical activity study of lapatinib (GW572016), a reversible dual inhibitor of epidermal growth factor receptor tyrosine kinases, in heavily pretreated patients with metastatic carcinomas. J Clin Oncol 2005, 23: 5305-5313. Phase I safety, pharmacokinetics, and clinical activity study of lapatinib (GW572016), a reversible dual inhibitor of epidermal growth factor receptor tyrosine kinases, in heavily pretreated patients with metastatic carcinomas. J Clin Oncol 2005, 23: 5305-5313. to decreased sensitivity to trastuzumab [5]. Decreased interaction between trastuzumab and its target receptor HER2, due to steric hindrance of the HER2 receptor by cell surface proteins, such as MUC- 4 [6], or by the presence of a truncated HER2 protein [7], may block inhibitory actions of trastuzumab. Novel therapies targeted against these aberrant molecular pathways are being studied in laboratory and clinical settings, and offer hope that the efficacy and duration of response to trastuzumab can be greatly improved. to decreased sensitivity to trastuzumab [5]. Decreased interaction between trastuzumab and its target receptor HER2, due to steric hindrance of the HER2 receptor by cell surface proteins, such as MUC- 4 [6], or by the presence of a truncated HER2 protein [7], may block inhibitory actions of trastuzumab. P2 7. Scaltriti M, Rojo F, Ocana A, Anido J, Guzman M, Cortes J, Di Cosimo S, Matias-Guiu X, Cajal S, Arribas J, Baselga J: Expres- sion of p95HER2, a truncated form of the HER2 receptor, and response to anti-HER2 therapies in breast cancer. J Natl Cancer Inst 2007, 99:628-638. Retreatment with trastuzumab in Her2-positive metastatic breast cancer patients: a clinical study F Carabantes-Ocón, E Saez-Lara, L Burgos-Garcia, E Villar-Chamorro, A Casaus-Hazañas, S Luna, C Martínez Unidad de Mama, Hospital Carlos Haya, Málaga, Spain Breast Cancer Research 2007, 9(Suppl 1):P2 (doi: 10.1186/bcr1708) Objective To study the benefit of trastuzumab in monotherapy or combined with different chemotherapeutic agents in the treatment for Her2+ metastatic breast cancer (MBC) patients after progression on prior trastuzumab therapy P4 Patients and methods The clinical evolution of patients with Her2+ MBC diagnosed by IHC3+/FISH+, and treated with trastuzumab in several lines for the metastatic disease, has been studied retro- spectively. A safety and efficacy study of bleomycin sulfate and electroporation in patients with metastatic or locally recurrent breast cancer Results The mean age of patients was 48.6 years (range 30–70). Of the 61 patients, BCS was achieved in 48 (79%) patients. On the core biopsy, four (6.5%) patients had grade I cancer, 26 (43%) had grade II cancer and 31 (51%) had grade III cancer. Final histology showed no invasive cancer in eight (13%) patients (seven DCIS, and complete pathological response in one patient). In the rest of the patients, four (6.5%) had grade I tumours, 26 (43%) had grade II tumours and 23 (38%) had grade III tumours. Overall, 14 patients (23%) showed a decrease in histological grade (see Table 1). Seven patients (11.5%) had a higher grade than the initial core. V Paramanov1, O Tyurin1, S Polenkov2, PM Goldfarb3 1Cherkassy Regional Oncologic Clinic, Cherkassy, Ukraine; 2Chernigiv Regional Oncology Centre, Chernigiv, Ukraine; 3Consulting Medical Director, Inovio Biomedical Corporation, San Diego, CA, USA Breast Cancer Research 2007, 9(Suppl 1):P5 (doi: 10.1186/bcr1711) V Paramanov1, O Tyurin1, S Polenkov2, PM Goldfarb3 1Cherkassy Regional Oncologic Clinic, Cherkassy, Ukraine; 2Chernigiv Regional Oncology Centre, Chernigiv, Ukraine; 3Consulting Medical Director, Inovio Biomedical Corporation, San Diego, CA, USA Breast Cancer Research 2007, 9(Suppl 1):P5 (doi: 10.1186/bcr1711) Objective The study evaluated bleomycin sulfate (BS) followed by electroporation (EP) treatment in patients with recurrent in-breast or chest-wall tumors. EP treatment following intralesional injection of BS has been studied in various cancers, and significantly increased destruction of cancer cells within the treatment field. The treatment has also been used as an intracellular delivery system for gene therapies and DNA vaccines. Table 1 (abstract P3) Initial histology (core) Final histology designation (post NC) No residual Grade n invasive cancer DCIS Grade I Grade II Grade III III 28 1 5 1 4 17 II 29 0 2 1 21 5 I 4 0 0 2 1 1 Table 1 (abstract P3) Retreatment with trastuzumab in Her2-positive metastatic breast cancer patients: a clinical study F C b O ó E S L L B G i p metastatic breast cancer patients: a clinical study F Carabantes-Ocón, E Saez-Lara, L Burgos-Garcia, E Villar-Chamorro, A Casaus-Hazañas, S Luna, C Martínez Unidad de Mama, Hospital Carlos Haya, Málaga, Spain Breast Cancer Research 2007, 9(Suppl 1):P2 (doi: 10.1186/bcr1708) 8. Nahta R, Yuan LX, Du Y, Esteva FJ: Lapatinib induces apoptosis in trastuzumab-resistant breast cancer cells: effects on insulin-like growth factor I signaling. Mol Cancer Ther 2007, 6: 667-674. Objective To study the benefit of trastuzumab in monotherapy or combined with different chemotherapeutic agents in the treatment for Her2+ metastatic breast cancer (MBC) patients after progression on prior trastuzumab therapy. 9. Burris HA, III, Hurwitz HI, Dees EC, Dowlati A, Blackwell KL, O’Neil B, Marcom PK, Ellis MJ, Overmoyer B, Jones SF, et al.: S17 Breast Cancer Research Vol 9 Suppl 1 VII Madrid Breast Cancer Conference reast Cancer Research Vol 9 Suppl 1 VII Madrid Breast Cancer Conference M Dani, T Tahmid, J McDonnell, V Jaffe Department of Surgery (Breast Firm), Chase Farm Hospital, London, UK Department of Surgery (Breast Firm), Chase Farm Hospital, London, UK Breast Cancer Research 2007, 9(Suppl 1):P4 (doi: 10.1186/bcr1710) Objective To demonstrate that saline instillation is a safe and simple procedure for volume replacement after wide local excision in breast surgery. Methods We performed a pilot study over a 12-month period at the Chase Breast Unit. One hundred and six patients who underwent wide local excision for breast cancer had saline instilled into the surgical cavity at the time of wound closure. This was to maintain the volume and shape of the breast after removal of significant amounts of tissue. We measured the volume instilled and monitored the wound and breast postoperatively at 1 week, 2 weeks and 3 months. As is our normal practice, all patients received perioperative antibiotics. Results The volume of fluid instilled varied between 30 and 180 ml. The weight of tissue removed was in the range of 12–116 g. The fluid was retained within the cavity. However, in one case the wide local excision cavity unexpectedly communicated with the axillary clearance cavity and all the fluid was evacuated spontaneously through the axillary suction drain with a resultant visible reduction in the volume of the breast. There were no complications of saline instillation. In particular, there was no early or late infection in any of the 106 patients. None of the patients reported any additional discomfort or pain. There were no visible abnormalities apart from a subjective enhancement in the shape and volume of the breast. This improvement in shape and volume was maintained for the entire length of assessment (3 months). Conclusion Saline instillation is a simple and safe method of replacing volume after removal of significant amounts of breast tissue. Surprisingly, the benefits seem to persist. We are now proceeding to fully evaluate this technique in a formal prospective trial. Methods We performed a pilot study over a 12-month period at the Chase Breast Unit. One hundred and six patients who underwent wide local excision for breast cancer had saline instilled into the surgical cavity at the time of wound closure. This was to maintain the volume and shape of the breast after removal of significant amounts of tissue. We measured the volume instilled and monitored the wound and breast postoperatively at 1 week, 2 weeks and 3 months. Do breast cancer tumours downsize as well as downgrade with neoadjuvant chemotherapy? M Dani1, J McDonnell1, S Karp2, V Jaffe1 1Department of Surgery (Breast Firm) and 2Department of Oncology, Chase Farm Hospital, London, UK Breast Cancer Research 2007, 9(Suppl 1):P3 (doi: 10.1186/bcr1709) Objective Neoadjuvant chemotherapy (NC) is increasingly being used for large primary breast carcinomas with the aim of improving breast- conservation surgery (BCS) rates. This study was conducted to assess the tumour response following NC. Methods In this retrospective study over a 4-year period, 61 women with large operable invasive breast cancers (T2–4 N0–2 M0), unsuitable for BCS, were consecutively treated with NC (5-FU, epirubicin, cyclophosphamide and Taxotere). Pathological response was monitored, comparing original core biopsy histology with final excisional histology. M Dani, T Tahmid, J McDonnell, V Jaffe We are now proceeding to fully evaluate this technique in a formal prospective trial. Saline instillation into the cavity after conservation surgery for breast cancer is a safe way of improving cosmesis y Results Twenty-four patients with Her2+ MBC were treated with several regimens containing trastuzumab alone or associated with chemotherapy and/or hormonotherapy. In the first line of treatment 12 RR (50%), 11 SD (45%), with a 95% clinical benefit, was observed. The patients received a second line obtaining 8 RR (33%), 15 SD (62%), with a clinical benefit of 95%. Seventeen patients were treated with a third line, 5 RR (29.4%) and 11 SD (64%) being observed, with a clinical benefit of 93%. Seven patients received a fourth line. In these, 2 RR and 4 SD, with a clinical benefit of 85%, were observed. Fifteen of the patients, with RE+, received hormonotherapy plus trastuzumab alone or with chemotherapy in one or more lines of treatment, obtaining 8 RR (53%) and 4 SD (26%), with a clinical benefit of 79%. M Dani, T Tahmid, J McDonnell, V Jaffe M Dani, T Tahmid, J McDonnell, V Jaffe As is our normal practice, all patients received perioperative antibiotics. Results The volume of fluid instilled varied between 30 and 180 ml. The weight of tissue removed was in the range of 12–116 g. The fluid was retained within the cavity. However, in one case the wide local excision cavity unexpectedly communicated with the axillary clearance cavity and all the fluid was evacuated spontaneously through the axillary suction drain with a resultant visible reduction in the volume of the breast. There were no complications of saline instillation. In particular, there was no early or late infection in any of the 106 patients. None of the patients reported any additional discomfort or pain. There were no visible abnormalities apart from a subjective enhancement in the shape and volume of the breast. This improvement in shape and volume was maintained for the entire length of assessment (3 months). Conclusion Saline instillation is a simple and safe method of replacing volume after removal of significant amounts of breast tissue. Surprisingly, the benefits seem to persist. We are now proceeding to fully evaluate this technique in a formal prospective trial. Conclusion The association of herceptin with chemotherapy and/or hormonotherapy demonstrates a very active treatment in patients with Her2+ MBC. The benefit seems to continue in patients who already have received treatment with trastuzumab even in more than one regimen. Results The volume of fluid instilled varied between 30 and 180 ml. The weight of tissue removed was in the range of 12–116 g. The fluid was retained within the cavity. However, in one case the wide local excision cavity unexpectedly communicated with the axillary clearance cavity and all the fluid was evacuated spontaneously through the axillary suction drain with a resultant visible reduction in the volume of the breast. There were no complications of saline instillation. In particular, there was no early or late infection in any of the 106 patients. None of the patients reported any additional discomfort or pain. There were no visible abnormalities apart from a subjective enhancement in the shape and volume of the breast. This improvement in shape and volume was maintained for the entire length of assessment (3 months). Conclusion Saline instillation is a simple and safe method of replacing volume after removal of significant amounts of breast tissue. Surprisingly, the benefits seem to persist. P7 Objective To evaluate the feasibility, acute complications and cosmetic results of partial breast cancer radiotherapy in initial tumors, with an electron beam, in patients submitted to conservative surgery and sentinel lymph-node dissection. Resveratrol downregulates acetyl-CoA carboxylase alpha and fatty acid synthase by activating AMP-activated protein kinase and suppressing the mammalian target of rapamycin signal pathway S Yoon, B-W Park, K-S Kim 1REH Oncology Center of Latvia, Riga, Latvia; 2University of Latvia, Riga, Latvia Breast Cancer Research 2007, 9(Suppl 1):P6 (doi: 10.1186/bcr1712) Breast Cancer Research 2007, 9(Suppl 1):P6 (doi: 10.1186/bcr1712) Objective To determine the predictive value of HER2 expression for the selection of adjuvant chemotherapy (antracycline or nonantra- cycline based) in patients with breast cancer. Yonsei University College of Medicine, Seoul, Korea Breast Cancer Research 2007, 9(Suppl 1):P8 (doi: 10.1186/bcr1714) Expression of HER2 is reported to be increased in approximately 30% of human breast carcinoma. Fatty acid synthase (FASN) was expressed higher in HER2-overexpressing breast cancer cells. Resveratrol (3,5,4′- trihydroxystilbene), a red-wine-derived polyphenol, has been shown to suppress cancer cell proliferation, and to interfere with the several signaling pathways and induce apoptosis. We investigated the effects of resveratrol on the expression of lipogenic enzymes in BT-474 cells, in which HER2 overexpresses cells. Resveratrol treatment to BT-474 cells resulted in inhibition of acetyl-CoA carboxylase alpha (ACCalpha) and FASN expression and a strong activation of AMP-activated protein kinase (AMPK), which could be mimicked by the changes caused by AICAR treatment or forced expression of constitutively active AMPK mutant. The decreased ACCalpha and FASN expressions by resveratrol were abolished by overexpression of dominant-negative AMPK mutant. The activation of AMPK was accompanied with the reduction of the mammalian target of rapamycin (mTOR), which plays a key role in the upregulation of ACCalpha and FASN expression in BT-474 cells. These results indicate that downregulation of HER2- mediated ACCalpha and FASN expression by resveratrol is regulated through suppressing the mTOR signaling pathway resulting from the activation of AMPK in breast cancer cells. y Methods Medical records of primary breast cancer patients treated at the Oncology Center of Latvia from January 2002 to August 2005 were retrospectively reviewed. One hundred and ninety-two patients with histopathologically confirmed breast cancer, who had undergone radical surgery and adjuvant chemotherapy (CT), were identified. Tumor characteristics were: ER+ in 62.5%, ER– in 37.5%, HER2-positive (3+ by IHC) in 30.2% (58 patients), HER2-negative (0, 1+) in 54.7% (105 patients); patients with score 2+ (15.1%, 29 patients) were excluded from analysis. Staging: I (20.8%), II (51%), III (29.2%); 43.8% patients were node negative, 56.2% were node positive. Median age at diagnosis was 52.4 (30–75) years. P9 g Conclusion In patients with HER2 overexpression, antracycline-based CT produced a significantly lower relapse rate when compared with nonantracycline-based CT. In HER2-negative patients there was no benefit from antracycline-based CT compared with nonantracycline- based CT. However, a longer follow-up period is needed to confirm the results. Partial breast irradiation with an electron beam for patients submitted to conservative surgery MAC Maia, MM Netto, MS Maciel, JC Donoso, JV Salvajoli, ACA Pellizzon, RC Fogaroli, KW Bocalletti, C Tanous Hospital AC Camargo, São Paulo – SP, Brazil Breast Cancer Research 2007, 9(Suppl 1):P9 (doi: 10.1186/bcr1715) Breast Cancer Research 2007, 9(Suppl 1):P9 (doi: 10.11 Axillary study before surgery in patients with breast cancer M Izquierdo, R Fabregas, J Feu, L Lopez Marin, B Navarro, C Ara, M Cusido, F Tresserra M Izquierdo, R Fabregas, J Feu, L Lopez Marin, B Navarro, C Ara, M Cusido, F Tresserra Resveratrol downregulates acetyl-CoA carboxylase alpha and fatty acid synthase by activating AMP-activated protein kinase and suppressing the mammalian target of rapamycin signal pathway S Yoon, B-W Park, K-S Kim g y Results Antracycline-based CT (FAC or FEC) was received by 62.9% patients in the HER2-negative group and 41.4% in the HER2-positive group, nonantracycline based (CMF) CT in 37.1% and 58.6% respec- tively. The median follow-up time was 21.3 (6.3–51.7) months. A total of 25.2% tumor recurrences were observed. Median time to relapse was 12.5 (1.4–34) months. In the HER2-positive group the relapse rate was 20.8% in patients receiving antracycline-based CT versus 29.4% in patients receiving nonantracycline-based CT (P = 0.09). In the HER2-negative group the relapse rate was 33.3% in patients receiving antracycline-based CT versus 15.8% in patients receiving nonantracycline-based CT (P = 0.2). The subgroup of patients with HER2-negative, ER-negative tumors had the worst prognosis (relapse rate 45%) regardless of the CT received. Table 1 (abstract P3) Methods Patients with histologically confirmed recurrent breast cancer following partial or complete mastectomy received BS intratumorally, 1 unit/cm3 tumor volume, followed by EP under general anesthesia. A CE-marked medical device (MedPulser®) and a six-needle array applicator were used to electroporate each tumor. Safety evaluations and monitoring for local tumor recurrence were performed periodically up to 24 weeks. Results Ten female patients with 11 tumors were enrolled. The mean age was 58.0 years (range 43.6–67.7). The mean tumor volume was 1.81 cm3 (0.01–6.60), the mean BS dose was 1.80 U (range 0.50–5.08), and the mean number of EP applications was 11 (range 7–16). No treatment-related serious adverse event was observed. Nonserious adverse events were unremarkable during the 30-day follow-up period. At 24 weeks, the complete response rate was 75% (6/8) among Conclusion In our series of patients receiving NC for breast cancer, there is not only a significant downsizing (permitting BCS) but also a trend of downgrading of the tumour, and this is seen particularly in poorly differentiated tumours. The higher grade on final histology compared with the core could be due to an unrepresentative core biopsy in large tumours prior to NC. S18 Available online http://breast-cancer-research.com/supplements/9/S1 Results In 54 patients (33.96%) FNA was positive. When we conducted axillary lymphadenectomy, 13 patients (24%) were found to have one positive node, seven patients (13%) two positive nodes, nine patients (16%) three positive nodes, and 25 patients (45%) more than three positive nodes. patients with evaluations for treated lesions; two patients were inevaluable for tumor response. Conclusion Treatment with this drug–device combination was well tolerated in these patients, a majority of whom were free of local disease at 24 weeks. These results suggest a promising potential for BS/EP as a new treatment for controlling local disease in patients with recurrent breast cancer. Conclusion Axillary study with ultrasound and FNA before surgery allows exclusion of a group of patients from sentinel node biopsy. M Izquierdo, R Fabregas, J Feu, L Lopez Marin, B Navarro, C Ara, M Cusido, F Tresserra Methods From 2005 to 2007 we evaluated 40 patients, median age 63 years, with T1–T2 (maximum 3 cm) breast cancer, clinically N0 axilla, submitted to conservative surgery and intraoperative radiotherapy with an electron beam. Institut Universitari Dexeus, Barcelona, Spain Breast Cancer Research 2007, 9(Suppl 1):P7 (doi: 10.1186/bcr1713) Breast Cancer Research 2007, 9(Suppl 1):P7 (doi: 10.1186/bcr1713) Objective Axillary study with ultrasound and cytological puncture with fine-needle aspirate (FNA) in patients with invasive breast cancer is a diagnostic method included in protocols. Results Of 40 patients, we had 73% T1 and 27% T2; 82% N0, 8% N1micro, and 10% N1. As for acute complications we had 2% infection and 5% fat necrosis. Regarding cosmetic results, 85% were good and excellent, 10% were fair and 5% were bad. Methods We studied 159 patients with invasive breast cancer with axillary ultrasound and cytological puncture with fine needle of suspicious nodes before surgery. Suspicious nodes were those with at least one of the following signs: long to short axis ratio less than 1.5, absence of hilius and cortical disruption. If the results were compatible with metastasis, then we performed axillary lymphadenectomy; if it was found to be benign, then we conducted sentinel node biopsy. Conclusion This is a quite feasible method for partial breast irradiation, with satisfactory results in terms of acute complications and cosmetic results. For local control and late complications we still have to wait longer and wait for the reports of the open trials. It also should be emphasized that these patients should be treated only in a study protocol. S19 Breast Cancer Research Vol 9 Suppl 1 VII Madrid Breast Cancer Conference S Menjón-Beltrán, R Olivencia, E Gonzalez S Menjón-Beltrán, R Olivencia, E Gonzalez Unidad de Ginecología Oncológica, Hospital Universitario Virgen de las Nieves, Granada, Spain Methods From January 2000 to March 2007, 356 breast cancer patients were operated on in the Department of General Surgery of General Hospital ‘Sveti Vracevi’ in Bijeljina. We selected 173 (48.6%) cases with pT1–pT3 node-positive breast cancer. The prognostic significance of ECE of axillary metastases was evaluated with respect to disease-free survival, overall survival, and the patterns of disease recurrence. Such prognostic significance was then compared with that of other clinical and pathologic factors. Breast Cancer Research 2007, 9(Suppl 1):P12 (doi: 10.1186/bcr1718) Objective To study the efficacy of different regimens of treatment based on trastuzumab in patients with Her2+ metastatic breast cancer (MBC). Patients and methods Medical records of 47 Her2+ MBC patients were retrospectively studied in our center between December 1999 and February 2004. The Her2 status was determined by immuno- histochemistry (Herceptest), FISH being used to discriminate the doubtful cases. Results Ninety-five patients (26.68%) presented with ECE. Thirty patients (31.57%) were identified as having three or less lymph nodes involved, 26 patients (27.36%) patients four to six nodes, 18 patients (18.9%) seven to nine nodes, and 22.16% patients 10 or more nodes, respectively. With a median follow-up of 86 months, factors with independent prognostic value for disease-free survival by multivariate analysis included absence of estrogen receptors (P < 0.005), pN category (P < 0.01), presence of lymphovascular invasion (LVI; P < 0.005), and ECE (P < 0.001). An independent negative prognostic effect on overall survival was observed for absence of estrogen and progesterone receptors (P < 0.05), pN category (P < 0.05), and presence of LVI (P < 0.005) and ECE (P < 0.001). Results Forty-seven patients with MBC of bad prognosis treated previously have been evaluated, a complete response (CR) being observed in 30% of the cases with a response rate (RR) (CR + partial response (PR)) of 63%. The stable disease rate (SD) was 12%, which provides a clinical benefit (CR + RP + SD) of 75%. Thirteen of these patients received a new scheme of treatment based on trastuzumab after progression to the first regimen containing trastuzumab, 76% of RR (CR + PR) and 30% of CR being observed. Three of these patients received a third regimen of treatment with trastuzumab, still obtaining one CR (RR 33%), and one SD. S Maksimovic1, Z Gojkovic2, M Opric3 1General Hospital “Sveti Vracevi” in Bijeljina, Bosnia and Herzegovina; 2Clinic for Oncology Clinical Center Banja Luka, Bosnia and Herzegovina; 3Clinical Center “Bezanijska Kosa” Belgrade, Serbia Breast Cancer Research 2007, 9(Suppl 1):P10 (doi: 10.1186/bcr1716) Background Extracapsular extension (ECE) of axillary metastases has 1General Hospital “Sveti Vracevi” in Bijeljina, Bosnia and Herzegovina; 2Clinic for Oncology Clinical Center Banja Luka, Bosnia and Herzegovina; 3Clinical Center “Bezanijska Kosa” Belgrade, Serbia Breast Cancer Research 2007, 9(Suppl 1):P10 (doi: 10.1186/bcr1716) S Menjón-Beltrán, R Olivencia, E Gonzalez Conclusion The association of trastuzumab with chemotherapy constitutes a very active regimen in previously treated patients with MBC of poor prognosis. This activity even continues in patients who have already received a previous treatment based on trastuzumab. Conclusion ECE demonstrated a stronger statistical significance in predicting prognosis than the pN category and was also related to an increased risk of distant recurrences. We suggest that the decision on adjuvant therapy should consider the presence of ECE of axillary metastases and peritumoral LVI as indicators of high biological aggressiveness. Balancing the risks and benefits of irradiation, we continue to recommend that complete axillary irradiation is not routinely indicated after adequate axillary dissection. P10 previous treatment with trastuzumab alone or in combination, four additional responses and four stable disease responses being observed, which produces an 88% clinical benefit. One patient discontinued treatment because of LVEF decline (>50%). Conclusion Trastuzumab and gemcitabine constitutes a well-tolerated and very active regimen for Her2+ MBC heavily pretreated patients, including patients who have been previously treated with trastuzumab. Conclusion Trastuzumab and gemcitabine constitutes a well-tolerated and very active regimen for Her2+ MBC heavily pretreated patients, including patients who have been previously treated with trastuzumab. S Maksimovic1, Z Gojkovic2, M Opric3 BRCA1a has antitumor activity in triple-negative breast cancers C Yuli, N Shao, G Oprea-llies, J Okoli, ESP Reddy, VN Rao Cancer Biology Program, Department OB/GYN, Morehouse School of Medicine, Atlanta, GA, USA C Yuli, N Shao, G Oprea-llies, J Okoli, ESP Reddy, VN Rao Cancer Biology Program, Department OB/GYN, Morehouse School of Medicine, Atlanta, GA, USA Clinical role of trastuzumab in metastatic breast cancer: experience of a center Background Extracapsular extension (ECE) of axillary metastases has importance as a risk factor for local or distant recurrence. Poorer survival in breast cancer has been suggested, but its prognostic value has not been uniformly confirmed. Study of the combination gemcitabine and trastuzumab in the treatment of HER2+ metastatic breast cancer Breast Cancer Research 2007, 9(Suppl 1):P13 (doi: 10.1186/bcr1719) Most BRCA1-related breast cancers are high-grade, basal-like, estrogen receptor (ER)-negative, progesterone receptor (PR)-negative, and Her2-negative. Triple-negative breast cancers are more common in younger black women than their white counterparts, have higher rates of distant metastasis, and currently there are no effective treatments against these cancers. We have previously characterized a splice variant of BRCA1 (BRCA1a/p110), which is expressed at reduced levels in several breast tumors. Stable expression of BRCA1a resulted in inhibition of growth in vitro of human breast, ovarian, prostate and colon cancer cells, and only those cells with wild-type Rb were sensitive to BRCA1a-induced growth suppression and the status of p53 did not affect the ability of BRCA1a to suppress growth of tumor cells. We have introduced BRCA1a into the CAL51 cell line, which is negative for ER and PR, and our results using semiquantitative immunocytochemistry show CAL51 to be negative for HER2. These transfectants were analyzed for BRCA1a protein expression by western blot analysis. The BRCA1a transfectants were slow growing and 98% inhibited in their growth in soft agar. BRCA1a also significantly inhibited CAL-51 triple-negative breast cancer xenografts in nude mice. These results suggest that the majority of exon 11 sequences lost in BRCA1a are not required for the tumor suppressor breast cancer S Menjón-Beltrán, R Olivencia-Dueso, E González-Jiménez, MJ Titos Frequency of febrile neutropenia in patients treated with taxane/epirubicine and colony-stimulating growth factors for breast cancer: comparison of filgrastim/lenograstim with pegfilgrastim W Schippinger, R Holub, N Dandachi, T Bauernhofer Hellmut Samonigg Medical University Graz, Department of Internal Medicine, Division of Medical Oncology, Graz, Austria Breast Cancer Research 2007, 9(Suppl 1):P16 (doi: 10.1186/bcr1722) Frequency of febrile neutropenia in patients treated with taxane/epirubicine and colony-stimulating growth factors for breast cancer: comparison of filgrastim/lenograstim with pegfilgrastim W Schippinger, R Holub, N Dandachi, T Bauernhofer Hellmut Samonigg Medical University Graz, Department of Internal Medicine, Division of Medical Oncology, Graz, Austria Breast Cancer Research 2007, 9(Suppl 1):P16 (doi: 10.1186/bcr1722) Methods The multiplex PCR was conducted on DNA from 71 archive breast tissue samples and 13 blood samples. We compared different paraffin removal procedures and DNA extraction procedures in combination in order to optimize and compare the results of the multiplex PCR with morphological and immunohistochemical para- meters for diagnosis of breast cancers. Objective This report describes a single centre’s experience on the efficacy of pegfilgrastim compared with filgrastim or lenograstim in reducing the incidence of febrile neutropenia in patients receiving combination chemotherapy with docetaxel or paclitaxel and epirubicin in neoadjuvant and adjuvant settings. Results The highest proportion of successful gene amplifications was obtained with a DNA isolation procedure using proteinase K digestion with followed boiling. Three 5382insC mutations were identified from 16 archival familial patients (19%) and five mtDNA4977 deletions were detected from nine blood familial breast cancers by the multiplex PCR. The mtDNA4977 deletion was highly prevalent in peripheral blood (56%), but it was absent in the breast tissue of cancer cases. Furthermore, familial breast cancer tumors exhibited higher mitotic activity, higher polymorphism, lower necrosis, lower tubules, higher ER- negatives and PR-negatives and lower TP53-positives than nonfamilial cancers. j j g Methods A total of 118 patients with breast cancer were treated with either epirubicin 75 mg/m2 and docetaxel 75 mg/m2 or with epirubicin 90 mg/m2 and paclitaxel 200 mg/m2 every 3 weeks; 88 patients received G-CSF support with daily filgrastim or lenograstim and 30 patients with pegfilgrastim once per cycle. Results Eight (9.1%) patients with prophylactic filgrastim or leno- grastim support developed febrile neutropenia, and one (3.3%) patient in the pegfilgrastim group (P = 0.445). Febrile neutropenia occurred in 13 (2.7%) of 476 filgrastim or lenograstim supported chemotherapy cycles, and in two (1.2%) of 172 cycles with pegfilgrastim support (P = 0.376). The frequency of chemotherapy delays and dose reduc- tions was not significantly different between the two G-CSF treatment groups. Conclusion Our results demonstrate that DNA extracted by the simple boiling method with a microwave yielded higher proportions of successful gene amplifications than the DNA extraction kit. Frequency of febrile neutropenia in patients treated with taxane/epirubicine and colony-stimulating growth factors for breast cancer: comparison of filgrastim/lenograstim with pegfilgrastim W Schippinger, R Holub, N Dandachi, T Bauernhofer Hellmut Samonigg Medical University Graz, Department of Internal Medicine, Division of Medical Oncology, Graz, Austria Breast Cancer Research 2007, 9(Suppl 1):P16 (doi: 10.1186/bcr1722) Furthermore, differences in successful gene amplification may be related to the size and number of the gene fragments amplified. Our analysis shows that testing of mtDNA4977 deletions and 5382insC in combination with morphological and immunohistochemical parameters may be an extremely effective and inexpensive tool in testing breast cancer patients aimed to identify individuals with high risk of familial breast cancers. Conclusion The results of this study demonstrate a trend towards superiority of pegfilgrastim over filgrastim or lenograstim in reducing the frequency of febrile neutropenia in patients treated with taxane and epirubicin chemotherapy regimens for breast cancer. P16 National Medical Academy of Post-Graduate Education, Kiev, Ukraine Breast Cancer Research 2007, 9(Suppl 1):P14 (doi: 10.1186/bcr1720) Objective Mutation analysis of mtDNA and nDNA are helpful in the determination of developmental potential, early diagnosis and gene therapy for breast cancer. In our study, we optimized the multiplex PCR in AFPBT and compared the results with morphological and immuno- histochemical parameters for detection of BRCA mutations and mtDNA4977 deletion in familial and nonfamilial breast cancers. S Menjón-Beltrán, R Olivencia-Dueso, E González-Jiménez, MJ Titos Unidad de Ginecología Oncológica, Hospital Universitario Virgen de las Nieves, Granada, Spain las Nieves, Granada, Spain Breast Cancer Research 2007, 9(Suppl 1):P11 (doi: 10.1186/bcr1717) Objective Trastuzumab and gemcitabine are two active drugs for meta- static breast cancer (MBC) treatment. We conducted a retrospective study of this combination in patients with Her2+ MBC in our hospital. Patients and methods Retrospective assessment of the efficacy of trastuzumab and gemcitabine association in patients with MBC previously treated with different lines of chemotherapy. Breast Cancer Research 2007, 9(Suppl 1):P11 (doi: 10.1186/bcr1717) Objective Trastuzumab and gemcitabine are two active drugs for meta- static breast cancer (MBC) treatment. We conducted a retrospective study of this combination in patients with Her2+ MBC in our hospital. Patients and methods Retrospective assessment of the efficacy of trastuzumab and gemcitabine association in patients with MBC previously treated with different lines of chemotherapy. Results Seventeen heavily pretreated patients with poor prognosis were evaluated. Fourteen patients (82%) had received prior adjuvant chemotherapy and 11 patients (64%) prior adjuvant radiotherapy; 12 patients (70%) had received prior chemotherapy for MBC. All the patients had received taxanes and 15 (88%) had received anthra- cyclines also. We observed a 41% rate of response, two complete responses, five partial responses, and 41% stable disease, obtaining 82% clinical benefit. The time-to-progression was 7.2 months. Median survival has not yet been reached. Nine of the patients had received S20 Available online http://breast-cancer-research.com/supplements/9/S1 Available online http://breast-cancer-research.com/supplements/9/S1 Available online http://breast-cancer-research.com/supplements/9/S1 function. This is the first report showing antitumor activity of BRCA1a in triple-negative breast cancers. function. This is the first report showing antitumor activity of BRCA1a in triple-negative breast cancers. intervention it is impossible to remove all the microscopic lymphatic vessels and nodes that could be damaged by the tumor micro- metastasis. In the field of high-frequency electromagnetic waves, the magnetite nanoparticles give us possibilities of local, distance hyperthermia on micrometastatic lesions. This minimizes the potentiality of development of postoperative recurrence and secondary metastatic processes. Identification of individuals with hereditary predisposition to breast cancer and other breast cancer-associated forms of malignant neoplasms: data of the Belarusian Cancer Registry Conclusion We have demonstrated that: (a) patients who received high-dose and standard chemotherapy showed similar results, (b) patients with a low-risk of relapse had markedly lower values as compared with high-risk primary breast cancer patients, and (c) micrometastases persist in a substantial proportion of patients. Table 1 (abstract P18) Incidence of positivity Standard High-dose chemotherapy chemotherapy All high risk T1N0 (n = 22) (n = 17) (n = 39) (n = 20) QPCR 58/138 (42%) 41/94 (44%) 103/238 (43%) 26/100 (26%) ICC 50/110 (45%) 37/80 (46%) 90/198 (45%) 27/96 (28%) Both 34/110 (34%) 26/80 (32%) 44/198 (22%) 14/96 (15%) Either 73/138 (53%) 42/94 (45%) 127/238 (53%) 39/100 (39%) Table 1 (abstract P18) Incidence of positivity Standard High-dose chemotherapy chemotherapy All high risk T1N0 (n = 22) (n = 17) (n = 39) (n = 20) QPCR 58/138 (42%) 41/94 (44%) 103/238 (43%) 26/100 (26%) ICC 50/110 (45%) 37/80 (46%) 90/198 (45%) 27/96 (28%) Both 34/110 (34%) 26/80 (32%) 44/198 (22%) 14/96 (15%) Either 73/138 (53%) 42/94 (45%) 127/238 (53%) 39/100 (39%) Minimal residue disease (MRD) A Zaidi1, G Tripuraneni1, S Weller2, B Ward2, HD Sinnett3, RC Coombes1, MJ Slade1 1Department of Oncology, Imperial College, London UK, 2Department of Medical Oncology and 3Department of Surgery, Charing Cross Hospital, London UK Objective To develop and introduce into clinical practice a diagnostic algorithm targeted at active identification of hereditary BC and other BRCA1/2 mutation-associated forms of malignant neoplasms. Breast Cancer Research 2007, 9(Suppl 1):P18 (doi: 10.1186/bcr1724) Introduction: We developed a quantitative PCR (QPCR) technique for the detection of cytokeratin 19 (CK19) transcripts in blood and bone marrow and compared this with immunocytochemistry (ICC). The aim was to monitor these assays for micrometastases in primary breast cancer patients. Methods Questionnaires for patients, and molecular genetic blood analysis. Results Questionnaires for patients with BC, ovarian cancer or colon cancer reveal individuals with cancer-burdened heredity, whose blood is analysed for the presence of germinal mutations. Relatives of BRCA1/2 mutation-positive patients are subject to examination for both germinal mutation-carrying and tumour markers. A cohort is formed for targeted prophylactic medical examinations. Materials and methods Bone marrow (BM) aspirates were taken 2–4 years following chemotherapy from 39 patients who had entered a trial comparing high-dose chemotherapy (n = 17) with standard chemotherapy (n = 22). The BM was tested for the presence of CK19- positive cells using ICC and QPCR. We compared this with 20 low- risk (T1N0) patients. Conclusion (1) An original method is developed for active identifica- tion of hereditary BC and other BC-associated malignant neoplasms. (2) The proposed method makes it possible to conduct targeted and efficient prophylatic medical examinations of germinal mutation carriers. Results See Table 1. A total of 232 aspirates were analysed from the high-risk patients and 100 from the T1N0 patients. The number of patients positive was therefore approximately the same irrespective of therapy in the high-risk patients, and the levels of positivity were not significantly different (PCR, P = 0.078; and ICC, P = 0.253). The levels of positivity, however, were higher in the high-risk patients than in the low-risk patients. IV Zalutsky, NN Antonenkova, GM Porubova NN Alexandrov Research Institute of Oncology and Medical Radiology, Minsk, Belarus NN Alexandrov Research Institute of Oncology and Medical Radiology, Minsk, Belarus Conclusion Although large numbers of side effects were reported, tamoxifen was well tolerated and no new safety concerns were identified. For most side effects no significant difference between treatment groups was found after ceasing tamoxifen. Breast Cancer Research 2007, 9(Suppl 1):P19 (doi: 10.1186/bcr1725) Introduction Hereditary cancers make up 5–15% of all forms of breast cancer (BC). BRCA1/2 mutations determine about 50% of hereditary BC forms and other pathologic BRCA1/2 genotype-associated forms of malignant neoplasms. Meta-analysis has demonstrated a high probability for development of ovarian cancer, gastric cancer, colon cancer, endometrial cancer, pancreatic cancer, skin melanoma, urinary bladder cancer, and head and neck tumours with pathologic BRCA1/2 genotype, which should be taken into consideration in medicogenetic consulting and monitoring of patient carriers. Minimal residue disease (MRD) Minimal residue disease (MRD) A Zaidi1, G Tripuraneni1, S Weller2, B Ward2, HD Sinnett3, RC Coombes1, MJ Slade1 Novel method of sentinel lymph node detection in malignant tumors using preparation ‘UNIMAG’ B Surguladze, R Zhorzoliani, T Tskitishvili , , , 1Cancer Research UK, Centre for Epidemiology, Mathematics and Statistics, London, UK; 2Department of Surgical Oncology, Newcastle Mater Hospital, University of Newcastle, UK “Magnetic Fluids in Medicine and Biology”, ATT Ltd, Georgia, USA Breast Cancer Research 2007, 9(Suppl 1):P15 (doi: 10.1186/bcr1721) Preparation ‘UNIMAG’ is registered by the pharmacological committee of Georgia (Cert.: DA Nr-000142, 08.04.2005; Author – PhD, ScD, Professor Besiki Surguladze; Owner – ‘ATT’ Ltd, Sci. Laboratory ‘Magnetic fluids in Medicine and Biology’). Preparation ‘UNIMAG’ represents a magnet-sensitive stabile suspension of magnetite nano- particles (magnetic fluid). After the peritumoral injection of ‘UNIMAG’, magnetite nanoparticles will be absorbed by macrophages, which deliver them through the lymphatic capillaries to regional lymph nodes. This mechanism of magnetite nanoparticle transport underlies the new method of sentinel lymph node detection. Diversely to the other dyestuff agents used in this direction, magnetite nanoparticles actively filled lymphatic nodes and colored them in bold black. This significantly facilitates the atraumatic separation of lymph nodes during lymph- adenectomy. Besides the intraoperative indication of lymphatic nodes, ‘UNIMAG’ provides the possibility of X-ray and ultrasound imaging of sentinel lymph nodes and performing their biopsy in the preoperative period. This assuredly helps in a better planning of the operation and considerably improves its radicalism. Even in the case of ideal surgical Breast Cancer Research 2007, 9(Suppl 1):P17 (doi: 10.1186/bcr1723) Objective Tamoxifen is an effective drug but its role in prevention is limited by its side-effect profile, particularly related to endometrial problems and thrombotic events. We present updated results on the comparison of side effects between active treatment and the follow-up period incorporating four additional years of follow-up. Methods In the International Breast Cancer Intervention Study I (IBIS-I) study, 7,154 women at increased risk of breast cancer were either randomised to tamoxifen 20 mg/day or placebo for 5 years. Women gave detailed information of any side effects at each 6-monthly follow- up visit and once a year after active treatment. Results During active treatment, large numbers of side effects were reported by participants in both treatment arms. However, the only major categories that showed differences were vasomotor and gynaecological side effects, which were about 12% higher in the tamoxifen group than the placebo group. Novel method of sentinel lymph node detection in malignant tumors using preparation ‘UNIMAG’ B Surguladze, R Zhorzoliani, T Tskitishvili Thromboembolic events were significantly higher in the tamoxifen group compared with the placebo S21 Breast Cancer Research Vol 9 Suppl 1 VII Madrid Breast Cancer Conference Proffered Papers group (85 versus 42, P < 0.001), whereas myocardial infarctions were reduced in women on tamoxifen during active treatment (2 versus 7, P = 0.09). Women on tamoxifen developed significantly more endo- metrial cancer during active treatment than women on placebo (12 versus 3, P = 0.02). After active treatment, most menopausal-like side effects decreased and the differences between the two treatment arms were not significant. Reports on vaginal discharge decreased after stopping tamoxifen. Thromboembolic events were similar between the two treatment arms after active treatment (32 versus 26, P = 0.4). No significant difference in endometrial cancer between the two treatment groups was found during follow-up time (8 versus 5, P = 0.4). Table 1 (abstract P22) Patients and methods Twenty-five patients with breast cancer were included in this prospective study. All patients underwent breast surgery including lumpectomy or mastectomy followed by axillary lymph node dissection (Berg Level I and II). In 13 patients, a surgical haemo- stasis sponge Tachosil® was applied from the apex axillae to the thoracic longus nerve and 12 had standard closure of the axillary lymphadenectomy area. Patients did not differ with respect to general characteristics, such as age, body mass index, treatment modality, and tumor stage distribution. There were six mastectomy and five lumpectomy operations in the Tachosil® group, and seven mastectomy and four lumpectomy surgeries in the control group. The mean number of removed lymph nodes was 11.9 in the Tachosil® group with a mean of 1.2 metastatics nodes, and was 12.2 in the control group with a mean of 2.5 metastatic nodes. intraoperative imaging probes, the time has come to quantify the clinical benefit of these devices. Results The mean duration of axillary drainage was 3.3 days in the Tachosil® group and 5.5 days in the control group. The mean total drainage volume was 246 ml in the Tachosil® group and 470 ml in the standard closure group. The mean length of postoperative hospital stay was 3.5 days in the Tachosil® group and 5.5 days in the standard closure group. Seroma formation after drain removal was found in four patients with a mean of drainage volume of 161 ml in the Tachosil® group, and in five patients with a mean of drainage volume of 517 ml in the standard closure group. No intolerance of the product was described. Objective To compare the hand-held camera (POCI) with a lymphoscintigraphy in order to precisely localize SLNs in patients with breast cancer requiring SLN biopsy. q g p y Methods The SLN protocol consisted of a peritumoral injection of 160 MBq 99mTc-labeled nano-colloids performed 2 hours before the lymphoscintigraphy, which is realized preoperatively using a conven- tional single-head gamma camera and then the hand-held POCI camera (field-of-view, 13 cm2) (Figure 1). The day after, in the operating room, the patients undergo one blue-dye subcutaneous tumoral injection. Before incision the surgeon first uses the POCI to perform an intraoperative lymphoscintigraphy and then the counting probe (Europrobe CsI, Eurorad) for transcutaneous SLN prelocalizations. The POCI camera was slowly moved on the skin until SLN identification. Incidence of positivity NN Alexandrov Research Institute of Oncology and Medical Radiology, Minsk, Belarus Breast Cancer Research 2007, 9(Suppl 1):P20 (doi: 10.1186/bcr1726) Introduction Nowadays, all developed countries spend enormous resources on breast cancer (BC) treatment. However, no significant reduction in the population’s BC rates due to the improvement of treatment methods can be expected in the near future. p Objective To use the data of the Belarusian Cancer Registry (BCR) for active identification of individuals with hereditary predisposition to BC and other BC-associated forms of malignant neoplasms. p Objective To use the data of the Belarusian Cancer Registry (BCR) for active identification of individuals with hereditary predisposition to BC and other BC-associated forms of malignant neoplasms. Materials and methods To make the analysis, 4,442 patients with second primaries (SP), one of which was BC, were selected, as well as BC patients with disease origination before age 40. There were 7,137 such patients. Conclusion We have demonstrated that: (a) patients who received high-dose and standard chemotherapy showed similar results, (b) patients with a low-risk of relapse had markedly lower values as compared with high-risk primary breast cancer patients, and (c) micrometastases persist in a substantial proportion of patients. Results BC was most commonly associated with skin cancer (24.9%), endometrial cancer (11.8%), ovarian cancer (9%), gastric cancer (7.8%), cervical cancer (5.9%), rectum cancer (5.2%), thyroid cancer (5%), colon cancer (4.8%), renal cancer (3.6%), lung cancer (3.4%), skin melanoma (2.3%) and other cancers (16.3%). The fate of each patient was retraced. Conclusions (1) BCR data enable one to identify individuals potentially predisposed to hereditary BC and other BC-associated forms of malignancies, according to two features. (2) Using the risk factors for Conclusions (1) BCR data enable one to identify individuals potentially predisposed to hereditary BC and other BC-associated forms of malignancies, according to two features. (2) Using the risk factors for S22 Available online http://breast-cancer-research.com/supplements/9/S1 Available online http://breast-cancer-research.com/supplements/9/S1 Figure 1 (abstract P22) Preoperative image by POCI showing two hot sentinel nodes. Figure 1 (abstract P22) development of hereditary BC and other BC-associated forms of malignant neoplasms makes it possible to draw the relatives of the identified individuals into genetic consulting. (3) The rates of BC association with other forms of malignancies in SP patients were evaluated in the Belarusian population. Table 1 (abstract P22) The axillary area was scanned with the POCI camera with 10-second acquisition images. SLN detection was defined as positive on the basis of a local radioactivity increase in the intraoperative images of the surveyed region. The SLN excision biopsy was guided, following the established procedure, using the counting probe and blue dye. Finally, a complementary scanning procedure was performed with the POCI directly in the operative wound before skin closure to confirm complete excision or to detect some residuals SLNs. The number and the localization of SLNs obtained by both intraoperative detectors are compared with those obtained the day before with the conventional gamma camera. Conclusion The use of Tachosil® in the axillary area in patients with breast cancer requiring axillary dissection seems to reduce the volume and duration of postoperative axillary drainage, the duration of hospital stay, and the frequency and volume of seroma after axillary drainage. Larger series are required to confirm these results. Incidence of positivity (4) The use of BCR data and active involvement of relatives of persons with a cancer burden in oncogenetic consulting offer opportunities for early diagnosis and prevention of hereditary BC and other BC-associated forms of malignant neoplasms. Tachosil® to reduce the morbidity of axillary lymph node dissection in breast cancer E Barranger, O Morel, G Akerman, C Malartic, D Clement Département de Gynecologie-Obstétrique, Hôpital Lariboisière, Paris, France Breast Cancer Research 2007, 9(Suppl 1):P21 (doi: 10.1186/bcr1727) Background Seroma formation after axillary lymph node dissection in breast cancer remains a problem despite many efforts to reduce surgery-related morbidity. The aim of the present study was to evaluate the impact of a fibrin-glue-coated collagen patch (Tachosil®; Nycomed Laboratory, Denmark) to reduce the volume and duration of post- operative axillary drainage, the duration of hospital stay, and procedural safety. Preoperative image by POCI showing two hot sentinel nodes. Preoperative image by POCI showing two hot sentinel nodes. Table 1 (abstract P22) Results of SLN identification with lymphoscintigraphy and POCI Total SLNs Mean 95% CI Range Preoperative lymphoscintigraphy 54 1.6 1.28–1.99 0–4 POCI camera the day before surgery 59 1.8 1.46–2.12 0–4 POCI intraoperative imager 61 1.9 1.55–2.15 0–4 P23 Results Twelve patients have so far been randomized. Preliminary safety data will be presented at the meeting. Acknowledgement Supported by GlaxoSmithKline. Preliminary safety data of preoperative chemotherapy plus trastuzumab, lapatinib or both in HER2-positive operable breast cancer V Guarneri1, A Frassoldati1, F Piacentini1, K Cagossi2, L Cavanna3, A Michelotti4, G Jovic1, S Giovannelli1, G Ficarra5, C Oliva6, P Conte1 First clinical results on the potential of intraoperative imaging for sentinel lymph node biopsy in breast cancer by the POCI in 32/33 patients (97%) (P = 0.13). At least one SLN (mean: 2.3; range: 1–4) was identified in all patients during the surgery. Concerning the duration of these procedures, the mean acquisition time was 15 minutes (range: 10–30) with lymphoscintigraphy, 8.8 minutes (range: 3–18) with the POCI used the day before surgery and 3.35 minutes (range: 2–6.5) with preoperative POCI. Conclusion These preliminary clinical results show that the POCI camera is able to predict the number and localization of breast cancer SLNs. The POCI camera appears to be a promising tool to complete or replace the preoperative standard lymphoscintigraphy, especially in surgical centers without an onsite nuclear medicine department. Sample size Assuming a 50% increase in the pCR rate for the CT + T + L arm versus CT + T or CT + L, 52 patients will be enrolled in the first stage; in case we observe 13 pCR, an additional 68 patients will be enrolled, for a total of 120 patients (Simon’s design). Results Twelve patients have so far been randomized. Preliminary safety data will be presented at the meeting. Acknowledgement Supported by GlaxoSmithKline. Sample size Assuming a 50% increase in the pCR rate for the CT + T + L arm versus CT + T or CT + L, 52 patients will be enrolled in the first stage; in case we observe 13 pCR, an additional 68 patients will be enrolled, for a total of 120 patients (Simon’s design). First clinical results on the potential of intraoperative imaging for sentinel lymph node biopsy in breast cancer E Barranger1, K Kerrou2, S Pitre3, MA Duval3, C Coutant1, R Siebert3, J Talbot2, Y Charon3, S Uzan1 1Gynécologie Obstétrique, Hôpital Lariboisière, Hôpital Tenon AP-HP, Paris, France; 2Médecine Nucléaire, Hôpital Tenon AP-HP, Paris, France; 3Laboratoire IMNC–CNRS Paris 7–Paris 11, Orsay, France Breast Cancer Research 2007, 9(Suppl 1):P22 (doi: 10.1186/bcr1728) Background The growing interest in the sentinel lymph node (SLN) detection concept has led to the development of several prototypes of hand-held gamma cameras. After the first successful clinical trials with E Barranger1, K Kerrou2, S Pitre3, MA Duval3, C Coutant1, R Siebert3, J Talbot2, Y Charon3, S Uzan1 1Gynécologie Obstétrique, Hôpital Lariboisière, Hôpital Tenon AP-HP, Paris, France; 2Médecine Nucléaire, Hôpital Tenon AP-HP, Paris, France; 3Laboratoire IMNC–CNRS Paris 7–Paris 11, Orsay, France Results To date, 33 patients (mean age: 64 years) have been included in this clinical protocol. A SLN was identified by lymphoscintigraphy in 31/33 patients (94%), versus 30/33 (90.9%) with the POCI (P > 0.3). The mean number of SLNs identified by lymphoscintigraphy was 1.6 (range: 0–4), versus 1.8 (range: 0–4) with the POCI (Table 1). The day of the surgery, at least one SLN (mean: 1.9; range: 0–4) was identified Background The growing interest in the sentinel lymph node (SLN) detection concept has led to the development of several prototypes of hand-held gamma cameras. After the first successful clinical trials with S23 Breast Cancer Research Vol 9 Suppl 1 VII Madrid Breast Cancer Conference Breast Cancer Research Vol 9 Suppl 1 VII Madrid Breast Cancer Conference by the POCI in 32/33 patients (97%) (P = 0.13). At least one SLN (mean: 2.3; range: 1–4) was identified in all patients during the surgery. Concerning the duration of these procedures, the mean acquisition time was 15 minutes (range: 10–30) with lymphoscintigraphy, 8.8 minutes (range: 3–18) with the POCI used the day before surgery and 3.35 minutes (range: 2–6.5) with preoperative POCI. for 12 weeks, followed by four courses of FEC (5-FU 600 mg/m2 + epirubicin 75 mg/m2 + cyclophosphamide 600 mg/m2 i.v.) every 3 weeks. T is administered at the dose of 2 mg/kg weekly in arms A and C; L is administered at 1,500 mg p.o. daily in arm B, and at 1,000 mg p.o. daily in arm C. Both T and L are administered throughout the duration of CT. The following biomarkers are evaluated at baseline, and at surgery: EGFR, HER2, pTEN, pAKT, pMAPK, apoptosis (TUNEL Test), Ki67. Significance of nuclear medicine: 99mTc-MDP in the detection of breast cancer bone metastasis 1Department of Oncology and Hematology, University of Modena and Reggio Emilia, Modena, Italy; 2Division of Medical Oncology Ramazzini Hospital, Carpi, Italy; 3Division of Medical Oncology, Hospital of Piacenza, Italy; 4Division of Medical Oncology, S. Chiara University Hospital, Pisa, Italy; 5Department of Pathology, University of Modena and Reggio Emilia, Modena, Italy; 6Oncology Medicine Development Center, GlaxoSmithKline, Greenford, UK Nuclear Medicine, Department of Radiation Oncology, Kamla Nehru Hospital, Gandhi Medical College, Bhopal, India Nuclear Medicine, Department of Radiation Oncology, Kamla Nehru Hospital, Gandhi Medical College, Bhopal, India Breast Cancer Research 2007, 9(Suppl 1):P24 (doi: 10.1186/bcr1730) Finding bone metastases is frequently important for clinical decisions affecting quality of life. Detection of such bone metastatic lesions allows radiation therapy or surgical interventions to prevent pathologic fractures from disabling the patients. We investigated 53 breast cancer patients referred by surgeons, radiotherapists and chemotherapists. Adsorption of 99mTc-MDP occurs in the mineral phase of the bone. High-sensitivity bone scanning in determining the presence and extent of metastatic disease is an extremely important tool in decision-making. Bone scans demonstrate metastatic lesions much earlier than X-ray, CT or MRI. 99mTc-MDP 22–25 mCi was injected i.v. In total, 53 patients were investigated. On visual analysis there were positive scan findings (bone mets) in 17 patients (33.4%) and negative scan findings (normal bone scan) in 34 patients (66.6%). Significance of bone mets in the management of disease occurs when clinically stage I and scintigraphically stage IV; here a change of treatment is needed. Sensitivity for detecting bone mets is quoted as 95% or above. Bone scintigraphy is a noninvasive technique used for detection of breast cancer bone metastasis. Quality of life will be good if diagnosed early and accurately and if managed properly. and Reggio Emilia, Modena, Italy; Oncology Medicine Development Center, GlaxoSmithKline, Greenford, UK Breast Cancer Research 2007, 9(Suppl 1):P23 (doi: 10.1186/bcr1729) Introduction and objective Lapatinib (L) is a tyrosine kinase inhibitor of EGFR and HER2. The inhibition of these two pathways can affect tumor growth by reducing the EGFR-dependent proliferative stimulus, by restoring apoptosis, and possibly by enhancing sensitivity to chemotherapy (CT). On these premises, the combination of L with CT or with CT and trastuzumab (T) is promising. We have therefore designed a phase II randomized trial to evaluate the activity and safety of this combination as preoperative therapy for HER2+ operable breast cancer (BC). The primary endpoint was percentage of pathologic complete response (pCR). Significance of nuclear medicine: 99mTc-MDP in the detection of breast cancer bone metastasis Secondary aims were the breast objective response, breast conservative surgery, safety, molecular responses, and gene expression related to pCR. Patients and methods After a core biopsy for diagnosis, biomarker evaluation and storage of fresh tissue for molecular analyses, patients with HER2+ stage II–IIIA BC are randomized to: arm A, CT + T; arm B, CT + L; arm C, CT + T + L. CT consists of: paclitaxel 80 mg/m2 weekly Breast Cancer Research 2007, 9(Suppl 1):P23 (doi: 10.1186/bcr1729) Introduction and objective Lapatinib (L) is a tyrosine kinase inhibitor of EGFR and HER2. The inhibition of these two pathways can affect tumor growth by reducing the EGFR-dependent proliferative stimulus, by restoring apoptosis, and possibly by enhancing sensitivity to chemotherapy (CT). On these premises, the combination of L with CT or with CT and trastuzumab (T) is promising. We have therefore designed a phase II randomized trial to evaluate the activity and safety of this combination as preoperative therapy for HER2+ operable breast cancer (BC). The primary endpoint was percentage of pathologic complete response (pCR). Secondary aims were the breast objective response, breast conservative surgery, safety, molecular responses, and gene expression related to pCR. Patients and methods After a core biopsy for diagnosis, biomarker evaluation and storage of fresh tissue for molecular analyses, patients with HER2+ stage II–IIIA BC are randomized to: arm A, CT + T; arm B, CT + L; arm C, CT + T + L. CT consists of: paclitaxel 80 mg/m2 weekly S24
https://openalex.org/W2027792960	https://www.scielo.br/j/fm/a/bngHBvBbXS93d57V6LX8vxN/?lang=pt&format=pdf	Portuguese	null	Avaliação da atividade eletromiográfica com ou sem o uso de diversos tipos de calçado, em diferentes planos de locomoção	Fisioterapia em Movimento	2,012	cc-by	5,564	Evaluation of eletromyographic activity with or without the use of various types of shoes, in different plans Geraldo Fabiano de Souza Moraes[a], Adriana Papini Antunes[b], Elaine Souza Rezende[c], Poliana Cardoso Ribeiro de Oliveira[d] [a] Fisioterapeuta, mestre em Ciências da Reabilitação, especialista em Fisioterapia Ortopédica e Esportes, professor assistente do curso de Engenharia de Saúde e Segurança da Universidade Federal de Itajubá – Câmpus Itabira - Be Horizonte, MG - Brasil, e-mail: geraldmoraes@terra.com.br [b] Fisioterapeuta, especialista em Fisioterapia Ortopédica e Desportiva - Belo Horizonte, MG - Brasil, e-mail: dripantunes@yahoo.com.br [c] Fisioterapeuta, Belo Horizonte, MG - Brasil, e-mail: enialerezende@yahoo.com.br [d] Fisioterapeuta, Belo Horizonte, MG - Brasil, e-mail:polianacro@yahoo.com.br [a] Fisioterapeuta, mestre em Ciências da Reabilitação, especialista em Fisioterapia Ortopédica e Esportes, professor assistente do curso de Engenharia de Saúde e Segurança da Universidade Federal de Itajubá – Câmpus Itabira - Belo Horizonte, MG - Brasil, e-mail: geraldmoraes@terra.com.br ISSN 0103-5150 Fisioter. Mov., Curitiba, v. 25, n. 3, p. 507-516, jul./set. 2012 Licenciado sob uma Licença Creative Commons Avaliação da atividade eletromiográfica com ou sem o uso de diversos tipos de calçado, em diferentes planos de locomoção Evaluation of eletromyographic activity with or without the use of various types of shoes, in different plans Abstract Introduction: It is very common women’s complaints of discomfort in the lumbar region, caused by the use of high heeled shoes. Objective: The objective of this study was to evaluate the electromyographyc activity of the muscles of the leg and spine associated with different types of shoes and barefoot, in various plans of locomotion, before and after fatigue. Materials and methods: This is a cross-sectional study, in which was used a sample of convenience with 15 young women, aged between 18 and 35 years, sedentary. The examined muscles were tibialis anterior, gastrocnemius medialis and lateralis and erector spinae. The muscular activity was assessed during locomotion on flat surface, up and down stairs and in an inclined surface. The shoes were high-heeled, small heeled, snickers, slipper, and it was also made an evaluation in barefoot. Results: The results of electromyographyc analysis showed that the muscles of the leg had greater electromyographyc activity in the position of pre-fatigue of the lower limbs and erector spinae in the post-fatigue of the lower limbs, in most of the conditions and situations. Conclusion: Adaptive conditions associated with regular use of high heels has been widely questioned in relation to the incidence of injuries. In post-fatigue of the lower limbs, there was a decline in the activities of the muscles of the leg and increased recruitment of the erector muscles, justified by the need for greater stability of the spine and pelvis during the locomotion of young women. [K] Keywords: Electromyography. Gait. Evaluation. Muscle fatigue. Resumo Introdução: São muito recorrentes as queixas de mulheres com desconforto na região lombar durante o uso de sapato de salto. Objetivo: O objetivo deste estudo foi avaliar a atividade eletromiográϐica de músculos da perna e eretores da espinha associada a tipos de calçados e ao andar descalço, nos diferentes planos de locomoção, nas situações pré e pós-fadiga. Materiais e métodos: Trata-se de um estudo ob- servacional transversal, no qual foi utilizado uma amostra de conveniência com 15 mulheres jovens, com idades entre 18 e 35 anos, sedentárias. Os músculos analisados foram: tibial anterior, gastrocnêmio me- dial e lateral e eretores. A atividade muscular foi avaliada durante a marcha em superϐície plana, subindo e descendo escada e rampa. Os calçados utilizados foram salto alto, salto baixo, tênis, chinelo, e também foi feita a avaliação sem o uso deles, ou seja, descalço. Resultados: Os resultados da análise eletromiográϐica mostrou que os músculos da perna tiveram maior atividade eletromiográϐica pré-fadiga e os eretores, pós- -fadiga dos membros inferiores, na maioria das condições e situações. Conclusão: Condições adaptativas associadas ao uso regular de salto alto foram amplamente questionadas em relação à incidência de lesões. Após fadiga de membros inferiores, houve declínio na atividade dos músculos da perna e aumento do Fisioter Mov. 2012 jul/set;25(3):507-16 Moraes GFS, Antunes AP, Rezende ES, de Oliveira PCR. 508 recrutamento dos músculos eretores, justiϐicado pela necessidade de maior estabilidade da coluna e da pelve durante a locomoção de mulheres jovens. [P] recrutamento dos músculos eretores, justiϐicado pela necessidade de maior estabilidade da coluna e da pelve durante a locomoção de mulheres jovens. [P] [ ] Palavras-chave: Eletromiograϐia. Marcha. Avaliação. Fadiga muscular. Fisioter Mov. 2012 jul/set;25(3):507-16 Keywords: Electromyography. Gait. Evaluation. Muscle fatigue. Introdução 2012 jul/set;25(3):507-16 Avaliação da atividade eletromiográfica com ou sem o uso de diversos tipos de calçado, em diferentes planos de locomoção 509 áreas do corpo. Outra boa prática é alongar periodi- camente os músculos da coxa e da panturrilha (10). áreas do corpo. Outra boa prática é alongar periodi- camente os músculos da coxa e da panturrilha (10). Hoje em dia, as mulheres estão mais inseridas no mercado de trabalho, e o uso de salto é frequente, principalmente associado à questão estética. Devido a relatos de incômodos como dores, alterações pos- turais e deformidades nos pés, é extremamente im- portante veriϐicar qual tipo de calçado seria o mais adequado e o mais confortável para o uso diário. refere-se à medida dos ângulos formados nas arti- culações pelos segmentos corporais. Ao utilizar um goniômetro universal, o examinador obtém essas medidas colocando as partes do instrumento junto aos segmentos imediatamente proximal e distal à ar- ticulação que está sendo avaliada. É um equipamento de medida do ângulo articular que apresenta validade e conϐiabilidade já descritas na literatura (12, 13). Hoje em dia, as mulheres estão mais inseridas no mercado de trabalho, e o uso de salto é frequente, principalmente associado à questão estética. Devido a relatos de incômodos como dores, alterações pos- turais e deformidades nos pés, é extremamente im- portante veriϐicar qual tipo de calçado seria o mais adequado e o mais confortável para o uso diário. Foi utilizado o eletromiógrafo de superϐície da marca EMG System, modelo EMG 1600 (EMG System, São José dos Campos, SP, Brasil), conectado a um microcomputador com oito eletrodos de superϐície ativos, bipolares, Ag/AgCl. Os resultados dos testes foram armazenados e analisados por um programa especíϐico, seguindo o manual do fabricante. O objetivo deste estudo foi avaliar a atividade ele- tromiográϐica dos músculos da perna e eretores da espinha associados aos tipos de calçados e sem eles (descalço), nos diferentes planos de locomoção, nas situações pré e pós-fadiga. A eletromiograϐia (EMG) possibilita o registro dos sinais elétricos gerados pelas células musculares, o que permite a análise da atividade muscular durante o movimento simples ou associado à realização de uma tarefa. Introdução A validade e a precisão da avaliação da atividade muscular, por meio da utilização da EMG, estão relacionadas ao processo de detecção dos sinais e à correta condução dos procedimentos de análise, bem como a conhecimentos de possíveis interferên- cias durante o processo de coleta (14, 15). Locais de análise da locomoção Utilizou-se um plano com uma distância retilínea de seis metros, uma rampa de um metro e sessenta centímetros, com uma inclinação de 16º, e uma esca- da com três degraus, com altura de 16 centímetros e largura de 24 centímetros. Procedimentos Inicialmente, todas as voluntárias foram escla- recidas quanto aos objetivos e aos procedimentos do estudo e, em seguida, assinaram o termo de con- sentimento livre e esclarecido. Em seguida, foi feita uma avaliação padronizada, por uma avaliadora de- vidamente treinada, para identiϐicação, anamnese e exame ϐísico de cada voluntária. Esse estudo foi aprovado pelo Comitê de Ética em Pesquisa do Centro Universitário Newton Paiva, com o número interno do projeto 0082, de 6 de março de 2008. Materiais e métodos Trata-se de um estudo observacional transversal, no qual foi utilizada uma amostra de conveniência com 15 mulheres jovens, com idades entre 18 e 35 anos, sedentárias. Os dados foram coletados entre fevereiro e junho de 2008. Os critérios de exclusão foram mulheres que possuíssem rigidez articular, dor que incapacite a realização dos testes, discrepância de MMII (acima de um centímetro), terem tido entorse de tornozelo há menos de seis meses do estudo, terem sido diag- nosticadas com ruptura ligamentar nos tornozelos, terem sido submetidas a cirurgias ou ϐisioterapia nos membros inferiores há menos de seis meses. Também foram excluídas mulheres obesas – IMC ≥ 30 (11), grávidas, diabéticas, que utilizassem auxílio à marcha e/ou palmilhas, com presença de edema de tornozelo e pés, hipotroϐia acima de dois centímetros de pantur- rilha e coxa, que possuíssem hálux valgo, joanete do V metatarso e neuroma de Morton que impossibilitasse a realização dos procedimentos. Introdução Neptune et al. (7) mostraram que o gastrocnêmio medial é o único ϐlexor plantar que auxilia no início da fase de balanço. Em síntese, há pouco consenso sobre o papel funcional dos músculos individualmen- te durante a fase de balanço da marcha. Um dos aspectos mais prevalentes são queixas de mulheres com desconforto na região lombar durante o uso de sapato de salto (1). Snow e Williams (2) des- creveram que pode haver uma relação entre a postura assumida e os desconfortos presentes, geralmente associados à fadiga muscular e ao aumento das forças de reação do solo durante o uso desse tipo de calçado. Durante a marcha, o corpo se movimenta de for- ma semelhante a um pêndulo invertido. Na primeira metade da fase de apoio, a energia cinética é trans- formada em energia potencial gravitacional, que é parcialmente recuperada; o corpo inclina para frente e para baixo na segunda metade da fase de apoio. Quando uma perna atinge o chão, as energias cinética e potencial gravitacional são armazenadas tempora- riamente como energia elástica em músculos, tendões e ligamentos e, então, é quase totalmente recuperada durante a fase de impulso (8). Alguns estudos (2-4) demonstraram que as maio- res alterações que ocorrem nos membros inferiores, de acordo com a altura do salto, são o aumento signi- ϐicativo na ϐlexão plantar, na mudança da orientação relativa das estruturas ósseas das articulações do tornozelo, metatársica e metatarsofalangeanas, al- teração dos ângulos de inserção dos músculos do pé e do tornozelo e diminuição do ângulo máximo do joelho durante a fase de balanço e na velocidade de extensão do joelho. O trabalho estático é altamente fatigante e, sempre que possível, deve ser evitado. Quando isso não for possível, pode ser aliviado por meio de mudanças de postura, uso de sapatos adequados ou por meio de apoios para partes do corpo, com o objetivo de reduzir as contrações estáticas dos músculos (9). Trabalhos anteriores sugerem que alterações na atividade muscular terão um efeito previsível sobre as forças de reação do solo. Tais trabalhos demonstra- ram que os ϐlexores plantares são músculos principais que contribuem para o impulso propulsor na última metade do apoio da marcha (5, 6). Dentre os melhores recursos para a prevenção, está a opção pelos calçados mais baixos, que dão mais sustentação e equilíbrio e não forçam determinadas Fisioter Mov. Redução dos dados Para análise eletromiográϐica, utilizou-se o pro- grama AqDanalysis, seguindo as recomendações do fabricante. Para normalização dos dados, utilizou-se o cálculo da Root Mean Square (RMS) e cálculo da porcentagem da CVMI. O processamento dos dados eletromiográϐicos incluiu retiϐicação e ϐiltragem dos sinais. Os dados foram coletados a uma frequência de 1000 Hz e foram ϐiltrados com um ϐiltro passa alta de 10 Hz e um ϐiltro passa baixa de 500 Hz. Após a colocação dos eletrodos, observou-se o “silêncio” muscular, e, em seguida, solicitou-se a con- tração dos músculos a serem analisados, durante os respectivos movimentos realizados, a ϐim de se con- ϐirmar o posicionamento correto do equipamento. Posteriormente, foi realizada a contração voluntária máxima isométrica (CVMI) de cada músculo, para normalização dos dados e posterior análise da ati- vidade muscular. Os dados foram armazenados no programa especíϐico. Instrumentos Após avaliação, a voluntária realizou um sorteio, por meio de envelope selado, para aleatorização dos planos de locomoção e dos calçados a serem utiliza- dos durante a coleta da atividade muscular, antes e após o protocolo de fadiga. Foi utilizado, para mensurar ϐlexibilidade, um go- niômetro universal (Baseline®, Aurora, IL, EUA), tendo duas hastes de 31,5 cm de comprimento e um fulcro com precisão de medida de um grau. A goniometria Fisioter Mov. 2012 jul/set;25(3):507-16 Moraes GFS, Antunes AP, Rezende ES, de Oliveira PCR. 510 ininterruptamente, até a sensação de queimação e de não suportar mais executar as repetições (16, 17). Após a aplicação desse protocolo, foram feitas as mesmas medidas pré-fadiga, sendo que a sequên- cia de calçados e as atividades foram previamente aleatorizadas. Todos os dados de análise da loco- moção nos diversos planos, utilizando os diversos calçados e de maneira descalça, foram armazenados no programa especíϐico do equipamento, para pos- terior análise. ininterruptamente, até a sensação de queimação e de não suportar mais executar as repetições (16, 17). Foi demarcada uma distância de seis metros para locomoção no plano. Durante a análise, desprezaram- -se os dois metros iniciais e ϐinais, a ϐim de se evitar interferências na coleta da eletromiograϐia durante a aceleração e a desaceleração da marcha. Após a aplicação desse protocolo, foram feitas as mesmas medidas pré-fadiga, sendo que a sequên- cia de calçados e as atividades foram previamente aleatorizadas. Todos os dados de análise da loco- moção nos diversos planos, utilizando os diversos calçados e de maneira descalça, foram armazenados no programa especíϐico do equipamento, para pos- terior análise. Uma avaliadora realizou a tricotomia dos respecti- vos pontos de inserção dos eletrodos, seguindo as re- comendações (14), e a higienização do local por meio de fricção com gaze e álcool. Os músculos analisados foram tibial anterior (TA), gastrocnêmio medial (GM) e lateral (GL) e eretores (ERET), todos bilateralmente. Foram colocados dois eletrodos na região em que a avaliadora detectava, por palpação, a região de maior contração muscular (14). Os eletrodos foram aϐixados a uma distância de dois centímetros de cada um. Foi colocado um eletrodo “terra” sobre o acrômio direito, como forma de padronização das medidas. Análise estatística Estatísticas descritivas, testes de normalidade (Shapiro-Wilk) e testes de igualdade de variância (Levene) foram realizados para todas as variáveis, utilizando-se o pacote estatístico Statistical Package of Social Science for Windows (SPSS), na sua versão 15.0 (SPSS Inc©, Chatanoga, IL). A atividade muscular foi avaliada durante a loco- moção em solo plano, subindo e descendo escadas (três degraus com 15 centímetros de altura cada), aclive e em declive (16). Os sapatos utilizados foram: salto alto tipo Chanel, salto baixo tipo Chanel, tênis, chinelo modelo Havaianas®, além de ter sido feita a avaliação sem sapatos, ou seja, descalço. Foram consi- derados sapato de salto alto aqueles com altura entre nove e onze centímetros e, para salto baixo, foram considerados aqueles entre cinco e sete centímetros. Para o tênis, foi necessário que ele tivesse uma eleva- ção de calcanhar de dois a quatro centímetros. Foi usado o teste Post Hoc com correção de Scheffé para comparações múltiplas, a ϐim de se determinar o efeito de interação dos calçados. Para análise da inϐlu- ência das situações de pré e pós-fadiga, nos diversos planos utilizados e com os diferentes calçados e de maneira descalça, utilizou-se o t Student para amos- tras pareadas. O nível de signiϐicância estabelecido foi de α igual a 5%. Foi utilizado foot switch para determinar as fases da marcha, flash sincronizado com o eletromiógrafo, a ϐim de se determinar o início do movimento e o registro da marcha. Um metrônomo foi utilizado para cadenciar e padronizar a velocidade de marcha em 110 passos por minuto, no plano. Fisioter Mov. 2012 jul/set;25(3):507-16 Resultados Após a primeira coleta, a participante foi enca- minhada para um protocolo de fadiga dos múscu- los do tríceps sural, bilateralmente. Esse protocolo correspondeu à participante executar movimentos de ϐlexão plantar concêntrica e excentricamente, Participaram deste estudo 15 voluntárias, com idade variando entre 18 e 31 anos (23,2 ± 3,36 anos), sedentárias. Das 15 voluntárias, 13 eram destras e duas eram sinistras. A descrição das voluntárias está demonstrada na Tabela 1. Fisioter Mov. 2012 jul/set;25(3):507-16 Avaliação da atividade eletromiográfica com ou sem o uso de diversos tipos de calçado, em diferentes planos de locomoção 511 Tabela 1 - Descrição das participantes Voluntária Idade Tamanho do calçado Dominância Atividade física 1 22 36 D Não 2 21 36 D Não 3 23 35 D Não 4 18 36 D Não 5 20 37 E Não 6 21 36 D Não 7 22 33 D Não 8 22 36 E Não 9 25 37 D Não 10 23 38 D Não 11 24 37 D Não 12 31 36 D Não 13 30 36 D Não 14 22 35 D Não 15 27 36 D Não Fonte: Dados da pesquisa. Neste estudo, foram analisados os músculos tibial anterior direito (TAD), tibial anterior esquerdo (TAE), gastrocnêmio medial direito (GMD), gastrocnêmio medial esquerdo (GME), gastrocnêmio lateral direito (GLD), gastrocnêmio lateral esquerdo (GLE), eretor da espinha direito (ERETD), eretor da espinha es- querdo (ERETE), os quais apresentaram diferenças signiϐicativas, demonstradas na Tabela 2. disso, observou-se, também, um aumento na ativi- dade eletromiográϐica pós-fadiga, exceto para o TAE descalço-subir rampa. O músculo TA controla o posicionamento do pé tanto na fase de apoio, com um aumento da estabili- dade da base de suporte do segmento em contato com o solo, quanto na fase de balanço, durante o movimen- to de dorsiϐlexão, que prepara o pé para o próximo apoio do calcâneo, durante o ciclo da marcha (19, 20). Isso pode ser um fator que justiϐicaria a diferença signiϐicativa encontrada nesses músculos ao subir e descer rampa, durante a fase de balanço da marcha. Fisioter Mov. 2012 jul/set;25(3):507-16 Discussão Os resultados da análise eletromiográϐica dos músculos avaliados demonstram que os músculos da perna apresentaram maior atividade eletromio- gráϐica na situação de pré-fadiga dos MMII, e os ere- tores da espinha, na situação pós-fadiga dos MMII, na maioria das condições e situações. Bogey, Perry e Gitter (20) descrevem uma rápida ativação do músculo TA, durante a fase inicial de ba- lanço da marcha, como forma de preparação para o descolamento do segmento distal do MI do solo, bem como na fase terminal do balanço, com o objetivo de manter a dorsiϐlexão do tornozelo, para aumentar a estabilidade para o início da nova fase de contato do segmento distal no duplo apoio. O músculo TA é o motor primário para a dorsiϐle- xão de tornozelo e ajuda em sua estabilização (18). No presente estudo, os músculos TAD e TAE apresen- taram diferenças signiϐicativas quando as voluntá- rias estavam descalças nas condições descer rampa e subir rampa, respectivamente, durante a fase de balanço da marcha, e com o uso de salto baixo, no plano, durante a fase de balanço da marcha. Além Com a coativação dos músculos da perna durante o apoio do segmento distal do MI e em função do protocolo de fadiga, uma maior ativação do TA tam- bém pode se manifestar como forma de recrutamento desse músculo, para maior estabilidade do segmento distal do MI durante os ciclos da marcha. Fisioter Mov. 2012 jul/set;25(3):507-16 Moraes GFS, Antunes AP, Rezende ES, de Oliveira PCR. Discussão 512 Tabela 2 - Comparação da atividade eletromiográfica muscular Músculo Calçado Condição Valor de p TAD - apoio MIE* Descalça Descer rampa 0,001 Salto baixo Plano 0,010 TAE - apoio MID** Descalça Subir rampa 0,005 Salto baixo Plano 0,028 GMD - apoio MID Chinelo Descer escada 0,007 Tênis Plano 0,049 GMD - apoio MIE Salto alto Plano 0,002 GME - apoio MID Descalça Descer escada 0,009 Descalça Subir rampa 0,039 Salto alto Subir escada 0,049 GME - apoio MIE Tênis Subir escada 0,032 Salto alto Plano 0,008 Salto alto Descer rampa 0,036 GLD - apoio MID Descalça Plano 0,007 GLE - apoio MIE Descalça Subir rampa 0,001 Salto baixo Subir rampa 0,036 ERETD - apoio MID Descalça Subir escada 0,011 Descalça Descer escada 0,001 Chinelo Plano 0,049 Chinelo Descer escada 0,001 Chinelo Subir rampa 0,001 Chinelo Descer rampa 0,015 Tênis Plano 0,008 Tênis Descer escada 0,022 Tênis Subir rampa 0,022 Tênis Descer rampa 0,016 Salto baixo Subir escada 0,033 Salto baixo Subir rampa < 0,001 Salto alto Plano 0,008 Salto alto Descer rampa 0,015 ERETD - apoio MIE Descalça Subir escada 0,034 Descalça Descer escada 0,014 Descalça Descer rampa 0,002 Chinelo Plano 0,002 Chinelo Subir escada 0,002 Chinelo Subir rampa < 0,001 Chinelo Descer rampa 0,002 Tênis Plano 0,016 Tênis Descer escada 0,001 Tênis Subir rampa < 0,001 Salto baixo Descer escada 0,001 Salto baixo Subir rampa 0,005 Salto baixo Descer rampa 0,041 Salto alto Subir escada 0,048 (Contin Tabela 2 - Comparação da atividade eletromiográfica muscular Fisioter Mov. Discussão O GLD apresentou diferença signiϐicativa na con- dição descalça, no plano, na fase de apoio da mar- cha. O GLE apresentou diferença signiϐicativa na condição descalça subindo rampa, na fase de apoio da locomoção. Não foram observadas diferenças signiϐicativas para os ERET bilateralmente: nas condições descalço no plano e ao subir rampa, tanto com apoio de MID quanto de MIE, e ao descer rampa com apoio de MID; com o uso de tênis, ao subir escadas, tanto com apoio de MID quanto de MIE, e descer rampa com apoio de MIE; com o uso de salto baixo, no plano, tanto com apoio de MID quanto de MIE; ao subir escadas com apoio de MIE, descer escadas com apoio de MID e descer rampa com apoio de MID; com o uso de salto alto, no plano apoio de MIE, subir escadas com apoio de MID e descer escadas com apoio de MIE. O Gastrocnêmio ajuda na propulsão durante a locomoção do indivíduo nas AVD e nas atividades proϐissionais. A prevalência de maior atividade desse músculo durante a fase de apoio da marcha pode ser compreendida em virtude da necessidade de manu- tenção da estabilidade da massa corporal e absorção e dissipação de forças reativas, pois atua tanto na fase do impulso quanto no apoio inicial para controle da ϐlexão do joelho, por ser um músculo biarticular (21). Em relação à atividade eletromiográϐica dos mús- culos eretores da espinha, houve diferença signiϐica- tiva nas condições de pré e pós-fadiga, na maioria das condições de estudos com todos os tipos de calçados e sem o uso deles (descalço). Tais achados contradizem os encontrados no estudo de Bendix et al. (24), no qual os músculos eretores da espinha foram avalia- dos e não foram observadas diferenças estatísticas signiϐicativas com o uso sapatos de salto alto. Gefen et al. (22) demonstraram um aumento na atividade eletromiográϐica do GL em relação ao GM com o uso de salto alto em mulheres que tinham hábi- tos regular e não regular de uso. Avaliaram, ainda, que a fadiga muscular do Gastrocnêmio foi preponderante na porção lateral em relação à medial durante o uso de sapato de salto alto. Discussão 2012 jul/set;25(3):507-16 Avaliação da atividade eletromiográfica com ou sem o uso de diversos tipos de calçado, em diferentes planos de locomoção 513 Tabela 2 - Comparação da atividade eletromiográfica muscular Músculo Calçado Condição Valor de p Salto alto Descer rampa 0,021 ERETE – apoio MID Descalça Subir escada 0,037 Descalça Descer escada 0,002 Chinelo Plano < 0,001 Chinelo Subir escada 0,015 Chinelo Descer escada 0,022 Chinelo Subir rampa 0,004 Chinelo Descer rampa 0,007 Tênis Plano < 0,001 Tênis Descer escada < 0,001 Salto baixo Subir rampa 0,011 Salto alto Plano 0,026 Salto alto Descer escada 0,011 Salto alto Subir rampa 0,022 Salto alto Descer rampa < 0,001 ERETE - apoio MIE Descalça Subir escada 0,03 Descalça Descer escada 0,003 Descalça Descer rampa 0,004 Chinelo Plano < 0,001 Chinelo Subir escada 0,025 Chinelo Descer escada 0,036 Chinelo Subir rampa 0,009 Chinelo Descer rampa < 0,001 Tênis Plano < 0,001 Tênis Descer escada < 0,001 Tênis Subir rampa 0,003 Salto baixo Descer escada 0,005 Salto baixo Subir rampa 0,001 Salto baixo Descer rampa 0,039 Salto alto Subir escada 0,004 Salto alto Subir rampa 0,004 Salto alto Descer rampa 0,001 Fonte: Dados da pesquisa (Conclusão) Tabela 2 - Comparação da atividade eletromiográfica muscular Fonte: Dados da pesquisa. Legenda: MIE – membro inferior esquerdo; MID – membro inferior direito. Legenda: MIE – membro inferior esquerdo; **MID – membro inferior direito. O GME apresentou diferenças signiϐicativas na condição descalça descendo escadas e subindo ram- pa, na fase de apoio de MID da locomoção, com o uso de tênis subindo escadas, na fase de apoio de MIE, de salto alto no plano e descendo rampa, na fase de apoio de MIE, e subindo escadas, na fase de apoio de MID. Observou-se, também, um aumento da Em relação à ativação do músculo GMD, observou- -se diferenças signiϐicativas quando as voluntárias estavam de chinelo descendo escadas e de tênis no plano, na fase de apoio, e de salto alto no plano, na fase de balanço da marcha. Observou-se, também, um aumento na atividade eletromiográϐica pós-fadiga, apenas com o uso do chinelo. Fisioter Mov. 2012 jul/set;25(3):507-16 Moraes GFS, Antunes AP, Rezende ES, de Oliveira PCR. 514 ao subir e descer rampa com apoio de MID, e com uso de salto baixo, ao subir escadas com apoio de MID. atividade eletromiográϐica pós-fadiga, apenas com o uso do tênis. Discussão A análise eletromiográϐica indicou menor ativi- dade dos ERET quando comparado aos músculos da perna na situação de pré-fadiga, em função da menor exigência dos músculos lombares após as voluntárias alcançarem o equilíbrio necessário, calçando os sa- patos de salto alto. Pelo fato de ter sido utilizado o protocolo de fadiga, pode-se inferir que os eretores da espinha foram mais recrutados para manter a pos- tura durante a locomoção, compensando a redução da ativação dos outros músculos avaliados. Como sugerido por Lee et al. (25), os músculos eretores se adaptam à situação de fadiga compensando a redução da ativação dos músculos dos MMII. Neste estudo, as porções mediais do músculo Gastrocnêmio apresentaram maior atividade ele- tromiográϐica que as porções laterais. Isso pode ser explicado pelo fato de o movimento de inversão dos pés aumentar com o desequilíbrio corporal acentua- do pelo uso de sapatos com pouca estabilidade, como chinelo e salto alto, e pelo pouco uso de sapatos com o calcanhar elevado (18, 22). Os ERET são importantes músculos antigravita- cionais que atuam predominantemente de forma es- tática. Os tecidos passivos, como cápsulas, ligamentos e discos da coluna vertebral, são mais sobrecarre- gados após os elementos ativos tornarem-se menos efetivos, em detrimento da fadiga muscular (23). O chinelo foi o calçado que promoveu maior ativi- dade eletromiográϐica dos músculos eretores da es- pinha, tanto à direita como à esquerda. Tal fato pode ser justiϐicado por ser um calçado instável, sem apoio nas laterais do pé e na região posterior do calcâneo, quando comparado aos outros calçados utilizados. A maioria das condições e situações pré e pós- -fadiga dos músculos da perna, avaliadas com o uso dos diversos sapatos e sem o uso deles (descalço), apresentou diferenças signiϐicativas para os músculos ERET bilateralmente. Os ERETE isoladamente apresentaram diferenças signiϐicativas apenas com o uso de chinelo subindo esca- das, na fase de apoio de MID, e descendo escadas, na fase de apoio de MIE da locomoção. Com o uso de salto alto, apresentou diferenças signiϐicativas ao descer escadas, com apoio do MID, e ao subir rampa, tanto com o apoio do MID quanto do MIE. Os ERETD isoladamente apre- sentaram diferenças signiϐicativas com o uso de tênis, Fisioter Mov. 2012 jul/set;25(3):507-16 Relevância clínica O uso regular de salto, principalmente o alto, pode levar ao aprendizado motor da mulher que usa esse tipo de calçado com frequência, pois a repeti- ção de uma tarefa é um fator determinante para a Fisioter Mov. 2012 jul/set;25(3):507-16 Avaliação da atividade eletromiográfica com ou sem o uso de diversos tipos de calçado, em diferentes planos de locomoção 515 4. Hong WH, Lee YH, Chen HC, Pei YC, Wu CY. Inϐluence of heel height and shoe insert on comfort perception and biomechanical performance of young female adults during walking. Foot Ankle Int. 2005;26(12):1042-8. PMid:16390637. plasticidade cortical, que, por sua vez, gera automa- tismo do gesto (26). Consequentemente, pode haver menor recrutamento de ϐibras para realizar uma ta- refa e gasto energético reduzido. Condições adaptativas, tais como fraqueza, en- curtamento e desequilíbrios musculares, associa- das ao uso regular de calçados com salto têm sido amplamente questionadas em relação à incidência de lesões como entorses de joelho e tornozelo, além de alterações posturais, como aumento da lordose lombar ou sua retiϐicação. 5. Neptune R, Zajac F, Kautz S. Muscle force redistributes segmental power for body progression during walk- ing. Gait Posture. 2004;19(2):194-205. doi:10.1016/ S0966-6362(03)00062-6. 6. Liu MQ, Anderson FC, Pandy MG, Delp SL. Muscles that support the body also modulate forward progression during walking. J Biomech. 2006;39(14):2623-30. doi:10.1016/j.jbiomech.2005.08.017. Portanto, medidas preventivas, tais como imple- mentação de um programa de atividade ϐísica regular, com reforço muscular e alongamentos, devem ser prescritas e orientadas às mulheres com esse hábito, além de serem implantadas em empresas, a ϐim de se prevenirem lesões e quadros álgicos. Dessa forma, poder-se-ia evitar a diminuição na capacidade pro- dutiva, bem como diminuir a taxa de absenteísmo, desonerando o sistema de saúde vigente no país. 7. Neptune R, Kautz S, Zajac F. Contributions of the individual ankle plantar ϐlexors to support, for- ward progression and swing initiation during walk- ing. J Biomech. 2001;34(11):1387-98. doi:10.1016/ S0021-9290(01)00105-1. 8. Cappellini G, Ivanenko Y, Poppele V, Lacquaniti F. Motor patterns in human walking and running. J Neurophysiol. 2006;95(6):3426-37. doi:10.1152/jn.00081.2006. 9. Moraes GFS, Antunes AP, Rezende ES, Oliveira PCR. Uso de diferentes tipos de calçados não interfere na postura ortostática de mulheres hígidas. Fisioter Mov. 2010;23(4):565-74. Conclusão A avaliação eletromiográϐica demonstrou, na maioria das condições utilizadas, uma maior ativi- dade dos músculos da perna na condição pré-fadiga, principalmente com sapato de salto alto e chinelo, provavelmente pela instabilidade e pelo não hábito de uso regular desses calçados. 10. Potério-Filho J, Silveira F, Potério G, Almeida F, Mene- zes F. The effect of walking with high-heeled shoes on the leg venous pressure. Angiology. 2006;57(4):424-30. doi:10.1177/0003319706290744. 11. Anjos L. Body mass index as a tool in the nutritional assessment of adults: a review. Rev Saúde Pública. 1992;26(6):431-6. Na situação pós-fadiga dos MMII, houve um de- clínio nas atividades dos músculos da perna e maior recrutamento dos músculos eretores, justiϐicados pela necessidade de mais estabilidade da coluna e da pelve durante a locomoção de mulheres jovens. 12. Gajdosik R, Bohannon R. Clinical measurement of range of motion. Review of goniometry emphasizing reli- ability and validity. Phys Ther. 1987;67(12):1867-72. PMid:3685114. 13. Rothstein J, Miller P, Roettger R. Goniometric reliability in a clinical setting. Elbow and knee measurements. Phys Ther. 1983;63(10):1611-5. PMid:6622536. Fisioter Mov. 2012 jul/set;25(3):507-16 Referências 1. Frey C. Foot health and shoewear for women. Clin Orthop. 2000;372(3):32-44. PMid:7978696. 14. Wirth DP, Cram JR. Multisite surface electromyography and complementary healing intervention: a comparative analysis. J Altern Complement Med. 1997;3(4):355-64. doi:10.1089/acm.1997.3.355. 2. Snow R, Williams K. High heeled shoes: their effect on center of mass position, posture, three-dimensional kine- matics, rear foot motion and ground reaction forces. Arch Phys Med Rehabil. 1994;75(5):568-76. PMid:8185452. 2. Snow R, Williams K. High heeled shoes: their effect on center of mass position, posture, three-dimensional kine- matics, rear foot motion and ground reaction forces. Arch Phys Med Rehabil. 1994;75(5):568-76. PMid:8185452. 15. Fridlund A, Cacioppo J. Guidelines for human electromyo- graphic research. Psycophysiology. 1986;23(5):567-98. doi:10.1111/j.1469-8986.1986.tb00676.x. 3. Bertoncello D, De Sá CSC, Calapodópulos AH, Lemos VL. Equilíbrio e retração muscular em jovens estu- dantes usuárias de calçado de salto alto. Fisioter Pesq. 2009;16(2):107-12. 16. Silva S, Gonçalves M. Comparação de protocolos para veriϐicação da fadiga muscular pela eletromiograϐia de superϐície. Motriz. 2003;9(1):51-8. Fisioter Mov. 2012 jul/set;25(3):507-16 Moraes GFS, Antunes AP, Rezende ES, de Oliveira PCR. 516 17. Silva S, Gonçalves M. Análise da fadiga muscular pela amplitude do sinal eletromiográϐico. Rev Bras Ciência Mov. 2003;11(3):15-20. 23. Roy SH, De Luca CJ, Emley M, Oddsson LI, Buijs RJ, Levins JA, et al. Classiϐication of back muscle impairment based on the surface eletromyographic signal. J Rehabil Res Dev. 1997;34(4):405-14. PMid:9323644. 18. Henderson P, Piazza S. A biomechanical evaluation of standing in high-heeled shoes; [citado 12 ago. 2012]. Disponível em: www. gradsch.psu.edu. 24. Bendix T, Sorensen SS, Klausen K. Lumbar curve, trunk muscles and line of gravity with different heel heights. Spine. 1984;9(2):223-7. doi:10.1097/00007632- 198403000-00016. 19. Li J, Hong Y. Kinematic and electromyographic analysis of the trunk and lower limbs during walking in negative- heeled shoes. J Am Podiatr Med Assoc. 2007;97(6):447- 56. PMid:18024839. 25. Lee K, Shieh J, Matteliano A, Smiehorowski T. Electro- myographic changes of leg muscles with heel lifts in women: therapeutic implications. Arch Phys Med Re- habil. 1990;71(1):31-3. PMid:2297307. 25. Lee K, Shieh J, Matteliano A, Smiehorowski T. Electro- myographic changes of leg muscles with heel lifts in women: therapeutic implications. Arch Phys Med Re- habil. 1990;71(1):31-3. PMid:2297307. 20. Bogey RA, Perry J, Gitter AJ. An EMG-to-force processing approach for determining ankle muscle forces during normal human gait. IEEE Trans Neural Syst Rehabil Eng. 2005;13(3):302-10. doi:10.1109/TNSRE.2005.851768. 26. Janacsek K, Nemeth D. Predicting the future: from im- plicit learning to consolidation. Int J Psychophysiol. Referências 2012;83(2):213-21. doi:10.1016/j.ijpsycho.2011.11.012. 26. Janacsek K, Nemeth D. Predicting the future: from im- plicit learning to consolidation. Int J Psychophysiol. 2012;83(2):213-21. doi:10.1016/j.ijpsycho.2011.11.012. 21. Stefanyshyn D, Nigg B, Fisher V, O'Flynn B, Liu W. The inϐluence of high heeled shoes on kinematics, kinetics, and muscle emg of normal female gait. J Appl Biomech. 2000;16(3). PMid:11757569. Recebido: 16/08/2010 Received: 08/16/2010 Recebido: 16/08/2010 Received: 08/16/2010 22. Gefen A, Megido-Ravid M, Itzchak Y, Arcan M. Analysis of muscular fatigue and foot stability during high-heeled gait. Gait Posture. 2002;15(1):56-63. doi:10.1016/ S0966-6362(01)00180-1. Aprovado: 15/05/2012 Approved: 05/15/2012 Aprovado: 15/05/2012 Approved: 05/15/2012 Fisioter Mov. 2012 jul/set;25(3):507-16
https://openalex.org/W3180331899	https://www.nature.com/articles/s41598-021-93385-4.pdf	English	null	Geochemical characterization and assessment of fluoride sources in groundwater of Siloam area, Limpopo Province, South Africa	Scientific reports	2,021	cc-by	15,858	Tobiloba Onipe1, Joshua N. Edokpayi1* & John O. Odiyo1,2 Siloam’s groundwater is reportedly characterized by high fluoride. In response to the reported high incidence of dental fluorosis in the area, sources of elevated fluoride in the groundwater were investigated. Total fluoride (TF) was determined using Ion Chromatograph and Fluoride Ion Selective Electrode. The mineral composition of rocks and soils were determined using X-ray Fluorescence and X-ray diffraction, respectively. Results revealed that groundwater fluoride concentration ranged from 3.92 to 4.95 mg/L. Na-Cl water type was found to be dominant in the water samples. TF content of the rocks and soils ranged from 10 to 2000 mg/L. Leachates were obtained by making a slurry from the samples at a predetermined temperature and time. TF in leachates ranged between 0.27 and 14.88 mg/L and 0.05 to 10.40 mg/L at induced, and non-induced temperatures, respectively. The possible source of fluoride has been previously inferred to be caused by fluorite minerals occurring at greater depth. However, this study proves that fluoride decreases with depth and the elevated fluoride in the groundwater is caused by smectite-kaolinite clay, muscovite and chlorite minerals abundant in the area. Geothermal temperature exhibited by the groundwater in the area is a major factor enhancing the release of fluoride from the clay materials. Water is an important basic resource necessary for human development and economic growth. The supply of clean and safe water over the last decade has improved progressively globally1. Despite this recorded success, millions of people are not connected to water supply infrastructure and often resort to several alternative sources for their domestic water needs2,3. One of such sources include the exploitation of groundwater4. Groundwater is the largest deposit of freshwater on earth and is a dependable alternative source of water with more than 75% of African population depending on it for survival5. However, there are various misconceptions about groundwater that it is free from chemical contamination and pathogens owing to its aesthetic property. Over the years, studies have shown that groundwater is exposed to various chemical contaminants from natural geological processes and anthropogenic activities6–8. Groundwater are often exploited in the form of natural springs, wells and drilling of boreholes. p g g Several contaminants have been reported in the groundwater which are of potential risk to public health. Amongst which are the presence of elevated levels of arsenic, lead, cadmium, mercury, fluoride, nutrients and microorganisms9–11. Some of these contaminants are more linked to geogenic sources than others. www.nature.com/scientificreports www.nature.com/scientificreports 1Department of Hydrology and Water Resources, University of Venda, Private bag X5050, Thohoyandou 0950, South Africa. 2DVC: RICl, Vaal University of Technology, Vanderbiljpark, South Africa. email: Joshua.Edokpayi@ univen.ac.za Scientific Reports \| (2021) 11:14000 Geochemical characterization and assessment of fluoride sources in groundwater of Siloam area, Limpopo Province, South Africa OPEN Tobiloba Onipe1, Joshua N. Edokpayi1 & John O. Odiyo1,2 Tobiloba Onipe1, Joshua N. Edokpayi1* & John O. Odiyo1,2 Fluoride is believed to be caused majorly by fluoride bearing minerals with little or no contribution from human activities. However, groundwater contamination from lead, nutrients and microorganisms are more linked to anthropo- genic activities such as mining and agriculture10,11. g g g Fluoride is very important in the healthy development of the skeletal and dental framework of the body, if present in levels < 0.5 mg/L, can lead to dental caries while concentration exceeding 1.5 mg/L can lead to fluorosis and no fluorosis diseases. The levels of fluoride vary globally and various climatic, hydrological and geochemical properties often determine and influence its levels in groundwater. The consumption of fluoride rich water has been linked to various public health burden notable among which are dental and skeletal fluorosis12–14. Cases of several non-fluorosis diseases such as Alzheimer’s disease, loss of mobility, infertility, hearing difficulty, retarded growth, low intelligence quotient and cancer have also been linked to the consumption of fluoride rich water9,15–20. 1Department of Hydrology and Water Resources, University of Venda, Private bag X5050, Thohoyandou 0950, South Africa. 2DVC: RICl, Vaal University of Technology, Vanderbiljpark, South Africa. email: Joshua.Edokpayi@ univen.ac.za \| https://doi.org/10.1038/s41598-021-93385-4 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 1. Map of the study area (Figure drawn using ArcGIS version 10.4, licensed to the University of Venda, South Africa). Figure 1. Map of the study area (Figure drawn using ArcGIS version 10.4, licensed to the University of Vend South Africa). Occurrence of elevated fluoride in groundwater is an emerging world-wide threat that affects over 200 million people worldwide and over 80 million people in East Africa21.hl The occurrence of high levels of fluoride exceeding the permissible level of 1.5 mg/L by the World Health Organization has been recorded in many regions of the world. The East Africa rift valley system have been renowned for high levels of fluoride in groundwater22–24. In most parts of South Africa, elevated levels of fluoride have been reported in groundwater. The Northern region of the Limpopo Province which is semi-arid consist of numerous springs which are also known to contain high fluoride concentration9,25,26.f p g gl Globally, several efforts have been channeled towards the monitoring of groundwater quality for compliance to regulatory standard due to the number of people that depends on it for survival27–29. Fluoride compliance study have been greatly reported in literature7,8,26. However, very few studies have reported the source of fluoride in groundwater9,18,25. Tobiloba Onipe1, Joshua N. Edokpayi1 & John O. Odiyo1,2 The most important factor controlling the occurrence of fluoride naturally in groundwater is the rock types (geology) of the area. Fluorine has been reported in all the major rock types (igneous, sedimen- tary and metamorphic)9,18. Notable geogenic sources that have been reported include fluoride bearing minerals such as fluorite, villiaumite, apatite, biotite, amphibole, micas, topaz, cryolite, muscovite and fluorspar9,18,25,30,31. ll In Siloam and the Northern part of South Africa, studies from our research group and other researchers have shown the occurrence of elevated fluoride concentrations in wells, boreholes (cold and hot) and geothermal springs25,26,31–34. Siloam area is known for a large-scale occurrence of dental fluorosis amongst children and adults and according to Odiyo and Makungo35, over 85% of the population in the study area could be affected by dental fluorosis. The groundwater chemistry of this region has not been fully explored or reported. Therefore, in this study, we report mainly on the occurrence of fluoride bearing minerals in the region and some factors that could favor its dissolution. Description of the study areah p y Location. The study area (Fig. 1) is located within Makhado Municipality, Vhembe District in Limpopo Province of South Africa. This includes the northern flank of Tswime Mountain where Mphephu thermal spring is located. It is 60 km northeast of Makhado and is approximately 45 km west of Thohoyandou. Siloam falls under surface water and groundwater quaternary catchment A80A of the Nzhelele River catchment, which is in the northern region of Limpopo Province, South Africa36. The Nzhelele River flows in the northwest direction, towards Nzhelele Dam26. The prevailing seasons are winter and summer, with average winter temperatures rang- ing between 16 and 22 °C and average summer temperatures varies between 22 to 40 °C37. Siloam experiences seasonal rainfall, ranging from 350 – 400 mm per annum in the summer months of September to March36,38. Rainfall in Siloam is largely influenced by its position on the leeward side of Soutpansberg mountain which plays an important role in the groundwater recharge of the area35. Evaporation rate is higher than the precipitation rate at 1300–1400 mm/annum. Siloam is dominated by two major geothermal springs and other hot private and https://doi.org/10.1038/s41598-021-93385-4 Scientific Reports \| (2021) 11:14000 \| www.nature.com/scientificreports/ community borehole Boreholes in the study area varies in depth and are usually within the range of 65–85 m Figure 2. Geological map of the study area32, permission granted by Dr. Durowoju). Figure 2. Geological map of the study area32, permission granted by Dr. Durowoju). Figure 2. Geological map of the study area32, permission granted by Dr. Durowoju). community borehole. Boreholes in the study area varies in depth and are usually within the range of 65–85 m and the mean depth to groundwater is about 15–25 m33. ommunity borehole. Boreholes in the study area varies in depth and are usually within the range of 65–85 m nd the mean depth to groundwater is about 15–25 m33. Geology. Siloam lies within the Soutpansberg group (Fig. 2). Soutpansberg group is a Mokolian age exten- sively faulted succession that provides a link between the pre-and post-Bushveld age (younger than 1800 mya) rocks39. The Soutpansberg succession is an east to west trending asymmetrical rift overlying the Palala shear belt39. Soutpansberg group is subdivided into six formations namely: Tshifhefhe formation, Sibasa formation, Fundudzi formation, Wyllie’s formation, Musekwa formation and Nzhelele formation40,41. The Palala shear belt separates the Kaapval craton in the South and the Limpopo belt in the North39. Description of the study areah The area has been through differ- ent post-deposition tectonic and erosional activities. The evolution of Soutpansberg started with the deposition of basaltic lava as a result of volcanic activities, followed by the deposition of syn-rift sequence of sedimentary rock. The area was subjected to an extensive erosional period of non-deposition after which resistant sandstone- quartzite was deposited42. Unconformable deposition of Karoo supergroup sediments occurred and altogether, went through the process of block-faulting43. The sequence is best developed from north of Siloam fault to Musekwa mountains44. Materials and methods The EC of the samples were below 500 µs/cm hence dilution was not done. Trace elements analysis for groundwater and rock samples. Major cations in the acidified ground- water water samples were analyzed using Inductively coupled plasma optical emission spectrophotometer (ICP- OES) while Inductively coupled plasma mass spectrophotometer (ICP-MS) were used for the analysis of trace metals. Similarly, Trace and major metals in the soil and rock samples were analyzed using the same analytical instruments outlined above after microwave digestion. The procedure reported by Pais and Jones47 for soil and rock sample digestion were adhered to. Briefly: The soil/rock samples were digested using a microwave digestion system (SR ISO 11,466: 1999). Approximately 1.0 g of pre-treated samples were digested with 9 mL HNO3 and 1 mL H2O2. The solutions were allowed to stay overnight at room temperature and then, placed in the microwave for 30 min, followed by cooling. The solutions were diluted to 50 mL with distilled water. Preparation of soil and rock samples. The rocks and soil samples for X-ray diffractometer (XRD) analy- ses were prepared according to the standardized PANalytical backloading system, which provides nearly random distribution of the particles, while the samples for X-ray fluorescence (XRF) were prepared as pressed powders48. The rock samples were split, washed, dried, crushed and pulverized. Splitting into chips was done by a hydraulic splitter after which the split chip was washed and treated with ultrasound wave to remove any loose impure particles. After washing, the samples were dried overnight in an oven to remove excess water introduced dur- ing washing of the samples. A jaw crusher was used to crush the samples which were further reduced to finer grain (< 40 μm in diameter) by pulverizing them for about five (5) min using a Retsch RS 200 miller. The milled samples were transferred to a paper bag and oven dried at 110 °C for 6 h. After oven drying, the samples were further milled to less than 20 µm48.l µ Samples for total fluoride in soil and rocks were prepared by weighing 0.2 g of the samples in a nickel cru- cible, 2 g of CaCO3 and Na2O2 was added, respectively. The mixture was fused manually to aid leaching, using a low flame bunsen burner in a fume box. Auto fusion was avoided to prevent burning of the nickel crucibles and ensure that Na2O2 does not get moist. Materials and methods Sampling of soil, water and rock. One soil and rock samples were collected from the surface in the study area while twelve borehole cuttings from the subsurface were sampled for analysis. Four water samples were col- lected across the area. The samples were collected using plastic cups as recommended by Harvey45. For each loca- tion, the samples were collected three times in a 1 L sampling bottle and were transported to the laboratory for analysis. As suggested by Weaver et al.46, the bottles were pre-rinsed with the water to be sampled, to avoid cross contamination. The groundwater samples for anion analysis were preserved in a refrigerator at a temperature of 4 °C and analyzed within seven days of collection. However, the samples for cation and trace metals analysis were preserved with concentrated nitric acid. Table 1 below summarizes the sample types and number. Measurement of basic water quality parameters. The pH, electrical conductivity (EC) and tempera- ture were measured insitu using a multimeter (Multi 340i/SET). The instrument was calibrated prior to measure- ment in adherence to the manufacturer’s guideline. https://doi.org/10.1038/s41598-021-93385-4 Scientific Reports \| (2021) 11:14000 \| www.nature.com/scientificreports/ Table 1. Description of Sample types. N/A not available. Sample type Sample ID Depth (m) Location No of samples Water samples Monitoring borehole 1 BH1 > 60 22° 53′ 34.008″ S 30° 11′ 47.22″ E 1 Monitoring borehole 2 BH2 > 60 22° 53′ 38.148″ S 30° 11′ 18.52″ E 1 Private Borehole 1 H1 > 60 22° 53′ 39.43" S 30° 11′ 43.94" E 1 Private Borehole 2 H2 > 60 22°53′43.39"S 30°11′32.98"E 1 Rocks and soil samples Surface Soil Sample S1 N/A 22° 53′ 34.83″ S 30° 11′ 45.56″ E 1 Surface Rock Sample 1 SR2 N/A 22° 54′ 22.8″ S 30° 10′ 44.0″ E 1 Surface Rock Sample 2 SR3 N/A 22° 52′ 02.5″ S 30° 12′ 09.7″ E 1 Borehole 1 Cuttings X 1–65 22° 53′ 34.008″ S 30° 11′ 47.22″ E 6 Borehole 2 Cuttings Y 1–40 22° 53′ 38.148″ S 30° 11′ 18.528″ E 6 Table 1. Description of Sample types. N/A not available. Major anions analysis. Groundwater samples were analyzed for major anions content (fluoride, nitrate, phosphate, bicarbonate) using an Ion Chromatograph (Dionex Model DX 500). Prior to analysis, the samples were filtered using 0.45 μm syringe filter. The samples were placed in vails associated with an auto sampler1. www.nature.com/scientificreports/ ions, forming HF or HF2, which cannot be detected by the fluoride electrode49. Likewise, in a solution with pH above 9, the electrode responds to hydroxide ion as well as to fluoride ion giving an exaggerated reading. Sam- ples for leachable fluoride in soil and rocks were prepared by mixing 10 g of the sample with 100 mL of distilled water at a ratio of 1:10. The samples were prepared in duplicate to ensure that each sample is represented under induced temperature and room temperature. The first set of samples were shaken in a warm bath at normal room temperature for 6 h48. The second set of samples were shaken in a warm bath at a temperature of 42 °C. Because of the clay contents of the sample the mixture was centrifuged for 10 min at a speed of 3000 rpm. The centrifuged samples were decanted and filtered using a 9 cm filter paper. The resultant mixture was then further filtered using a 40 µm syringe filter to ensure that all impurities that could block the IC column were removed. The EC of the samples were below 500 µs/cm hence dilution was not required. Lithology and mineral phase identification. The minerals in the rocks and soil samples from Siloam were identified and quantified using X-ray diffraction method of analysis. The samples were analyzed using a PANalytical X’Pert Pro powder diffractometer in θ–θ configuration with an X’Celerator detector and variable divergence and fixed receiving slits with Fe filtered Co-Kα radiation (λ = 1.789 Å). The phases were identified using X’Pert Highscore plus software. The relative phase amounts (weight%) were estimated using the Rietveld method (Autoquan Program). The angles of the peaks were used to identify the mineral phase while the intensi- ties of the peaks indicated the abundance of each mineral50. Total oxide analysis. The total rock and soil oxides were measured using X-ray Fluorescence analytical method. The ARL Perform’X Sequential XRF instrument with Uniquant software was used for the analyses51. The software analysed for all elements in the periodic Table between Na and U, but only elements found above the detection limits were reported51. The intensities for all elements were corrected automatically for line inter- ference and absorption effects due to all the other elements using the fundamental parameter method49. Total fluoride analysis. www.nature.com/scientificreports/ Total fluoride analysis were performed on the groundwater samples as well as the leached samples from rocks and soil. The groundwater analysis was carried out using Fluoride Ion-Selective electrode Orion Versastar Advanced Electrochemistry meter, while the fluoride in solids were analyzed using Metrohm fluoride meter with reference electrode48. Calibration standards 0.1 ppm, 1 ppm, 10 ppm and 100 ppm were used to calibrate the equipment. Orion TISAB II was used as the ionic strength adjustment buffer and the TISAB II was mixed in equal volume of 50 mL with 50 mL of samples and buffer. The TISAB used were pre- mixed with standard at ratio 1:1. Labotec Magnetic stirrer was used to ensure that the mixture of TISAB and sample was thorough and accurate during measurement. Leaching experiment. A form of mini-leaching experiment was used to determine the leachability of fluo- ride and other major cation and anions from the rocks and soil samples. Ion chromatography (IC) method was used for the analysis of both normal samples and the temperature induced samples. The IC was stabilized for 15 min prior to analysis48. The eluent used was Dionex AS22 which is made up of 45 mM Na2CO3 and 1.4 mM NaHCO3. The pH, EC and alkalinity of the water, soil and rock samples were pre-measured using Mantech Titra- sip Autotitrator to ensure that the unknown samples fall within the recommended pH and EC limit for using Ion chromatograph1. The analysis was carried out using Dionex Model DX 500 Ion Chromatograph. Statistical analysis. Experimental data were analyzed using the Statistical Package for Social Sciences (SPSS) (IBM Version 22) and Microsoft Excel 2013 (Microsoft Corp., Santa Rosa, CA). Descriptive statistics using tables, charts and graphs were used to present the chemical analysis data of the groundwater. Data recorded from the geochemical processes were subjected to Piper chart to identify the main chemical compositions of the groundwater. Correlation analysis was performed using a suitable package in SPSS. Materials and methods 250 mL plastic beakers were pre-washed in a heated Eco bath using concentrated HCl. The heated crucibles were transferred into 250 mL plastic beakers where 50 mL of de-ionized water and 8 mL of HCl were added49. The mixtures were covered with a plastic lid and placed on warm bath until the samples were leached out of the crucibles. The crucibles were washed with de-ionized water to make sure that all the leachate was removed from the crucibles. The solutions were transferred into 100 mL volumetric flask and diluted to mark. Blank was prepared by fusing 2 g of CaCO3 and Na2O2. The fused mixture was transferred into a 250 mL beaker and 100 mL of de-ionized water and 16 mL of concentrated HCl were added48. The mixture was allowed to leach out and the crucible was rinsed with distilled water. The leachate was transferred into a 200 mL volumetric flask and filled to mark with de-ionized water. Calibration was carried out using 0.1 ppm, 1 ppm, 10 ppm, 100 ppm and 1000 ppm fluoride standards. The standards, samples and blanks were all mixed in equal proportion with TISAB II in a 1:1 ratio.fh p p Calibration of pH meter was done using buffers 6.00 and 4.00. The Slope was 97.9% which fell within the recommended slope limit of 95 to 105%. The pH of the samples and blanks were adjusted to 6.00 using 50% NaOH and HCl. The pH was adjusted because at pH below 5, hydrogen ions complex a portion of the fluoride https://doi.org/10.1038/s41598-021-93385-4 Scientific Reports \| (2021) 11:14000 \| www.nature.com/scientificreports/ Results and discussion Fluoride levels in groundwater samples of the study area. Fluoride levels in the groundwater sam- ples ranged between 3.92–4.95. These levels exceeded the South African National standards (SANS) for drinking water and the guidelines (1.5 mg/L) of the52,53. The levels recorded can lead to dental and skeletal fluorosis on the consumers of this waters and they are also at risk of non-fluorosis diseases. Previous studies by Odiyo and Makungo35 have reported fluoride levels in the range of 1.7–5.6 mg/L. Similarly, a more recent publication by the authors also reported fluoride levels exceeding 1.5 mg/L. Table 2 presents the fluoride levels results of this study and other related studies on groundwater around the study area. pH levels in groundwater of the study area. The pH of all the samples were in the range of 8.10–9.19 and complied with the South African (2014) National Standards for drinking water of 5.0 to 9.5. The samples with higher temperature (45–48 °C) in this study recorded slightly higher pH (8.86–9.10) than those with a lower temperature (25–27 °C) but this difference was not statistically significant (p > 0.05). Odiyo and Makungo35 reported pH levels in the range of 6.9–8.7 for groundwater around Siloam area. Durowoju et al.32 reported mean pH levels between 7.89 and 9.39 for geothermal springs in Siloam, Tshipise, Mpephu and Sagole areas of North- ern Limpopo province. High groundwater pH is a proxy indicator of long residence time of groundwater-rock interaction54,55. Although pH affects fluoride mineralization differently, it is widely reported that alkaline pH favors the dissolution of fluoride from most fluoride bearing minerals9,25. Previous results on the monitoring of fluoride in the groundwater of Vhembe district in Limpopo Province shows the prevalence of alkaline pH26,32. In https://doi.org/10.1038/s41598-021-93385-4 Scientific Reports \| (2021) 11:14000 \| www.nature.com/scientificreports/ Table 2. Mean fluoride, pH, temperature and calcium levels of groundwater in the study area. Nr not reported. Results and discussion Sample code Groundwater type Study location Fluoride (mg/L) pH Temperature (oC) Ca (mg/L) Reference H1 Borehole Siloam 4.55 ± 0.00 8.86 ± 0.01 48.00 ± 0.01 3.54 ± 0.01 This study H2 Borehole Siloam 4.95 ± 0.01 9.19 ± 0.06 45.00 ± 0.00 0.82 ± 0.00 This study BH1 Borehole Siloam 4.50 ± 0.00 8.17 ± 0.00 25.00 ± 0.01 27.80 ± 1.71 This study BH2 Borehole Siloam 3.92 ± 0.01 8.10 ± 0.01 27.00 ± 0.00 12.80 ± 0.61 This study BH3 Borehole Siloam 5.60 ± 0.85 8.97 ± 0.49 Nr 0.33 ± 0.71 Odiyo and Makungo33 BH4 Borehole Siloam 5.76 ± 0.55 8.26 ± 0.40 Nr 0.08 ± 0.07 Odiyo and Makungo33 BH5 Borehole Siloam 3.37 ± 1.56 7.54 ± 0.18 Nr 0.47 ± 0.46 Odiyo and Makungo33 BH6 Borehole Siloam 1.51 ± 0.12 7.34 ± 0.27 Nr 1.41 ± 1.25 Odiyo and Makungo33 BH7 Borehole Siloam 1.44 ± 0.18 7.17 ± 0.13 Nr 1.04 ± 0.71 Odiyo and Makungo33 BH8 Borehole Siloam 1.49 ± 0.06 7.44 ± 0.44 Nr 1.62 ± 2.43 Odiyo and Makungo33 BH9 Borehole Siloam 5.59 ± 0.16 8.11 ± 0.35 Nr 0.07 ± 0.02 Odiyo and Makungo33 BH10 Borehole Siloam 5.37 ± 0.48 8.62 ± 0.47 Nr 0.07 ± 0.06 Odiyo and Makungo33 BH11 Borehole Siloam 4.25 ± 0.90 7.30 ± 0.30 Nr 2.95 ± 2.58 Odiyo and Makungo33 BH12 Borehole Siloam 2.21 ± 1.24 7.35 ± 0.16 Nr 1.81 ± 1.65 Odiyo and Makungo33 BH13 Borehole Siloam 5.11 ± 0.49 7.44 ± 0.22 Nr 1.30 ± 0.68 Odiyo and Makungo33 BH14 Borehole Siloam 5.10 ± 1.09 8.70 ± 0.33 35.30 ± 1.53 4.40 ± 2.42 Odiyo and Makungo35 BH15 Borehole Siloam 1.70 ± 0.60 6.90 ± 0.01 26.90 ± 0.64 70.40 ± 13.59 Odiyo and Makungo35 GS1 Geothermal spring Siloam 5.50 ± 0.35 8.40 ± 0.15 63.86 ± 18.19 2.40 ± 0.86 Odiyo and Makungo35 GS2 Geothermal spring Siloam 6.51 ± 0.08 9.39 ± 0.06 67.7 ± 1.68 5.69 ± 0.05 Durowoju et al.32 GS3 Geothermal spring Mphepu 3.43 ± 1.25 8.10 ± 0.05 42 ± 1.12 12.05 ± 0.14 Durowoju et al.32 GS4 Geothermal spring Tshipise 5.50 ± 0.36 8.47 ± 0.22 55 ± 2.24 2.81 ± 0.09 Durowoju et al.32 GS5 Geothermal spring Sagole 1.69 ± 0.59 8.38 ± 0.93 43.60 ± 1.79 2.28 ± 3.36 Durowoju et al.32 Table 2. Results and discussion Parameters Sampling sites WHO (2011) (mg/L) H1 H2 BH1 BH2 NO3 − 0.17 1.31 3.22 83.95 44.0 Cl− 153.30 38.90 80.14 103.23 250 SO4 2− 16.45 10.55 17.56 25.88 500 PO4 3− 1.15 1.52 3.29 4.59 N/A CO3 2− 1.80 2.40 0.00 2.70 20.0 HCO2 − 8.54 9.76 31.11 15.86 N/A Na+ 118.00 62.70 124.00 170.00 400 K+ 2.73 2.21 5.15 4.67 400 Mg2+ Bdl Bdl 15.80 3.72 100 TDS 306.00 130.00 296.00 423.00 450.00 EC (µS/m) 63.00 33.00 69.00 73.00 150.00 Table 3. Chemical parameters of groundwater. STD N/A Not available, H1 & H2 individual borehole 1 & 2, BH1 & BH2 monitoring borehole 1 & 2, Bdl Below detection limit. Parameters Sampling sites WHO (2011) (mg/L) H1 H2 BH1 BH2 NO3 − 0.17 1.31 3.22 83.95 44.0 Cl− 153.30 38.90 80.14 103.23 250 SO4 2− 16.45 10.55 17.56 25.88 500 PO4 3− 1.15 1.52 3.29 4.59 N/A CO3 2− 1.80 2.40 0.00 2.70 20.0 HCO2 − 8.54 9.76 31.11 15.86 N/A Na+ 118.00 62.70 124.00 170.00 400 K+ 2.73 2.21 5.15 4.67 400 Mg2+ Bdl Bdl 15.80 3.72 100 TDS 306.00 130.00 296.00 423.00 450.00 EC (µS/m) 63.00 33.00 69.00 73.00 150.00 Table 3. Chemical parameters of groundwater. STD N/A Not available, H1 & H2 individual borehole 1 & 2, BH1 & BH2 monitoring borehole 1 & 2, Bdl Below detection limit. Table 3. Chemical parameters of groundwater. STD N/A Not available, H1 & H2 individual borehole 1 & 2 BH1 & BH2 monitoring borehole 1 & 2, Bdl Below detection limit. between 41.3–67.7 in thermal springs around the study area. Kirkpatrick57 reported higher fluoride levels in Malawi which were associated with higher geothermal temperature. His report shows that higher fluoride levels (17 mg/L and 20 mg/L) were determined at 65 and 79 °C when compared to 3–12 mg/L recorded within the temperature range of 32–54 °C. Results from several other studies agrees that geothermal temperature generally leads to more fluoride mineralization in groundwater14,23. Influence of calcium on fluoride levels in groundwater. In this study, Ca varied differently in the sites (Table 2). Generally, the sites H1 and H2 with the lowest Ca concentration recorded the highest fluoride level. Although BH2 recorded lower Ca levels, the fluoride level was lower than that of BH1. It has been widely reported that low calcium concentration in groundwater often increases the dissolution of CaF2 which increases fluoride levels in groundwater26,33. Results and discussion Mean fluoride, pH, temperature and calcium levels of groundwater in the study area. Nr not reported this study, high fluoride levels were also associated with alkaline pH values. A Pearson’s correlation analysis per- formed on the data in Table 2 between fluoride and pH yielded a positive correlation with R2 = 0.72. This shows that alkaline pH could have contributed to the mineralization of fluoride from their host rocks. Temperature levels in groundwater of the study area. The mean temperature ranged from 25 to 48 °C. Generally, temperature has a direct effect on chemical reactions as it speeds up the rate of chemical reaction and dissolution. Fluoride concentration was higher in hot water aquifer (H1 and H2) than in cold water aquifer (BH1 and BH2). At H1 and H2, the temperature of the groundwater was 45 °C and 48 °C yield- ing fluoride concentrations of 4.55 mg/L and 4.95 mg/L, respectively, whereas lower levels of fluoride (4.50 and 3.92 mg/L) were determined at groundwater temperatures of 27 °C and 25 °C, respectively. The temperature change of 23 °C resulted in 1.03 mg/L increase in fluoride concentration in groundwater. This is a significant increase in fluoride concentration. A similar trend was reported in Ethiopia by Tekle-Haimanot et al.56, where 80% of the groundwater samples obtained in the geothermal springs of the rift valley area contained fluoride concentrations above 3.0 mg/L and 30% contained fluoride concentrations above 13.0 mg/L compared to the cold springs in the same area with maximum fluoride concentration of < 3.0 mg/L. High geothermal temperature causes an increased dissolution of fluoride bearing minerals and thus increases the fluoride concentration in groundwater35. However, existence of geothermal springs does not necessarily mean fluoride will always be high. Therefore, water chemistry and local geology also plays an important role in the occurrence of high groundwater fluoride in any area. A positive correlation (R2 = 0.6) was computed between fluoride and temperature from the data presented in Table 2. p Previous studies conducted within the region have also reported varying temperatures. Temperature in the range of 26.9–63.86 °C have been reported for boreholes in Siloam area35. Durowoju et al.32 reported temperatures https://doi.org/10.1038/s41598-021-93385-4 Scientific Reports \| (2021) 11:14000 \| www.nature.com/scientificreports/ Table 3. Chemical parameters of groundwater. STD N/A Not available, H1 & H2 individual borehole 1 & 2, BH1 & BH2 monitoring borehole 1 & 2, Bdl Below detection limit. Results and discussion Chae et al.58 stated that there is a thermodynamic relationship between Ca2+ and F- ions which is controlled by the equilibrium of fluorite. Odiyo and Makungo35 reported an inverse relation- ship between fluoride and calcium levels in groundwater around Siloam. A negative correlation (R2 = − 0.30) was computed between fluoride and calcium levels in the groundwater of the study area reported in Table 2.l l Nezli et al.59 however reported that when there is groundwater saturation with respect to fluorite, low calcium levels in groundwater can yield high fluoride concentrations. Odiyo and Makungo33 reported a mean level of calcium in the range of 0.38–1.44 mg/L associated with high levels of fluoride (1.34–6.74 mg/L) from 10 borehole samples from Siloam area. The chemistry of the groundwater at Siloam partially agrees with the opposing factor of solubility between calcium and fluoride because it appears that geothermal temperature plays an important role in the inverse solubility trend between fluoride and calcium. Sulphate, nitrate and electrical conductivity levels in groundwater samples. The levels of sul- phate recorded in this study complied with the regulatory standards for drinking water. Similarly, the levels of sodium, potassium, chloride all complied with regulatory standards (Table 3). Sulphate, nitrate and electrical conductivity levels in groundwater samples. The levels of sul- phate recorded in this study complied with the regulatory standards for drinking water. Similarly, the levels of sodium, potassium, chloride all complied with regulatory standards (Table 3). p p g y ( ) Nitrate contamination is a growing problem and ingestion by infants can cause methemoglobinemia and death60,61. High concentration of nitrate in groundwater above the permissible limit of 44 mg/L is considered an indicator of anthropogenic pollution62. Nitrate source is mostly from septic tanks, pit latrines and fertilizers. The water table in well BH2 occurs at 28 m below the surface, and this makes it easy for infiltration of sewage from nearby pit latrines located less than 20 m uphill from the well. The nitrate levels of the other boreholes complied with the regulatory guideline limit.h g y g The electrical conductivity (EC) and total dissolved solids (TDS) values for all the locations sampled within iloam were within the recommended limits of < 150 µS/m and < 450 mg/L, respectively52. Geochemical classification of groundwater. Groundwater chemistry is directly linked to its quality therefore it is imperative to understand the complex geochemical processes occurring below the surface of the earth through different geochemical facies present. The Major cations (Ca2+, Mg2+, Na+ and K+) were plotted against major anions (SO4 2-, HCO3 - and Cl-) on a piper trilinear diagram, to understand the water type dominant in the groundwater of Siloam (Fig. 3).h g g Piper diagram divides water into six basic types based on the dominating cation and anion. The divisions are: (1) Ca-HCO3 type, (2) Na-Cl type, (3) Ca–Mg–Cl type, (4) Ca-Na-HCO3 type, (5) Ca–Cl type, and (6) Na-HCO3 type63. From the piper diagram (Fig. 3), all the samples were plotted within the sodium chloride (Na-Cl) quadrant. Na-Cl water type is dominated by Na+ and Cl- derived from Na-Cl which could be linked to the underlying geology emanating from gneissic rocks. This water type is typical of marine and deeper ancient groundwater influenced by ion exchange. Although exceptions can be made in situations where dissolution fac- tors like residence time, climate and rainfall, are not favorable for fluoride migration from the rock to ground- water as experienced in Eswatini Kingdom32. https://doi.org/10.1038/s41598-021-93385-4 Scientific Reports \| (2021) 11:14000 \| www.nature.com/scientificreports/ Figure 3. Sulphate, nitrate and electrical conductivity levels in groundwater samples. The levels of sul- phate recorded in this study complied with the regulatory standards for drinking water. Similarly, the levels of sodium, potassium, chloride all complied with regulatory standards (Table 3). Piper Trilinear diagram of major anions and cations in Siloam groundwater (Figure drawn using GW Chart version 1.30: U.S. Geological Survey Software Release, 26 June 2020, https://doi.org/10.5066/P9Y29U1H). Figure 3. Piper Trilinear diagram of major anions and cations in Siloam groundwater (Figure drawn using GW Chart version 1.30: U.S. Geological Survey Software Release, 26 June 2020, https://doi.org/10.5066/P9Y29U1H). A previous study based on the water type of thermal groundwaters around Siloam area indicated that the dominant water type is Na-Cl26,32. Groundwater mixing from different sources do not occur in the study area because the water is predominantly and 100% Na+–Cl− type. A piper plot by Durowoju64 shows that the geother- mal springs of Siloam in both wet and dry periods were dominated by Na-Cl ions. Therefore, seasonal changes do not significantly impact on the groundwater type and dominant cation and anions. However, groundwater with high fluoride in Korea occurs mostly within the basement aquifer and are dominantly Na-HCO3 water type influenced by granitoids and metamorphic rocks58. Elevated Na+ in groundwater could be from the weathering and dissolution of plagioclase tectosilicate mineral end member (albite). Na–Cl water type indicates ancient groundwater with ample residence time with associated overburden and aquifer material. bb d d f h b h f h h Th bb d l g q Gibbs diagram was used to further buttress the source of the Na enrichment. The Gibbs diagram gave a clear indication that the groundwater enrichment was as a result of rock-water interaction and weathering (Fig. 4). The total dissolved solids were plotted against major anions and cations on Gibbs diagram to determine the geo- chemical process responsible for the chemical constituents of groundwater at Siloam using the Eqs. (1) and (2)65. (1) Anions = Cl Cl + HCO3 (2) Cations = Na + K Na + K + Ca (1) (2) However, the occurrence of Cl- > Na+ hydro-chemical process in borehole H1 (Table 2) denotes that although the prevailing water type is Na+—Cl-, the dominance of the water type is as a result of ion exchange and reverse ion exchange. The occurrence of sulphate (SO4 2-) in groundwater is mostly assumed to be influenced by the dissolution of gypsum or the neutralization of acid water by limestone or dolomite66. Gibbs plot showed the geological process responsible for the mineralization process in the groundwater at Siloam.h The plot of Na/Cl (Fig. Sulphate, nitrate and electrical conductivity levels in groundwater samples. The levels of sul- phate recorded in this study complied with the regulatory standards for drinking water. Similarly, the levels of sodium, potassium, chloride all complied with regulatory standards (Table 3). 5) is a good indicator of the type of weathering responsible for the dominance of Na in groundwater. Mayback67 noted that Na/Cl ratio greater than one is an indication of silicate weathering, otherwise the source of Na is from another source other than silicate weathering. From the Na/Cl ratio plot, 75% of the samples (BH2, BH1 and H2) show predominant silicate weathering while 25% (H1) of the samples show other sources of Na+ other than silicate weathering. This other source is the reverse ion exchange. The dominant Na+ https://doi.org/10.1038/s41598-021-93385-4 Scientific Reports \| (2021) 11:14000 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ A 1 10 100 1000 10000 100000 0 0.2 0.4 0.6 0.8 1 TDS (mg/L) Cl/Cl+HCO3 Rock-Water Interacon & Evaporaon Precipitaon B 1 10 100 1000 10000 100000 0 0.2 0.4 0.6 0.8 1 TDS (mg/L) Na+K/Na+Ca+K Rock-Water Interacon & Weathering Evaporaon Precipitaon Figure 4. Gibbs plot showing dominant anion (A) and cation (B) mineralisation process of groundwater in Siloam. A 1 10 100 1000 10000 100000 0 0.2 0.4 0.6 0.8 1 TDS (mg/L) Cl/Cl+HCO3 Rock-Water Interacon & Evaporaon Precipitaon A A B 1 10 100 1000 10000 100000 0 0.2 0.4 0.6 0.8 1 TDS (mg/L) Na+K/Na+Ca+K Rock-Water Interacon & Weathering Evaporaon Precipitaon Evaporaon B Figure 4. Gibbs plot showing dominant anion (A) and cation (B) mineralisation process of groundwater in Siloam. BH1 BH2 H1 H2 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 0 1 2 3 4 5 Na/Cl Sample No Figure 5. Plot showing Na/Cl ratio. Figure 5. Plot showing Na/Cl ratio. in the groundwater at Siloam is thus as a result of the weathering and chemical alteration of tectosilicate miner- als in the form of Plagioclase feldspar (Na rich feldspar) or sheet silicate minerals in the form of chlorite, due to rock-water interaction over a period resulting in base ion exchange between the rocks, soil and groundwater. in the groundwater at Siloam is thus as a result of the weathering and chemical alteration of tectosilicate miner- als in the form of Plagioclase feldspar (Na rich feldspar) or sheet silicate minerals in the form of chlorite, due to rock-water interaction over a period resulting in base ion exchange between the rocks, soil and groundwater. p g g g Chloro-Alkaline Indexes (CAI) were calculated from Eqs. Sulphate, nitrate and electrical conductivity levels in groundwater samples. The levels of sul- phate recorded in this study complied with the regulatory standards for drinking water. Similarly, the levels of sodium, potassium, chloride all complied with regulatory standards (Table 3). (3) and (4) to determine if the Na+ is from base ion exchange or from reverse ion exchange. In ion exchange process, the Ca and Mg from the groundwater is exchanged with the Na+ and K+ from the host rock or surrounding rocks. CAI 1 and CAI 2 were plotted in Fig. 6a,b. (3) CAI 1 = Cl− Na + + K + Cl (4) CAI 2 = [Cl−− Na + + K + ] SO2− 4 + HCO− 3 + CO2− 3 + NO− 3 (3) (4) CAI 1 and CAI 2 will result in a negative value if the process of Na+ enrichment is by ion exchange process, oth- erwise, the process of reverse ion exchange is responsible68. CAI 1 and CAI 2 from Fig. 6 shows that the boreholes BH1, BH2 and H2 exhibited a sodium enrichment process by normal ion exchange while borehole H1 shows that the sodium enrichment process is by reverse ion exchange process. CAI 1 and CAI 2 will result in a negative value if the process of Na+ enrichment is by ion exchange process, oth- erwise, the process of reverse ion exchange is responsible68. CAI 1 and CAI 2 from Fig. 6 shows that the boreholes BH1, BH2 and H2 exhibited a sodium enrichment process by normal ion exchange while borehole H1 shows that the sodium enrichment process is by reverse ion exchange process. https://doi.org/10.1038/s41598-021-93385-4 https://doi.org/10.1038/s41598-021-93385-4 Scientific Reports \| (2021) 11:14000 \| www.nature.com/scientificreports/ a b BH1 BH2 H1 H2 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0 1 2 3 4 5 CAI 1 Sample no BH1 BH2 H1 H2 -1.5 -1 -0.5 0 0.5 1 1.5 0 1 2 3 4 5 CAI 2 Sample no Figure 6. Chloro-Alkaline Index CAI 1 (a) and CAI 2 (b) for enrichment process. a BH1 BH2 H1 H2 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0 1 2 3 4 5 CAI 1 Sample no b Figure 6. Chloro-Alkaline Index CAI 1 (a) and CAI 2 (b) for enrichment process. Lithology and mineral composition of soils and rocks in the study area. The lithologic descrip- tion of the samples is given in Table 4. Chlorite minerals can be found in any major rock group, but they are known to be predominant in low grade metamorphic rocks. Chlorite is also the index mineral for green schist facie69. Sulphate, nitrate and electrical conductivity levels in groundwater samples. The levels of sul- phate recorded in this study complied with the regulatory standards for drinking water. Similarly, the levels of sodium, potassium, chloride all complied with regulatory standards (Table 3). Chlorite is found as a common constituent of igneous rocks resulting from hydrothermal alteration of pyroxenes, amphiboles, biotite and garnet69. In sedimentary basins, Clay-rich sediments derived from the weathering of igneous rocks are often rich in chlorite69. This is basically the source of chlorite enrichment in sedimentary terrain.h y The abundance of chlorite can be seen in Siloam which is made up of volcano sedimentary sequence. Table 5 gives the weight percent (wt.%) and the constituent minerals in all the soil and rock samples collected in Siloam as analysed by X-ray Diffraction (XRD). The abundance of chlorite is seen especially in borehole cuttings of BH1 at depths 40–65 m where chlorite is seen as much as 38 wt.% of the total rock mass at some depths (Table 5). However, the occurrence of chlorite determines the colour of the total rock mass given that the wt.% of chlorite is more than 20% as seen in cuttings X40 and X65 (Table 5). From depths 5–35 m in borehole BH1, the pink–brown colour could be due to the presence of smectite and occurrence of hematite. The brown colour may also be due to the presence of plagioclase. p p g However, occurrences of quartz and muscovite mostly do not determine the colour of the whole rock mass because they are mostly colourless to white in colour. The pinkish colour of rock SR2 could signify the occurrence of hematite (iron oxide). The occurrence of chlorite in the sedimentary terrains of Siloam and the high abundance of smectite and hematite group shows that the present geologic sequence has undergone a series of weathering and tectonic activities. This explains the abundance of Na+ in groundwater at Siloam because of ion exchange and possible reverse ion exchange during the process of weathering of silicate minerals like plagioclase and chlorites.h From the mineralogy (Table 5), silicate minerals as well as clay minerals are abundant in the study area. The area is dominated by clay minerals such as chlorite and smectite, as well as silicate minerals such as muscovite, biotite and plagioclase. Clay can adsorb and desorb fluoride under favorable condition. Clay in the soil can func- tions as a natural barrier to protect groundwater from fluoride pollution due to its strong adsorption potential70. The adsorption capabilities of clay are possible in the acidic pH conditions. Sulphate, nitrate and electrical conductivity levels in groundwater samples. The levels of sul- phate recorded in this study complied with the regulatory standards for drinking water. Similarly, the levels of sodium, potassium, chloride all complied with regulatory standards (Table 3). However, the adsorbed fluoride ion can also be released into the groundwater under alkaline conditions.hl g The adsorption properties of fluoride by clay are proportional to the amount of clay minerals present. Clay desorption is a faster geochemical process when compared to adsorption process71, thus it is easy for fluoride to migrate from clay to groundwater when in contact with small volume of alkaline water. The abundance of clay minerals show that extensive weathering altered the chemical and physical composition of the silicate minerals to form clay minerals. The fluoride enrichment of the groundwater in the area is as a result of ion exchange between the fluoride bearing minerals and groundwater. However, muscovite also contains fluoride as an accessory ion in its chemistry, which at favorable conditions, is released into the groundwater. Scientific Reports \| (2021) 11:14000 \| https://doi.org/10.1038/s41598-021-93385-4 www.nature.com/scientificreports/ Table 4. Lithologic Description of Sampled Rocks and Soils. S surface soil, SR surface rocks, BH1 borehole 1, BH2 borehole 2. Location Sample ID Depth (M) Associated group Lithology Brief description S S1 Soil Clay Brown color with very fine texture SR2 Metamorphic Epitomized grey wacke This rock type shows a low-grade metamorphism characteristic and it exhibits an aphanitic texture. It has a green colour due to the abun- dance of chlorite SR3 Sedimentary Sandstone Pinkish Sandstone due to the presence of hematite BH1 X5 5 Sedimentary Argillaceous Sediment Topsoil with brittle texture and pinkish-brown color. Presence of Smectite and hematite noticeable X15 15 Sedimentary Argillaceous Sediment Dark brown color with reduced pinkness probably due to the hematite content X20 20 Sedimentary Argillaceous Sediment Light pink color due to the heavy presence of clay mineral Smectite and Hematite X30 30 Sedimentary Argillaceous Sediment Reduction in the pink color and traces of darker brown color. Smectite and Hematite still present although at a reduced concentration X40 40 Sedimentary Gray Wacke Green in color probably due to the presence of Chlorite mineral and the rock is slightly saturated due to its function being the aquifer rock. Total disappearance of clay minerals of smectite and hematite BH2 X65 65 Sedimentary Gray Wacke Green in color probably due to the presence of Chlorite mineral and the rock is slightly saturated due to its function being the aquifer rock. Total disappearance of clay minerals of smectite and hematite noticeable. Sulphate, nitrate and electrical conductivity levels in groundwater samples. The levels of sul- phate recorded in this study complied with the regulatory standards for drinking water. Similarly, the levels of sodium, potassium, chloride all complied with regulatory standards (Table 3). Index mineral noticed are quartz and chlorite Y5 5 Soil Clay Light brown in color with more than 60 wt.% Smectite Y10 10 Soil Clay Light brown color with increased smectite content of more than 70 wt.% Y20 20 Soil Clay Reduced lightness with two different groups of Smectite namely smectite 1 and 2. Overall smectite content is greater than 75 wt.% Y25 25 Soil Sandy Clay Dark brown color with intercalation of clay and sand. Although smectite content is still above 50% but there is reduction in its wt.% Y28 28 Igneous Basalt Dark color with dominantly plagioclase feldspar and diopside mineral Y40 40 Igneous Basalt Dark color with dominantly plagioclase feldspar and diopside mineral Table 4. Lithologic Description of Sampled Rocks and Soils. S surface soil, SR surface rocks, BH1 borehole 1, BH2 borehole 2. Table 5. Mineral constituents and percentage weight of rocks and soils. S surface soil, SR surface rock, X borehole BH1 depth, Y borehole BH2 depth S1 SR2 SR3 X5 X15 X20 X30 X40 X65 Y5 Y10 Y20 Y25 Y28 Y40 Weight (%) Diopside 2.76 20.5 28.7 29.87 Chlorite 28.41 4.21 5.42 4.59 5.42 38.92 26.98 4.69 4.72 Hematite 1.7 0.17 0.67 9.41 7.87 5.07 7.27 0.78 Muscovite 1.6 32.41 33.77 33.07 41.15 12.33 7.49 4.56 3.29 Orthoclase 8.04 2.65 1.87 7.7 5.61 7.97 14.99 Plagioclase 32.38 14.82 17.35 16.31 11.08 6.86 9.75 22.93 39.49 38.27 Quartz 8.36 14.47 97.73 35.99 34.44 44.76 30.04 25.99 20.72 12.19 8.34 5.53 9.72 7.69 9.00 Smectite 44.26 15.33 16.63 12.51 16.1 69.02 78.41 52.36 5.88 7.1 Smectite1 41.2 Smectite 2 35.55 Sepiolite 2.49 Epidote 21.63 12.04 Titanite 5.4 7.25 Actinolite 9.22 2.08 2.6 Enstatite 3.02 Ilmenite 3.89 5.18 Table 5. Mineral constituents and percentage weight of rocks and soils. S surface soil, SR surface rock, X borehole BH1 depth, Y borehole BH2 depth Muscovite is an important fluoride bearing mineral because it contains fluorine in its crystal lattices and hydroxyl groups which can also substitute for fluoride because of their similar ionic charges70. This fluorine can be released as fluoride ion into the groundwater under favorable conditions. Muscovite occurs at about 1.62 wt. % (Table 5) in SR3. Onipe48 noted that muscovite in the surface rocks at Siloam contributes to the groundwater fluoride rather than fluorite as earlier inferred by McCaffrey and Willis25 and Odiyo and Makungo35. Sulphate, nitrate and electrical conductivity levels in groundwater samples. The levels of sul- phate recorded in this study complied with the regulatory standards for drinking water. Similarly, the levels of sodium, potassium, chloride all complied with regulatory standards (Table 3). Fluorine affiliated muscovite increases with depth from 5 to 15 m, slows down at depth 20 m and picks up its increasing trend again at depth 30 m below ground level before drastically reducing as the depth increases. Figure 7C shows the XRD peaks for the epitomized gray-wacke which is the aquifer rock for borehole BH1. Although muscovite is present, the abundance is not as much as at depths 5 to 30 m. The grey wacke is dominated by chlorite which explains why the color is greenish grey (Table 4). The primary minerals present in the rock materials include chlorite, quartz and plagioclase. The intensities and abundance of each mineral are identified in Fig. 7. The combined presence of muscovite and chlorite at depth X40 and X 65 in borehole BH1 may accounts for the slightly high total fluoride concentration of the aquifer due to the exchange of OH with F in their structure. Figure 7 also shows a high abundance of plagioclase which denotes that the level of weathering is either absent or low at depth 40 m below the earth surface, therefore there has been no chemical alteration of plagioclase to smectite clay or kaolin as seen at shallow depths before 40 m. Two main dynamic geochemical processes occur in clay, they are the enrichment and leaching processes. Under the dynamic geochemical process of leaching, areas with clay formations and arid to semi-arid climatic conditions, experience high groundwater and surface water fluoride concentration72. Dynamic geochemical process of leaching occurs when fluoride is leached out of clay formation into groundwater through infiltration and percolation of water. Fluorine can easily migrate from clay formations to groundwater during migration of groundwater73.l y g g g g In borehole BH2 at Siloam, muscovite which is the predominant fluoride bearing mineral in the area is seen to be absent until the depth of 28 m (Table 5). Muscovite occurrence was found at depth 28 to 40 m although at a volume less than 5 wt.%. The peaks of smectite at depths Y5, Y10, Y15 and Y20 occur at almost the same count and intensity (Fig. 7). Smectite is the most abundant mineral at depths Y5–Y20. Clay could also act as a migration agent for fluoride enrichment of groundwater at Siloam. The abundance of major minerals in borehole BH2 at depths Y28 and Y40 are also described in Table 5. Sulphate, nitrate and electrical conductivity levels in groundwater samples. The levels of sul- phate recorded in this study complied with the regulatory standards for drinking water. Similarly, the levels of sodium, potassium, chloride all complied with regulatory standards (Table 3). Onipe48 also argued that the muscovite present in the surface sandstone at Siloam can never be the sole contributor of fluoride to groundwater of the area.h g Surface rock SR2 contains high proportions of chlorite and epidote (Fig. 6). The occurrence of clinochlore n end member of chlorite is predominant in Siloam. Chlorite has been linked to fluoride contribution to Scientific Reports \| (2021) 11:14000 \| https://doi.org/10.1038/s41598-021-93385-4 www.nature.com/scientificreports/ B A B C 0 5 10 15 20 25 30 35 40 45 0 4100 8200 12300 0 5 10 15 20 25 30 35 40 45 0 4500 9000 13500 Plagioclase Chlorite Muscovite Chlorite Intensities (Count) X40 Chlorite Muscovite Quartz Plagioclase Plagioclase Quartz Chlorite Chlorite Intensities (Count) 2-theta (Degrees) X65 Epidote Figure 7. XRD plot showing major minerals in borehole 1 at X15, X20 & X30 (A) and surface soil SR2 & SR3 (B) borehole 1 at X40 and X65. 0 5 10 15 20 25 30 35 40 45 0 4100 8200 12300 0 5 10 15 20 25 30 35 40 45 0 4500 9000 13500 Plagioclase Chlorite Muscovite Chlorite Intensities (Count) X40 Chlorite Muscovite Quartz Plagioclase Plagioclase Quartz Chlorite Chlorite Intensities (Count) 2-theta (Degrees) X65 Epidote B C A Figure 7. XRD plot showing major minerals in borehole 1 at X15, X20 & X30 (A) and surface soil SR2 & SR (B) borehole 1 at X40 and X65. groundwater mostly due to its hydroxyl content which is displaced by fluoride ion. Generally, chlorite contains fluoride but not as much as its ripidolite. This reason is attributed to the fact that clinochlore has an appreciable number of aluminium in its octahedral layer therefore limiting its fluoride adsorption properties unlike ripidolite that has aluminium in its tetrahedral layer. However, at varying depth in borehole BH1, muscovite occurs as an abundant rock forming mineral rather than its accessory form as found in SR2. The concentration of muscovite in wt. % increases from topsoil to 30 m below the surface (Table 5). The major minerals present in borehole BH1 from depth 5 m to 30 m are muscovite, quartz and smectite (Table 5). The abundance of major dominating minerals of borehole BH1 from depth 15–30 m are shown in Fig. 7. Oxide wt percent × Conversion factor = Elemental wt percent (5) The conversion factor is the sum of the atomic weights of the elements in the required formula divided by the sum of the atomic weights in the original formula. Silica (SiO2) and quartz (SiO2) have the same chemical composition, but they are different in nature. Silica refers to the total pure silicon dioxide in the rock mass while quartz refers to a mineral with a very high concentration of silica. However, often, quartz occurs as silica with little amount of impurities. Therefore, the name is used interchangeably. Total rock silica could be from quartz and other silicate minerals like feldspars, chlorites and other aluminium and silicate rich minerals. The SiO2 refers to the total silica content of the rock and soil. Silica has the highest wt. % in soils and rocks of Siloam. This means that the area is dominated by felsic rock and the soil with high silica content. Felsic rocks, which contain relatively high concentrations of SiO2, tend to have higher concentrations of fluorine than mafic rock74. bl h h l l l d fl f k d l l k li Table 6 shows the total major elemental and fluorine compositions of rocks and soils at Siloam in mg/kg. Although fluorine is regarded as a trace element, its concentration in some rocks and soil in Siloam is higher than expected. The total fluorine content of rocks and soil ranges from 10 to 2000 mg/kg (Table 6). The average fluorine concentration is about 762 mg/kg. This is above the permissible limit of 30 mg/kg concentration of fluorine in soil for agriculture75. The highest fluorine concentration of 2000 mg/kg occurs at a depth of 5 m in borehole BH2. The lowest concentration occurs in borehole BH1 at a depth of 30 m (Table 6).hlh h The fluorine concentration in clay is typically in the range of 20–500 mg/kg76. The surface clay deposit S1 has a total fluorine concentration of 431 mg/kg (Table 6). However, the clay formation of borehole BH2 from 5 m to around 20 m depth has the highest concentrations of fluorine. The Soil of BH2 at depths 5 m and 10 m have more fluorine concentration than the surface rock formations and aquifer rocks in Siloam as observed from Table 6. This is different from the findings of McCaffrey and Willis25 that stated that rock samples have higher fluorine content than soil samples. Oxide wt percent × Conversion factor = Elemental wt percent The clay deposit and argillaceous sediments of Siloam are from chemical alteration and weathering of plagioclase of parent rock materials therefore giving more room for OH and F replacements. This ion displacement gives a higher room for more storage of fluoride in the crystal lattices of the resultant clay. It should be noted, however, that clays formed under hydrothermal conditions are mostly rich in fluoride77 as observed in Siloam, although this is not always the case as observed in Sagole, Tshipise and Evangelina area of Limpopo, South Africa78. Fluoride leaching and other chemical constituents from rocks and soils. The fluoride concentra- tion emigrating out of the rock and soil samples of Siloam ranges from 0.27 to 14.88 mg/L (Table 7). Leachate with the lowest fluoride concentration occurs at surface rock SR2 and leachate with the highest fluoride concen- tration occurs at a depth of 5 m of borehole BH2 (Table 7). Fluoride concentrations show a decreasing trend from the surface down to the aquifer. Figure 9 shows the fluoride concentration trend in the borehole BH1 leachates.h Fluoride leaching and other chemical constituents from rocks and soils. The fluoride concentra- tion emigrating out of the rock and soil samples of Siloam ranges from 0.27 to 14.88 mg/L (Table 7). Leachate with the lowest fluoride concentration occurs at surface rock SR2 and leachate with the highest fluoride concen- tration occurs at a depth of 5 m of borehole BH2 (Table 7). Fluoride concentrations show a decreasing trend from the surface down to the aquifer. Figure 9 shows the fluoride concentration trend in the borehole BH1 leachates. The fact that from boreholes BH1 and BH2, the sub-surface rocks and soils of Siloam show decreasing trends in fluoride concentrations, means that the aquifer rocks release less fluorine into the groundwater compared to the regolith and overburden soils and clays. This may partially be due to the loose state of the overburden. The ions in the leachate were correlated against each other to determine the relationship between them in relation to tration occurs at a depth of 5 m of borehole BH2 (Table 7). Fluoride concentrations show a decreasing trend from the surface down to the aquifer. Figure 9 shows the fluoride concentration trend in the borehole BH1 leachates. Sulphate, nitrate and electrical conductivity levels in groundwater samples. The levels of sul- phate recorded in this study complied with the regulatory standards for drinking water. Similarly, the levels of sodium, potassium, chloride all complied with regulatory standards (Table 3). The abundance of chlorite and plagioclase were also revealed from the results. Y28 and Y40 is the basaltic aquifer material of borehole BH2. Chemical composition of rocks and soils in the study area. In sedimentary terrain, the most abun- dant fluorine bearing mineral is associated with mica mineral group and clay minerals especially montmoril- lonite and kaolinite. Total rock chemistry is important to determine the total rock composition in oxides and elemental properties. The graphical representation of major oxides in the rocks and soil of Siloam are presented in Fig. 8. The rocks and soil are dominated by high silica content followed by aluminium oxides. The highest concentration of silica occurs at surface sandstone rock of location SR3. The formula for conversion of oxides to elemental weight. percent is given in Eq. (5). https://doi.org/10.1038/s41598-021-93385-4 Scientific Reports \| (2021) 11:14000 \| www.nature.com/scientificreports/ d f l l 0 20 40 60 80 100 120 S1 SR2 SR3 X5 X15 X20 X30 X40 X65 Y5 Y10 Y20 Y25 Y28 Y40 Major Oxides in wt.% Sample ID CaO Na₂O MgO Al₂O₃ SiO₂ P₂O₅ K₂O MnO Fe₂O₃ Figure 8. Major oxide abundance in sampled rocks and soils. 0 20 40 60 80 100 120 S1 SR2 SR3 X5 X15 X20 X30 X40 X65 Y5 Y10 Y20 Y25 Y28 Y40 Major Oxides in wt.% Sample ID CaO Na₂O MgO Al₂O₃ SiO₂ P₂O₅ K₂O MnO Fe₂O₃ Figure 8. Major oxide abundance in sampled rocks and soils. CaO Na₂O MgO Al₂O₃ SiO₂ P₂O₅ K₂O MnO Fe₂O₃ Figure 8. Major oxide abundance in sampled rocks and soils. (5) Oxide wt percent × Conversion factor = Elemental wt percent Oxide wt percent × Conversion factor = Elemental wt percent The fact that from boreholes BH1 and BH2, the sub-surface rocks and soils of Siloam show decreasing trends in fluoride concentrations, means that the aquifer rocks release less fluorine into the groundwater compared to the regolith and overburden soils and clays. This may partially be due to the loose state of the overburden. The ions in the leachate were correlated against each other to determine the relationship between them in relation to l The fact that from boreholes BH1 and BH2, the sub-surface rocks and soils of Siloam show decreasing trends in fluoride concentrations, means that the aquifer rocks release less fluorine into the groundwater compared to the regolith and overburden soils and clays. This may partially be due to the loose state of the overburden. The ions in the leachate were correlated against each other to determine the relationship between them in relation to https://doi.org/10.1038/s41598-021-93385-4 Scientific Reports \| (2021) 11:14000 \| www.nature.com/scientificreports/ Table 6. Major elemental compositions of rocks and soils (mg/kg) in the study area. S surface soil, SR surface rock, X borehole BH1 depth, Y borehole BH2 depth, ion units, mg/kg depth: m. Sample ID Ca Na Mg Al Si P K Mn Fe F S1 25,429.08 23,889.18 21,587.40 90,880.81 241,417.00 3500.73 11,206.35 1541.26 95,607.98 431.00 SR2 58,286.88 20,253.87 48,240.00 87,863.80 225,241.50 969.03 305.48 1657.43 101,972.52 1400.00 SR3 415.72 242.60 940.68 12,650.27 448,332.50 484.52 3137.78 41.82 6329.57 160.00 X5 658.58 5749.73 20,019.60 100,461.14 271,477.25 281.54 33,619.05 569.26 91,551.46 450.00 X15 2021.47 2559.56 22,672.80 102,101.97 271,617.50 471.42 33,536.04 723.38 86,096.14 330.00 X20 2521.48 1409.61 21,406.50 94,744.70 296,628.75 475.79 35,030.22 749.72 62,806.12 290.00 X30 4057.22 1468.96 23,215.50 108,877.01 264,231.00 659.12 43,580.25 836.46 79,941.42 10.00 X40 14,143.14 10,757.55 59,094.00 103,848.66 231,926.75 1169.82 11,621.40 1773.61 97,706.18 850.00 X65 36,000.72 11,944.59 54,330.30 88,340.17 232,815.00 999.59 11,123.34 1618.71 100,084.14 770.00 Y5 13,500.27 10,905.93 29,486.70 86,911.06 267,129.50 2330.91 8301.00 1184.99 91,551.46 2000.00 Y10 13,785.99 12,389.73 27,376.20 85,587.81 265,166.00 2099.57 8633.04 1239.20 95,188.34 1500.00 Y20 17,143.20 8457.66 26,954.10 97,444.13 260,163.75 2549.16 10,210.23 1215.97 89,872.90 1200.00 Y25 31,214.91 14,021.91 24,602.40 86,805.20 255,442.00 3727.71 17,017.05 1394.10 87,145.24 800.00 Y28 51,572.46 20,847.39 29,124.90 82,041.50 239,593.75 4932.45 19,922.40 1014.60 74,695.92 620.00 Y40 54,429.66 19,437.78 31,959.00 76,960.22 236,181.00 4234.05 16,851.03 1177.24 81,340.22 620.00 Table 6. Major elemental compositions of rocks and soils (mg/kg) in the study area. S surface soil, SR surface rock, X borehole BH1 depth, Y borehole BH2 depth, ion units, mg/kg depth: m. Table 7. Oxide wt percent × Conversion factor = Elemental wt percent Chemical parameters in the leachate obtained from leaching experiment. BDL Below detection Lim S surface soil, SR surface rock, X borehole BH1 depth, Y borehole BH2 depth Sample K EC pH Ca Mg Cl NO3 NO2 PO4 Na B F mg/kg µs/cm mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg S 1 62.50 24.00 7.57 135.00 891.00 36,03 1,65 0.00 1,89 929.00 1.75 10,52 SR 2 11.60 3.00 7.17 BDL 40.00 2,75 0,12 0.00 0,67 36.10 0.05 0,27 SR 3 27.80 3.00 6.83 BDL BDL 2,77 0,21 0.00 3,17 2.26 0.04 0,44 X 5 379.00 42.00 7.10 19.80 773.00 114,43 24,66 0.00 1,55 1020.00 2.07 3,85 X 15 125.00 7.00 6.91 2.26 172.00 10,59 2,41 0.00 0,78 110.00 0.46 2,29 X 20 118.00 3.00 6.95 1.88 150.00 2,15 0,60 0.00 0,65 52.40 0.28 1,10 X 30 164.00 3.00 7.03 11.90 183.00 1,61 0,72 0,03 0,59 35.90 0.38 0,78 X 40 33.40 4.00 7.16 18.20 179.00 1,43 0,58 0,13 0,30 72.00 0.26 0,68 X 65 31.90 9.00 7.37 36.30 41.20 2,34 0,56 0,71 0,50 82.40 0.02 0,53 Y 5 31.90 32.00 8.00 186.00 675.00 11,57 0,78 0.00 0,39 886.00 0.93 14,88 Y 10 45.60 76.00 7.97 145.00 438.00 171,79 8,10 0.00 1,00 1620.00 1.11 9,51 Y 20 26.00 6.00 7.16 68.00 178.00 2,12 0,85 0.00 0,39 129.00 0.26 3,79 Y 28 61.90 9.00 7.43 94.80 97.90 1,93 0,29 0,33 0,90 151.00 0.15 1,02 Y 25 25.30 5.00 7.10 19.30 61.60 1,59 0,15 0,01 0,70 84.20 0.14 2,60 Y 40 54.50 10.00 7.50 110.00 104.00 2,46 0,27 0,49 0,80 166.00 0.17 0,72 Table 7. Chemical parameters in the leachate obtained from leaching experiment. BDL Below detection Limit, S surface soil, SR surface rock, X borehole BH1 depth, Y borehole BH2 depth the release of fluoride. Table 8 shows the correlation coefficients in the leachate at Siloam. Fluoride concentrations in groundwater of boreholes BH1 and BH2 show high positive correlations to pH, Ca, Na (Table 8).hh the release of fluoride. Table 8 shows the correlation coefficients in the leachate at Siloam. Fluoride concentrations in groundwater of boreholes BH1 and BH2 show high positive correlations to pH, Ca, Na (Table 8).hh This means that at an increasing F concentration, pH, Ca and Na increases. The concentrations of these highlighted parameters (pH, Ca, Na) are directly proportional to each other. Oxide wt percent × Conversion factor = Elemental wt percent Fluoride concentration of (a) borehole 1 and (b) borehole 2. Table 8. Correlation of physico-chemical constituent of leachates. K EC pH Ca Mg Cl NO3 NO2 PO4 Na B F K 1.00 EC 0.25 1.00 pH − 0.27 0.69 1.00 Ca − 0.26 0.60 0.94 1.00 Mg 0.44 0.64 0.50 0.56 1.00 Cl 0.41 0.94 0.44 0.33 0.55 1.00 NO3 0.85 0.60 0.02 − 0.03 0.56 0.74 1.00 NO2 − 0.20 − 0.19 0.17 0.09 − 0.35 − 0.24 − 0.21 1.00 PO4 0.15 0.11 − 0.22 − 0.09 0.17 0.19 0.22 − 0.22 1.00 Na 0.26 0.97 0.72 0.67 0.80 0.89 0.59 − 0.27 0.15 1.00 B 0.62 0.70 0.36 0.39 0.95 0.70 0.76 − 0.37 0.27 0.82 1.00 F − 0.06 0.67 0.77 0.82 0.80 0.44 0.16 − 0.35 0.01 0.79 0.64 1.00 Table 8. Correlation of physico-chemical constituent of leachates. K EC pH Ca Mg Cl NO3 NO2 PO4 Na B F K 1.00 EC 0.25 1.00 pH − 0.27 0.69 1.00 Ca − 0.26 0.60 0.94 1.00 Mg 0.44 0.64 0.50 0.56 1.00 Cl 0.41 0.94 0.44 0.33 0.55 1.00 NO3 0.85 0.60 0.02 − 0.03 0.56 0.74 1.00 NO2 − 0.20 − 0.19 0.17 0.09 − 0.35 − 0.24 − 0.21 1.00 PO4 0.15 0.11 − 0.22 − 0.09 0.17 0.19 0.22 − 0.22 1.00 Na 0.26 0.97 0.72 0.67 0.80 0.89 0.59 − 0.27 0.15 1.00 B 0.62 0.70 0.36 0.39 0.95 0.70 0.76 − 0.37 0.27 0.82 1.00 F − 0.06 0.67 0.77 0.82 0.80 0.44 0.16 − 0.35 0.01 0.79 0.64 1.00 Table 8. Correlation of physico-chemical constituent of leachates. Table 8. Correlation of physico-chemical constituent of leachates. groundwater at Siloam. The study area is dominated by rocks of high silica content (Table 6). The silica content is a combined value of all the silicate minerals (quartz, plagioclase and muscovite). Rocks with high silica content often have high fluorine concentration74. This agrees with this study because there seem to be a decreasing trend in the fluorine content towards the mafic rocks at depth. Also, a decreasing trend could be noticed as opposed to a general norm that fluoride increases with depth. This is possibly due to the clay and argillaceous overburden. At BH1 and BH2, fluoride decreases from depth X5 to X65 and Y5 to Y40. This coincides with the decreasing concentration of smectite and chlorite in the mineralogy of the rocks. Oxide wt percent × Conversion factor = Elemental wt percent Effect of temperature on fluoride release from soil and rocks. Siloam is located on Siloam fault which acts as a channel for transport of heated groundwater up to some near surface aquifer. This gives Siloam a very high geothermal gradient. Therefore, the effect of temperature is very important when investigating the source of fluoride from the dissolution of minerals irrespective of the temperature of the water at present. The effect of temperature in the dissolution of fluoride from the mineral constituents in the rocks and soil into the groundwater was experimented in the laboratory. The average water temperature of 42 °C from past literature focusing on Siloam was used as the base temperature. Although, the effect of temperature was simulated for less than 10 h. Temperature plays an important role by increasing the fluoride concentration in the leachates. This difference can be observed in Fig. 10 where the comparison can be seen between fluoride release at room tem- perature and fluoride release at 40 °C.hfh pl The temperature effect can be seen at almost all sampling points except for Y28 (Fig. 10). The sampling point Y5 shows a distinct effect of temperature by a concentration difference of almost 5 mg/L. The groundwater tem- perature is evident to be one of the driving factors favoring the dissolution of fluorine from the rocks and soils at Siloam, into the groundwater in the area. Oxide wt percent × Conversion factor = Elemental wt percent EC is also positively correlated to F, which means that increase in fluoride results in an increase in pH, Ca and Na which in turn increases the conductivity of the groundwater of boreholes BH1 and BH2. The very weak correlation between fluoride and nitrate confirms that the possible source of high nitrate in groundwater of BH2 is not from a natural source rather from anthropogenic sources which were previously inferred.hl g y The weak negative correlation between fluoride and potassium connotes that most of the occurrence does not originate from K bearing minerals. The groundwater fluoride of the study area is controlled by the desorption and adsorption properties of clay and partly by chlorite. Therefore, the groundwater hydrochemistry is dominated by Na-Cl water type signifying the importance of Na weathering and dissolution to investigating groundwater fluoride source. The process is simplified by Eq. (6). (6) (6) NaAlSiO8 + 2H+ + 9HO →AlSiO5(OH) + 4HSiO + 2Na The hydroxyl ion in the clay is displaced by fluoride in the structure lattice of the kaolin-smectite clay due to the similarity in their charge and radius. The resultant Na in the equation explains the abundance of Na in The hydroxyl ion in the clay is displaced by fluoride in the structure lattice of the kaolin-smectite clay due to the similarity in their charge and radius. The resultant Na in the equation explains the abundance of Na in https://doi.org/10.1038/s41598-021-93385-4 Scientific Reports \| (2021) 11:14000 \| ww.nature.com/scientificreports/ Figure 9. Fluoride concentration of (a) borehole 1 and (b) borehole 2. Table 8. Correlation of physico-chemical constituent of leachates. K EC pH Ca Mg Cl NO3 NO2 PO4 Na B F K 1.00 EC 0.25 1.00 pH − 0.27 0.69 1.00 Ca − 0.26 0.60 0.94 1.00 Mg 0.44 0.64 0.50 0.56 1.00 Cl 0.41 0.94 0.44 0.33 0.55 1.00 NO3 0.85 0.60 0.02 − 0.03 0.56 0.74 1.00 NO2 − 0.20 − 0.19 0.17 0.09 − 0.35 − 0.24 − 0.21 1.00 PO4 0.15 0.11 − 0.22 − 0.09 0.17 0.19 0.22 − 0.22 1.00 Na 0.26 0.97 0.72 0.67 0.80 0.89 0.59 − 0.27 0.15 1.00 B 0.62 0.70 0.36 0.39 0.95 0.70 0.76 − 0.37 0.27 0.82 1.00 F − 0.06 0.67 0.77 0.82 0.80 0.44 0.16 − 0.35 0.01 0.79 0.64 1.00 www.nature.com/scientificreports/ w.nature.com/scientificreports/ Figure 9. Fluoride concentration of (a) borehole 1 and (b) borehole 2. Figure 9. Conclusions Fluoride levels exceeding 1.5 mg/L recommended by the WHO was determined in the groundwater of the study area. The level is in the range that could cause both fluorosis and non-fluorosis diseases to the consumer of the groundwater resource. Positive correlation was determined between fluoride levels with pH and temperature while a negative correlation was determined with calcium. The major water type present in the study area is the https://doi.org/10.1038/s41598-021-93385-4 Scientific Reports \| (2021) 11:14000 \| www.nature.com/scientificreports/ 0 2 4 6 8 10 12 14 16 S 1 SR 2 SR 3 X 5 X 15 X 20 X 30 X 40 X 65 Y 5 Y 10 Y 20 Y 28 Y 25 Y 40 Fluoride Concentraon mg/L Sample ID F+ Temp F- Temp Figure 10. Fluoride leachate behaviour at room temperature and at 40 °C, F + Temp = Fluoride concentration at induced temperature and F-Temp = fluoride at concentration at room temperature. 0 S 1 SR 2 SR 3 X 5 X 15 X 20 X 30 X 40 X 65 Y 5 Y 10 Y 20 Y 28 Y 25 Y 40 Sample ID F+ Temp F- Temp igure 10. Fluoride leachate behaviour at room temperature and at 40 °C, F + Temp = Fluoride concentration at nduced temperature and F-Temp = fluoride at concentration at room temperature. NaCl water type. The geological process responsible for the enrichment of groundwater is rock-water interac- tion and weathering. However, chemical processes activated by the rock-water interaction were identified as ion exchange and reverse ion exchange. The host minerals determined from this study are the muscovite, chlorite and smectite. The study also revealed a decreasing trend in the fluoride concentrations from the surface down to the aquifer. From the hydrogeochemical analysis, a final conclusion can be drawn that muscovite, smectite and chlorite minerals are the main contributor of fluoride to groundwater in the study area. Groundwater temperature was also determined as a major driving force for fluoride dissolution. Data availabilityh The data used in this study has been included in the manuscript. Received: 5 September 2020; Accepted: 24 June 2021 Received: 5 September 2020; Accepted: 24 June 2021 Received: 5 September 2020; Accepted: 24 June 2021 References 1. Edokpayi, J. N. et al. 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https://openalex.org/W2892177915	https://phs.brage.unit.no/phs-xmlui/bitstream/11250/2563618/1/diverging_assessments%20.pdf	English	null	Diverging assessments of learning organizations during reform implementation	Learning organization/The Learning organisation	2,018	public-domain	5,828	Reference to this paper in APA (6th): Borge, B. H., Filstad, C., Olsen, T. H. & Skogmo, P. Ø. (2018). Diverging assessments of learning organizations during reform implementation. The Learning Organization. doi: https://doi.org/10.1108/TLO-02-2018-0024 Open Access implies that scientific publications are made freely accessible on the web. The author or originator keeps the copyright to the publication, but gives the users permission to read, download, copy, distribute, print out, search or link to the full text without a claim for compensation. Open Access implies that scientific publications are made freely accessible on the web. The author or originator keeps the copyright to the publication, but gives the users permission to read, download, copy, distribute, print out, search or link to the full text without a claim for compensation. Reference to this paper in APA (6th): Borge, B. H., Filstad, C., Olsen, T. H. & Skogmo, P. Ø. (2018). Diverging assessments of learning organizations during reform implementation. The Learning Organization. doi: https://doi.org/10.1108/TLO-02-2018-0024 Reference to this paper in APA (6th): Borge, B. H., Filstad, C., Olsen, T. H. & Skogmo, P. Ø. (2018). Diverging assessments of learning organizations during reform implementation. The Learning Organization. doi: https://doi.org/10.1108/TLO-02-2018-0024 Reference to this paper in APA (6th): Reference to this paper in APA (6th): Reference to this paper in APA (6th): Borge, B. H., Filstad, C., Olsen, T. H. & Skogmo, P. Ø. (2018). Diverging assessments of learning organizations during reform implementation. The Learning Organization. doi: https://doi.org/10.1108/TLO-02-2018-0024 This is the final text version of the article, it may contain minor differences from the publisher’s pdf version. Diverging assessments of learning organizations during reform implementation Baard H. Borge, Cathrine Filstad, Trude Høgvold Olsen & Per Ø. Skogmo Baard H. Borge, Cathrine Filstad, Trude Høgvold Olsen & Per Ø. Skogmo Introduction The purpose of this paper is to explore whether hierarchical position and organizational size affect perceptions of a learning organization (LO) during reform implementation in four Norwegian police districts. This contributes to the LO literature in two ways. First, it builds on the importance of national and sector contexts (Örtenblad, 2013; 2017) and suggests public sector reform implementation as an additional dimension of context. In the Norwegian police reform, one of the objectives is to develop the police towards working in a more knowledge- based manner and a number of governmental reports implicitly and explicitly acknowledge the LO as an ideal (NOU 2009; NOU 2012; NOU 2013). Second, it explores diverging views within an organization about its learning characteristics. Previous research has identified challenges regarding LO in the police service (Filstad and Gottschalk, 2010; 2011; Wathne, 2012), but has relied on leaders’ accounts. In a reform context, however, we might expect leaders and employees to have diverging perceptions. By including employees’ accounts, we point to challenges of developing LOs, which may guide practitioners in their implementation work. It is perhaps not surprising that organizational reforms adopt the idea of an LO as an ideal, implying expectations to organizations to develop learning practices, processes, structures and cultures. For example, studies of the police show a lack of a vision and strategy for organizational learning across organizational units (Filstad and Gottschalk, 2010; Wathne, 2012). In addition, police leaders’ espoused values correspond only to a limited extent with the values that are assumed to be important to develop an LO (Filstad and Gottschalk, 2011). However, Wathne (2012) acknowledges that the police does have some well-functioning local learning practices but that there is scant knowledge sharing across organizational boundaries. If each organization is unique and must be given necessary flexibility to develop its own version of the learning organization (Pedler et al., 1991; Senge et al., 1994), we might expect diverging views of whether a large organization shares LO characteristics, because organizational members assess this from their particular position in the organization. An LO consists of local learning practices and practices to tie these together across organizational boundaries. This implies that local learning practices and learning processes may diverge considerably between organizational units in large organizations. Abstract Purpose – This study explores whether hierarchical position and organizational size affect perceptions of a learning organization during reform implementation. Design/methodology/approach – An electronic survey was distributed in four Norwegian police districts at an early stage of reform implementation. One of the objectives of the reform was to develop the police towards being more knowledge-based and there had been specific calls for the police to become a learning organization. The respondents were 753 top managers, middle managers and employees. Findings – Respondents rated their organizations lower than benchmark scores on supportive learning environment, learning processes and practices, and leadership that reinforces learning. The perceptions diverged across hierarchical levels: middle managers and top managers gave higher scores to the organization as a learning one than employees did. Respondents from large police districts gave higher scores to their organizational units as learning organizations than respondents from small police districts. Research limitations/implications – The study captures perceptions of characteristics of a learning organization at one point in reform implementation, and further studies are needed to fully understand explanations of diverging views within an organization as to whether it can be characterized as a learning organization. Practical implications – Actual differences in local learning practices or different assessments of learning practices within the organization should be considered when developing learning organizations. Originality/value – The study contributes to our knowledge of learning organizations by showing diverging views within the same organization in a context of reform implementation. Keywords: learning organization, learning practices, reform implementation, police services Keywords: learning organization, learning practices, reform implementation, police services Introduction The research reported in this paper answers a call for more pragmatic and divergent approaches to the LO by exploring how hierarchical position and organizational size affect the assessment of the learning practices and 2 processes. This is especially important in reform implementation where a shared understanding of the status quo of the organization’s learning potential could facilitate development work. The paper is structured as follows. First, the literature on LOs is briefly reviewed and hypotheses are developed. Second, the research methods and data analysis are described. Finally, findings and contributions are discussed. Literature review The learning organization concept is based on the idea of an organization as a dynamic system that promotes continuous learning in response to various pressures (Grieves, 2008; Senge 1990), where individuals learn and transform themselves into a learning unit (Pedler et al., 1991). The concept still relies heavily on the originators’ contributions; Pedler et al.’s (1991) learning company, Senge’s (1990) fifth discipline and Argyris and Schön’s (1978) single and double-loop learning. The key point for learning organizations has traditionally been outcomes rather than mechanisms and processes of learning, and a unilateral focus on normative models for change, reconstruction and best practice of organizations (Elkjaer and Wahlgren, 2006; Laursen, 2006). The literature on the learning organization has been criticized for not sufficiently addressing how and why learning occurs (Senge and Kofman, 1995) but instead describing learning as more prescriptive than practical. First and foremost, a learning organization has been described as a set of actions that purport to ensure learning capabilities such as experimentation, continuous improvement, team work and group problem solving (Alegre and Chiva, 2008; Rebelo and Gomes, 2008). The learning organization concept, however, suffers from a lack of a clear definition that can be tested, and contested (Garvin, 2000; Grieves, 2008), and a lack of a clear description of the challenges of transforming an organization into a learning one (Bui and Baruch, 2010). However, the concept of the learning organization ‘survives’. New contributions argue for the idea that each organization needs to create its own version of a learning organization (Örtenblad, 2004). The concept of a learning organization brings valuable insights to learning processes and a context for institutions to prove and reflect upon their incapacity (Örtenblad, 2011). Even though the literature shows differences concerning what constitutes an LO and whether LOs can be compared across cultures and sectors, there seems to be general agreement on local learning practices and learning processes being at the heart of the learning organization. Such practices and processes may vary considerably between organizational units in large organizations. This implies that 3 3 there may be diverging views of the learning characteristics of large organizations and this may hamper attempts to improve learning. Literature review Örtenblad (2013) suggests that four factors characterize the LO: workplace learning (continuous learning through practice, informal learning based in social practice, and knowledge sharing), organizational learning (learning based in organizational routines, shared understandings, organizational knowledge and organizational memory), climate for learning (leaders facilitating learning, workplace that initiates and stimulates learning), and structures for learning. Garvin et al. (2008) discussed three building blocks and developed the Learning Organization Survey (LOS), a diagnostic tool for organizations to rate the degree to which they share the characteristics of an LO: supportive learning environment, concrete learning processes and practices, and leadership that reinforces learning. The Dimensions of the Learning Organization Questionnaire (DLOQ) (Kim et al., 2015; Marsick and Watkins, 2003; Watkins and Dirani, 2013; Watkins and O’Neil, 2013; Yang et al., 2004) identifies seven factors characterizing an LO: it creates continuous learning opportunities, promotes inquiry and dialogue, encourages collaboration and team learning, creates systems to capture and share learning, empowers people toward a collective vision, connects the organization to its environment, and provides strategic leadership for learning. Even though these examples illustrate different factor structures of the learning organization, there seem to be similarities between them. For example, supportive learning environment (Garvin et al., 2008) could be compared to climate for learning (Örtenblad, 2013), concrete learning processes and practices (Garvin et al., 2008) resemble workplace learning and organizational learning (Örtenblad, 2013), and leadership that reinforces learning is reflected in climate and structures for learning (Örtenblad, 2013). The study reported in this paper uses the three central building blocks for organizational learning and adaptability developed by Garvin and colleagues (2008, p. 110): ‘a supportive learning environment, concrete learning processes and practices, and leadership behavior that provides reinforcement… Each block and its discrete subcomponents, though vital to the whole, are independent and can be measured separately.’ Senge’s (1994) and Pedler and colleagues’ (1991) work focused on businesses as LOs; however, in recent decades the concept has also led to considerable research and practical interest in several parts of the public sector. For example, the LOS (Garvin et al., 2008) has been adapted for use in health care (Singer et al., 2012) and schools (Higgins et al., 2012). The 4 DLOQ has been adapted for non-profit organizations, higher education, government and the army (Watkins and O’Neil, 2013). Literature review However, organizational learning presupposes that information and knowledge can be shared easily in the organization and division of work, size and geographical dispersion could be structural barriers to learning (Lipshitz et al., 2007). This question is of particular relevance for the Norwegian police reform as the number of police districts was reduced from 27 to 12. Naturally, this resulted in larger police districts, in terms of both the geographic region they cover and the number of employees. We have not been able to find studies of LOs that explore whether there are different perceptions across organizational units of different size. However, organizational learning presupposes that information and knowledge can be shared easily in the organization and division of work, size and geographical dispersion could be structural barriers to learning (Lipshitz et al., 2007). This question is of particular relevance for the Norwegian police reform as the number of police districts was reduced from 27 to 12. Naturally, this resulted in larger police districts, in terms of both the geographic region they cover and the number of employees. Hypothesis 4: Large police districts score higher on all three dimensions of the learning organization than do small police districts. Literature review The police in different parts of the world have been studied as learning organizations (Crank and Giacomazzi, 2009; Filstad and Gottschalk, 2010; 2011; 2013; Wathne, 2012). A meta-analysis of the DLOQ (Watkins and Dirani, 2013, p. 155) concluded that government respondents rated their organizations consistently below non- government respondents, but that ‘the pattern of high and low dimensions is relatively consistent across government and nongovernment respondents’. Filstad and Gottschalk (2011; 2013) found a medium score on ‘learning structures’, and low score on ‘learning at work’, ‘climate for learning’, and ‘organizational learning’ among managers in the police. As the police is part of the public domain, we expect the respondents to rate their organization lower than benchmark scores for an LO: Hypothesis 1: Respondents give their organization low ratings on the dimensions of learning organizations. A meta-analysis of the DLOQ showed that non-managers rated five of seven dimensions significantly higher than managers did (Watkins and Dirani, 2013, p. 156): continuous learning, embedded systems, dialogue and inquiry, system connection and strategic leadership. This is interesting because we would expect that leaders and managers responsible for developing an LO would tend to be positive about the results of their efforts. However, these results also underscore that there may be large variations between organizational units, and that managers’ scores could reflect a mean for the organization, while non-manager scores reflect their organizational unit. Studies of the preparations for the implementation of the Norwegian police reform, however, suggest that more managers than employees expressed positive views on the consequences of the reform because they were more involved in the implementation work (Renå et al., 2016) or because they gave their ratings according to espoused theory rather than their theory in use (Filstad and Gottschalk, 2011). Because elements of the learning organization are intertwined in the reform, it is likely that managers give their organization a higher rating for learning than employees. Hypothesis 2: Middle managers give their organization a higher learning rating than employees in all three dimensions. Hypothesis 3: Top managers give their organization a higher learning rating than middle managers in all three dimensions. 5 5 We have not been able to find studies of LOs that explore whether there are different perceptions across organizational units of different size. Research methods and data analysis An electronic survey was sent to top managers, middle managers and employees in four local police districts in January 2017. The survey was based on the LOS (Garvin et al., 2008) because it provided benchmark scores (Garvin et al., 2008, p. 14) that were necessary to test the hypotheses. The study was approved by the participating police districts, reported to and approved by the Norwegian privacy protection commission for research. Respondents were informed about the objectives of the study, that it was voluntary to participate, and that data provided could not be traced back to them personally or to their police district. The 55 items developed by Garvin et al. (2008, pp. 112-113) were used. In line with research in particular contexts or organizations (Higgins et al., 2012; Singer et al., 2012) some items were modified to make the language specific to context. For example, market specific terms such as ‘competitors’ were changed to ‘other police districts’ as the police frequently compare results across police districts. The survey was translated into Norwegian and pretested to ensure that the respondents would understand the questions. The building blocks ‘supportive learning environment’ and ‘concrete learning processes and practices’ were measured by 47 items that respondents were asked to rate on a seven-point scale. Instead of providing a description for each of the seven points as in the original survey, a scale where 1 indicated ‘poor description’ and 7 indicated ‘perfect description’ was chosen. ‘Supportive learning environment’ was measured by items reflecting psychological safety, appreciation of differences, openness to new ideas and time for reflection. Items reflecting experimentation, information collection, analysis, education and training, and information transfer measured ‘concrete learning processes and practices’. Eight items that 6 respondents were asked to state whether they agreed or disagreed with on a five-point scale measured the third building block ‘leadership that reinforces learning’. The items described how managers facilitate learning and how they act as learning role models for example by inviting input from others, acknowledging their own limitations and asking probing questions. A factor analysis (see Table 1) showed that the items in the translated survey load on two common factors that correspond with two of the components that the survey was intended to measure: ‘supportive learning environment’ and ‘concrete learning processes and practices’. However, ‘leadership that reinforces learning’ loaded on the factor ‘supportive learning environment’. Garvin et al. Research methods and data analysis (2008) did not report factor analysis, Higgins et al. (2012) extracted one subscale from each of the building blocks and Singer et al. (2012) developed a short version of the survey with only 27 items. This makes it difficult to compare the results of our factor analysis with previous studies. However, Cronbach’s alphas for the three factors are satisfactory and do not indicate problems with the Norwegian translation of the items supportive learning environment (0.838), concrete learning processes and practices (0.846), and leadership that reinforces learning (0.925). Table 1. Factor analysis Rotated Component Matrixa Component = C 1 2 Factor 1 Appreciation of differences (C1) .901 Openness to new ideas (C1) .858 Psychological Safety (C1) .855 Analysis (C2) .650 .445 Leadership that reinforces learning (C3) .602 .430 Time for reflection (C1) .527 Factor 2 Information collection (C2) .843 Information transfer (C2) .841 Experimentation (C2) .311 .718 Education and training (C2) .502 .626 Extraction Method: Principal Component Analysis. Rotation Method: Varimax with Kaiser Normalization. a. Rotation converged in 3 iterations. Rotated Component Matrixa 7 7 Background questions about the respondent’s position in the police district and the size of the police district where the respondent worked were included, allowing for the testing of hypotheses 2-4. Even though the reduction to 12 police districts was formally decided in 2016, the practical mergers of the police districts were not finalized at the time of the study. Two large and two small police districts from the old structure of 27 police districts were invited to participate. The size of the police districts was defined with reference to the Norwegian context where a large police district had more than 500 employees and a small one had fewer than 500. Originally distributed to 2340 individuals in four police districts in January 2017, the survey yielded a response rate of 32.2% (N=753). While the response rate was not particularly high, the number of respondents was sufficient to allow careful statistical inferences about the population, given that we assumed the sample to be fairly representative, i.e. that nonresponse was random and not systematic. The survey was open for 30 days, and two reminders were sent to respondents. Considering that this period was a busy one regarding the reform implementation and that several police districts had experienced a large increase in research interest and invitations to surveys because of the reform, this response rate was satisfactory. Research methods and data analysis The respondents included employees at all three levels of authority (29 top managers, 125 middle managers and 599 employees) and from small and large police districts, with a total of 216 and 535 respondents respectively. Results Hypothesis 1. Table 2 shows the median scores from the police survey. Overall, findings strengthened H1 that respondents give their organization a low rating on the dimensions of the LO. Within all the three building blocks, median scores from the survey were lower (67.1, 49.1 and 70) than the corresponding benchmark scores (71, 74 and 76) (Garvin et al., 2008, p. 114). The police survey median scores were lower than the benchmark scores on seven of the nine underlying components of the building blocks, two notable exceptions being ‘appreciation of differences’ and ‘time for reflection’. Regarding these seven components, five of the median scores from the police survey (openness to new ideas, experimentation, information gathering, education and training, information exchange) fell within the lowest quartile of the benchmark scores and two (psychological safety, analysis) fell within the second quartile. Table 2. Median scores from the police survey. 8 SURVEY MEDIAN SCORES A supportive learning environment: - Psychological safety 74.3 - Appreciation of differences 64.3 - Openness to new ideas 71.4 - Time for reflection 60.0 Average score 67.1 Learning processes and practices: - Experimentation 42.9 - Information gathering 52.4 - Analysis 60.0 - Education and training 52.4 - Information exchange 42.9 Average score 49.1 Leadership that reinforces learning Average score 70.0 Hypothesis 2. This hypothesis was only partly supported by the data. As shown in Table 3, the survey findings indicated that middle managers on two of the three dimensions did tend to rate their organization as more of a learning organization than employees. Regarding ‘leadership that reinforces learning’, the opposite pattern was identified. However, t-tests showed that only one of the three differences in scores between the two groups, i.e. on the ‘supportive learning’ dimension, was statistically significant (p<0.01). When comparing the two groups’ answers to the individual factors underlying the dimensions, t-tests demonstrated that ‘time for reflection’, ‘experimentation’ and ‘information’ stood out as there was no significant difference between employees and middle managers. Table 3. Relationship between job level and size of police districts and scores on the three building blocks. Results A supportive learning environment Learning processes and practices Leadership that reinforces learning Job level Operating unit manger Average scores 77.8638 62.2762 76.8103 9 9 N 27 25 29 Middle manager with personnel responsibility Average scores 70.3205 50.8584 64.4612 N 117 112 116 Employee without managerial responsibility Average scores 65.0872 48.7396 67.2671 N 565 510 569 Total Average scores 66.4374 49.6294 67.1989 N 709 647 714 Size of police district Small (less than 500 employees) Average scores 64.9064 46.5561 63.4587 N 206 186 206 Large (more than 500 employees) Average scores 66.9935 50.8617 68.6215 N 501 461 506 Total Average scores 66.3854 49.6239 67.1278 N 707 647 712 Hypothesis 3. As expected, average scores in Table 3 show a clear difference between top managers and middle managers. On all three counts, the top managers gave their organization significantly higher scores than the middle managers, as demonstrated by t-tests (p<0.01). Thus, H3 was strengthened. Among the individual factors, ‘time for reflection’ was found to be the only one where the difference in the two groups’ ratings was not significant (p>0.10). Hypothesis 4. Average scores also indicated that respondents in large police districts rated their local organization on all three dimensions as more of a learning organization than their colleagues in smaller districts (see Table 3). On all three dimensions, the difference in scores was statistically significant (p<0.01). Consequently, H4 was strengthened by the data. Again, ‘time for reflection’ was the only underlying factor where the difference was not significant (p>0.10). Discussion The findings suggest some challenges regarding an LO, which confirms previous studies of police managers (Filstad and Gottschalk, 2010, 2011, 2013; Gottschalk et al., 2009; Wathne, 2012). Even though the LO is one of the important goals of the police reform, substantial challenges for learning and knowledge-based policing need to be addressed. When accounting 10 for the whole organization and not only police leaders, as has been common in previous research, becoming an LO seems even more at a distance. Our findings indicate major concerns regarding a supporting learning environment, concrete learning processes and leadership that reinforces learning. In fact, these factors mostly scored lower than the benchmark scores, with the only notable exceptions being appreciation of differences and time for reflection, both addressed as important variables for a supporting learning environment. What constitutes an LO is argued to be continuing learning in practices that represent structures, such as leadership and organization in teams, enabling these continuing learning processes for change and the creation of new knowledge (see original contributions from Argyris and Scön (1978); Pedler et al. (1991) and Senge et al. (1994)). However, organizations can prove and reflect upon their incapacity and capacity provided by learning structures, learning at work, climate for learning and organizational learning (Örtenblad, 2011). The factors of time for reflections and appreciation of differences found in this study are, however, not enough, but could represent a potential. Openness to new ideas and psychological safety, which scored quite low in our study, are needed for a supporting environment, as a climate for learning (Örtenblad, 2011). The low scores on information gathering and sharing provide serious challenges for organizational learning, where knowledge sharing bridges individual learning and organizational learning (Elkjaer and Wahlgren, 2006; Filstad, 2016). The acknowledgement of leaders as facilitators for learning and knowledge sharing and the importance of learning for the creation of a learning organization are not evident in our study, especially in the lower levels of policing. It might be that the police is typical of the public sector. Watkins and Dirani (2013) find that managers in the public sector give their organizations a lower score on learning than managers in the private sector. However, their study does not account for diverging views within the organizations. The present study is an important contribution, adding knowledge by identifying diverging views on whether managers and employees consider their organization as an LO. Discussion This is in accordance with the understanding of a learning organization as built up of local learning practices and learning processes that may vary considerably within a large organization. The findings suggest that the higher up in the hierarchy the respondents were, the more positive they were in characterizing the organization’s learning practices and processes. The present Norwegian reform context in the police conveys great expectations that leaders will develop an LO (NOU 2009, NOU 2012, NOU 2013). At the time of the study, the reform work and implementation was in an early stage with significant uncertainty for employees and 11 managers regarding their future responsibilities and job roles. This may have influenced the respondents’ responses because learning practices and learning processes may have had less focus, but on the other hand, their awareness of the need for learning due to the reform might also be higher. In addition, the lack of a clear vision and strategy on what constitutes a learning organization might explain the conflicting views we found among employees and managers on how good their organization is at learning. Managers may base their understanding on formal structures such as learning routines and reporting structures that may or may not operate as intended. Employees, on the other hand, may take as their baseline the actual learning practices that they engage in and their daily practice. International research suggests that police mergers involve risks of competence loss at all levels (Holmberg, 2010; Mendell et al., 2017). Risk of competence loss is not measured in this study, but in light of previous research, the finding that large police districts are seen as providing more learning than small police districts is interesting. More knowledge is needed of whether and how an LO can mitigate the risks of competence loss in the context of mergers. In larger police districts, there are possibilities of greater specialization. While specialization answers the calls in the reform for more knowledge-based police services, it also demands coordination across specialities. Concrete learning processes and practices beyond organizational units could be established and encouraged to remedy the negative effects of specialization. In addition, local variation within the organization can be a source of learning because it can initiate discussions of good learning practices across organizational units. Conclusions and implications This study identifies a tendency that managers at higher levels are more positive to the idea of a learning organization than middle managers are, and that middle managers are more positive than employees are. The study also shows that respondents across hierarchical levels in large police districts give their organization a higher rating for learning than respondents in small police districts. The findings contribute to the literature on learning organizations by showing that there may be diverging views of the learning characteristics within large organizations. The definition of small and large police districts was tailored to the Norwegian context, and may not necessarily generalize to other countries. Therefore, we encourage more research to explore the issues of both hierarchy and organizational size and their impact on organizational learning. In our own data, regression analysis indicated that hierarchy overall had a stronger 12 effect than size but the question remains to be answered by further research. The study’s primary contribution is linked to the call for context in studies of LO (Örtenblad, 2017), suggesting that public sector reform implementation is a specific contextual dimension in addition to national culture, religion and sector affiliation, which have been studied in previous research. 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https://openalex.org/W2100272656	https://agricultureandfoodsecurity.biomedcentral.com/track/pdf/10.1186/s40066-016-0086-0	English	null	Optimizing yield of improved varieties of millet and sorghum under highly variable rainfall conditions using contour ridges in Cinzana, Mali	Agriculture & food security	2,017	cc-by	10,191	© The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Optimizing yield of improved varieties of millet and sorghum under highly variable rainfall conditions using contour ridges in Cinzana, Mali Kalifa Traore1* , Daouda Kalifa Sidibe1, Harouna Coulibaly1 and Jules Bayala2 Kalifa Traore1* , Daouda Kalifa Sidibe1, Harouna Coulibaly1 and Jules Bayala2 Abstract Background: Low productivity of cereals, the staple food, in Sahelian zone of Cinzana in Mali is caused by a range of factors including but not limited to inherent low soil fertility, and insufficient and inadequate distribution of the rainfall due to high climate variability. In addition, the small amount of rain falls as heavy storms in very short periods of time leading to water losses by runoff which in turn causes a lot of erosion. The two phenomena therefore call for a combination of both strategic (combating erosion) and tactical (coping with inter- and intra-annual rainfall vari- ability) measures to cope with the production uncertainties in such risk-prone environment. As opposed to most farmers’ practice of using the same variety, a tactical solution of using varieties of different cycles for different rainfall amounts/patterns was thought to be worth testing. Varieties of different cycles for different rainfall amounts/patterns were combined with a well-known soil and water conservation practice which is the contour ridge tillage (CRT). The combined effects of the two measures on the production of different varieties of sorghum and millet as well as on soil water content were assessed in on-farm participatory trials in five villages. The experiment was run during three consecutive years (2012, 2013 and 2014). Results: A key finding of this research is that regardless of the yearly rainfall amount and provided CRT is used, there were large differences in yields between improved varieties and local ones. This is a result of higher soil water conser- vation and better response of the improved varieties. Conclusion: The use of CRT increases considerably the yields of improved varieties of the most important staple crops of the Cinzana commune which are millet and sorghum. Thus, the use of these early maturing improved varie- ties, along with CRT, could be an accessible adaptation strategy to climate variability by farmers. Keywords: Climate variability, Mitigation, Soil moisture, Improved varieties *Correspondence: ibosimon_1@yahoo.fr 1 Institut d’Economie Rurale (IER), BP 262, Bamako, Mali Full list of author information is available at the end of the article Agriculture & Food Security Agriculture & Food Security Traore et al. Agric & Food Secur (2017) 6:11 DOI 10.1186/s40066-016-0086-0 Open Access © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Background The combined effects of all the above-mentioned factors will induce low water infiltration rates [12] with only 10–15% of rainfall that will be used by crops for transpiration [6, 13]. Thus, water availability to crop roots in the soil pro- file constitutes a major constraint for sustainable produc- tion systems [14–17]. Because of unpredictable rainfall [18] and decreased agricultural productivity, many soil and water conservation technologies such as stone lines, half-moons, contour hedgerows, rock bunds, filter walls, zaï, agroforestry, contour ridges, benches and no-tillage have been developed and are now widespread [19–23]. Tested technologies have shown that they can reduce runoff [16, 24–26] and soil erosion [27–29], improve water infiltration [12, 22, 30] and increase soil moisture [20, 23, 31–33]. However, their adoption did not always meet the expectations probably due to the promotion of a limited number of options everywhere as if “one size fits all.” Actually, some analysis has revealed that the effects of such technologies depend on a number of factors such as rainfall (some performed better than others in drier areas, while others performed better in humid zones), soil productivity potential (some performed better in less fertile soils) and labor availability [31, 32]. All these observations call for local testing and adaptation of even the proven practices to make them context specific [34]. Background Indeed, improved varieties along with CRT could help in addressing both the short- and long-term climate-related stresses and improve the resilience of the agro-ecosystems and farmers’ income. We therefore hypothesized that improved crop varieties as opposed to the local ones will make a better use of a higher soil water storage using the CRT technique in the Sahelian zone of Cinzana in Mali. Centre de Coopération Internationale en Recherche Agronomique pour le Développement (CIRAD). Accord- ing to Kablan et al. [12], the innovation of CRT resides in the fact that it is a holistic landscape level method for managing surface water on farmers’ fields. Indeed, the contoured ridges decrease runoff, increase water infiltra- tion and, therefore, capture rainfall close to the crop root system. The technology has been applied in the Sudanian area with rainfall varying from 600 to 1200 mm in south- ern Mali where runoff still occurs in fields with a slope as low as 1 to 2%, [6]. As a consequence of increased water infiltration due to CRT [8, 12], an increase in crop yields of 30–50% was reported for millet, sorghum, maize, groundnut and cotton [30, 36]. This is probably due to the fact that water availability is important for evapo- transpiration, but also for releasing nutrients in the root- ing zone of the crops. Without CRT, runoff varied from 25–55% (unavailable water for crops) while its implemen- tation reduced runoff to 10% of annual rainfall. Although the CRT was introduced since early 1990s [35], its effects on crop yield in the Sahelian area of Mali are not well documented. Indeed, improved varieties along with CRT could help in addressing both the short- and long-term climate-related stresses and improve the resilience of the agro-ecosystems and farmers’ income. We therefore hypothesized that improved crop varieties as opposed to the local ones will make a better use of a higher soil water storage using the CRT technique in the Sahelian zone of Cinzana in Mali. erratic rainfall patterns leading to water stress at some critical plant-growth stages [7] and inherent low soil fertility [6]. In addition, rain storms generate runoff and associated soil erosion [8–11]. This later phenomenon reduces top soil layer depth and soil organic matter con- tent leading to weak soil water-holding capacity. Background average yield of 768 kg ha−1. Equivalent figures for sor- ghum (Sorghum spp) were 986,367 ha and 943 kg ha−1 [2]. Millet occupies a larger area than sorghum because of its higher plasticity. Average yields for millet and sor- ghum in Cinzana commune, Mali (Ségou region), were, respectively, 822 and 926 kg ha−1 reflecting national trends [3]. Obviously the yield values mentioned above are below the potential of most grown varieties of these staple crops [4, 5]. The low yields are attributed not only to climate change-related decreases in rainfall amount but also to variability of water availability related to the In sub-Saharan Africa region, 97% of the agricultural land is rainfed with crop yields of about 0.5–1 t ha−1 [1]. The most common crops in the Sahelian part of this region are cereals (millet, sorghum, maize, rice, etc.) as a staple food. For instance in 2008 in Mali, 1,615,450 ha were planted with pearl millet (Pennisetum spp) with an Traore et al. Agric & Food Secur (2017) 6:11 Page 2 of 13 Page 2 of 13 Centre de Coopération Internationale en Recherche Agronomique pour le Développement (CIRAD). Accord- ing to Kablan et al. [12], the innovation of CRT resides in the fact that it is a holistic landscape level method for managing surface water on farmers’ fields. Indeed, the contoured ridges decrease runoff, increase water infiltra- tion and, therefore, capture rainfall close to the crop root system. The technology has been applied in the Sudanian area with rainfall varying from 600 to 1200 mm in south- ern Mali where runoff still occurs in fields with a slope as low as 1 to 2%, [6]. As a consequence of increased water infiltration due to CRT [8, 12], an increase in crop yields of 30–50% was reported for millet, sorghum, maize, groundnut and cotton [30, 36]. This is probably due to the fact that water availability is important for evapo- transpiration, but also for releasing nutrients in the root- ing zone of the crops. Without CRT, runoff varied from 25–55% (unavailable water for crops) while its implemen- tation reduced runoff to 10% of annual rainfall. Although the CRT was introduced since early 1990s [35], its effects on crop yield in the Sahelian area of Mali are not well documented. Materials and methods Study siteh i Because the farmers of Cinzana collaborate with the Climate Change Agriculture and Food Security (CCAFS) project and are therefore well aware of the advantages of soil and water conservation techniques and crop diver- sity to buffer climate change effects, a diagnosis including key stakeholders was realized in this village. Despite the awareness of the contour ridge tillage technique (CRT), there is a weak adoption of this practice and a need for training. The participatory selection of the technologies (the selection of technologies was done with the active participation of farmers) to be tested led to the choice of contour ridge tillage technique (CRT) and the use of improved sorghum and millet varieties among several adaptation strategies proposed to mitigate the observed erratic rainfall patterns. These improved varieties were chosen because of their performance on station trials, although local varieties are sometimes considered bet- ter adapted to high stress and low productivity condi- tions typical of smallholder farms. In Mali, CRT, referred to as “Aménagement en courbes de niveau” [12, 6, 35], is a water conservation technique locally developed in the early 1990s by Institut d’Economie Rurale (IER) and This on-farm trial was conducted in the Cinzana rural commune, which belongs to the Sahelian agroecologi- cal zone of Mali (Fig. 1). The trial area is located between 13°53′N and 13°14′N latitude and 5°63′W–6°15′W longi- tude. Participating villages are distributed within a radius of 30 km and are represented by the stars in Fig. 1. Rain- fall was measured in Cinzana Research Station which is the closest meteorological weather station. Rainfall is uni-modal with the maximum of rain events occur- ring in July and August (Fig. 2) with a long-term average annual rainfall of 680 mm (Fig. 3b). The first useful rains (which provide enough soil moisture at time of planting without prolonged dry spells that could prevent the sur- vival of seedlings after sowing [37]) occur in May and the rainy season ends in October. An analysis of rainfall pat- terns in Cinzana was performed using decadal (10-day) intervals in the month (Fig. 3b). Low temperatures occur in December through February (18 °C monthly aver- age low), and high temperatures occur in April and May (40 °C monthly average high) (Fig. 2a). The daily evapo- transpiration is 6–7 mm day−1 in the dry season and Traore et al. Agric & Food Secur (2017) 6:11 Page 3 of 13 Fig. Cultural operations 0 5 10 15 20 25 30 35 40 45 0 50 100 150 200 250 300 350 400 J F M A M J J A S O N D Temperature (°C) Rainfall (mm) 2012 2013 2014 Average PET Max T Min T For both millet and sorghum, planting dates were 2–3, 11–12 and 3–4 July in 2012, 2013 and 2014, respectively. Sorghum and millet seeds were placed at 0.5-m inter- vals within rows and 0.8-m intervals between rows, and seedlings were thinned to two plants per hill 15 days after emergence to ensure the targeted population of 50,000 plants ha−1 which is the density advised by extension services in Cinzana area. Immediately after thinning (i.e., the same day), the trials were hand-weeded using a hoe and again 30 days after crop germination. Fertilizer was uniformly applied to the trial at the rate of 37.5 kg ha−1 of NPK (15–15–15) and 37.5 kg ha−1 of urea (46% of nitrogen), respectively, 15 and 30 days after germination. Fertilizer was buried in microdoses (1.5 g per hole) 5 cm below and 5 cm away the plant rows. Fig. 2 Monthly rainfall average, maximum (Max T), minimum (Min T) temperatures and potential evapotranspiration (PET) of three con- secutive years (2012, 2013 and 2014) in Cinzana commune, Mali Sorghum and millet grains were harvested on 20–21 October, 5–6 November and 25–26 October in 2012, 2013 and 2014, respectively, and sun-dried straw meas- ured 21 days after. 4 mm day−1 during the rainy season. The main soil types of the area are classified as leached tropical ferruginous soils with spots and concretions [38] and Alfisols accord- ing to U.S. Soil Taxonomy [39], with many Paleustalfs and frequent Plinthustalfs [12, 40]. Ustalfs are highly weath- ered and highly leached soils. Plinthustalfs are of special concern because they contain a plinthite layer of soft iron (Fe) and aluminum (Al) oxides that will harden irrevers- ibly into lateritic stone if exposed. Materials and methods Study siteh 1 The Cinzana commune and the location of the on-farm trial sites where the effects of contour ridge tillage on the production of local and improved millet and sorghum varieties were tested in 2012, 2013 and 2014 in Mali Fig. 1 The Cinzana commune and the location of the on-farm trial sites where the effects of contour ridge tillage on the production of local and improved millet and sorghum varieties were tested in 2012, 2013 and 2014 in Mali 0 5 10 15 20 25 30 35 40 45 0 50 100 150 200 250 300 350 400 J F M A M J J A S O N D Temperature (°C) Rainfall (mm) 2012 2013 2014 Average PET Max T Min T Fig. 2 Monthly rainfall average, maximum (Max T), minimum (Min T) temperatures and potential evapotranspiration (PET) of three con- secutive years (2012, 2013 and 2014) in Cinzana commune, Mali Data collection and analysis Composite soil samples were collected only in May 2012 at 0–20 cm soil depth before establishing the tri- als on each site. Composite samples were made of 20 soil samples taken in an asterisk shape pattern in the field. Samples were analyzed for both physical and chemical properties. Particle size (soil texture) analysis was per- formed by the hydrometer method [41]; pH was deter- mined by the electrometric method in a soil solution with a soil/water ratio of 1:2.5; soil organic C was determined by the modified Walkley–Black wet oxidation method as outlined by Nelson and Sommers [42]; total nitrogen was determined by the modified Kjeldahl digestion method [43]; bases, effective cation exchange capacity (CEC) and available P were determined as described in Page et al. [44]. 1981 1982 1983 1984 1985 1986 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 Year 0 20 40 60 80 100 120 140 160 180 200 Rainfall (mm) 2012 2013 2014 b b Jan1 Jan2 Jan3 Feb1 Feb2 Feb3 Mar1 Mar2 Mar3 Apr1 Apr2 Apr3 May1 May2 May3 Jun1 Jun2 Jun3 Jul1 Jul2 Jul3 Aug1 Aug2 Aug3 Sep1 Sep2 Sep3 Oct1 Oct2 Oct3 Nov1 Nov2 Nov3 Dec1 Dec2 Dec3 Decades In 2012 and 2013, soil moisture was measured first in the dry season (mean dry soil) and in the rainy season at 15-day interval period in Sorobougou village to illustrate water conservation related to the use of CRT. Soil was sampled using an Edelman Combination Auger (4 cm core) of 1.2 m length at three locations in both plots in each field. Soil was sampled at four depths (0–10, 10–20, 20–40 and 40–60 cm) until a hardpan layer was reached, which in general was at about 60 cm depth. Soil samples were sent to the laboratory after securing them in a dou- ble plastic bag to avoid moisture loss. Gravimetric soil water content was determined at the same soil depths by weighing soil samples to obtain their wet mass, followed by oven-drying at 105 °C during 24 h until constant mass and weighing them again for their dry mass. Gravimetric soil water content was calculated as: Fig. Experimental designhi The field of each farmer was divided into two parts: one with contour ridges and the second part without contour ridge. The trial was established as a randomized complete block design with farmer fields or sites as replicates. This disposal allows a first examination of the global trend of the results for the control and the CRT plots. This pair Traore et al. Agric & Food Secur (2017) 6:11 Page 4 of 13 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1981 1982 1983 1984 1985 1986 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 Rainfall (mm) Year 0 20 40 60 80 100 120 140 160 180 200 Jan1 Jan2 Jan3 Feb1 Feb2 Feb3 Mar1 Mar2 Mar3 Apr1 Apr2 Apr3 May1 May2 May3 Jun1 Jun2 Jun3 Jul1 Jul2 Jul3 Aug1 Aug2 Aug3 Sep1 Sep2 Sep3 Oct1 Oct2 Oct3 Nov1 Nov2 Nov3 Dec1 Dec2 Dec3 Rainfall (mm) Decades 2012 2013 2014 a b Fig. 3 Long-term annual rainfall a and decadal (10-day interval) rainfall (Month1 first decade, Month2 second decade, Month3 third decade) of three consecutive years (2012, 2013 and 2014), b in Cin- zana commune, Mali 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 00 01 02 03 04 05 06 07 08 09 10 11 12 13 14 Rainfall (mm) a farmers for each of the two crop species (millet and sor- ghum), giving a total 20 farmers involved. Tested varie- ties were local Boboni, Soxat, improved Toroniou and HKD for millet and local Kenikeni, CSM 219, Seguifa and Sangatigui for sorghum. Data collection and analysis 3 Long-term annual rainfall a and decadal (10-day interval) rainfall (Month1 first decade, Month2 second decade, Month3 third decade) of three consecutive years (2012, 2013 and 2014), b in Cin- zana commune, Mali of plots was treated the same way with regard to sowing dates, crop species and other cropping operations, except the ridging mode which was tested. Varieties were ran- domly distributed in each part of the experimental field. Plot sizes were 42 m2 in 2012 and 2013 and 98 m2 in 2014 for demonstrative purpose.h The trial started in 2012 with three farmers in each vil- lage (Moussawere, Sorobougou and Ngakoro), and two factors were studied which are field preparation (con- tour ridging and control) and varieties for each crop. Two varieties were tried in 2012 for both sorghum (local Jacumbe and Seguifa) and millet (local Toronion and Syn 0006) and the trial replicated three times. These varieties were chosen by farmers during a field visit in Cinzana research station. In 2013, two more villages (Tongo and Dougakoungo) were included in the trial as well as two more fodder crop “stay green” varieties, i.e., they remain green as fodder after harvesting the panicles (Seguifa and Tiandougou). The trial was therefore replicated five times for three varieties of sorghum (local Jacumbe, Seguifa and Tiandougou) and three varieties of millet (local Toro- nion, Syn 0006 and Soxat). Based on the outcomes of the first two years of trial and to facilitate the monitor- ing, the experience was conducted in two villages (Tongo and Ngakoro) in the third year, but was expanded to ten Gravimetric soil water content (GSW) g g−1 Gravimetric soil water content (GSW) g g−1 = wet mass −dry mass /dry mass = wet mass −dry mass /dry mass From the data collected throughout the rainy season, dates were chosen to represent the beginning, the middle and the end of the rainy season and are plotted in Fig. 4. As soil bulk density changes very little with time, we decided to use values obtained in a previous study in 2011 along the 0–60 cm profile which includes most of the root system of cereals [45, 46]. The values for soil bulk density were 1.67 ± 0.02, 1.61 ± 0.01, 1.48 ± 0.01 and 1.45 ± 0.00 g cm−3 for 0–10, 10–20, 20–40 and 40–60 cm depths, respectively [46]. Soil moisture storage (SMS, mm) for each layer was calculated from gravimetric soil Traore et al. Agric & Food Secur (2017) 6:11 Page 5 of 13 Fig. 4 Vertical distribution of gravimetric soil water content in the 0–60 cm depth in Contour ridge tillage plots (CRT) and plots without CRT (Ctrl) in Sorobougou village in Cinzana commune in 2012 and 2013; a beginning of the growing season (July 22, 2012; July 20, 2013); b middle of the grow- ing season (August 7, 2012; August 5, 2013); c end of the growing season (September 7, 2012; September 4, 2013); dry soil soil during the dry season (May 5, 2012); bars indicate standard errors of the means Fig. 4 Vertical distribution of gravimetric soil water content in the 0–60 cm depth in Contour ridge tillage plots (CRT) and plots without CRT (Ctrl) in Sorobougou village in Cinzana commune in 2012 and 2013; a beginning of the growing season (July 22, 2012; July 20, 2013); b middle of the grow- ing season (August 7, 2012; August 5, 2013); c end of the growing season (September 7, 2012; September 4, 2013); dry soil soil during the dry season (May 5, 2012); bars indicate standard errors of the means Fig. Gravimetric soil water content (GSW) g g−1 4 Vertical distribution of gravimetric soil water content in the 0–60 cm depth in Contour ridge tillage plots (CRT) and plots without CRT (Ctrl) in Sorobougou village in Cinzana commune in 2012 and 2013; a beginning of the growing season (July 22, 2012; July 20, 2013); b middle of the grow- ing season (August 7, 2012; August 5, 2013); c end of the growing season (September 7, 2012; September 4, 2013); dry soil soil during the dry season (May 5, 2012); bars indicate standard errors of the means water content (GSW), soil bulk density (Db) and soil layer depth (H) as follows: Crop yields were measured in central rows of each plot by discarding two rows along the border of each side of the plot. At harvest, total panicles, grain and stems dry weight were recorded in the central subplot as indicated above and data extrapolated from the subplot size to ha. SMSi = SGWi × Dbi × H (i = 1, 2, 3 . . . n) where SMSi is the soil moisture storage for a certain soil layer depth (mm), SGWi is the gravimetric soil water content (g g−1) at such soil depth, Dbi is the soil bulk density (g cm−3) at such depth, H is the soil layer depth (mm), i is the soil sequence and n is the number of meas- ured layers. At the beginning of data processing, each part of the experiment plot was analyzed as a simple trial and the means and residual values used to pool the trial fol- lowing two hierarchized factors (CRT-Ctrl; varieties) to just determine the global significance of data using Page 6 of 13 Page 6 of 13 Traore et al. Agric & Food Secur (2017) 6:11 Soil characteristicsh STATBOX 7.4.4. Then, data were analyzed using Gen- stat statistical software (Release 14 for Windows) where crop data were subjected to a factorial analysis consid- ering the individual effects of CRT and crop variety as well as their interaction. Because the numbers of rep- lications and crop varieties varied from year to year, the data were analyzed separately per year. Finally, the three years were pooled to statistically assess the inter- action between CRT and varieties. The effects of the treatments were considered significant at the probabil- ity threshold of P < 0.05. Newman–Keuls test was used to separate means for significant differences between treatments. The soils of Ngakoro and Tongo sites displayed more silt content and less clay than those of other villages, whereas mean sand content was less variable between villages with a value around 90% (Table 1). Soil pH (water) of the study sites was generally slightly acid, but more acid at the Sorobougou village site. The Ca and Mg contents at this latter site had double the values observed on the other sites. However, considering other physicochemical characteristics, Moussawere village appeared to have the poorest soil with phosphorus content at least twice lower compared to other sites. The CEC was also higher in all sites in comparison with Moussawere, except Tongo. All soils showed very low values in organic matter, nitrogen and phosphorus (Table 1). Results Figure 4 shows that gravimetric soil water content (GSW) was frequently greater in CRT than in the control (P = 0.04). In general, soil was drier in 2012 compared to 2013. Rainfall was more abundant in 2012 but less well distributed compared to 2013 because of two heavy rain events of 70 and 80 mm in less than two hours (Fig. 3b). In July at the beginning of the rainy season, CRT exhib- ited greater differences from control in 2013 compared to 2012. Differences were only noticeable at the 10–20 cm soil depth in 2012 but were noticeable in the whole soil profile in 2013. CRT differences from control were also more noticeable at the 10 to 20 cm soil depth in early August 2012 and 2013 when rainfall events were still frequent and differences were more pronounced at the 10–20 cm soil depth in 2012 when soils were drier com- pared to 2013. This difference appeared at 10 cm and was maintained up to 60 cm in 2012. In 2013, when rainfall was much lower, CRT displayed only higher soil water content in the 10–20 cm depth. Rainfall pattern in the study area Maximum amount of 166 mm of rain was received in the first decadal (10-day) interval of August in 2012, where July and August represented 77% of the total amount of rainfall (Fig. 3b), with no significant dry spell during this period. Dry spell of 6 days occurred once in the second decade of June. In September, dry spells of 7 and 8 days occurred in the second and third decades, respectively. The dry spells coincided with the development of repro- ductive organs of millet and sorghum. In 2013, the long- est dry spell lasted 14 days and occurred in second and third decades of June, before crop sowing. A 9-day dry spell also occurred in July (second and third decades) and a 11-day one in September (second and third decades). In 2014, the longest dry spell was 5 days and occurred in the second decade of September, suggesting that rainfall was evenly distributed throughout the cropping season. This relatively short dry spell was adequate to allow weeding, mounding and other crop management operations. Table 1 Soils characteristics in the 0–20 cm soil depth of five sites under trial in 2012 in Cinzana commune, Mali Sites Sorobougou N’Gakoro Moussawere Tongo Dougakoungo pH (water) 5.75 6.54 6.44 6.50 6.48 pH (KCl) 4.04 5.10 4.44 5.37 5.34 OC (g kg−1) 3 4 4 5 6 Total N (g kg−1) 0.2 0.03 0.2 0.4 0.05 P av (ppm) 5.32 4.20 1.96 3,75 3,67 CEC meq 100 g−1 5.80 5.80 4.35 4.40 4,65 Ca meq 100 g−1 3.86 1.58 1.58 1.41 1.18 Mg meq 100 g−1 1.80 0.95 0.84 0.81 0.77 K meq 100 g−1 0.41 0.13 0.15 0.16 0.18 Na meq 100 g−1 0.03 0.03 0.03 0.02 0.04 Sand % 0.05 mm 92 89 92 90 92 Silt % 0.05–0.002 mm 4 9 4 8 6 Clay % 0.002 mm 4 2 4 2 2 ristics in the 0–20 cm soil depth of five sites under trial in 2012 in Cinzana commune, Mali ble 1 Soils characteristics in the 0–20 cm soil depth of five sites under trial in 2012 in Cinzana commu Traore et al. Agric & Food Secur (2017) 6:11 Page 7 of 13 Water storage reached a maximum of 42 mm in August 2012 and a maximum of 68 mm in August 2013 (Fig. 5). Discussion Sorghum yield did not differ significantly under the CRT technique in 2012 as opposed to the two following years during which CRT displayed statistically higher values compared to the control plot (all P < 0.05). Yield increases related to the CRT technique were 613 kg ha−1 in 2013 and 616 kg ha−1 in 2014 or, respectively, 85 and 58% yield increases compared to the control plot An increasing but non-consistent trend in both grain and straw biomass yields was observed for millet (Table 2) and sorghum (Table 3) from the first to the third year. Such trend cannot be easily related to the amount of rain- fall recorded during the three years of testing. Indeed, the amount of rainfall recorded in 2013 (692 mm) and in 2014 (725 mm) was almost half of the volume in 2012 (1051 mm), yet better yields were recorded in 2014. Bet- ter production in 2014 might therefore be due to bet- ter rainfall distribution during the rainy season of that year. Decadal rainfall analysis revealed the occurrence of several dry spells in September during the grain fill- ing period which might have affected the yields in both 2012 and 2013 (two very contrasting years in terms of rainfall amount) independently of the total amount of rainfall received in a given year. These observations are supported by Sivakumar [18] who widely reported simi- lar trends when studying the relation between climate and soil productivity in Sudanian and Sahelian zones of Africa. Indeed, high inter-annual rainfall and within-sea- son variability of rainfall typify the West Africa Sahel cli- mate [47–49]. A supplementary explanation may be the cumulative effect of fertilization which would make the 0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 20-7 3-8 17-8 31-8 14-9 28-9 12-10 26-10 9-11 23-11 7-12 Soil water storage (mm) Time CRT 2013 Ctrl 2013 CRT 2012 Ctrl 2012 Fig. 5 Soil water storage in the 60 cm profile in the Sorobougou village of the Cinzana commune in 2012 and 2013. CRT contour ridge tillage plots; Ctrl plots without Contour ridge tillage 0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 20-7 3-8 17-8 31-8 14-9 28-9 12-10 26-10 9-11 23-11 7-12 Soil water storage (mm) Time CRT 2013 Ctrl 2013 CRT 2012 Ctrl 2012 Time Fig. 5 Soil water storage in the 60 cm profile in the Sorobougou village of the Cinzana commune in 2012 and 2013. Rainfall pattern in the study area Water storage decreased regularly until November when both CRT plot and control displayed similar values of 13.4 mm in 2012 and 21.8 mm in 2013. Water storage was always higher in CRT plot compared to control plot with a surplus of 0.23 mm day−1 in 2012 and 0.43 mm day −1 in 2013 in the CRT plots over the monitoring period. (Table 3a). In the overall, average sorghum grain yield increased consistently from 461 kg ha−1 in 2012 to 1378 kg ha−1 in 2014. Improved varieties produced on average 55% more yield than the local ones (Fig. 6). There were significant effects of CRT on sorghum grain pro- duction in 2013 (P < 0.04) and 2014 (P < 0.001). There was, also, significant interaction in 2014 between variety and tillage mode (P = 0.001). Table 3b shows sorghum straw production for which significant differences were only observed in 2014 for both varieties and the tillage modes but the interaction between these two factors was not significant (P > 0.05). Straw biomass increase due to CRT was 3363 kg ha−1 in 2014, representing 61% more biomass, while increase due to improved varieties was much lower (17%). Millet and sorghum yield Average millet grain yield was 783 kg ha−1 in 2012, 1424 kg ha−1 in 2013 and 1301 kg ha−1 in 2014 (Table 2a). Millet grain yield in 2012, 2013 and 2014 was statisti- cally higher in CRT plots compared to the control (all P < 0.01) with yield difference ranging from 301 kg ha−1 in 2012 to 622 kg ha−1 in 2013. These values correspond to an increase of 60 and 56%, respectively. Improved vari- eties produced more than the local ones, and the average increase was +25% in 2012 and +35% in 2014 (Table 2a). There were significant interactions between variety and tillage mode in 2012 and 2014 (P < 0.029 in 2012 and P < 0.002 in 2014).f For both crops, the statistical analysis showed incon- sistent interactions between variety and tillage mode each year which is characteristic of on-farm trials. How- ever, when plotting grain yield against the tillage modes, another interaction is revealed, which is an interaction between CRT mode and improved varieties. In general, without CRT there were no much differences between local and improved varieties of both millet (Table 2a) and sorghum (Table 3a). However, improved varieties produced better under CRT compared to the local ones. Moreover, such difference is larger in sorghum compared to millet, particularly for Sangatigui (Fig. 6) which out- yielded the other sorghum varieties. Millet straw production differed statistically according to varieties only in 2014 (P = 0.01) during the three years (Table 2b). Straw production was statistically greater in CRT plots compared to control plots in 2013 and 2014, but not in 2012. The effect of CRT on straw yield varied from 881 kg ha−1 in 2012 to 2654 kg ha−1 in 2014 corre- sponding to an increase of 32–61%.fi Discussion This finding corroborates Gigou et al. [36] who estimated water supply through modeling and concluded that more significant water was available in CRT field when compared to the control in South Mali. The use of CRT can result in reducing soil erosion by reducing pre- cipitation water runoff. It allows more time than the control for rainwater to infiltrate, therefore increasing water storage. This leads to better growth and higher yield during cropping seasons with unpredictable rain- fall or low total rainfall as reported by many authors [12, 29, 30, 52].h comparison between years inappropriate in the absence of soil data for each year.h The overall low grain yield of the tested varieties for both millet and sorghum, with highest grain yield infe- rior to 1500 kg ha−1, might stem from a combination of limited water availability (due to poor within-sea- son distribution) and poor soil fertility. The labora- tory analyses have revealed that the soils of the study sites have a low fertility and are slightly acid. They all showed a very high proportion of sand of about 90% and very low nitrogen and soil organic matter contents as well as low CEC values (Table 1). Tropical ferrugi- nous soils are characterized by a high water infiltration rate (leading to high drainage) in sandy soils and a low water-holding capacity unless soil organic matter con- tent is improved [9, 11]. In fact, low activity of kaolinite (1:1 clay type) which is the dominant clay in these soils suggests that other fine elements (e.g., silt) intervene in complexes with soil organic matter and play a cer- tain role in soil chemical (CEC) properties by influenc- ing its capacity for storage and exchange of nutrients as reported by previous workers [9, 50]. In such sandy soils, an increase of 1 g kg−1 of organic carbon leads to an increase of 4.3 mol kg−1 of CEC [51]. Improved vari- eties as expected performed better than the local ones for both crops (Tables 2 and 3). Indeed, Fig. 6 shows that a marginal difference was found between improved varieties and local ones with the control tillage mode and a much larger difference was found between these two types of varieties under CRT. Discussion CRT contour ridge tillage plots; Ctrl plots without Contour ridge tillage Page 8 of 13 Traore et al. Agric & Food Secur (2017) 6:11 Table 2 Effects of contour ridge tillage on the production of millet varieties in Cinzana commune, Mali Mean values and comparisons Year Conservation technique Variety Variety Technique Variety × technique a. Grain yield (kg ha−1) 2012 CRT Syn 006 1094a 963a Toronion (local) 831b Control Syn 006 641c 602b Toronion (local) 563c SE 45.6 Probability value 0.002 0.001 0.029 CRT yield increase 301 2013 CRT Syn 006 1837a 1735a Toronion (local) 1497a Soxat 1871a Control Syn 006 1063a 1113b Toronion (local) 1280a Soxat 997a SE 309.4 Probability value 0.97 0.002 0.125 CRT yield increase 622 2014 CRT Toroniou 1716a 1585a Soxat 1646a Boboni (local) 1166b HKP 1811a Control Toroniou 1064b 1017b Soxat 1009b HKP 1105b Boboni (local) 891b SE 102.9 Probability value 0.001 0.001 0.002 CRT yield increase 568 Mean values and comparisons Year Technique Varieties Variety Technique Variety × technique b. Straw dry mass (kg ha−1) 2012 CRT Syn 006 4048a a Toronion (local) 3238a Control Syn 006 3048a a Toronion (local) 2476a SE 404 Probability 0.611 0.641 0.691 CRT yield increase 881 2013 CRT Syn 006 3514a a Toronion (local) 2886a Soxat 4314a Control Syn 006 2200a b Toronion (local) 2657a Soxat 2514a SE 524 Probability 0.270 0.001 0.148 Table 2 Effects of contour ridge tillage on the production of millet varieties in Cinzana commune, Mali ridge tillage on the production of millet varieties in Cinzana commune, Mali Mean values and comparisons Page 9 of 13 Traore et al. Agric & Food Secur (2017) 6:11 Table 2 continued Mean values and comparisons Year Technique Varieties Variety Technique Variety × technique CRT yield increase 1114 2014 CRT Toroniou 7389a a Soxat 7034ab Boboni (local) 6112b HKP 7563a Control Toroniou 4460c b Soxat 4530c HKP 4588c Boboni (local) 4460c SE 312 Probability 0.01 0.001 0.001 CRT yield increase 2654 NB For a given year, values with different letters are statistically different at P = 0.05. Column means represent tillage techniques and row means the varieties. SE standard error of the mean Table 2 continued Mean values and comparisons higher yield. Accumulated water in CRT may delay water stress [6, 30, 52] while being more available for the crop to accomplish its physiological processes of biomass accumulation and grain filling [53, 54]. Discussion Improved varie- ties with a higher yield potential likely took advantage of the higher available water under CRT to produce The higher grain and straw biomass yields could also be attributed to growth and genetic characteristics of the crop. Improved varieties have a greater ability to convert assimilates to grain and biomass. In fact, genetic char- acteristics could explain variability of sensitivity of crop to water deficit or availability. There is a very large plant genetic variability of growth sensitivity related to water deficit as reported by Tardieu [55], who mentioned that sensitivity of leaf growth to evaporative demand and soil water deficit can be translated into biomass accumula- tion in the field. The different responses of improved vs local varieties to water availability could be attributed to this difference in biomass accumulation both in grain and straw.h The substantial response of millet and sorghum to the use of CRT indicated that in Sahelian area such as the Cinzana zone, this soil and water conservation technique Page 10 of 13 Traore et al. Agric & Food Secur (2017) 6:11 Table 3 Effects of contour ridge tillage on the production of sorghum varieties in Cinzana commune, Mali Mean values and comparisons Year Technique Varieties Variety Technique Variety × technique a. Grain yield (kg ha−1) 2012 CRT Seguifa 621a a Jacumbe (local) 381b Control Seguifa 545a a Jacumbe (local) 297b SE 43.5 Probability 0.01 0.56 0.97 CRT yield increase 96 2013 CRT Jacumbe(local) 1174a a Seguifa 1231a Tiandougou 1600a Control Jacumbe(local) 677b b Seguifa 704b Tiandougou 786b SE 287 Probability 0.39 0.04 0.62 CRT yield increase 613 2014 CRT Seguifa 1957b a CSM 219 1353c Sangatigi 2300a Kenikeni (local) 1134cd Control Seguifa 1200cd b CSM 219 928de Sangatigi 1333c Kenikeni (local) 819e SE 81.6 Probability 0.001 0.001 0.001 CRT yield increase 616 Mean values and comparisons Year Technique Varieties Variety Technique Variety × technique Straw dry biomass (kg ha−1) 2012 CRT Jacumbe 3524a a Seguifa (local) 6857a Control Jacumbe 3943a a Seguifa (local) 5000a SE 1407 Probability 0.08 0.49 0.29 CRT yield increase −167 2013 CRT Jacumbe 5057a a Seguifa (local) 4457a Tiandougou 4143a Control Jacumbe 3314a a Seguifa(local) 3429a Tiandougou 3486a SE 958 Probability 0.92 0.06 0.88 Table 3 Effects of contour ridge tillage on the production of sorghum varieties in Cinzana commune, Mali Technique Variety × technique Page 11 of 13 Traore et al. Discussion Agric & Food Secur (2017) 6:11 Table 3 continued Mean values and comparisons Year Technique Varieties Variety Technique Variety × technique CRT yield increase 1142 2014 CRT Seguifa 10452a a CSM 219 8007a Sangatigi 9543a Kenikeni (local) 7558a Control Seguifa 6027b b CSM 219 4937b Sangatigi 5873b Kenikeni (local) 5273b SE 445 Probability <0.001 <0.001 0.97 CRT yield increase 3363 NB For a given year, values with different letters are statistically different at P = 0.05. Column means represent tillage techniques and row means the varieties. SE standard error of the mean Table 3 continued NB For a given year, values with different letters are statistically different at P = 0.05. Column means represent tillage techniques and row means the varieties. SE standard error of the mean NB For a given year, values with different letters are statistically different at P = 0.05. Column means represent tillage techniques and row means the varieties. SE standard error of the mean varieties requires improved water management. How- ever, as mentioned by Mcauley et al. [56], additional sup- port is required to strengthen production and delivery systems of improved seed varieties while encouraging farmers to better manage their natural resources with the CRT technique. 0 200 400 600 800 1000 1200 1400 1600 1800 2000 CRT Control Millet grain yield (kg ha-1) Technique HKP Soxat Syn 006 Toroniou Boboni (local) Toronion (local) 0 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 CRT Control Sorghum grain yield (kg ha-1) Technique CSM 219 Sangatigui Seguifa Tiandougou jacumbe (local) Kenikeni (local) a b Fig. 6 Interactions between contour ridge tillage (CRT) and millet a varieties (improved: Soxat, Soxat1, HKP, Synthetic 006, Toroniou; local: Boboni, Toronion) and sorghum b varieties (improved: CSM219, Sangatigui, Seguifa, Tiandougou; local: Jacumbe, Kenikeni) observed from 2012 to 2014 in Cinzana commune, Mali. CRT contour ridge till- age plots; Ctrl plots without contour ridge tillage 0 200 400 600 800 1000 1200 1400 1600 1800 2000 CRT Control Millet grain yield (kg ha-1) Technique HKP Soxat Syn 006 Toroniou Boboni (local) Toronion (local) a Acknowledgements This work was funded by the CGIAR Program on Climate Change, Agriculture and Food Security (CCAFS), which is a strategic partnership of the CGIAR and Future Earth. The CCAFS Program is carried out with funding by CGIAR Fund Donors, the Canadian International Development Agency (CIDA), the Danish International Development Agency (DANIDA) and the European Union (EU), with technical support from the International Fund for Agricultural Develop- ment (IFAD). The world Agroforestry Centre (ICRAF) is also acknowledged for coordinating the participatory action research on climate smart agriculture (PAR-CSA) in West Africa. Additional funding from the Institut d’Economie Rurale du Mali is also gratefully acknowledged. We are grateful to AMEDD and ARCAD NGOs for supporting farmer exchange visit in the PAR site. We are grateful to farmers of Nkakoro, Tongo, Sorobougou, Moussawèrè and Dougakoungo. We thank a lot Youssouf Coulibaly (extension agent in Cinzana) and Oudou Cissé (technician of the project) for facilitating the implementa- tion of research activities and data collection. Dr. Coe Richard is acknowledged for his support for the data analysis and comments on the earlier version of this article. 6. Doumbia M, Jarju A, Sene M, Traore K, Yost R, Kablan R, et al. Sequestra- tion of organic carbon in West African soils by Aménagement en Courbes de Niveau. Agron Sust Dev. 2008;29:267–75. 6. Doumbia M, Jarju A, Sene M, Traore K, Yost R, Kablan R, et al. Sequestra- tion of organic carbon in West African soils by Aménagement en Courbes de Niveau. Agron Sust Dev. 2008;29:267–75. 7. Chakraborty S, Newton AC. Climate change, plant diseases and food security: an overview. Plant Pathol. 2011;60:2–14. 7. Chakraborty S, Newton AC. Climate change, plant diseases and food security: an overview. Plant Pathol. 2011;60:2–14. 8. Casenave A, Valentin C. Soil surface status in the Sahelian zone. Influence on infiltration. Paris: Office de Recherche Scientifique des Territoires d’Outre Mer; 1989. 8. Casenave A, Valentin C. Soil surface status in the Sahelian zone. Influence on infiltration. Paris: Office de Recherche Scientifique des Territoires d’Outre Mer; 1989. 9. Pieri C. Fertilité des terres de savanes. Bilan de trente ans de recherche et de développement agricoles au sud du Sahara. Ministère de la Coopéra- tion Et du Développement-CIRAD-IRAT, Paris. 1989. 10. Roose E, Barthes B. Organic matter management for soil conservation and productivity restoration in Africa: a contribution from francophone research. Nutr Cycl Agroecosyst. 2001;61:159–70. 11. Van Der Pol F. Acknowledgements L’épuisement des terres, une source de revenu pour les paysans au Mali-Sud. In: Pieri C, editor. Savanes d’Afrique, terres fertiles?. Montpellier: France CIRAD; 1991. p. 403–19. Competing interests The authors declare that they have no competing interests. 12. Kablan R, Yost RS, Brannan K, Doumbia MD, Traoré K, Yoroté A, et al. “Amé- nagement en courbes de niveau”, Increasing rainfall capture, storage, and drainage in soils of Mali. Arid Land Res Manag. 2008;22(1):62–80. Author details 1 1 Institut d’Economie Rurale (IER), BP 262, Bamako, Mali. 2 World Agroforestry Centre (ICRAF), West and Central Africa Regional Office - Sahel Node, Bamako, Mali. 1 Institut d’Economie Rurale (IER), BP 262, Bamako, Mali. 2 World Agroforestry Centre (ICRAF), West and Central Africa Regional Office - Sahel Node, Bamako, Mali. 5. Goudou D, Traoré-Gue JN, Ouedraogo M, Segda Z, Diakité L, Kebe Z et al. Village Baseline Study—Site Analysis Report for Segou—Cinzana, Mali (MA0109). CGIAR Research Program on Climate Change, Agriculture and Food Security (CCAFS), Copenhagen, Denmark Available online at: wwwccafscgiarorg. 2012. 5. Goudou D, Traoré-Gue JN, Ouedraogo M, Segda Z, Diakité L, Kebe Z et al. Village Baseline Study—Site Analysis Report for Segou—Cinzana, Mali (MA0109). CGIAR Research Program on Climate Change, Agriculture and Food Security (CCAFS), Copenhagen, Denmark Available online at: wwwccafscgiarorg. 2012. Ethical approval and consent to participate IER: Institut d’Economie Rurale; ICRAF: World Agroforestry Centre; CRT: contour ridge tillage; CCAFS: Climate Change, Agriculture and Food Security; CIRAD: Centre de Coopération Internationale en Recherche Agronomique pour le Développement; Fe: iron; Al: aluminum; NPK: nitrogen–phosphorus–potas- sium; ECEC: effective cation exchange capacity; SM: soil moisture content; SMS: soil moisture storage; Db: soil bulk density; H: soil depth; D1: first decade; D2: second decade; D3: third decade; Ca: calcium; Mg: magnesium; CGIAR: Consultative Group on International Agricultural Research; CIDA: Canadian International Development Agency; DANIDA: Danish International Develop- ment Agency; EU: European Union; IFAD: International Fund for Agricultural Development; PAR-CSA: participatory action research on climate smart agriculture; AMEDD: Association Malienne d’Eveil pour le Developpement Durable; ARCAD: Association pour le Renforcement des Capacités pour une Agriculture Durable; NGO: Non-Governmental Organization; ENSAM: Ecole Normale Supérieure Agronomique de Montpellier; FAO: Food and Agricul- ture Organization; SSAC: Sous-Secteur d’Agriculture de Cinzana; IRAT: Institut de Recherche en Agronomie Tropicale; IRD: Institut de Recherche pour le Développement; ORSTOM: Office de la Recherche Scientifique et Technique Outre-mer; USDA: United States Department of Agriculture; USA: United States of America; DC: District of Columbia. Generating scientific knowledge, technological innovations and decision sup- port tools for improving agricultural sector in Mali is part of the mandate and missions of Institut d’Economie Rurale (IER, http://www.ier.gouv.ml/), institu- tion to which belong Dr. Kalifa Traore, Dr. Daouda Kalifa Sidibe and Dr. Harouna Coulibaly. Such research involves active participation of local stakeholders to help implementing trials. The protocol of the current study was submitted to the scientific and ethical committee of IER and was approved. Before starting the field work, farmers in the community were informed about the context of the study and the willingness to participate. They have all given their consent and participated to all field works. Authors’ information y 2. FAOSTAT. Food and agriculture organization of the United Nations, FAOSTAT database. 2008. http://faostat.fao.org/site/362/DesktopDefault. aspx?PageID=362 y 2. FAOSTAT. Food and agriculture organization of the United Nations, FAOSTAT database. 2008. http://faostat.fao.org/site/362/DesktopDefault. aspx?PageID=362 KT is a researcher in the Soil–Water–Plant Laboratory of the Institut d’Economie Rurale (IER) in Mali. He holds a doctorate degree in Soil Sciences from the University of Montpellier II, ENSAM, France. DKS is a researcher in the Program of Forest Resources of the Institut d’Economie Rurale (IER) in Mali. He holds a PhD in Agroforestry from the University of Bangor, UK. HC is a researcher in the Soil–Water–Plant Laboratory of the Institut d’Economie Rurale (IER) in Mali. He holds a doctorate degree in Regional Human Geography and Economy from the University of “Paris Ouest Nanterre La Defense,” France. 3. Témé B, Niangado O, Traoré S, Kanté S. De la création d’une station de recherche au renforcement des capacités des producteurs: L’experience de la Fondation Syngenta au Mali. 2011. http://www.future-agricultures. org/farmerfirst/files/T2a_Teme.pdf. Accessed 22 Aug 2016. 4. Coulibaly H. Role des organisations paysannes dans la diffusion de semences de céréales: articulation des réseaux semenciers étatiques et traditionnels paysans pour une conservation in situ des variétés. Le cas des mils et sorghos au Mali. Thèse de Doctorat en géographie humaine, économique et régionale de l’université de Paris ouest Nanterre, France. 2010. p. 295. Funding h k This work was funded by the CGIAR Program on Climate Change, Agriculture and Food Security (CCAFS), which is a strategic partnership of the CGIAR and Future Earth. Received: 5 March 2016 Accepted: 24 December 2016 Conclusionh The current investigation highlighted the importance of rainfall variability during the cropping season in com- parison with inter-annual variability of total rainfall. Fortunately, there are management practices like the CRT that can help buffering the effects of the uneven distribution of rainfall within a season. The results also showed that the effects of the efforts and resources put in realizing the CRT may be optimized by using improved varieties of the most important staple crops of the Cinzana rural commune and its region which are millet and sorghum. Thus, the use of the tested early maturing varieties of the two crops could be an accessi- ble adaptation strategy to climate variability by farmers. Ridging being already traditionally practiced in dif- ferent farmer communities, the additional step will be the implementation of ridge tilling in contour lines and mechanization of the operation to reduce human labor (otherwise this operation can be done by hand hoe but takes more time and effort in this case). This finding is very important but may gain more attention if the social acceptability of the tested practice (CRT) is assessed through a cost–benefit analysis. Such investigation also needs to be validated on a larger scale by involving more farmers of different wealth status and by including more agroecological zones. Technique 0 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 CRT Control Sorghum grain yield (kg ha-1) Technique CSM 219 Sangatigui Seguifa Tiandougou jacumbe (local) Kenikeni (local) b b Fig. 6 Interactions between contour ridge tillage (CRT) and millet a varieties (improved: Soxat, Soxat1, HKP, Synthetic 006, Toroniou; local: Boboni, Toronion) and sorghum b varieties (improved: CSM219, Sangatigui, Seguifa, Tiandougou; local: Jacumbe, Kenikeni) observed from 2012 to 2014 in Cinzana commune, Mali. CRT contour ridge till- age plots; Ctrl plots without contour ridge tillage should be largely recommended as a sustainable agro- nomic practice. Indeed, results clearly showed that realizing some of the increased potential of these new Page 12 of 13 Page 12 of 13 Traore et al. Agric & Food Secur (2017) 6:11 References KT, DKS and HC designed the experiments, the data collection instruments and gathered the data, and helped in analysis and write-up. 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https://openalex.org/W1600153004	https://cienciasmarinas.com.mx/index.php/cmarinas/article/download/1270/1857	Spanish; Castilian	null	Influence of winter waves on the longitudinal growth of the Punta Banda Estuary sandbar, Todos Santos Bay, Baja California, Mexico	Ciencias marinas	2,008	cc-by	7,015	Resumen El Estero de Punta Banda, localizado dentro de la Bahía de Todos Santos, en el noroeste de la Península de Baja California, se encuentra delimitado hacia el mar por una barra arenosa de 8 km de longitud. De 1972 a 2003 la barra tuvo un crecimiento longitudinal neto de 420 m, lo que redujo el ancho de la boca del estero en más de 400 m. A pesar de dicho crecimiento, se ha sugerido que la boca del estero no se ha cerrado debido a la existencia de un balance anual en los volúmenes de sedimento que se transportan en ambas direcciones a lo largo de la barra. Mientras que mediciones directas indican que en las épocas de primavera, verano y otoño la dirección del transporte litoral de sedimento es hacia el noreste, se ha planteado la hipótesis de que durante la época invernal el transporte litoral en la barra debería ser hacia el suroeste En este trabajo se analizan mediciones de oleaje realizadas de noviembre de 2004 a marzo de 2005 frente a la barra del Estero de Punta Banda y se infiere por primera vez la magnitud y dirección del trasporte litoral de sedimento en esta barra durante la época invernal. Los resultados muestran que durante la época invernal el oleaje dominante que arriba a la bahía proviene del oeste-noroeste, aproximándose a la barra del estero con dirección oeste franco, lo que en combinación con la orientación de la barra induce un transporte litoral de sedimento en dirección noreste, hacia la boca del estero. Este resultado contradice la hipótesis de que durante el invierno la dirección del transporte litoral es hacia el sur y permite establecer que a lo largo de un año típico la dirección dominante del transporte litoral de sedimento en la barra del estero es hacia el noreste. El hecho de que la boca del estero no se haya cerrado debe ser atribuible a otros procesos físicos, como por ejemplo a aportes pluviales extraordinarios durante la época de lluvias o posibles desigualdades entre las velocidades del flujo y reflujo de la marea al estero. Palabras clave: oleaje invernal, refracción, transporte de sedimento, Estero de Punta Banda. Abstract Punta Banda Estuary, located within Todos Santos Bay, on the northwest coast of the Baja California Peninsula, is separated from the bay by a single sandbar, approximately 8 km long. From 1972 to 2003 this sandbar showed a longitudinal net growth of 420 m, which decreased the width of the estuary mouth by more than 400 m. Despite the bar’s rapid growth it has been suggested that the estuary mouth remains open because in spring, summer and autumn the longshore sand transport is northeastward, but during winter the longshore sand transport should be toward the southwest, away from the mouth, establishing a balance between the amount of sediment transported both ways. In this study we analyze wave data acquired from November 2004 to March 2005 on the bay side of the sandbar, and for the first time provide the magnitude and direction of the longshore sand transport during winter. The results show that during this season the dominant waves approach the bay from the west-northwest and the sandbar from the west, inducing a net longshore sand transport of 817 m3 day–1 to the northeast. This result contradicts the hypothesis of a longshore sand transport balance, and shows that throughout a typical year the net longshore sand transport is to the northeast. The fact that the estuary mouth has not closed up should be attributed to other causes, such as extraordinary amounts of rain during the rainy season or inequalities between the ebb and flood tidal current velocities to the estuary. Key words: winter waves, refraction, sediment transport, Punta Banda Estuary. A Martínez-Díaz-de-León, R Lizárraga-Arciniega, O Delgado-González, L Galindo-Bect, CR Torres-Navarrete, R Blanco-Betancourt, E Gil-Silva, R Cano-Cetina A Martínez-Díaz-de-León, R Lizárraga-Arciniega, O Delgado-González, L Galindo-Bect, CR Torres-Navarrete, R Blanco-Betancourt, E Gil-Silva, R Cano-Cetina Instituto de Investigaciones Oceanológicas, Universidad Autónoma de Baja California, Apartado postal 453, Ensenada 22800, Baja California, México. * E-mail: asdrubal@uabc.mx Ciencias Marinas (2008), 34(1): 69–76 Ciencias Marinas (2008), 34(1): 69–76 http://dx.doi.org/10.7773/cm.v34i1.1270 Introduction Punta Banda Estuary (PBE), one of the northernmost coastal lagoons on the Pacific coast of Mexico, is located within Todos Santos Bay, on the northwest side of the Baja California Peninsula (fig. 1). One of the estuary’s most notable El Estero de Punta Banda (EPB), una de las lagunas coste- ras más norteñas en el litoral Pacífico mexicano, se localiza dentro de la Bahía de Todos Santos, en el noroeste de la Penín- sula de Baja California (fig. 1). Una de sus características más 69 Ciencias Marinas, Vol. 34, No. 1, 2008 features is the approximately 8-km-long and 1-km-wide sandbar that separates it from the bay. This sandbar consists of well-classified quartz sands of approximately 2φ in diameter (Blanco-Betancourt 1987) and numerous dunes, and on the bay side, a moderately wide (~250 m) beach slopes gently (0.02) to the sea. These characteristics make it highly attractive for recreational use and for the construction of tourism-related infrastructure. importantes es su barra arenosa, de aproximadamente 8 km de longitud y 1 km de ancho, que lo separa de la bahía. Consti- tuida de arenas de cuarzo bien clasificadas cuyo diámetro medio es de 2φ (Blanco-Betancourt 1987), la barra cuenta con numerosas dunas y presenta hacia el mar una playa moderada- mente ancha (~250 m), de pendiente suave (0.02), lo que la hace sumamente atractiva para el desarrollo de actividades recreativas y la construcción de infraestructura turística. Entre 1972 y 2003 la barra del EPB mostró un crecimiento longitudinal neto de 420 m, lo que redujo el ancho de su boca de 600 a 180 m (Delgado-González et al. 2005). El crecimiento longitudinal de la barra se ha atribuido a que, durante prima- vera, verano y otoño, el transporte litoral dominante de sediNSmento a lo largo de la barra es hacia el noreste; esto es, en dirección a la boca del estero, según se infiere de mediciones de oleaje realizadas durante tales estaciones (Torres-Navarrete 1991). Introduction Por otro lado, se ha sugerido que, a pesar del importante crecimiento de la barra, la boca del estero no se ha cerrado debido a que durante la época invernal, cuando el oleaje arriba a la bahía con sus máximas alturas y del noroeste, la dirección del transporte litoral de sedimento debiera ser de mayor magnitud que el resto del año y hacia el sur, lo que permite establecer un balance anual entre las canti- dades de sedimento que se trasportan en ambas direcciones a lo largo de la barra (Torres-Navarrete 1991). Between 1972 and 2003, the PBE sandbar showed a longitudinal net growth of 420 m and the width of the estuary mouth decreased from 600 to 180 m (Delgado-González et al. 2005). This longitudinal growth has been attributed to the fact that in spring, summer and autumn the longshore sand transport along the bar is predominantly northeastward, in the direction of the mouth, as inferred from wave measurements made during those seasons (Torres-Navarrete 1991). Despite the bar’s rapid growth, it has been suggested that the mouth remains open because during winter, when waves approach from the northwest and maximum wave heights occur in the bay, the longshore sand transport should be toward the south and of greater magnitude than at other times of the year, estab- lishing an annual balance between the amounts of sediment that are transported in both directions along the bar (Torres- Navarrete 1991). The objective of this study was to analyze wave measure- ments taken during winter, from November 2004 to March 2005, off the PBE sandbar. This is the first time that the charac- teristics of the waves approaching the sandbar and that the magnitude and direction of the wave-induced longshore sedi- ment transport are inferred for this season. Moreover, the data were compared with existing measurements to determine the possible occurrence of an annual balance between the amounts of sediment that are transported in both directions along the sandbar. En este trabajo se analizan mediciones de oleaje realizadas durante la época invernal, de noviembre de 2004 a marzo de 2005, frente a la barra del EPB. Se infieren por primera vez para esta época las características del oleaje que arriba a la barra del estero, y la magnitud y dirección del trasporte litoral de sedimento inducido por este oleaje. Introduction Se investiga también, comparando con mediciones existentes, el posible balance anual entre las cantidades de sedimento que se transportan en ambas direcciones a lo largo de la barra del EPB. Resultados Las características generales del oleaje registrado por el instrumento instalado frente a la boca del estero durante el periodo de medición se muestran en la figura 2. Las series de tiempo para las otras dos localidades no mostraron diferencias relevantes, excepto para el caso de Hs, que resultó ser en pro- medio 20% mayor en el punto de medición ubicado al norte de la boca del estero. Esto concuerda con lo reportado por Martínez-Díaz-de-León (2004), quien encontró que las alturas del oleaje que arriban a la zona conocida como El Ciprés (fig. 1) pueden ser hasta 2.5 veces mayores que en otros sitios den- tro de la bahía. Durante el periodo de mediciones se observó (fig. 2a) variabilidad en la altura del oleaje típica para esta región durante de la época invernal (Martínez-Díaz-de-León et al. 1989, Martínez-Díaz-de-León 2004). El promedio de la altura fue de 0.7 m, con mínimos de 0.3 m. Destaca el arribo de varias tormentas, cuatro de ellas importantes, con altura significante Figure 1. Geographic location of the wave sampling stations off the Punta Banda Estuary sandbar. The broken lines indicate the bathymetric configuration (depth in meters), and the square corresponds to the area considered to obtain the wave refraction pattern shown in figure 4. Figure 1. Geographic location of the wave sampling stations off the Punta Banda Estuary sandbar. The broken lines indicate the bathymetric configuration (depth in meters), and the square corresponds to the area considered to obtain the wave refraction pattern shown in figure 4. Figura 1. Ubicación geográfica de las estaciones de medición de oleaje frente a la barra del Estero de Punta Banda. Las líneas segmentadas muestran la configuración batimétrica (profundidades en metros), y el recuadro frente al estero corresponde al área considerada para obtener el patrón de refracción del oleaje mostrado en la figura 4. Figura 1. Ubicación geográfica de las estaciones de medición de oleaje frente a la barra del Estero de Punta Banda. Las líneas segmentadas muestran la configuración batimétrica (profundidades en metros), y el recuadro frente al estero corresponde al área considerada para obtener el patrón de refracción del oleaje mostrado en la figura 4. Figure 2. Time series of wave height, period and direction measured at the Boca station (fig. 1) in front of the mouth of Punta Banda Estuary from November 2004 to March 2005. Resultados The time series were interrupted in late January because the instruments were removed for maintenance purposes. Figura 2. Series de tiempo de altura, periodo y dirección del oleaje medido en la estación Boca (fig. 1) frente a la boca del Estero de Punta Banda de noviembre de 2004 a marzo de 2005. La interrupción de las series de tiempo a finales de enero se debe a que los instrumentos fueron recuperados para realizar maniobras de mantenimiento. instruments were installed at an average depth of 15 m and operated from 9 November 2004 to 30 March 2005, a period corresponding to the winter season. The measurements were interrupted from 18 to 24 January 2005 for the instruments to undergo maintenance. instruments were installed at an average depth of 15 m and operated from 9 November 2004 to 30 March 2005, a period corresponding to the winter season. The measurements were interrupted from 18 to 24 January 2005 for the instruments to undergo maintenance. Martínez-Díaz-de-León et al.: Winter growth of the Punta Banda Estuary sandbar Figure 1. Geographic location of the wave sampling stations off the Punta Banda Estuary sandbar. The broken lines indicate the bathymetric configuration (depth in meters), and the square corresponds to the area considered to obtain the wave refraction pattern shown in figure 4. frente a la barra, y el tercer instrumento al norte de la boca (fig. 1). Los tres se instalaron a una profundidad media de 15 m y operaron del 9 de noviembre de 2004 al 30 de marzo de 2005, periodo correspondiente a la época invernal. Las mediciones se interrumpieron del 18 al 24 de enero de 2005 para realizar maniobras de mantenimiento a los equipos de medición. Material and methods Para establecer durante la época invernal el clima de oleaje que arriba al EPB y la dirección del transporte litoral de sedi- mento a lo largo de su barra se realizaron mediciones costeras de oleaje en tres localidades. Las estaciones de medición se identificaron como Ciprés (31º45.5′ Ν, 116º37.9′ W), Boca (31º47.0′ N, 116º38.6′ W) y Barra (31º45.5′ Ν, 116º39.4′ W) (fig. 1). Se utilizaron instrumentos de medición tipo ADP (Acoustic Doppler Profiler), con capacidad para resolver el campo direccional del oleaje, dos de 1000 kHz (SonTek) y uno de 600 kHz (RD Instruments). Estos instrumentos fueron pro- gramados para registrar cada hora 2048 muestras de presión (p) y de las componentes horizontales del campo de velocidad (u, v), con un intervalo de muestreo de 0.5 s. Las rutinas de cómputo de los propios instrumentos, que combinan p, u y v, proporcionan para cada registro de oleaje valores de altura sig- nificante (Hs), periodo del pico espectral (Tp) y dirección media de propagación del oleaje (θ). Uno de los medidores quedó ubicado frente a la boca del estero, otro al sur de la boca To determine the winter wave climate off PBE and the direction of the longshore sand transport along the sandbar, coastal wave measurements were taken at three stations, identi- fied as Ciprés (31º45.5′ Ν, 116º37.9′ W), Boca (31º47.0′ Ν, 116º38.6′ W) and Barra (31º45.5′ Ν, 116º39.4′ W) (fig. 1). Measurements were taken with acoustic doppler profilers (ADPs) capable of resolving the directional wave field. One 600 kHz (RD Instruments) and two 1000 kHz (SonTek) ADPs were used. The instruments were programed to record, every hour, 2048 samples of pressure (p) and of the horizontal com- ponents of the velocity field (u, v), with a sampling interval of 0.5 s. The software of these instruments, which combine p, u and v, provide significant wave height (Hs), spectral peak period (Tp), and wave propagation direction (θ) data for each measurement. One of the ADPs was located in front of the estuary mouth, another to the south of the mouth, off the sand- bar, and the third to the north of the mouth (fig. 1). The three 70 Martínez-Díaz-de-León et al.: Winter growth of the Punta Banda Estuary sandbar Martínez-Díaz-de-León et al.: Winter growth of the Punta Banda Estuary sandbar Results The wave data recorded during the sampling period by the instrument deployed off the estuary mouth are shown in figure 2. The time series for the other two sites did not show relevant differences, except in the case of Hs, which was on average 20% higher at the station located to the north of the mouth. This result concurs with that reported by Martínez- Díaz-de-León (2004), who found that the height of the waves reaching the area known as El Ciprés (fig. 1) can be up to 2.5 times higher than at other sites within Todos Santos Bay. Figure 2. Time series of wave height, period and direction measured at the Boca station (fig. 1) in front of the mouth of Punta Banda Estuary from November 2004 to March 2005. The time series were interrupted in late January because the instruments were removed for maintenance purposes. Figure 2a shows a typical variation in the wave height for this region during the winter season (Martínez-Díaz-de-León et al. 1989, Martínez-Díaz-de-León 2004). The mean Hs value was 0.7 m and the minimum value was 0.3 m. The arrival of several storms, four of them significant, is noticeable, with Hs values greater than 1.5 m, but greater than 2 m in the case of p p Figura 2. Series de tiempo de altura, periodo y dirección del oleaje medido en la estación Boca (fig. 1) frente a la boca del Estero de Punta Banda de noviembre de 2004 a marzo de 2005. La interrupción de las series de tiempo a finales de enero se debe a que los instrumentos fueron recuperados para realizar maniobras de mantenimiento. 71 Ciencias Marinas, Vol. 34, No. 1, 2008 the storm in early December 2004. The number and severity of the storms recorded is not unusual, since numerous cold fronts occur in winter over the Pacific Ocean of the Northern Hemisphere. Most of these fronts affect the coasts of California and Baja California (Allan and Komar 2000, 2002). mayor a 1.5 m. La tormenta de principios de diciembre de 2004 rebasó inclusive los 2 m de altura. El número y severidad de las tormentas registradas no es algo sorprendente, ya que durante la época invernal se tiene la ocurrencia de numerosos frentes fríos sobre el Océano Pacífico del hemisferio norte. Estos frentes en su mayoría afectan las costas de California y Baja California (Allan y Komar 2000, 2002). Results The wave period (fig. 2b) also showed high variability, with minimum and maximum values of 3.6 and 21 s, respec- tively. The period can be observed to increase drastically on several occasions and then decrease slowly until reaching, in most cases, its initial value. It is interesting to note the clear association between the peaks in wave period and the peaks in wave height; this behaviour indicates that most of the peaks in height are related to the arrival of waves generated by distant storms. El periodo del oleaje (fig. 2b) mostró igualmente una gran variabilidad, con valores mínimos de 3.6 s y máximo de 21 s. Se observa como en repetidas ocasiones el periodo se incre- menta drásticamente para luego decrecer de manera paulatina hasta alcanzar, en la mayoría de los casos, su valor inicial. Es interesante notar la marcada asociación entre los picos de periodo y los picos de altura del oleaje, comportamiento que indica que la mayoría de los picos de altura están asociados al arribo de olas generadas por tormentas lejanas. The predominant wave direction (fig. 2c) at the three sam- pling stations was from the west, with a standard deviation of only 17º. To identify the association between wave height and approach direction, joint histograms were calculated (Hs, θ) and are presented as wave roses in figure 3. In these charts the thickness of the bars corresponds to wave height according to the scale shown. The length of the bars (individual or cumula- tive) represents the percentage of occurrence of the pairs of values (Hs, θ), whose scale is indicated by the concentric cir- cles. Note how regardless of height, more than 70% of the waves approach from the west, while only a small percentage (<15%) approach from other directions. La dirección dominante del oleaje en los tres puntos de medición (fig. 2c) resultó ser del oeste franco, con una desviación estándar de tan sólo 17 grados. Para identificar la asociación entre la altura del oleaje y su dirección de arribo se calcularon histogramas conjuntos (Hs, θ) que se presentan como rosas de oleaje en la figura 3. En estas figuras el grosor de las barras corresponde a la altura del oleaje según la escala mostrada en la parte inferior derecha. Martínez-Díaz-de-León et al.: Winter growth of the Punta Banda Estuary sandbar Martínez-Díaz-de-León et al.: Winter growth of the Punta Banda Estuary sandbar rompiente. Para su propagación se utilizó el modelo numérico de refracción y difracción REFDIF-1 (Kirby y Dalrymple 1984). Este modelo, ligeramente no lineal, utiliza una expan- sión en series de Stokes para resolver el problema analítico del oleaje e incluye una corrección de tercer orden a la velocidad de fase de las olas. La derivación matemática del modelo supone que los cambios de profundidad se suceden en distan- cias más grandes que la longitud de onda de las olas. correction to the wave phase velocity. The model’s mathemati- cal deduction assumes that the changes in depth occur in distances greater than the wave wavelengths. Different triads of wave height, period and direction were used as model input parameters; nevertheless, as already men- tioned, the predominant wave direction was from the west (fig. 3). The refraction pattern of one of these triads (Hs = 0.75 m, Tp = 15 s, θ = 270º) is shown as sea surface height contours in figure 4. This example is representative of the dif- ferent trials carried out, since the refraction pattern proved to be more sensitive to the wave incidence angle than to the varia- tions in wave height or period. In general, the refraction trials revealed that in the breaker zone the dominant wave direction was from the southwest, generating a longshore transport along the sandbar towards the northeast, in the direction of the estu- ary mouth. Como parámetros de entrada al modelo se utilizaron dife- rentes triadas de altura, periodo y dirección del oleaje. Sin embargo, como ya se comentó, la dirección dominante de las olas resultó ser del oeste franco (fig. 3). El patrón de refracción para una de estas triadas (Hs = 0.75 m, Tp = 15 s, θ = 270º) se ilustra como contornos de la elevación de la superficie del mar en la figura 4. Cabe mencionar que este ejemplo es representa- tivo de los diferentes ejercicios realizados, ya que el patrón de refracción resultó ser más sensible al ángulo de incidencia de las olas que a las variaciones en su altura o periodo. Martínez-Díaz-de-León et al.: Winter growth of the Punta Banda Estuary sandbar En general, los ejercicios de refracción mostraron que en la zona de rom- piente la dirección dominante del oleaje es suroeste, lo que induce que el transporte litoral a lo largo de la barra sea hacia el noreste, esto es, hacia la boca del estero. To calculate the magnitude of longshore transport, expressed as volume per unit of time (m3 day–1), the following equation developed by the US Corps of Engineers (CERC 1984) was used: Q1 Il ρs ρ – ( ) g 1 p – ( ) ------------------------------------------- = Q1 Il ρs ρ – ( ) g 1 p – ( ) ------------------------------------------- = Para calcular la magnitud del transporte litoral, expresado como volumen por unidad de tiempo (m3 día–1), se utilizó la fórmula desarrollada por el Cuerpo de Ingenieros de los Estados Unidos (CERC 1984): where ρs is the relevant sediment density (2650 kg m–3), ρ is the density of seawater (1025 kg m–3), g is the constant of gravity (9.81 m s–2), p is the porosity of the substrate (= 0.4), and Il is the immersed weight sediment transport rate (N s–1) given by: Q1 Il ρs ρ – ( ) g 1 p – ( ) ------------------------------------------- = Q1 Il ρs ρ – ( ) g 1 p – ( ) ------------------------------------------- = Il KPl = donde ρs es la densidad relativa del sedimento (2650 kg m–3), ρ es la densidad del agua de mar (1025 kg m–3), g es la constante de gravedad (9.81 m s–2) y p es la porosidad del substrato (= 0.4), e Il es la tasa del transporte de sedimento como peso inmerso en N s–1, dada por: where K is an adimensional constant (= 0.77), and Pl is the longshore component of wave power (N s–1) given by: Pl ECn θ senθ cos ( )r = Pl ECn θ senθ cos ( )r = Il KPl = where E is the wave energy (N m–1), C is the celerity of the waves (m s–1), n represents the quotient between the group velocity and the celerity of the waves, and θ is the wave propagation direction. The subindex r indicates that all these parameters are evaluated at the depth where the waves begin to break. Results La longitud de las barras (individual o acumulativa) representa el porcentaje de ocurren- cia de las parejas de valores (Hs, θ), cuya escala es indicada por los círculos concéntricos. Se aprecia como, sin importar su altura, más del 70% de las olas provienen del oeste, mien- tras que sólo un pequeño porcentaje (<15%) arriba de otras direcciones. To be able to infer the magnitude and direction of sediment transport, wave height and direction at the breaker line have to be known. As the wave measurements were made in 15-m- deep waters, it was necessary to conduct a numerical propagation of the waves measured up to the breaker zone. The REFDIF-1 numerical refraction/diffraction model (Kirby and Dalrymple 1984) was used for the propagation. This weakly nonlinear model uses Stokes´ series expansion to resolve the analytical problem of waves and includes a third-order Para inferir la magnitud y dirección del transporte de sedi- mento se requiere conocer la altura y dirección del oleaje en la línea de rompiente. Debido a que las mediciones de oleaje se realizaron en aguas de 15 m de profundidad, fue necesario propagar numéricamente el oleaje medido hasta la zona de Figure 3. Wave, height and direction roses for the three sampling stations (fig. 1) off the Punta Banda Estuary sandbar. Figura 3. Rosas de oleaje, altura y dirección, en los tres puntos de medición (fig. 1) frente a la barra del Estero de Punta Banda. Figure 3. Wave, height and direction roses for the three sampling stations (fig. 1) off the Punta Banda Estuary sandbar. Figura 3. Rosas de oleaje, altura y dirección, en los tres puntos de medición (fig. 1) frente a la barra del Estero de Punta Banda. 72 Martínez-Díaz-de-León et al.: Winter growth of the Punta Banda Estuary sandbar donde K es una constante adimensional (= 0.77), y Pl es la componente a lo largo de la costa de la potencia del oleaje en N s–1, dada por: Pl ECn θ senθ cos ( )r = Based on the pair of height (Hs = 0.75 m) and direction (θ = 270º) values that occurred with most frequency (>70%, fig. 3) during the sampling period, the volume of sediment trans- ported along the PBE sandbar was 817 m3 day–1. This amount is much higher than the sum of the volumes reported by Torres- Navarrete (1991) for spring (384 m3 d–1), summer (10.5 m3 d–1), and autumn (–110.4 m3 d–1). The wave approach direction and orientation of the sandbar generate a sediment transport towards the estuary mouth (i.e. towards the northeast). donde E es la energía del oleaje en N m–1, C es la celeridad de las olas (m s–1), n representa el cociente entre la velocidad de grupo y la celeridad de las olas, y θ es la dirección de pro- pagación de las olas. El subíndice r indica que todos estos parámetros se evalúan en la profundidad donde inicia la rom- piente de las olas. Considerando la pareja más frecuente (>70%, fig. 3) de valores de altura (Hs = 0.75 m) y dirección (θ = 270º) durante el periodo de mediciones se obtiene que el volumen de This result contradicts the hypothesis presented by Torres- Navarrete (1991) that during winter the sediment along the 73 Ciencias Marinas, Vol. 34, No. 1, 2008 Figure 4. Wave refraction pattern off the Punta Banda Estuary sandbar, expressed as sea surface height contours. The refraction corresponds to the area indicated by the square in figure 1 and was obtained with the REFDIF-1 model, using Hs = 0.75 m, Tp = 14 s, and θ = 270º as model input data. Figura 4. Patrón de refracción del oleaje frente a la barra del Estero de Punta Banda, expresado como contornos de elevación de la superficie del mar. La refracción corresponde al área enmarcada en el recuadro de la figura 1 y se obtuvo utilizando el modelo REFDIF-1. Como información de entrada al modelo se utilizó: Hs = 0.75 m, Tp = 14 s y θ = 270º. Figure 4. Wave refraction pattern off the Punta Banda Estuary sandbar, expressed as sea surface height contours. Martínez-Díaz-de-León et al.: Winter growth of the Punta Banda Estuary sandbar The refraction corresponds to the area indicated by the square in figure 1 and was obtained with the REFDIF-1 model, using Hs = 0.75 m, Tp = 14 s, and θ = 270º as model input data. Figura 4. Patrón de refracción del oleaje frente a la barra del Estero de Punta Banda, expresado como contornos de elevación de la superficie del mar. La refracción corresponde al área enmarcada en el recuadro de la figura 1 y se obtuvo utilizando el modelo REFDIF-1. Como información de entrada al modelo se utilizó: Hs = 0.75 m, Tp = 14 s y θ = 270º. sandbar is transported southward. On the other hand, for the first time it allows us to suggest that during a typical annual cycle the dominant transport along the sandbar is in the direc- tion of the estuary mouth. This result could partially explain the significant longitudinal growth of the sandbar and the rele- vant decrease of more than 400 m in the width of the PBE mouth observed over the last decades (Delgado-González et al. 2005). sedimento transportado a lo largo de la barra del estero es de 817 m3 día–1, el cual resultó ser mucho mayor que la suma de los volúmenes reportados por Torres-Navarrete (1991) para primavera (384 m3 d–1), verano (10.5 m3 d–1) y otoño (–110.4 m3 d–1). La dirección de aproximación del oleaje y la orientación de la barra inducen un trasporte de sedimento hacia la boca del estero, esto es, hacia el noreste. Este resultado contradice la hipótesis planteada por Torres- Navarrete (1991) en la que durante la época invernal el sedimento en la barra es transportado hacia el sur. Sin embargo, al mismo tiempo permite sugerir por primera vez que a lo largo de un ciclo anual típico el transporte dominante a lo largo de la barra es en dirección a la boca del EPB. Este resultado pudiera explicar en parte el importante crecimiento longitudinal de la barra y la relevante disminución en más de 400 m del ancho de la boca del estero, observados en las últimas décadas (Delgado-González et al. 2005). Discussion Coastal waves are the main physical agent responsible for changes in the morphology of the coastline, so monitoring and analyzing their characteristics is of the utmost importance, especially when sandy beaches are involved. The wave characteristics analyzed in this study reflect a typical winter condition for Todos Santos Bay (Martínez-Díaz- de-León et al. 1987, Martínez-Díaz-de-León 2004, Lizárraga- Arciniega et al. 2007). The occurrence of some short-duration storms shows a clear association between the increase in both wave height and period. It can be assumed from this result that the dominant waves were generated by distant storms. Discusión Es de suma importancia medir y analizar las características del oleaje que arriba a las costas, ya que éste es el principal agente físico responsable de inducir cambios en la morfología costera particularmente cuando se trata de playas arenosas. On the other hand, a small standard deviation (~17º) was observed in the direction that waves approach the PBE sandbar. This suggests that the sediment transport direction determined in this study is a typical condition of winter waves, especially if we consider that the refraction pattern showed greater sensitivity to the wave approach angle than to wave Las características del oleaje analizado en este trabajo reflejan una condición invernal típica para la Bahía de Todos Santos (Martínez-Díaz-de-León et al. 1987, Martínez-Díaz-de- León 2004, Lizárraga-Arciniega et al. 2007). La ocurrencia de 74 Martínez-Díaz-de-León et al.: Winter growth of the Punta Banda Estuary sandbar height and period, increasing the relevance of the results presented here. algunos eventos de tormenta de corta duración muestra una marcada asociación entre la creciente altura del oleaje y el incremento en el periodo, lo que permite suponer que las olas dominantes registradas fueron generadas por tormentas lejanas. The length of the sandbar has been increasing over the past two decades and though the width of the estuary mouth has been considerably reduced it has not closed up. This has been explained by inferring a balance between the sediment trans- ported southwestward during spring, summer and autumn, and that transported northeastward during winter. The wave data analyzed here indicates that this hypothesis is not necessarily true. Even though the amount of sediment transported along the sandbar is much greater in winter (817 m3 day–1) than at other times of the year, the winter wave characteristics, particu- larly direction, indicate that sediment is transported in the direction of the estuary mouth. This result suggests that during a typical year the dominant sediment transport direction along the sandbar is to the northeast, and this has undoubtedly con- tributed to increase the length of the sandbar. Por otro lado, se observa una pequeña desviación estándar (~17º) en la dirección de aproximación del oleaje a la barra del estero. Discusión Esto sugiere que la dirección del transporte de sedi- mento determinada en este trabajo es una condición típica del oleaje durante la época invernal, sobretodo si se considera que el patrón de refracción resultó ser más sensible al ángulo de aproximación de las olas que a su altura y periodo. Esto incre- menta la relevancia de los resultados aquí presentados. La longitud de la barra del EPB se ha estado incrementando durante las últimas dos décadas, y aunque esto conlleva a una reducción considerable del ancho de la boca del estero, ésta no se ha cerrado. Esto se ha explicado infiriendo un balance entre el sedimento que se transporta hacia el oeste durante prima- vera, verano y otoño, y el que se transporta hacia el noreste durante la época invernal. La información de oleaje analizada aquí sugiere que dicha hipótesis no es necesariamente cierta. Si bien durante el invierno la cantidad de sedimento que se trans- porta a lo largo de la barra es mucho mayor (817 m3 día–1) que las cantidades que se reportan para las otras épocas del año, las características del oleaje en invierno, particularmente su direc- ción, inducen un transporte de sedimento en dirección a la boca del estero. Esto parece sugerir que durante un año típico la dirección dominante del transporte de sedimento a lo largo de la barra es hacia el noreste, lo que sin duda ha contribuido a incrementar más rápidamente la longitud de la barra del estero. The fact that the PBE mouth still remains open must be attributed to other physical processes and not to an annual balance in the magnitudes and directions of sediment transport along the sandbar. For example, recent studies (Ortiz et al. 2003, Delgado-González et al. 2005) suggest that the extraor- dinary input of sediment during the extreme rainfall events of 1978 and 1983 caused significant silting in the PBE basin, which considerably reduced the self-dredging ability of the mouth and facilitated the longitudinal growth of the sandbar, but kept the mouth open. It is important to mention that the longitudinal growth of the PBE sandbar has not been continuous. During the winter of 1978/1979, the width of the estuary mouth increased from 444 to 756 m as a result of severe erosion at the tip of the sandbar. Discusión This erosion was attributed to the effects caused by the extreme wave storms during the El Niño winter of 1977/1978 (Dormurat 1978, González-Calvillo and Cupul-Magaña 1986, Lizárraga-Arciniega et al. 2003). El hecho de que la boca del estero no se haya cerrado aún puede ser atribuible a otros procesos físicos y no a un balance en un ciclo anual en las magnitudes y direcciones del trans- porte de sedimento a lo largo de la barra. Por ejemplo, trabajos recientes (Ortiz et al. 2003, Delgado-González et al. 2005) sugieren que los aportes extraordinarios de sedimento durante las lluvias extremas de 1978 a 1983 asolvaron considerable- mente la cuenca del estero, lo que redujo considerablemente la capacidad de autodragado de la boca del estero y facilitó el cre- cimiento longitudinal de la barra pero manteniendo la boca del estero abierta. Another aspect that may be playing an important role in maintaining the estuary mouth open is a possible inequality between the magnitudes of the ebb and flood tidal currents. Ortiz et al. (2003) used a hydrodynamic model to simulate the maximum speeds attained during ebb and flood tides in PBE, and their results show that the velocities during ebb tide are much greater than during flood tide, thus preventing the accu- mulation of sediment at the mouth and increasing the estuary’s self-dredging ability. Es importante mencionar que el crecimiento longitudinal de la barra no ha sido continuo. Durante el invierno 1978/1979 el ancho de la boca del estero pasó de 444 a 756 m, como con- secuencia de la erosión extrema en la punta de la barra del estero. Tal erosión fue atribuida a los efectos ocasionados por las tormentas con oleaje extremo registradas durante el invierno 1977/1978, reportado como invierno Niño (Dormurat 1978, González-Calvillo y Cupul-Magaña 1986, Lizárraga- Arciniega et al. 2003). It can therefore be concluded that during winter, the dominant waves that reach Todos Santos Bay come from the west-northwest and approach the sandbar from the west. The characteristics of these waves together with the orientation of the sandbar can generate a net longshore sediment transport of up to 817 m3 day–1 towards the northeast, in the direction of the estuary mouth. Discusión This result contradicts the hypothesis that the winter longshore transport direction is southeastward and allows us to establish, considering the results of Torres- Otro especto que pudiera contribuir de manera importante a mantener la boca del estero abierta es la existencia de una des- igualdad entre las magnitudes del flujo y reflujo de la marea. Ortiz et al. (2003), mediante el uso de un modelo hidrodiná- mico, simularon las velocidades máximas alcanzadas durante flujo y reflujo de la marea en el EPB. Sus resultados muestran 75 Ciencias Marinas, Vol. 34, No. 1, 2008 Ciencias Marinas, Vol. 34, No. 1, 2008 Navarrete (1991), that during a typical year the dominant long- shore sediment transport along the PBE sandbar is to the northeast. que las velocidades durante el reflujo son mucho mayores que durante el flujo, previniendo así la acumulación de sedimento en la boca e incrementando por otro lado la capacidad de auto- dragado del estero. References Allan JC, Komar PD. 2000. Are ocean wave heights increasing in the eastern North Pacific? EOS 81: 561, 566–567. Allan JC, Komar PD. 2002. Nearshore-process climates related to changing deep-water wave conditions. Shore & Beach 70: 31–40. Blanco-Betancourt R. 1987. Comparación de dos modelos de predicción de la corriente a lo largo de la playa, con datos reales obtenidos en la barra del estero de Punta Banda, B.C. Tesis de licenciatura, Facultad de Ciencias Marinas, Universidad Autónoma de Baja California, Ensenada, México, 36 pp. English translation by Christine Harris. English translation by Christine Harris. Acknowledgements Por lo anterior se puede concluir que durante la época invernal el oleaje dominante que arriba a la bahía proviene del oeste-noroeste, aproximándose a la barra del estero con dirección oeste franco. Las características de este oleaje en combinación con la orientación de la barra pueden inducir un transporte litoral neto de sedimento de hasta 817 m3 día–1 en dirección noreste, hacia la boca del estero. Este resultado con- tradice la hipótesis de que durante el invierno la dirección del transporte litoral es hacia el suroeste, y permite establecer, aunado a los resultados de Torres-Navarrete (1991), que a lo largo de un año típico de oleaje la dirección dominante del transporte litoral de sedimento en la barra del estero es hacia el noreste. We thank CONACYT for financial support through project SEP-2004-C01-47656. We thank CONACYT for financial support through project SEP-2004-C01-47656. Recibido en mayo de 2007; aceptado en febrero de 2008. Agradecimientos Agradecemos al CONACYT por el apoyo financiero a tra- vés del proyecto SEP-2004-C01-47656. CERC. 1984. Shore Protection Manual. Vicksburg, Mississippi: US Army Coastal Engineering Research Center, Corps of Engineers. 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https://openalex.org/W2112799145	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0058542&type=printable	English	null	Predator-Driven Nutrient Recycling in California Stream Ecosystems	PloS one	2,013	cc-by	12,063	Introduction The rate at which nutrients are recycled within a system depends heavily on the physical characteristics of that system and characteristics of the organisms that inhabit it. The productivity of primary producers in aquatic ecosystems is limited by a range of biotic and abiotic factors, with light and nutrient availability among the primary drivers [1–6]. Nutrient limitation can over-ride the importance of light, and even in low- light environments, increasing the availability of nutrients can increase primary producer biomass [7]. Nitrogen (N) and phosphorus (P) are key macronutrients that can individually or simultaneously limit growth depending on their availability and the requirements of the demand assemblage [1,8]. External ambient nutrient fluxes of N and P are driven by weathering, fixation, and runoff from terrestrial ecosystems [9] as organic and inorganic components of soils release dissolved mobile ions in surface waters [10]. In addition, internal biotic processes can subsidize fluxes into the system, increasing nutrient availability. Interactions among producers, herbivores, and predators are important determinants of nutrient dynamics in aquatic ecosys- tems. Changes in nutrient availability can have direct effects on producer biomass [16], as well as potential for future growth through nutrient storage [17]. As the base of the food web, producers in streams can drive ecosystem productivity and can be directly and indirectly affected by a range of animals. Herbivores can directly limit producer growth and biomass through grazing pressure [18]. Higher trophic level animals indirectly affect autotroph abundance by stimulating higher producer productivity through nutrient recycling and preying on herbivores, which in turn reduces grazing pressure [13,19–22]. To date, investigations of the effects of consumer identity and abundance on nutrient recycling in streams have focused mainly on fish assemblages (e.g. [12,15,23–30], but see [31–34]). While fish species represent a large fraction of animal biomass in many stream ecosystems, there are myriad other taxa that make up a substantial component of secondary production, including aquatic macroinvertebrates and other vertebrates (amphibians, reptiles, mammals, birds). Predatory amphibian populations in particular, often rival the abundance and biomass of fishes in many systems, In aquatic environments, animals process organic compounds through consumption, metabolism, and excretion into labile dissolved forms that can be taken up by autotrophs and other microbial organisms [11]. Abstract Nutrient recycling by consumers in streams can influence ecosystem nutrient availability and the assemblage and growth of photoautotrophs. Stream fishes can play a large role in nutrient recycling, but contributions by other vertebrates to overall recycling rates remain poorly studied. In tributaries of the Pacific Northwest, coastal giant salamanders (Dicamptodon tenebrosus) occur at high densities alongside steelhead trout (Oncorhynchus mykiss) and are top aquatic predators. We surveyed the density and body size distributions of D. tenebrosus and O. mykiss in a California tributary stream, combined with a field study to determine mass-specific excretion rates of ammonium (N) and total dissolved phosphorus (P) for D. tenebrosus. We estimated O. mykiss excretion rates (N, P) by bioenergetics using field-collected data on the nutrient composition of O. mykiss diets from the same system. Despite lower abundance, D. tenebrosus biomass was 2.5 times higher than O. mykiss. Mass-specific excretion summed over 170 m of stream revealed that O. mykiss recycle 1.7 times more N, and 1.2 times more P than D. tenebrosus, and had a higher N:P ratio (8.7) than that of D. tenebrosus (6.0), or the two species combined (7.5). Through simulated trade-offs in biomass, we estimate that shifts from salamander biomass toward fish biomass have the potential to ease nutrient limitation in forested tributary streams. These results suggest that natural and anthropogenic heterogeneity in the relative abundance of these vertebrates and variation in the uptake rates across river networks can affect broad-scale patterns of nutrient limitation. Citation: Munshaw RG, Palen WJ, Courcelles DM, Finlay JC (2013) Predator-Driven Nutrient Recycling in California Stream Ecosystems. PLoS ONE 8(3): e58542. doi:10.1371/journal.pone.0058542 Palen WJ, Courcelles DM, Finlay JC (2013) Predator-Driven Nutrient Recycling in California Stream Ecosystems. PLoS ONE 8(3): e58542 0058542 RG, Palen WJ, Courcelles DM, Finlay JC (2013) Predator-Driven Nutrient Recycling in California Stream Ecosystems. PLoS ONE 8(3): e58 one.0058542 Editor: Wayne M. Getz, University of California, Berkeley, United States of America Editor: Wayne M. Getz, University of California, Berkeley, United States of America Received October 13, 2012; Accepted February 5, 2013; Published March 8, 2013 Received October 13, 2012; Accepted February 5, 2013; Published March 8, 2013 Copyright: 2013 Munshaw et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Predator-Driven Nutrient Recycling in California Stream Ecosystems Robin G. Munshaw1, Wendy J. Palen1, Danielle M. Courcelles1, Jacques C. Finlay2 1 Earth to Ocean Research Group, Department of Biological Sciences, Simon Fraser University, Burnaby, British Columbia, Canada, 2 Department of Ecology, Evolution and Behavior, University of Minnesota, St. Paul, Minnesota, Unites States of America Robin G. Munshaw1, Wendy J. Palen1, Danielle M. Courcelles1, Jacques C. Finlay2 Abstract Funding: This work was supported by the National Science and Engineering Research Council of Canada’s USRA and Discovery Grant program (www.nserc-crsng. gc.ca), and the Canada Research Chairs program (www.chairs-chaires.gc.ca). No individuals employed or contracted by the funders played any role in study design, data collection and analysis, decision to publish, preparation of the manuscript. Competing Interests: This work has previously been reviewed by Dr. Mary Power, Dr. John Schade, Dr. Jill Welter, and Dr. James Hood. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. * E-mail: rgm1@sfu.ca * E-mail: rgm1@sfu.ca The rate at which nutrients are recycled within a system depends heavily on the physical characteristics of that system and characteristics of the organisms that inhabit it. PLOS ONE \| www.plosone.org Ethics Statement This work was conducted with the approval California Department of Fish and Game (#11077), NOAA (#14904), and Simon Fraser University Animal Care (920B-09) permits. All animals were anaesthetized using Tricaine methanesulfonate (MS- 222) before handling, and all efforts were made to minimize stress and suffering during this study. Here we examined the effects of species identity in nutrient recycling between two taxonomically diverse top-predators, D. tenebrosus and O. mykiss, and explored the relative importance of ecosystem-scale N and P recycling by these predators across a range of potential real-world densities. We conducted a field study to estimate diet composition, nutrient excretion rates (N, P), and densities for each species, as well as the elemental body composition (C, N, P) of their prey in a coastal stream in Northern California. We hypothesize that, due to their large body size and high abundance [42], D. tenebrosus would dominate predator-mediated nutrient recycling in tributary ecosystems where both species co-occur. We also hypothesize that changes in the relative abundance of each species could affect ecosystem- level nutrient availability. Study Site Like many coastal tributaries in Northern California, Oregon, and Washington, our study site (Fox Creek, South Fork Eel river watershed, UTM: 10S 445880E, 4399070N) has two classes of vertebrate predators, stream salamanders (Genus Dicamptodon) and juvenile salmonid fishes (Genus Oncorhynchus). Dicamptodon tenebrosus exhibits life-history plasticity; after two to three years as aquatic juveniles, D. tenebrosus can either remain aquatic as paedomorphic adults or metamorphose into terrestrial adults [52]. The geographic range of D. tenebrosus includes coastal watersheds from the southern extremes of British Columbia to central California with the exception of the Olympic Peninsula [53]. Oncorhynchus mykiss populations in Pacific coastal watersheds are usually comprised of a mix of anadromous (i.e. steelhead) and resident (i.e. rainbow trout) individuals, with juveniles rearing in freshwa- ters for one to two years typically followed by divergence into one of the two dominant life-histories [54,55]. To determine the mass- frequency distribution and total biomass represented by each species, we conducted a survey of D. tenebrosus and O. mykiss in a tributary typical of coastal watersheds in the Pacific Northwest. We conducted our survey on Fox Cr. (2.6 km2 drainage area), a perennial tributary of the South Fork Eel River (SF Eel), Mendocino County, California, whose watershed lies entirely within the University of California’s Angelo Coast Range Reserve. The Fox Cr. watershed is dominated by Douglas fir (Pseudotsuga menziesii), Coast redwood (Sequoia sempervirens), and mixed conifer- deciduous forests. The channel is moderately incised with steep banks and is heavily shaded, and frequent woody debris jams are indicative of high winter discharge (November-March) despite low dry-season base flow (April-October, 5–7 L?s21; [45]). During summer base flow, the stream is reduced to a series of short riffles and shallow pools. The streambed alluvium is a mixture of cobbles and boulders, embedded with sand and pebbles. Methods To quantify the magnitude and importance of nitrogen and phosphorus recycling by vertebrates in tributary biogeochemistry, we conducted a survey of the size distribution, abundance, and diet composition of D. tenebrosus and O. mykiss. We estimated mass- specific excretion rates by two methods; in situ incubations for D. tenebrosus to establish novel excretion rate estimates, and bioener- getics modeling for O. mykiss. Bioenergetics models are often substituted for direct excretion measurements when appropriate models and parameter estimates exist [13,46–49]. Using a bioenergetics model to estimate excretion rates has been shown to be a very equitable surrogate for direct measurement [50]. However, due to a lack of available data on D. tenebrosus, direct measurements were necessary to establish excretion estimates. Despite the uncertainties of using two methodologies for deter- mining excretion rates, both methods can be considered compa- rable if adequate care is taken with handling and incubation time to minimize the effects of stress and fasting [11,23,51]. By using a bioenergetics model for fish, we were able to avoid the error in estimating normal excretion rates caused by handling stress, and reduce the need to experimentally manipulate additional animals. Although aquatic vertebrates can produce similar daily quan- tities of N and P through egestion and excretion [49], nutrients in fecal matter must undergo further microbial processing before they are available for uptake by autotrophs. We therefore do not consider egestion as an instantaneous contribution to nutrient recycling in this study. As such, we acknowledge that our estimates of excretion represent underestimates of total nutrients recycled by Nutrient Recycling by Multiple Stream Predators and may have similarly large effects on nutrient recycling. While some previous work has estimated the potential for nutrient recycling by a limited number of amphibians at broad scales [11,35], excretion data for salamanders is non-existent for most species, and they have seldom been considered as potentially important nutrient recyclers [36]. In temperate tributary streams such as those in the Pacific Northwest, amphibian populations can reach extremely high densities [37–39], even surpassing those of fishes [40,41]. In this region, two main vertebrate groups dominate predator assemblages; larval salamanders of the Genus Dicamptodon and juvenile salmonids of the Genus Oncorhynchus. Larval salamanders can often occur at densities exceeding all other predators, accounting for up to 99% of the vertebrate biomass in some areas [42]. Dicamptodon tenebrosus (coastal giant salamander; [43]) is one of the largest salamanders in North America, and often co-occurs with Oncorhynchus mykiss (steelhead trout). Both verte- brates are similarly opportunistic predators [44,45]. As large bodied vertebrates, these predators have may fill similar ecological niches and may contribute similarly to ecosystem processes such as nutrient recycling. vertebrates. We parameterized an O. mykiss bioenergetics model using surveyed diet composition, prey whole-body nutrient compositions (carbon, nitrogen, phosphorus), and summer water temperatures collected from several tributary streams. We combined estimates of O. mykiss and D. tenebrosus population densities with mass-specific excretion rates to estimate the relative magnitude of nutrient recycling by each predator. Using these excretion estimates and literature values for local nutrient uptake rates, we determined total vertebrate contribution to nutrient demand, and lastly explore the consequences of different relative abundances of D. tenebrosus and O. mykiss for ecosystem level nutrient availability. Introduction This process of consumer-driven nutrient recycling subsidizes ambient nutrient levels, and can affect the community structure of phytoplankton in lakes [12,13], control the availability of nutrients in phytotelmata [14], and control the spatial distribution of nutrients in river systems [15]. March 2013 \| Volume 8 \| Issue 3 \| e58542 PLOS ONE \| www.plosone.org 1 March 2013 \| Volume 8 \| Issue 3 \| e58542 Nutrient Recycling by Multiple Stream Predators March 2013 \| Volume 8 \| Issue 3 \| e58542 Quantification of Nutrient Excretion Schindler and Eby [62] determined coefficients (a, b, c, d) for a generalized salmonid bioenergetics model through meta-analysis and literature review including several species of salmonids, and we applied those estimates to our model (N: a = 23.256, 0.084 SE, b = 0.893, 0.021 SE, c = 215.049, 5.526 SE, d = 0.014, 0.003 SE, P: a = 24.776, 0.068 SE, b = 0.902, 0.024 SE, c = 96.801, 19.288 SE, d = 0.008, 0.003 SE). Each time we applied the model to an individual, we selected coefficients from the error distribution associated with each estimate. To parameterize the remaining terms of our O. mykiss bioenergetics model, we surveyed the diet composition of O. mykiss (.50 mm TL) in Fox Cr. as described above. Diets were converted to biomass pooled by order (as described above), and the average C, N, and P composition for each order was estimated based on percent C, N, and P measured from invertebrates collected during a 2008 survey of tributary sites within the SF Eel watershed (J. Hood, unpublished data). Inverte- brate C and N composition was determined using an elemental analyzer on dried and homogenized individuals or composited samples of multiple individuals. Samples for P were ashed at 550uC and hydrolyzed with HCl followed by colorimetric determination of PO4 [63]. Multiple values for the same genus were averaged, and values for genera not represented in the survey were summarized from the literature (Table 1 footnote). Diet biomass and nutrient compositions (C, N, and P) were converted to wet-mass using length to wet mass regressions for terrestrial and aquatic diet items separately [64]. We used the combination of the diet composition from surveyed fishes and invertebrate body composition to determine the nitrogen and phosphorus compo- sition of an average steelhead diet, and used these values in the O. mykiss bioenergetics model. Hourly stream temperatures were D. tenebrosus field study. To estimate nutrient-specific excretion rates and ratios for larval D. tenebrosus, we incubated 18 individuals (SVL range: 56–133 mm) with one of three common diet items [45,60]; terrestrial invertebrates (Orthoptera adults), aquatic invertebrates (Odonata larvae), or aquatic vertebrates (young-of-the-year O. mykiss). Individuals for excretion trials were collected by hand from Fox Cr. and placed in window screen- covered flow-through buckets to prevent the introduction of additional prey. Minimum dietary throughput for D. Nutrient Recycling by Multiple Stream Predators present year round [38–40,56–58]. Animals were surveyed by serial depletion in 32 tributary reaches (total stream distance of 170 m) blocked with nets from the top of the upstream riffle to the downstream end of each pool, using a combination of methods including snorkeling, electro-shocking, and hand-capture using the Parker-stick method [45]. After capture, animals were anesthe- tized with MS-222, weighed (g), and measured for total length (TL), snout to vent length (SVL, for D. tenebrosus) and standard length (SL, for O. mykiss). We used SVL and SL measurements as the primary measures of body length due to the frequency of tail injuries that can bias TL measurements. To compare dietary intake of N and P, a subset of captured individuals were sampled for diet using non-lethal gastric lavage (O. mykiss n = 86, D. tenebrosus n = 55). We adopted a minimum size threshold of 100 mm SL for O. mykiss and 50 mm SVL for D. tenebrosus based on lavage apparatus to avoid injuring small individuals during diet collection. Individuals with extensive injuries were not dieted. All individuals in the diet survey were captured in the daytime in pools, controlling for diel and habitat variability in gut contents. Individuals were dieted opportunistically across several concurrent studies, which is reflected by the inconsistent sample sizes. Diets were preserved in 70% EtOH until enumeration, measurement, and identification to the highest level of taxonomic resolution possible (e.g. Genus or Family). Dry biomass of individual diet items was estimated using measured length (mm) and taxon- specific length-weight regressions developed from our survey data and the literature (Table S1). Diet data were supplemented with a longer-term data set from our study stream and nearby tributaries. Water content of prey items was determined from the literature [59], and used to convert invertebrate dry weight composition of C, N, and P to wet weight composition for use in the O. mykiss bioenergetics model. After processing and recovery from anesthe- sia, all animals were released live at the point of capture. processes, despite our care with filtration. After 120 minutes, approximately 100 mL of water was filtered (0.7 mm, Nalgene 190 syringe filter) to measure ammonium (NH4) and soluble reactive phosphorus (SRP), and animals were removed, weighed, measured as above and released. Water samples were kept dark in coolers with ice packs and were processed within 4 hours. Nutrient Recycling by Multiple Stream Predators SRP concentrations were determined using spectrophotometry, and ammonium concentrations were determined using fluorometry [61]. To evaluate the effects of mass and diet group on D. tenebrosus excretion rates, we compared the likelihood of several competing linear regression models using Akaike’s Information Criterion for small sample sizes (AICc). We fit linear models by maximum likelihood including all combinations of mass (log10(g)) and diet treatment (terrestrial invertebrate, aquatic invertebrate, aquatic vertebrate), including a mass by diet treatment interaction and an intercept only model, assuming normally distributed errors. We used the best-supported model by AICc to estimate daily excretion rates (N, P) for each individual from our survey reach and summed across all individuals (n = 348 D. tenebrosus) to estimate the total amount and ratio of N and P recycled by D. tenebrosus in the 1 km reach. O. mykiss bioenergetics model. We estimated O. mykiss mass-specific excretion rates using an established bioenergetics model for salmonids [62]. This model was built using a range of body masses that fully encompass those found in our study. Nutrient specific (total dissolved N, total dissolved P) bioenergetics models estimate mass-specific excretion rates (Ei) for each individual (i) in grams per day based on animal mass (Mi), water temperature (T), and N and P content of diet items (N). log10 Ei ð Þ~azb log10 Mi ð Þ ð Þzc N ð Þzd T ð Þ Abundance Survey To estimate the body size distribution and total biomass of D. tenebrosus and O. mykiss, we surveyed all individuals of both species in a sub-set of pools within the first 1.3 km of Fox Cr. Both O. mykiss and D. tenebrosus co-occur in Fox Cr. from the confluence with the SF Eel to approximately 1.3 km upstream, at which point fish passage is restricted, and only D. tenebrosus is present. Based on previous surveys, these two species constitute the vast majority of vertebrate biomass in tributary streams in the region and are Although aquatic vertebrates can produce similar daily quan- tities of N and P through egestion and excretion [49], nutrients in fecal matter must undergo further microbial processing before they are available for uptake by autotrophs. We therefore do not consider egestion as an instantaneous contribution to nutrient recycling in this study. As such, we acknowledge that our estimates of excretion represent underestimates of total nutrients recycled by March 2013 \| Volume 8 \| Issue 3 \| e58542 PLOS ONE \| www.plosone.org March 2013 \| Volume 8 \| Issue 3 \| e58542 2 Nutrient Recycling by Multiple Stream Predators Ecosystem Nutrient Recycling By combining measured excretion rates of D. tenebrosus, with bioenergetics estimates for O. mykiss, we predicted the total amount and ratios of N and P recycled by these two predators across the Table 1. Average elemental body composition (by dry mass) of common O. mykiss and D. tenebrosus diet items* by order. Table 1. Average elemental body composition (by dry mass) of common O. mykiss and D. tenebrosus diet items* by order. ental body composition (by dry mass) of common O. mykiss and D. tenebrosus diet items* by order. Table 1. Average elemental body composition (by dry mass) of common O. mykiss and D. tenebrosus diet items* by order. % elemental composition (dry mass±SD) Order % of O. mykiss diet % of D. tenebrosus diet C N P Coleoptera 9.25 0.50 53.661.0 8.760.7 0.560.1 Diptera 8.82 0.25 44.762.9 10.561.1 1.560.1 Ephemeroptera 26.82 75.53 46.264.2 10.761.0 1.260.2 Hemiptera1 0.45 1.15 50.764.2 11.760.9 1.060.3 Hymenoptera2,3 10.06 0.03 39.16 – 10.46 – 0.96 – Lepidoptera4,5 6.24 1.69 34.66 – 5.86 – 0.36 – Odonata – 0.59 45.062.4 12.360.3 1.060.1 Orthoptera – 0.54 46.861.7 9.660.3 0.760.1 Plecoptera6 0.37 0.85 52.960.4 10.360.2 1.160.1 Salmonid – 6.93 43.667.0 12.762.3 1.360.2 Trichoptera 5.11 1.40 47.362.8 9.761.3 1.160.3 Table 1. Average elemental body composition (by dry mass) of common O. mykiss and D. tenebrosus diet items* by order. % elemental composition (dry mass±SD) Order % of O. mykiss diet % of D. tenebrosus diet C N P Coleoptera 9.25 0.50 53.661.0 8.760.7 0.560.1 Diptera 8.82 0.25 44.762.9 10.561.1 1.560.1 Ephemeroptera 26.82 75.53 46.264.2 10.761.0 1.260.2 Hemiptera1 0.45 1.15 50.764.2 11.760.9 1.060.3 Hymenoptera2,3 10.06 0.03 39.16 – 10.46 – 0.96 – Lepidoptera4,5 6.24 1.69 34.66 – 5.86 – 0.36 – Odonata – 0.59 45.062.4 12.360.3 1.060.1 Orthoptera – 0.54 46.861.7 9.660.3 0.760.1 Plecoptera6 0.37 0.85 52.960.4 10.360.2 1.160.1 Salmonid – 6.93 43.667.0 12.762.3 1.360.2 Trichoptera 5.11 1.40 47.362.8 9.761.3 1.160.3 Elemental composition estimates from the literature for orders Lepidoptera and Hymenoptera did not include estimates of variability. Footnotes: Unaccounted for percentage of diet was comprised of diet items not covered by invertebrate CNP survey and for which values could not be found in literature. Contributions by these uncommon items were deemed inconsequential due to their small individual proportion of the wet mass of diets. Large and/or unique diet items (orders comprising ,0.5% of total items) were discounted in diets so as not to bias elemental estimates. Nutrient Recycling by Multiple Stream Predators Nutrient Recycling by Multiple Stream Predators 1 km study reach. We performed a bootstrap numerical simulation [72] on the data using R (version 2.12.1) to provide a mean and a 95% confidence interval for the estimate of total excreted nitrogen and phosphorus for each species. In each bootstrap simulation (n = 10,000), individuals of each species from our survey dataset were randomly selected with replacement until the summed biomass of selected individual (Mt-sim) equaled that observed for each species in our field survey of Fox Cr. for the study reach (O. mykiss Mt-obs = 1733 g wet mass, D. tenebrosus Mt-obs = 4542 g wet mass). Mass specific nutrient excretion rates were estimated for each selected individual as above, and summed to estimate the species-specific total N and total P recycled, as well as the N to P ratio. Each individual’s mass-specific excretion rates were estimated including parameter variability, as reported in the literature bioenergetics model in the case of O. mykiss, and from our empirically derived regressions in the case of D. tenebrosus. To assess the importance of the relative abundance of the two predators for nutrient recycling, we also ran simulations that varied the proportion of the predator biomass made up by salamanders and fish, assuming that the combined predator biomass we observed in Fox Cr. is a conservative estimate of the maximum sustainable regardless of predator identity. As such, we varied the predator composition from 100% O. mykiss to 100% D. tenebrosus biomass in 10% increments (n = 1,000 simulations for each increment). recorded in 11 pools within the 1.3 km study reach using iButton temperature loggers (Maxim Integrated Products Inc., Sunnyvale, CA) in Fox Cr. for a 7-week period (July-August) in 2010 during the peak season of biological productivity. Average daily temperatures at 11 stream locations were averaged to calculate stream-wide mean temperature (T). We used the average dietary nitrogen and phosphorus values (N, P), along with individual weight (Mi), and average summer stream temperature (T) to estimate the excretion rates of each individual sampled in our survey of Fox Cr. for both N and P. Excretion rates (N, P) for each individual were converted to daily rates (per 24 hr.) and summed across all individuals (n = 527 O. mykiss) to estimate the total amount and ratio of N and P recycled by O. mykiss in the 1 km study reach. Nutrient Recycling by Multiple Stream Predators In freshwater teleosts, urea and ammonium generally constitute the majority of the total nitrogenous end-products of metabolism [65], and in O. mykiss specifically, the greatest portion is excreted as ammonium [66,67]. To directly compare O. mykiss excretion to D. tenebrosus excretion (NH4), we considered in our results only the portion of total excreted nitrogen by O. mykiss that can be attributed to ammonium (59.3%, 3.99 SE in O. mykiss; [67]). Ammonium also constitutes the bulk of nitrogenous excretion in aquatic amphibians [68–70]. We therefore consider ammonium as a conservative proxy for total N excretion in this study. It is generally established that SRP constitutes the bulk of excreted P, and can therefore be considered an acceptable estimate of total excreted P (e.g. [20,24,71]). To evaluate the magnitude of nutrient recycling by top predators relative to ecosystem demand, we calculated rates of community nutrient uptake in the study reach. Areal uptake rates were calculated based on Schade et al. [61] to provide estimates of nutrient demand during the summer growth period. Uptake rate (U: mg?m22 s21) can be calculated by multiplying discharge rate Quantification of Nutrient Excretion tenebrosus in this system is estimated to be 60 hours (Munshaw unpublished data), therefore salamanders were incubated with food treatments for 60 hours (62 hours) to allow excretion rates and ratios to reflect dietary treatments. Food treatments were assigned to control for unknown diet composition that may introduce additional variation into our estimates of excretion rates and ratios. To allow study salamanders to feed ad libitum, we kept each enclosure stocked with an excess of treatment diet items. This reflects the high availability of prey items in the ecosystem, and low occurrence of individuals with empty stomachs [45]. After 60 hours, salamanders were removed from flow-through enclosures, gently rinsed with filtered water to remove adhered particles, and immediately placed in individual 2 L acid-washed containers with 1 L of 0.7 mm-filtered (Whatman GF-F) stream water. Containers were covered with loose plastic lids to prevent addition of airborne particles, and incubated in the stream margins (approx. 5 cm depth) to maintain ambient stream temperature for the duration of the incubation. In addition to incubating salamanders to measure nutrient excretion, we also incubated 3 control containers without salamanders to evaluate the change in nutrient concentrations due to microbial March 2013 \| Volume 8 \| Issue 3 \| e58542 March 2013 \| Volume 8 \| Issue 3 \| e58542 PLOS ONE \| www.plosone.org 3 mental composition estimates from the literature for orders Lepidoptera and Hymenoptera did not include estimates of variability. d f f d d f d d b b d f h h l Quantification of Excreted Nutrients D. tenebrosus field study. Control containers showed no significant change in nutrient concentration over time (N: t = 0.56, df = 2, p-value = 0.63, P: t = 0.10, df = 2, p-value = 0.93) indicating that any changes we observed in trials containing salamanders were attributable to animal excretion, and not microbial processes. Model selection by AICc of N and P excretion rates for D. tenebrosus from our experiment identified clear top models for both nutrients, with DAICc scores of all other models .3 (Table S2). The best-supported model for D. tenebrosus P (SRP) excretion rate included both intercept and mass (log10[mgP?- min21] = 23.12+1.60?log10[mass], SEintercept = 0.73, SEmass coeffi- cient = 0.56; Fig. 2), as did the best-supported model for N (NH4) excretion rate (log10[mgN?min21] = 22.04+1.41?log10[mass], SEin- tercept = 0.24, SEmass coefficient = 0.18; Fig. 2). We found little support for models that included diet treatment (terrestrial invertebrate, aquatic invertebrate, and aquatic vertebrate), where all models including diet had DAIC $3. Consequently, dietary treatment was disregarded for D. tenebrosus in subsequent analyses. Nutrient Recycling by Multiple Stream Predators (Q: Ls21) by nutrient concentration (C: mg?L21), and dividing by reach width (w: m) and nutrient-specific spiral length (Sw: m). max = 12.7% dry mass), and P (min = 0.3%, max = 1.5% dry mass) content varied substantially among orders (Table 1), and in total our analysis accounted for 90% of D. tenebrosus and 67% of O. mykiss diet items by mass (Table 1). The majority of unaccounted- for diet was composed of unidentifiable remains. We found that on average, D. tenebrosus diets were 8.5% C, 1.9% N, and 0.2% P by wet mass, and O. mykiss diets were composed of 7.9% C, 1.6% N, and 0.2% P by wet mass (Table 1). Only a small percentage of the salamanders surveyed had empty stomachs (5%), which is similar to the 1% occurrence of empty stomachs reported by Parker [45], which served as the basis for our decision to feed excretion trial salamanders ad libitum. U~ Q C ð Þ w Sw ð Þ The model was parameterized using measurements from 32 replicated pools (w, Q) measurements combined with data presented by Schade et al. (SwN: 200,SwP: 540, CN: 10.1, CP: 15) [61]. Aerial uptake rates (U) can be used to estimate the proportion of nutrient demand supplied by O. mykiss and D. tenebrosus excretion in the study reach by difference. These estimates consider the largest single component of dissolved inorganic nitrogen (i.e. NH4) and total dissolved phosphorus (i.e. SRP), and so can thus be considered a conservative estimate of uptake demand. Proportion of demand supplied by O. mykiss and D. tenebrosus recycling was determined by dividing areal recycling rate (total recycling rate divided by total study area) by uptake rate (U). Ecosystem Nutrient Recycling 1Frost PC, Tank SE, Turner MA, Elser JJ (2010) Elemental composition of littoral invertebrates from oligotrophic and eutrophic Canadian lakes. Journal of the North American Benthological Society 22:51–62. 2Elser JJ (2003) Biological stoichiometry: a theoretical framework connecting ecosystem ecology, evolution, and biochemistry for application in astrobiology. International Journal of Astrobiology 2:185–193. 3Woods HA, Fagan WF, and Elser JJ (2004) Allometric and phylogenetic variation in insect phosphorus content. Functional Ecology 18:103–108. 4Elser JJ, Fagan FF, Denno RF, Dobberfuhl DR, Folarin A, Huberty A, Interlandi S, Kilham SS, McCauley E, Schulz KL, Siemann EH, Sterner RW (2000) Nutritional constraints in terrestrial and freshwater food-webs. Nature 408:578–580. 5Slansky Jr. F, and Feeny P (1977). Stabilization of the rate of nitrogen accumulation by larvae of the cabbage butterfly on wild and cultivated food plants. Ecological Monographs 47:209–228. 6Cross WF, Benstead JP, Rosemond AD, and Wallace JB (2003) Consumer-resource stoichiometry in detritus-based streams. Ecology Letters 6:721–732. Monographs 47:209–228. 6Cross WF, Benstead JP, Rosemond AD, and Wallace JB (2003) Consumer-resource stoichiometry in detritus-based streams. Ecology Letters 6:721–732. doi:10 1371/journal pone 0058542 t001 Monographs 47:209 228. 6Cross WF, Benstead JP, Rosemond AD, and Wallace JB (2003) Consumer-resource stoichiometry in detritus-based streams. Ecology Letters 6:721–732. doi:10.1371/journal.pone.0058542.t001 March 2013 \| Volume 8 \| Issue 3 \| e58542 March 2013 \| Volume 8 \| Issue 3 \| e58542 March 2013 \| Volume 8 \| Issue 3 \| e58542 PLOS ONE \| www.plosone.org 4 Nutrient Recycling by Multiple Stream Predators Discussion This study suggests that differences in predator in abundance, body size distribution, and species identity directly influence nutrient recycling rates and ratios in headwater streams. These We found that varying the relative abundance of the two vertebrate predators (assuming constant total biomass) led to large differences in total excreted nutrients (N and P). When we simulated the predator biomass as being entirely comprised of D. tenebrosus, N and P excretion rates were estimated to be 0.34 and 0.06 g?day21 respectively (Fig. 5). These rates increased along the Figure 3. Daily excretion estimates for predators in our study reach. Estimated total daily excreted N (NH4) and P (SRP) by O. mykiss (filled), D. tenebrosus (grey), and both predators combined (open) within the Fox Cr. study reach. Bars represent mean 695%CI. Note the log scaled y-axis. doi:10.1371/journal.pone.0058542.g003 We found that varying the relative abundance of the two vertebrate predators (assuming constant total biomass) led to large differences in total excreted nutrients (N and P). When we simulated the predator biomass as being entirely comprised of D. tenebrosus, N and P excretion rates were estimated to be 0.34 and 0.06 g?day21 respectively (Fig. 5). These rates increased along the Figure 2. Excretion rates of D. tenebrosus. Nitrogen (NH4) and phosphorus (SRP) nutrient excretion rates (ug?min21) of D. tenebrosus. Lines represent the fit of the top model selected by AICc for P (log10[mgP?min21] = 23.12+1.60(log10[mass]), r2 = 0.31, P = 0.01), and N excretion rates (log10[mgN?min21] = 22.04+1.41(log10[mass]), r2 = 0.79, P,,0.001). doi:10.1371/journal.pone.0058542.g002 Figure 3. Daily excretion estimates for predators in our study reach. Estimated total daily excreted N (NH4) and P (SRP) by O. mykiss (filled), D. tenebrosus (grey), and both predators combined (open) within the Fox Cr. study reach. Bars represent mean 695%CI. Note the log scaled y-axis. doi:10.1371/journal.pone.0058542.g003 PLOS ONE \| www plosone org 6 March 2013 \| Volume 8 \| Issue 3 \| e58542 Figure 2. Excretion rates of D. tenebrosus. Nitrogen (NH4) and phosphorus (SRP) nutrient excretion rates (ug?min21) of D. tenebrosus. Lines represent the fit of the top model selected by AICc for P (log10[mgP?min21] = 23.12+1.60(log10[mass]), r2 = 0.31, P = 0.01), and N excretion rates (log10[mgN?min21] = 22.04+1.41(log10[mass]), r2 = 0.79, P,,0.001). Figure 3. Daily excretion estimates for predators in our study reach. Estimated total daily excreted N (NH4) and P (SRP) by O. mykiss (filled), D. Abundance Survey Density and biomass (abundance survey) and average wet weight elemental composition of diets (diet survey) for O. mykiss and D. tenebrosus in Fox Creek, California. Abundance survey Diet survey Species Density (m22) Biomass (gm22) n % of diet accounted for %C %N %P n O. mykiss 1.56 5.12 528 67.1 7.92 1.55 0.18 86 D. tenebrosus 1.03 13.42 348 89.5 8.51 1.88 0.23 55 Values calculated using total area of the study reach. doi:10.1371/journal.pone.0058542.t002 Table 2. Density and biomass (abundance survey) and average wet weight elemental composition of diets (diet survey) for O. mykiss and D. tenebrosus in Fox Creek, California. Table 2. Density and biomass (abundance survey) and average wet weight elemental composition of diets (diet survey) for O. mykiss and D. tenebrosus in Fox Creek, California. Table 2. Density and biomass (abundance survey) and average wet weight elemental composition of diets (diet survey) for O. mykiss and D. tenebrosus in Fox Creek, California. Values calculated using total area of the study reach. doi:10.1371/journal.pone.0058542.t002 relative abundance continuum to a maximum N and P excretion rate of 1.53 and 0.18 g?day21 respectively when O. mykiss comprised all the predator biomass. The overall N:P ratio of predator-recycled nutrients ranged from 6.04 when all biomass was D. tenebrosus, to 8.72 when all biomass was O. mykiss (Fig. 5). We calculated that uptake rates (U) for N and P were 0.1 mg?m22 s21 and 0.04 mg?m22 s21 respectively. At the ob- served densities of D. tenebrosus and O. mykiss, we estimated that nutrient recycling by predators in the study reach represented 20.3% of N (0.0023 mg?m22 s2161.11024 95% CI) and 7.3% of P (3.11024 mg?m22 s2161.51025 95% CI) demand by produc- ers in the study reach. these inputs, rate of excretion of N and P was calculated for each individual and used for the ecosystem analysis. these inputs, rate of excretion of N and P was calculated for each individual and used for the ecosystem analysis. relative abundance continuum to a maximum N and P excretion rate of 1.53 and 0.18 g?day21 respectively when O. mykiss comprised all the predator biomass. The overall N:P ratio of predator-recycled nutrients ranged from 6.04 when all biomass was D. tenebrosus, to 8.72 when all biomass was O. mykiss (Fig. 5). Ecosystem Nutrient Recycling We estimated the daily N and P recycled by individual D. tenebrosus and O. mykiss, by species, and by both species combined within the study reach. We estimated that O. mykiss recycled nearly twice as much N per day (0.42 g?day2160.028 95% CI) as that recycled by D. tenebrosus (0.25 g?day2160.017 95% CI, Fig. 3) within the study reach. The phosphorus recycled by O. mykiss (0.049 g?day2160.0021 95% CI) was approximately 19% greater than that recycled by D. tenebrosus (0.041 g?day2160.0039 95% CI). We found that when estimates for both species were summed, 0.67 g?day21 of N was recycled (60.032 95% CI, Fig. 3A), and 0.089 g day21 of P (60.0044 95% CI). The N:P ratio of nutrients recycled by O. mykiss (8.760.6 95% CI, Fig. 4) was higher than that of D. tenebrosus (6.060.2 95% CI) and the N:P ratio of both species combined (7.560.3 95% CI) was intermediate between the two. We calculated that uptake rates (U) for N and P were 0.1 mg?m22 s21 and 0.04 mg?m22 s21 respectively. At the ob- served densities of D. tenebrosus and O. mykiss, we estimated that nutrient recycling by predators in the study reach represented 20.3% of N (0.0023 mg?m22 s2161.11024 95% CI) and 7.3% of P (3.11024 mg?m22 s2161.51025 95% CI) demand by produc- ers in the study reach. Abundance Survey During the field survey of the study area in Fox Cr., we captured 356 D. tenebrosus and 595 O. mykiss, at an average density of 1.05 and 1.76 individuals/m2 respectively (Table 2). Despite lower numerical abundance, the total biomass of D. tenebrosus was 2.5 times higher than O. mykiss (13.7 and 5.4 g wet mass/m2 respectively; Table 2). While the length range of captured individuals of each species was quite similar (Fig. 1, left panels), mean D. tenebrosus body mass was nearly 6 times higher (Fig. 1, right panels; D. tenebrosus = 13.05 g wet mass, 0.79 SE, O. mykiss = 3.28 g wet mass, 0.37 SE). O. mykiss bioenergetics model. Results from the diet survey and the invertebrate body C, N, and P survey were used to establish the N and P composition of an average steelhead diet (Table S3; N = 1.8%, P = 0.2% wet weight). Diet information was combined with average stream temperature (14.2uC, 0.004 SE, min = 12.0uC, max = 16.5uC) in our bioenergetics model. Using Our summary of invertebrate C, N, and P composition and predator diet composition showed that both species of predator had diets with a high degree of taxonomic overlap and similar elemental composition. Both O. mykiss and D. tenebrosus diets were dominated by Order Ephemeroptera (.25% by mass; Table 1). Carbon (min = 34.6%, max = 53.6% dry mass), N (min = 5.8%, Figure 1. Histograms of size and mass of predators in our study reach. Size- and mass-frequency distributions for O. mykiss and D. tenebrosus from a 1 km study reach of Fox Creek. Standard length (mm) was used for fish (n = 528) and snout-vent length (mm) for salamanders (n = 348) to exclude the size variability generated by tail injuries. Note differences in y-axis scales. doi:10.1371/journal.pone.0058542.g001 Figure 1. Histograms of size and mass of predators in our study reach. Size- and mass-frequency distributions for O. mykiss and D. tenebrosus from a 1 km study reach of Fox Creek. Standard length (mm) was used for fish (n = 528) and snout-vent length (mm) for salamanders (n = 348) to exclude the size variability generated by tail injuries. Note differences in y-axis scales. doi:10.1371/journal.pone.0058542.g001 March 2013 \| Volume 8 \| Issue 3 \| e58542 March 2013 \| Volume 8 \| Issue 3 \| e58542 PLOS ONE \| www.plosone.org 5 Nutrient Recycling by Multiple Stream Predators Table 2. Discussion did provide 37% of the total N and 46% of the total P recycled by vertebrate predators, providing support for the importance of vertebrates other than fish in stream nutrient recycling. We also found that the relative abundance of salamander and fish biomass altered the total amount of nutrients recycled by top predators per day. Our simulations suggest that when predator biomass is comprised completely of D. tenebrosus, the amount of recycled N and P are only approximately half (51% N, 63% P relative to background) that recycled by the natural assemblage of salamanders and fish in Fox Cr. When the total predator biomass in our simulation was comprised entirely of O. mykiss, both N and P recycling rates were estimated to be approximately twice as high as background (2.36N, 2.06P). The large differences we found in estimated stream-wide excretion rates between O. mykiss and D. tenebrosus are in contrast to the many ecological similarities we found between the species. As ambush predators, D. tenebrosus feed mostly on aquatic benthic prey [73], whereas O. mykiss feed throughout the water column, and consume a larger proportion of allochthonous (e.g. terrestrial) prey. While each predator feeds in different stream microhabitats, they have a high degree of overlap in diet composition (Table 1, also see [44]), with similar elemental composition. Our data suggest that dietary or stoichiometric differences in prey are not likely to explain the observed differences in species-specific N:P ratios (Table S3). Similarly, data from a complementary study found that growth rates of D. tenebrosus and O. mykiss in the same study reach did not differ significantly over the summer growth period (Atlas et al., unpublished data), indicating that differences we identified in nutrient excretion rates are likely driven by differences Metabolic scaling theory predicts that as mass increases, mass specific excretion rate should decrease [77], which may further explain the disparity in total excreted nutrients between predator species. The higher numbers but lower biomass of O. mykiss as compared to D. tenebrosus (Table 2), resulted from a smaller average size of O. mykiss individuals in our study system (mean O. mykiss = 3.3 g wet mass, mean D. tenebrosus = 13.1 g wet mass). The size-frequency distribution of O. mykiss (Fig. 1), illustrates two distinct nodes representing young of the year (age 0) and one-year or older age-classes that make up the vast majority of O. mykiss individuals. Discussion Simulations assumed a fixed total biomass of predators (6275 g) within the study reach, and estimated total excretion rates (left y-axis) and ratios (right y- axis) by bootstrapped re-sampling of surveyed individuals. Predator relative abundance (x-axis) varies by 10% increments from 100% O. mykiss composition to 100% D. tenebrosus composition, expressed as the proportion of predator biomass (salamander:fish). Vertical line indicates the observed ratio of predators in Fox Cr. doi:10.1371/journal.pone.0058542.g005 Figure 5. Impacts on excretion of simulated tradeoffs between Figure 5. Impacts on excretion of simulated tradeoffs between predator biomasses. Estimated total recycled N (NH4) and P (SRP) excretion (g?day21) in Fox Cr. due to simulated changes in the relative abundance (by biomass) of O. mykiss and D. tenebrosus. Simulations assumed a fixed total biomass of predators (6275 g) within the study reach, and estimated total excretion rates (left y-axis) and ratios (right y- axis) by bootstrapped re-sampling of surveyed individuals. Predator relative abundance (x-axis) varies by 10% increments from 100% O. mykiss composition to 100% D. tenebrosus composition, expressed as the proportion of predator biomass (salamander:fish). Vertical line indicates the observed ratio of predators in Fox Cr. doi:10.1371/journal.pone.0058542.g005 Figure 4. Estimates of excreted N:P ratio for predators in our study reach. Estimates of the ratio of excreted N:P for O. mykiss (filled), D. tenebrosus (grey), and both predators combined (open). Bars represent mean 695%CI. doi:10.1371/journal.pone.0058542.g004 in metabolic requirements. At zero activity level, O. mykiss is estimated to have a 50% higher metabolic demand as measured by oxygen consumption [74] than an average salamander of the same weight [75]. D. tenebrosus is a sit-and-wait predator and is only marginally active at night and even less so during the day, whereas O. mykiss is an active water-column predator and expends large amounts of energy for locomotion and foraging when not resting or seeking cover in interstitial spaces [60]. Larger expenditures of energy by O. mykiss likely necessitate higher metabolic rates and faster production of metabolic wastes, which is excreted at a higher mass-specific rate than by D. tenebrosus. An additional factor that likely contributes to the difference in excretion ratios is species specific elemental requirements for growth and maintenance. Stoichiometric theory suggests that as the required N:P ratio of an organism increases at any given ingested N:P ratio, the ratio of excreted nutrients will decrease [76]. Though we do not have elemental composition data for D. Discussion tenebrosus from our study system, values from the literature show that eastern salamanders that fill a similar ecological niche have organismal N:P ratios of 2.4–4.8 [36], whereas juvenile steelhead in our study system had an average N:P ratio of 9.8. However, our findings that O. mykiss recycles N:P at a higher ratio than D. tenebrosus suggests that the body N:P ratio of D. tenebrosus may be much higher than eastern species. findings emphasize the importance of species identity in predator- driven nutrient recycling. As such, we found that biomass alone is not an adequate proxy for estimating species contributions to the total rate of nutrient recycling, which depends on physical, physiological, and ecological traits of different members of the predator guild. Despite more than two and a half times higher biomass, the total amount of nutrients recycled by D. tenebrosus in Fox Cr. was only a fraction (58% N, 84% P) of that recycled by O. mykiss. Despite a lower relative contribution, the D. tenebrosus population in Fox Cr. did provide 37% of the total N and 46% of the total P recycled by vertebrate predators, providing support for the importance of vertebrates other than fish in stream nutrient recycling. We also found that the relative abundance of salamander and fish biomass altered the total amount of nutrients recycled by top predators per day. Our simulations suggest that when predator biomass is comprised completely of D. tenebrosus, the amount of recycled N and P are only approximately half (51% N, 63% P relative to background) that recycled by the natural assemblage of salamanders and fish in Fox Cr. When the total predator biomass in our simulation was comprised entirely of O. mykiss, both N and P recycling rates were estimated to be approximately twice as high as background (2.36N, 2.06P). findings emphasize the importance of species identity in predator- driven nutrient recycling. As such, we found that biomass alone is not an adequate proxy for estimating species contributions to the total rate of nutrient recycling, which depends on physical, physiological, and ecological traits of different members of the predator guild. Despite more than two and a half times higher biomass, the total amount of nutrients recycled by D. tenebrosus in Fox Cr. was only a fraction (58% N, 84% P) of that recycled by O. mykiss. Despite a lower relative contribution, the D. tenebrosus population in Fox Cr. Discussion tenebrosus (grey), and both predators combined (open) within the Fox Cr. study reach. Bars represent mean 695%CI. Note the log scaled y-axis. doi:10.1371/journal.pone.0058542.g003 March 2013 \| Volume 8 \| Issue 3 \| e58542 PLOS ONE \| www.plosone.org 6 Nutrient Recycling by Multiple Stream Predators Figure 4. Estimates of excreted N:P ratio for predators in our study reach. Estimates of the ratio of excreted N:P for O. mykiss (filled), D. tenebrosus (grey), and both predators combined (open). Bars represent mean 695%CI. doi:10.1371/journal.pone.0058542.g004 Figure 5. Impacts on excretion of simulated tradeoffs between predator biomasses. Estimated total recycled N (NH4) and P (SRP) excretion (g?day21) in Fox Cr. due to simulated changes in the relative abundance (by biomass) of O. mykiss and D. tenebrosus. Simulations assumed a fixed total biomass of predators (6275 g) within the study reach, and estimated total excretion rates (left y-axis) and ratios (right y- axis) by bootstrapped re-sampling of surveyed individuals. Predator relative abundance (x-axis) varies by 10% increments from 100% O. mykiss composition to 100% D. tenebrosus composition, expressed as the proportion of predator biomass (salamander:fish). Vertical line indicates the observed ratio of predators in Fox Cr. doi:10.1371/journal.pone.0058542.g005 Figure 4. Estimates of excreted N:P ratio for predators in our study reach. Estimates of the ratio of excreted N:P for O. mykiss (filled), D. tenebrosus (grey), and both predators combined (open). Bars represent mean 695%CI. doi:10.1371/journal.pone.0058542.g004 Figure 5. Impacts on excretion of simulated tradeoffs between predator biomasses. Estimated total recycled N (NH4) and P (SRP) excretion (g?day21) in Fox Cr. due to simulated changes in the relative abundance (by biomass) of O. mykiss and D. tenebrosus. Simulations assumed a fixed total biomass of predators (6275 g) within the study reach, and estimated total excretion rates (left y-axis) and ratios (right y- axis) by bootstrapped re-sampling of surveyed individuals. Predator relative abundance (x-axis) varies by 10% increments from 100% O. mykiss composition to 100% D. tenebrosus composition, expressed as the proportion of predator biomass (salamander:fish). Vertical line indicates the observed ratio of predators in Fox Cr. doi:10.1371/journal.pone.0058542.g005 Figure 5. Impacts on excretion of simulated tradeoffs between predator biomasses. Estimated total recycled N (NH4) and P (SRP) excretion (g?day21) in Fox Cr. due to simulated changes in the relative abundance (by biomass) of O. mykiss and D. tenebrosus. Nutrient Recycling by Multiple Stream Predators salmonids [79–81]. By contrast, there is a near continuous distribution of D. tenebrosus body size, with no distinct nodes and a higher frequency of larger individuals than O. mykiss (Fig. 1). Coastal giant salamanders can achieve life spans in excess of 20 years, and are characterized by slow growth, leading to difficulty in assigning age based on body size. Ultimately, the larger average mass achieved by D. tenebrosus compared to O. mykiss is likely to contribute to the observed lower mass-specific excretion rate and lower total species-specific rate of nutrient recycling. As summer maximum temperatures increase with future climate change and continued land-cover change (e.g. forest clearing), amphibian populations may be able to persist in thermal environments that cause acute mortality or exclude salmonid populations. Our study suggests that in such cases, large salamander populations may act to partially buffer the impacts of declining fish populations via nutrient recycling. By not considering amphibians and other animals as nutrient recyclers, we may be missing critical components of nutrient dynamics in stream ecosystems. p p y g Our test of the influence of diet on excretion ratios in D. tenebrosus showed that despite the differing N:P ratios of the items fed to captive individuals (aquatic invertebrate = 12.7, terrestrial invertebrate = 13.4, aquatic invertebrate = 9.9), we found no support for differences in N or P excretion rates of D. tenebrosus fed these prey. Our results were likely inconsistent with our stoichiometric prediction for several reasons. Despite holding salamanders with food items for a full iteration of their estimated digestive throughput time (60 hours), individual excretion rates may not have had time to equilibrate to reflect the homogenous experimental diets. Additional factors may have included a wide range of body sizes (6.3–72.8 mm SVL), individuals at different stages of reproductive maturity [82], and small sample size (n = 18).Our findings demonstrate that vertebrate excretion can affect nutrient availability in light-limited forested streams. However, the repercussions of altering nutrient availability depend on the nutrient requirements of producers in the system. By increasing the availability of limiting nutrients, algal growth and elemental composition can be altered. Using nutrient addition experiments, Schade et al. [61] demonstrate that Fox Cr. is N- limited, and has an external dissolved inorganic NH4-N:SRP ratio of 0.67. The ratio of recycled nutrients from predators examined in this study is modestly higher than ambient levels (7.5; Fig. Table S2 AICc scores for models predicting N and P excretion rates. (DOCX) River networks are characterized by strong downstream gradients in light, temperature, disturbance frequency, and physical channel attributes, each of which can differentially affect the movement, growth, and persistence of riverine species [83,84]. Despite having highly overlapping regional distributions, O. mykiss and D. tenebrosus vary in their relative abundance across the heterogeneity present in river networks at local-scales [40]. Our simulation results suggest that such differences, combined with spatial and temporal variation in ecosystem nutrient demand, are likely to affect broad-scale patterns of nutrient limitation. While the study system we worked in is within a protected area, and has been well buffered from local anthropogenic impacts, the relative abundance of these two species across broader watersheds and larger coastal regions is likely affected by species-specific responses to anthropogenic stressors in the past as well as the future. In the past, steelhead abundance was very high along the entire west coast of North America, but overexploitation, especially along the California coastline, has drastically diminished abundance [85]. Future thermally limiting events may also affect abundances. Salmonids are known to have much lower critical thermal maxima, both as juveniles (26.2–27.9uC; [86]) and older classes (28.25–29.85; [87]), than many salamanders (30.1–37.3uC; [88]). Table S3 Raw excretion data from all Dicamptodon tenebrosus individuals used in the excretion study. Excretion rates were measured at t = 0 min and t = 120 min. Diet treatment codes are as follows: AM: Aquatic macroinvertebrate, YOY: young-of-the-year steelhead, TM: Terrestrial macroinver- tebrate. (DOCX) Table S3 Raw excretion data from all Dicamptodon tenebrosus individuals used in the excretion study. Excretion rates were measured at t = 0 min and t = 120 min. Diet treatment codes are as follows: AM: Aquatic macroinvertebrate, YOY: young-of-the-year steelhead, TM: Terrestrial macroinver- tebrate. (DOCX) Acknowledgments We thank Peter Steel and Mary Power for their tireless help and support at the University of California’s Angelo Coast Range Reserve, Grace Wilkinson, Jill Welter, and Sandra Brovold for their assistance with analytical chemistry, Zachary Monteith and Will Atlas for help wrangling salamanders, Camille McNeely for assistance with invertebrate taxonomy, John Schade for help designing the experiment, and Jim Hood for his contribution of unpublished elemental invertebrate data. Nutrient Recycling by Multiple Stream Predators 3), suggesting the potential for partial alleviation of the N-limitation experienced by autotrophs in Fox Cr. Due to the higher ambient concentrations of inorganic nutrients in Fox Cr. compared to other similar tributary streams [61], it is likely that the contribution by predators to nutrient availability in other streams that support similar predator densities is greater than in Fox Creek. This suggests that our estimates of the percent of nutrient demand supplied by vertebrates in Fox Cr. may be lower than other streams in the region. This study provides an estimate of excretion rates for a dominant stream predator in the Pacific Northwest that has not been previously studied. The bulk of literature in the field has examined highly productive tropical streams or lentic ecosystems (e.g. [15,25,29,30,89]). We show here that nutrient recycling by top predators is an important component of ecosystem nutrient dynamics even in small temperate streams. Though amphibians have been previously considered, their importance as nutrient recyclers has been discounted due to low densities compared to other vertebrate taxa (but see [35,90]). Salamanders in many stream ecosystems have strong top-down effects on prey abundance [91–93], and act as sources of energy for higher level predators [36,94,95]. This study demonstrates that amphibians can also contribute to ecosystem-level dynamics through recycling of nutrients, in this case accounting for 37% of N and 46% of P recycled by vertebrates. Our findings suggest that studies of consumer-driven nutrient recycling will continue to benefit by identifying additional key recyclers in various systems. Continued work in this field will help expand our knowledge of the ecological roles of many organisms, and foster a better understanding of nutrient dynamics in aquatic systems. Discussion This distribution is typical of populations consisting of primarily anadromous individuals that migrate out of freshwaters after approximately 1.5 years (summarized by [78]), and is also indicative of the consistent growth pattern exhibited by most March 2013 \| Volume 8 \| Issue 3 \| e58542 PLOS ONE \| www.plosone.org 7 Table S1 Sources for invertebrate length-weight regres- sion used to determine the mass of individual insects for the diet survey. (DOCX) Table S2 AICc scores for models predicting N and P excretion rates. (DOCX) Supporting Information Table S1 Sources for invertebrate length-weight regres- sion used to determine the mass of individual insects for the diet survey. 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Proceedings of the National Academy of Sciences 104: 4461. doi:10.1073/pnas.0608148104. 50. Whitledge GW, Hayward RS (1997) Laboratory Evaluation of a Bioenergetics Model for Largemouth Bass at Two Temperatures and Feeding Levels. Transactions of the American Fisheries Society 126: 1030–1035. doi:10.1577/ 1548-8659(1997)126,1030:LEOABM.2.3.CO;2. 26. Knoll LB, McIntyre PB, Vanni MJ, Flecker AS (2009) Feedbacks of consumer nutrient recycling on producer biomass and stoichiometry: separating direct and indirect effects. Oikos 118: 1732–1742. doi:10.1111/j.1600-0706.2009.17367.x. 51. Whiles MR, Huryn AD, Taylor BW, Reeve JD (2009) Influence of handling stress and fasting on estimates of ammonium excretion by tadpoles and fish: recommendations for designing excretion experiments. Limnology and Ocean- ography: Methods 7: 1–7. doi:10.4319/lom.2009.7.1. 27. Ville´ger S, Grenouillet G, Suc V, Brosse S (2012) Intra- and interspecific differences in nutrient recycling by European freshwater fish. Freshwater Biology 57: 2330–2341. doi:10.1111/fwb.12009. 28. Taylor JM, Jason M (2011) Nutrient enrichment effects on stream periphyton stoichiometry, algal and fish assemblage structure, and grazing fish-periphyton 52. Nussbaum RA, Clothier GW (1973) Population structure, growth, and size of larval Dicamptodon ensatus (Eschscholtz). Northwest Science 47: 218–227. March 2013 \| Volume 8 \| Issue 3 \| e58542 PLOS ONE \| www.plosone.org 9 Nutrient Recycling by Multiple Stream Predators Nutrient Recycling by Multiple Stream Predators Munro AF (1953) The ammonia and urea excretion of different species of Amphibia during their development and metamorphosis. Biochem J 54: 29–36. 90. Beard KH, Vogt KA, Kulmatiski A (2002) Top-down effects of a terrestrial frog on forest nutrient dynamics. Oecologia 133: 583–593. doi:10.1007/s00442-002- 1071-9. 69. Underhay EE, Baldwin E (1955) Nitrogen excretion in the tadpoles of Xenopus laevis Daudin. Biochem J 61: 544–547. 91. Davic RD (1983) An investigation of salamander guild predation in a North Carolina stream: an experimental approach [PhD Thesis]. Kent State University. 70. Wright PM, Wright PA (1996) Nitrogen Metabolism and Excretion in Bullfrog (Rana catesbelana) Tadpoles and Adults Exposed to Elevated Environmental Ammonia Levels. Physiological Zoology 69: 1057–1078. y 92. Parker MS (1992) Feeding ecology of the larvae of the Pacific giant salamander (Dicamptodon tenebrosus) and their role as top predator in a headwater stream benthic community [PhD Thesis]. Davis: University of California. y g gy 71. Small GE, Pringle CM, Pyron M, Duff JH (2011) Role of the fish Astyanax aeneus (Characidae) as a keystone nutrient recycler in low-nutrient Neotropical streams. Ecology 92: 386–397. doi:10.1890/10-0081.1. y [ ] y 93. Broadman R, Ogger J, Kolaczyk M, Pulver RA, Long AJ, et al. 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https://openalex.org/W1915497867	https://library.oapen.org/bitstream/20.500.12657/33307/1/507291.pdf	English	null	Ontological Catastrophe: Žižek and the Paradoxical Metaphysics of German Idealism	null	2,014	cc-by	138,496	Ontological Catastrophe: Žižek and the Paradoxical Metaphysics of German Idealism Joseph Carew Ontological Catastrophe: Žižek and the Paradoxical Metaphysics of German Idealism Joseph Carew Ontological Catastrophe: Žižek and the Paradoxical Metaphysics of German Idealism Joseph Carew Ontological Catastrophe: Žižek and the Paradoxical Metaphysics of German Idealism Joseph Carew Ontological Catastrophe New Metaphysics Series Editors: Graham Harman and Bruno Latour The world is due for a resurgence of original speculative metaphysics. The New Metaphys- ics series aims to provide a safe house for such thinking amidst the demoralizing caution and prudence of professional academic philosophy. We do not aim to bridge the analytic- continental divide, since we are equally impatient with nail-filing analytic critique and the continental reverence for dusty textual monuments. We favor instead the spirit of the intel- lectual gambler, and wish to discover and promote authors who meet this description. Like an emergent recording company, what we seek are traces of a new metaphysical ‘sound’ from any nation of the world. The editors are open to translations of neglected metaphysical classics, and will consider secondary works of especial force and daring. But our main inter- est is to stimulate the birth of disturbing masterpieces of twenty-first century philosophy. Joseph Carew Ontological Catastrophe Joseph Carew Ontological Catastrophe www.openhumanitiespress.org OPEN HUMANITIES PRESS First edition published by Open Humanities Press 2014 Freely available online at http://dx.doi.org/10.3998/ohp.12763629.0001.001 Copyright © 2014 Joseph Carew This is an open access book, licensed under a Creative Commons By Attribution Share Alike license. Under this license, authors allow anyone to download, reuse, reprint, modify, distribute, and/or copy this book so long as the authors and source are cited and resulting derivative works are licensed under the same or similar license. No permission is required from the authors or the publisher. Statutory fair use and other rights are in no way affected by the above. Read more about the license at creativecommons.org/licenses/by-sa/3.0 Copyright © 2014 Joseph Carew Design by Katherine Gillieson Cover Illustration by Tammy Lu The cover illustration is copyright Tammy Lu 2014, used under a Creative Commons By Attribution license (CC-BY). ISBN-13 978-1-60785-308-4 Open Humanities Press is an international, scholar-led open access publishing collective whose mission is to make leading works of contemporary critical thought freely available worldwide. Books published under the Open Humanities Press imprint at MPublishing are produced through a unique partnership between OHP’s editorial board and the University of Michigan Library, which provides a library-based managing and production support infrastructure to facilitate scholars to publish leading research in book form. www.publishing.umich.edu www.openhumanitiespress.org OPEN HUMANITIES PRESS www.openhumanitiespress.org OPEN HUMANITIES PRESS Contents Acknowledgements 9 Acknowledgements 9 Introduction 11 To the Memory of Joey Basha Introduction 11 1. The Madness of the Symbolic 19 2. Grasping the Vanishing Mediator Between the Real and the Ideal 37 3. Psychoanalysis and the Enigma of Transcendental Subjectivity 53 4. The Problem of Nature in the Lacanian Subject 67 5. Kant, Todestrieb, and Beyond the Pleasure Principle 85 6. From Transcendental Philosophy to Substance as Subject 108 7. The Logic of Transcendental Materialism 140 8. When the World Opens its Eyes 171 9. The Abyss of Unconscious Decision 193 10. Radicalizing the Subject 223 11. From Radical Idealism to Critical Metaphysics 259 12. The Deadlocks of Ontological Catastrophe 283 1. The Madness of the Symbolic 19 2. Grasping the Vanishing Mediator Between the Real and the Ideal 37 4. The Problem of Nature in the Lacanian Subject 67 12. The Deadlocks of Ontological Catastrophe 283 Bibliography 317 To the Memory of Joey Basha To the Memory of Joey Basha Acknowledgements Firstly, I would like to express my gratitude to two people without whom this project would not have been possible: Dr. Antoinette Stafford and Dr. Sean McGrath. Dr. Stafford is, in many ways, the reason why I am doing philosophy, and it is to her that I owe my knowledge of the history of modern philosophy and German Idealism, without which this book would never have gotten off the ground. Dr. McGrath sparked my interest in psychoanalysis and has been a constant source of inspiration for my reading of Schelling. I must thank him for stressing the notion of the ambiguity of the Real in Lacan and Žižek, which immensely shaped my own engagement with these authors, and for all the encouragement he has given me since. Secondly, I should thank four people who read the manuscript at various stages of its production. Dr. Peter Trnka and the late Dr. James Bradley read a very early version of the manuscript several years ago, and I am much indebted to their extremely kind and useful commentary. While Dr. Bradley gave me insight into further aspects of Žižek's philosophy—some of which has shown up in my other work on Žižek—and encouraged me to continue the project, Dr. Trnka's much appreciated criticism helped me avoid conceptual imprecision at several key points, as well as general difficulties in the presentation of psychoanalytical methodology. Specifically, Dr. Trnka's emphasis on the conflictual relation between materialism and idealism in many ways moulded my own views on the difficult Symbolic-Real relation in Acknowledgements 10 Žižek's transcendental materialism into the form they take here. Dr. Graham Harman's comments and suggestions were of great help in stylistically improving the manuscript's quality, in increasing its overall coherence, and also in avoiding several pitfalls of argumentation. I should also thank him for his always quick responses to various questions I had while preparing the manuscript. Last but certainly not least, I thank Kyla Bruff for her laborious proofreading of the final two drafts. Her careful eye was able to eliminate innumerable ambiguities and awkward turns of phrase, thus making the book much smoother. Thirdly, I would like to express my gratitude to the Social Sciences Research Council of Canada and Europhilosophie for their generous funding, which both directly and indirectly supported the project. Acknowledgements I would also like to thank the Memorial University of Newfoundland's Department of Philosophy and School of Graduate Studies for additional financing in the form of fellowships and awards, which enabled me to begin the first draft. An early version of three strands of argumentation that appear in what follows has been published as “The Grundlogik of German Idealism: The Ambiguity of the Hegel-Schelling Relation in Slavoj Žižek,” International Journal of Žižek Studies, 5, no.1 (2011); “'Why is There Nothing Rather Than Something?' Less Than Nothing's New Metaphysics,” International Journal of Žižek Studies, 8. no.1 (2014); and “Denaturalizing Nature, Dehumanizing Humanity: Lacan, Žižek, and the Metaphysics of Psychoanalysis,” in Natürlichkeit und Künstlichkeit zwischen Tatsache und Ideal, ed. Benedetta Bisol (Bielefeld: transcript, 2014). Introduction A Metaphysical Archaeology of the Psychoanalytico-Cartesian Subject This book is an investigation into Slavoj Žižek's return to German Idealism in the wake of Lacanian psychoanalysis. Its thematic crux is Žižek's attempt to develop, by reading the traditions against one another by means of their mutually compatible notions of Todestrieb, a highly original theory of subjectivity able to explain the subject's simultaneous freedom from and dependence upon its material ground. But it does not stop there: rather than just limiting itself to a recapitulation of Žižek's account of the eruptive, ontologically devastating birth of subjectivity out of nature, it also seeks to systematize the stark metaphysical consequences of this account. The fundamental thesis of this book is that, if the emergence of the Symbolic out of the Real—the passage from nature to culture enacted by the founding gesture of subjectivity—is the advent of a completely self-enclosed, self- sustaining structural system, then not only must its founding gesture withdraw from the scene in the very act of instituting the Symbolic, but further, even to explain this act we must posit the absolute as a fragile not-all wrought by negativity and antagonism. Or, to put it in terms of Žižek's Less Than Nothing (his latest magnum opus, or “big fat Hegel book,” as he says), as a series of less than nothings whose essence constitutes an ontologically incomplete field. By means of a metaphysical archaeology of the psychoanalytico- Cartesian subject, an archaeology that is the necessary supplement to Introduction 12 Freud's own archaeological investigations of the emergence of mind out of the conflict of unconscious drives and their vicissitudes,1 especially in the aftermath of Lacan's structuralist reworkings of it, what we will see is that Žižek's own ontology of the subject goes far beyond the normal constraints of psychoanalytical methodology (which is so concerned with psychogenesis and its various pathologies) and radically challenges our normal conception of self and world, a challenge summarized by the notion of ontological catastrophe, which I extract from it as its key operative moment. In the course of the book this concept takes on a number of different meanings. In a first moment, it refers to Žižek's interpretation of Todestrieb as that which incites the passage from nature to culture, a grotesque excess of life that is unable to control itself according to its own prescribed natural logistics and thus opens up room for the possibility of experience. Introduction Wondering how this void of nothingness could be broken so that creation itself could emerge, Žižek argues that the basic form of ontological catastrophe should be extended A Metaphysical Archaeology of the Psychoanalytico-Cartesian Subject 1 13 from that of the subject as the breakdown of nature in Todestrieb, or the incompletion of nature testified to by the latter's constitutive tension, to the world itself as the necessary disturbance of this void, whereby the classical terrain of metaphysics itself is inverted: “[f]or a true dialectician, the ultimate mystery is not 'Why is there something rather than nothing?' but 'Why is there nothing rather than something?': how is it that, the more we analyze reality, the more we find a void?”2 In broad strokes, this is the terrain we will investigate—a terrain that is not merely difficult because it is nuanced and challenging because it is new, but also primordially uncanny and traumatic, forcing us to encounter aspects of self and world that we not only normally do not acknowledge, or continually disavow, but that we even try to repress. To arrive at and evaluate this notion of ontological catastrophe, my metaphysical archaeology of the Žižekian psychoanalytico-Cartesian subject takes three paths: one that traverses the wider historical context informing Žižek's project, another that internally reconstructs its reactualization of German Idealism through psychoanalysis, and a final one that attempts to extract and problematize the intrinsic originality and daringness of Žižek's metaphysics. The first path consists of chapters 1 through 4. Chapter 1 outlines the ambiguity of the Real in Lacanian psychoanalysis. Displaying a form of radical idealism of a linguistic structuralist variety, it proclaims that the Symbolic operates as a self-enclosed system with no need of any external support. This not only means that human freedom is equivalent to an ontological madness, but also appears to foreclose the very possibility of explaining this passage into madness at its basis. Chapter 2 shows how, although Žižek believes himself able to find resources to overcome this difficulty in German Idealism, he can only do so by psychoanalytically tracing and reconstructing an unconscious history of struggle with the obscure origins of subjectivity he perceives throughout the tradition. Introduction In a second moment, it names the self-positing of subjectivity in nature tearing the latter apart into irreconcilable zones, which, although in a certain sense conditioned by a libidinal-material breakdown of the biological system, is ultimately irreducible to the latter as a pure act. Taken together, these two moments underlying the emergence of subjectivity demand that we delve into the naturephilosophy that this account implies, a nature that shows itself (due to the very extimate presence of Todestrieb and pure difference within its heart of hearts) to be predicated upon painful tension and self-sabotaging tendencies to such a degree that its very being is co- incidental not only with the existence of death, disease, and monstrosities, but also with the always possible unpredictable upsurge of disorder and complete collapse as it risks touching the void. In a third moment, the metaphysical archaeology of the subject is pushed to its ultimate limits. Delving into the question of how being could sustain itself despite its rampant and devastating negativity, what we will see is how the more we move towards the most fundamental level of the universe, the latter proves to be in its depths of depths not a dense, fully subsisting reality that exists by itself by means of a self-explanatory surplus, but a series of indeterminate proto-ontological states only minimally distinguishable from the void of nothingness that serves as its contrast. Introduction Given the psychoanalytical horror that is the basis of subjectivity, chapter 9 explains how Schelling, although the thinker of its abyssal origins, ultimately ends up recoiling just like Hegel from own great insights after coming face to face with its full trauma, which gives further support to the necessity of a psychoanalytical reconstruction of German Idealism. After this concrete exploration of Žižek's methodology, chapter 10 concludes the second path by bringing to the fore his three most significant theoretical contributions: a rich ontology of nature, a new metaphysics of the void developed through quantum mechanics, and a nuanced theory of The second path, which unfolds through chapters 5 to 10, comprises the substantiation of Žižek's claim that there is an identity between the founding insights of German Idealism and psychoanalysis by retroactively rewriting the former's unconscious history. Drawing upon the late Lacan's ruminations concerning the breakdown of nature as the pre-condition of the Symbolic, chapter 5 outlines how Kant finds also himself forced to posit a meta-transcendental ground of the transcendental in organic disorder, even going so far as to anticipate Lacan's mirror stage. Chapter 6 demonstrates how the early Hegel, led by insufficiencies in Kant's and Fichte's transcendentalism and Schelling's Naturphilosophie, attempts to reconcile idealist freedom and realist system by inscribing the subject into the fold of being as an eruptive, world-shattering event, thus radicalizing Kant's insight into the devastating origins of subjectivity. Chapter 7 then illustrates how the mature Hegel psychoanalytically recoils from the ontological catastrophe at the heart of the subject's essence by subsuming it under the self-mediation of the Notion. It is only with the middle-late Schelling, fighting against the perceived threats of Absolute Idealism, that the true kernel of truth unearthed by Kantian idealism is brought to the fore and along with it its stark, even horrifying implications for our understanding of nature, human historicity, and the absolute. Chapter 8 gives flesh to the Schellingian-Žižekian subject as the vanishing mediator between the Real and the Ideal. The symbolic universe of meaning is not a high point of evolutionary achievement, but rather a mistake, the outcome of something having gone horribly wrong in the order of things and to which it is only a defense mechanism. Introduction Insofar as the psychotic non-relation between the Real and the Symbolic is also a rethinking of the cogito, chapter 3 shows why Žižek feels the theoretical obligation to revitalize subjectivity in an intellectual milieu that attacks it from all sides. Chapter 4 tries to understand how the Real could have given rise to the Symbolic. Contra the early and middle Lacan, Žižek argues that the Symbolic cannot be an external parasite that attacks the Real from Introduction 14 nowhere, but must arise from some sort of self-sabotaging tendency always already implicit within it. already implicit within it. The second path, which unfolds through chapters 5 to 10, comprises the substantiation of Žižek's claim that there is an identity between the founding insights of German Idealism and psychoanalysis by retroactively rewriting the former's unconscious history. Drawing upon the late Lacan's ruminations concerning the breakdown of nature as the pre-condition of the Symbolic, chapter 5 outlines how Kant finds also himself forced to posit a meta-transcendental ground of the transcendental in organic disorder, even going so far as to anticipate Lacan's mirror stage. Chapter 6 demonstrates how the early Hegel, led by insufficiencies in Kant's and Fichte's transcendentalism and Schelling's Naturphilosophie, attempts to reconcile idealist freedom and realist system by inscribing the subject into the fold of being as an eruptive, world-shattering event, thus radicalizing Kant's insight into the devastating origins of subjectivity. Chapter 7 then illustrates how the mature Hegel psychoanalytically recoils from the ontological catastrophe at the heart of the subject's essence by subsuming it under the self-mediation of the Notion. It is only with the middle-late Schelling, fighting against the perceived threats of Absolute Idealism, that the true kernel of truth unearthed by Kantian idealism is brought to the fore and along with it its stark, even horrifying implications for our understanding of nature, human historicity, and the absolute. Chapter 8 gives flesh to the Schellingian-Žižekian subject as the vanishing mediator between the Real and the Ideal. The symbolic universe of meaning is not a high point of evolutionary achievement, but rather a mistake, the outcome of something having gone horribly wrong in the order of things and to which it is only a defense mechanism. 1. This, of course, being a constant metaphor throughout Freud’s corpus, span- ning from “The Aetiology of Hysteria” (1896) to “Construction in Analysis” (1937). See, respectively,The Standard Edition of the Complete Psychological Works of Sigmund Freud, translated from the German under the General Editorship of James Strachey, in collaboration with Anna Freud, assisted by Alix Strachey and Alan Tyson (London: Hogarth Press and the Institute of Psycho-analysis, 1953–1974) (hereafter SE), III, p. 192, and XXIII, p. 259. A Metaphysical Archaeology of the Psychoanalytico-Cartesian Subject 15 A Metaphysical Archaeology of the Psychoanalytico-Cartesian Subject 15 unconscious, each of which goes beyond a mere reactualization of German Idealism or psychoanalysis. The third and final path is summarized by the word “paradoxical” in the subtitle of the book—Žižek and the Paradoxical Metaphysics of German Idealism. Chapter 11 highlights that, instead of being opposed to metaphysics, radical idealism not only demands a metaphysics, for thinking in all of its intrinsic paradox and self-referentiality must be seen as existing in the world, but more primordially forces upon us a new domain of metaphysics, which first became explicit in German Idealism. Whereas all metaphysics prior to Kant is dogmatic insofar as it assumes thought's power to reach out and touch the truth of being in virtue of a special capacity (a gesture that is repeated by, amongst others, Badiou's and Meillassoux's elevation of mathematical formalization), what occurs in Schelling and Hegel is an intense reflection upon how the very process by which thought forms an image of being is inscribed into being as an event, whereby even the very philosophical position of theory formation is reflexively thematized both epistemologically and ontologically. What emerges is a metaphysics that can be baptized as critical because it is capable of developing a theory of reality that is maximally realist and idealist and therefore best suited to explicate the metaphysical whole of what is without falling into the downfalls of a theory that is one-sidedly one or the other.3 Chapter 12 explores the paradoxical nature of this endeavour as it articulates itself in the intrinsically original and daring character of Žižek's own variation upon this German Idealist leitmotif and the problems it potentially poses not only for his own philosophy, but perhaps for any radical idealism seeking to break the correlationist circle. Introduction Given the psychoanalytical horror that is the basis of subjectivity, chapter 9 explains how Schelling, although the thinker of its abyssal origins, ultimately ends up recoiling just like Hegel from own great insights after coming face to face with its full trauma, which gives further support to the necessity of a psychoanalytical reconstruction of German Idealism. After this concrete exploration of Žižek's methodology, chapter 10 concludes the second path by bringing to the fore his three most significant theoretical contributions: a rich ontology of nature, a new metaphysics of the void developed through quantum mechanics, and a nuanced theory of 3. Gabriel draws a distinction between critical and dogmatic metaphysics for similar, but different reasons. See Das Absolute und die Welt in Schellings Freiheitsschrift (Bonn: University Press, 2006), p. 8. I take up this distinction at length in chapter 11. 2. Žižek, Less Than Nothing: Hegel and the Shadow of Dialectical Materialism (London: Verso, 2012), p. 925. Chapter 1 The Madness of the Symbolic Transcendental Materialism and the Ambiguity of the Real Chapter 1 The Madness of the Symbolic Transcendental Materialism and the Ambiguity of the Real Re-interpreting Freud through structural linguistics, Lacan radically rethinks the unconscious: no longer a quasi-biological phenomenon centered in drives, it largely becomes associated with the differential system of the Symbolic responsible for the production of meaning. However, since the latter proves to be operationally closed and has no relationship to the world in itself, Lacan himself is forced to proclaim that the founding gesture of subjectivity is a passage through madness. This poses two difficulties that set up the entirety of Žižek's project. First, it points towards a transcendental materialism at the basis of the subject, a self-splitting of being into irreconcilable material and transcendental zones, but one that Lacan fails to systematize. Second, insofar as the Symbolic itself is self-enclosed, it seems methodologically impossible even to explain its own obscure origins, even if such is ultimately required if psychoanalysis is to find an adequate theoretical justification. In this regard, Žižek's primary task is to find a way to explain the madness of the Symbolic without overstepping the constraints of psychoanalysis. Notes 16 Introduction I Death Drive Death Drive The Madness of the Symbolic Žižek's metaphysics originates in his attempt to delve into the material origins preceding the psychoanalytical subject by focusing on this moment If madness is “freedom's most faithful companion” it is precisely because madness in its most primordial sense refers to the specific ontogenetic conditions for the irreducibility of language that makes us distinctly human: that is to say, to the state of affairs by means of which language can solipsistically relate to itself as a self-enclosed differential system of signification “with no an external support.”7 Just as in clinical cases of psychosis or madness, here too the subject has “lost touch” with reality, although reality must be understood in its natural (animalistic) rather than its sociopolitical (human) meaning. It is “the price the Lacanian subject pays for its 'transubstantiation' from being the agent of a direct animal vitality to being a speaking subject whose identity is kept apart from the direct vitality of passions,”8 that which guarantees that the subject is dominated by “non-natural” influences or which is, strictly speaking, at its zero- level abiological (wherein lies its freedom). This has two principal effects. First, because Lacan's self-given task is to formulate the autonomous structures that constitute human subjectivity in opposition to naturalist theories of psychiatry, his philosophy has the formal appearance of a retour to the modern transcendentalism of the cogito. The Lacanian subject is consequently haunted by similar problems as those of the Cartesian subject, both in terms of epistemology (since the relationship to the extra-conscious alterity of the world is problematized, how can we justify the propositions of science?) and the mind-body relation (what exactly is the relationship between symbolic thinking and natural processes?). Second, due to the internal constraints of his project, Lacan left unanswered how reality in itself could incite the generation of these quasi-transcendental structures that make up the universe of human meaning in its psychotic self-enclosure, with the concomitant problem of how we relate to this X that simultaneously precedes our emergence into language and forms its obscure ontologico- foundational basis. Seeing a structural identity with the immediate reactions to Kant, Žižek sees the possibility of confronting the fundamental concepts of psychoanalysis with those of German Idealism. The Madness of the Symbolic If madness is “freedom's most faithful companion” it is precisely because madness in its most primordial sense refers to the specific ontogenetic conditions for the irreducibility of language that makes us distinctly human: that is to say, to the state of affairs by means of which language can solipsistically relate to itself as a self-enclosed differential system of signification “with no an external support.”7 Just as in clinical cases of psychosis or madness, here too the subject has “lost touch” with reality, although reality must be understood in its natural (animalistic) rather than its sociopolitical (human) meaning. It is “the price the Lacanian subject pays for its 'transubstantiation' from being the agent of a direct animal vitality to being a speaking subject whose identity is kept apart from the direct vitality of passions,”8 that which guarantees that the subject is dominated by “non-natural” influences or which is, strictly speaking, at its zero- level abiological (wherein lies its freedom). This has two principal effects. First, because Lacan's self-given task is to formulate the autonomous structures that constitute human subjectivity in opposition to naturalist theories of psychiatry, his philosophy has the formal appearance of a retour to the modern transcendentalism of the cogito. The Lacanian subject is consequently haunted by similar problems as those of the Cartesian subject, both in terms of epistemology (since the relationship to the extra-conscious alterity of the world is problematized, how can we justify the propositions of science?) and the mind-body relation (what exactly is the relationship between symbolic thinking and natural processes?). Second, due to the internal constraints of his project, Lacan left unanswered how reality in itself could incite the generation of these quasi-transcendental structures that make up the universe of human meaning in its psychotic self-enclosure, with the concomitant problem of how we relate to this X that simultaneously precedes our emergence into language and forms its obscure ontologico- foundational basis. Seeing a structural identity with the immediate reactions to Kant, Žižek sees the possibility of confronting the fundamental concepts of psychoanalysis with those of German Idealism. 1.1 A (Transcendental) Materialism of the Psychoanalytical Subject Žižek's return to German Idealism is an investigation of the obscure origins of the Lacanian subject. Žižek is attracted not only to Lacanian Chapter 1 20 psychoanalysis' thematization of the non-coincident “gap” in the Symbolic and its consequences for politics, but also to the conflictual relationship between mind and body that it places at the foundation of psychogenesis. If symbolic structures of language display a radical autonomy from bodily forces and can construct their own world, the essence of human being must be constituted originally by a kind of biological “short circuit” that disrupts man's complete immersion in nature, eternally separating the Innenwelt and Aussenwelt (inner world and outer world), thereby making it so they can never coincide: that is, by a mal-adaptation that “represents the minimum of freedom, of a behaviour uncoupled from the utilitarian-survivalist attitude” insofar as “the organism is no longer fully determined by its environs, that it 'explodes/implodes' into a cycle of autonomous behaviour.”4 If, as conventionally defined in Freudian psychoanalysis, psychosis or madness is taken to be a withdrawal from the objective world into an inner, self- enclosed space (a loss of reality),5 then in Lacanian psychoanalysis psychosis or madness is paradoxically not a mere accidental state seen in certain “sick” individuals, but is the irreducible ontological background of all human existence. More disconcertingly, this is understood by Lacan not only to be the condition of possibility of human experience as such, but also that of freedom, so that the philosophical significance of the two is ultimately identified as dialectically interrelated aspects of the same phenomenon: Thus rather than resulting from a contingent fact—the frailties of his organism—madness is the permanent virtuality of a gap in his essence. And far from being an “insult” to freedom, madness is freedom's most faithful companion, following its every move like a shadow. Not only can man's being not be understood without madness, but it would not be man's being if it did not bear madness within itself as the limit of his freedom.6 For Lacan, the primary question in psychoanalysis is not how various forms of madness arise as a deviation from normal mental health, but how this more originary, irruptive state of nature as that within which freedom and madness magically emerge in a single brushstroke can be regulated so that what we regard as sanity and normality can themselves take hold. 1.1 A (Transcendental) Materialism of the Psychoanalytical Subject The Madness of the Symbolic 2 The Madness of the Symbolic Žižek's metaphysics originates in his attempt to delve into the material origins preceding the psychoanalytical subject by focusing on this moment Žižek's metaphysics originates in his attempt to delve into the material origins preceding the psychoanalytical subject by focusing on this moment of immanent rupture in being—which he links to the Todestrieb—as that which, by opening up a space separating us from nature in the latter, Chapter 1 Chapter 1 22 appears simultaneously to be linked to our freedom, that is, to our madness. Refusing to buy into the claim that all is ultimately reducible to the ebb and flow of matter, he sees his own endeavour as “the necessary step in the rehabilitation of the philosophy of dialectical materialism.”9 Yet this designation is inherently problematic, given what we have just seen: not only does it try to make Žižek's own form of materialism approach that of Marx and Engels without drawing the necessary distinctions between them, it more importantly fails to articulate the essentially paradoxical and innovative manner in which Žižek rearticulates the materialism-idealism debate and therefore risks obscuring his own originality. Consequently, I endorse Adrian Johnston's characterization of Žižek's theory of the subject as a form of transcendental materialism10 for four reasons, but differ in my own understanding in one important way that in turn distinguishes my own project from his. First, it has the benefit of allowing the reader to have a direct intuition of what is truly at stake in Žižek's parallax ontology and its metaphysical implications. Whereas dialectical materialism traditionally views the mind- body relationship as grounded within the dynamic interpenetration of the two as a complex self-unfolding identity within difference, transcendental materialism, by focusing on the ontogenetic conditions of the possibility of the transcendental subject, conventionally understood as in opposition to natural conditions, already suggests the immanent emergence of an irruptive negativity within being, an irreducible transcendence that paradoxically shatters the former's pure immanence from within. Second, a point not mentioned by Johnston, Fichte uses the expression in his 1794 Some Lectures Concerning the Scholar's Vocation to draw attention to the impossibility of explaining the (onto)genesis of the subject: “[i]t is certainly not true that the pure I is a product of the not-I [...]. The Madness of the Symbolic 23 autonomy. Writing in a similar vein, Žižek attempts to show not only how we can, but more primordially why we must develop a metaphysical account of reality that, instead of jeopardizing the (practical) primacy of idealism, would actually found it by inscribing this very dualism of I and not-I into the fold of being as that which makes the absolute divided within itself and whose non-coincidence to self thereby opens up the birthplace for human freedom. Transcendental idealism is—or better, must be said to always already spectrally refer to—transcendental materialism, the difference between them being only that of a parallax shift: the two are negatively linked to one another by an impossible in-between, a disjunctive “and,” the very name of which is for Žižek the subject, so that an idealism must convert itself into a materialism and vice versa if subjectivity is to be fully explained. It is precisely at this conceptual conjuncture of the role of a disjunctive “and” that my own understanding of Žižek's transcendental materialism distinguishes itself from that of Johnston. Although Johnston is right to claim that Žižek's own descriptions of the birth of subjectivity have a propensity to focus on its process as a self-instituting fiat “analogous to the cutting of the Gordian knot” (which in a certain sense obfuscates his project insofar as one of its major questions is how the closed circuit of drives in the Real could result in the transcendence of subjectivity),12 Johnston in my view has a tendency to downplay the intrinsically paradoxical nature of all such inquiry into the obscure origins of the psychoanalytico-Cartesian subject in Žižek's work in two ways. On the one hand, he emphasizes that the subject is rendered possible by a short-circuiting of its libidinal-material ground. But if an emergent breakdown in nature's inner being does give rise to the ontogenetic possibility of the subject, it in no way gives birth to the latter: nature's auto-laceration may be necessary for the self-positing freedom of transcendental subjectivity, but it is not sufficient, for there is no possible transition from nature to subjectivity, a point that—though also raised by Johnston—I believe must be radicalized. The Madness of the Symbolic The assertion that the pure I is a product of the not-I expresses a transcendental materialism which is completely contrary to reason.”11 Fichte's argument is simple: one cannot explain the material conditions of transcendental freedom insofar as that would equate two logically distinct fields irreconcilable with one another—namely, that of unbridled self- determination (the realm of acting) and that of dead contingency (the realm of being), thus causing us to lose sight of the radicality of human The Madness of the Symbolic The Madness of the Symbolic This is why on my reading the reference to Fichte is so important, for if Žižek's metaphysics is an attempt to show how a transcendental materialism can be developed, it nevertheless refuses to give up on the fundamental claim made by Fichte that the upsurge of the pure I in being is executed “by absolute freedom, not through a transition, but by means of a leap.”13 As a result, we can also understand Chapter 1 24 why Žižek displays hesitation concerning Johnston's and Malabou's shared project of merging philosophy and neuroscience—because there is ultimately no emergence of subjectivity possible within his parallax ontology.14 On the other hand, because Johnston attaches less importance than I do to the rupturing free leap into subjectivity, he is simultaneously silent concerning the necessarily mytho-poetic element of Žižek's transcendental materialism, which for me thus becomes central for understanding the nuance of his specific overcoming of radical idealism. This has two important consequences, one methodological and the other metaphysical. Methodologically, if the leap into subjectivity is an ontological passage through madness,15 then there is an upper limit to the power of thought to explain its own emergence in being. What we need is a capacity for fabulative mythologizing, for “the need for the form of mythical narrative arises when one endeavours to break the circle of the symbolic order and to give an account of its genesis ('origins') from the Real and its pre-symbolic antagonism.”16 Metaphysically, if subjectivity is the psychotic night of the world, then any investigation into its underlying ontology simultaneously requires a metaphysical archaeology of madness, that is, a theorizing of what the ultimate structure of reality must be like so that the subject's emergence could occur. In this manner, if Johnston is perhaps more interested in the paradoxical basis of transcendental subjectivity in nature, I am more interested in what occurs methodologically and metaphysically once we inscribe radical idealism as a form of madness into being, in such a way that two similar yet different views of Žižek's system emerge. The Madness of the Symbolic The Madness of the Symbolic 25 materialism, his Philosophies of Nature after Schelling17 offers an alternative account of the materialism-idealism relationship and the immanent emergence of the transcendental subject within nature that challenges not only Žižek's reading of Schelling, but more strongly his entire metaphysics. Zimmerman, a prolific German philosopher little known in the English-speaking world, was in fact the first person to use the concept of “transcendental materialism” in a contemporary context in his 1998 book Die Rekonstruktion von Raum, Zeit und Materie (The Reconstruction of Space, Time and Matter)18 and then fully develop it in his massive 2004 System des transzendentalen Materialismus (System of Transcendental Materialism).19 Zimmerman, like Grant, departs largely from Schelling, but also from Spinoza and Bloch, and offers an understanding of transcendental materialism in dialogue with contemporary science, especially physics, in stark opposition to the one presented here. For both, the thinking subject does not implicate an ontological trembling or pure difference within the field of being, and their respective accounts of the mind-body relationship can in no means be equated with what Žižek would perhaps be tempted to call a return to pre-modern cosmology. Moreover, considering how Žižek's own philosophy, similar in spirit to that of Zimmerman, does not possess mere implications or consequences for how we conduct empirical science, but directly engages with a broad range of disciplines such as quantum mechanics20 and cognitive science,21 by calling Žižek's philosophy a transcendental materialism I further hope to accomplish two things: to emphasize the systematic reach of Žižek's thinking and the exigence that metaphysics must also be an interlocutor with science. materialism, his Philosophies of Nature after Schelling17 offers an alternative account of the materialism-idealism relationship and the immanent emergence of the transcendental subject within nature that challenges not only Žižek's reading of Schelling, but more strongly his entire metaphysics. The Madness of the Symbolic Third, to return to the benefits of the characterization “transcendental materialism,” I endorse it because it draws our attention to Žižek's philosophical relevance outside of the fields of cultural studies, radical politics, and film theory by placing his thinking in direct contact with a series of other contemporary thinkers rethinking metaphysics, whether it be by representatives of the analytic tradition, object-oriented philosophy, new French materialism, or various other forms of the new speculative turn. Two thinkers deserve to be mentioned here by name, since they offer a transcendental materialism radically different from that of Žižek: Iain Hamilton Grant and Rainer E. Zimmerman. Although Grant would have reservations about his thinking being referred to as a “transcendental” The Madness of the Symbolic Zimmerman, a prolific German philosopher little known in the English-speaking world, was in fact the first person to use the concept of “transcendental materialism” in a contemporary context in his 1998 book Die Rekonstruktion von Raum, Zeit und Materie (The Reconstruction of Space, Time and Matter)18 and then fully develop it in his massive 2004 System des transzendentalen Materialismus (System of Transcendental Materialism).19 Zimmerman, like Grant, departs largely from Schelling, but also from Spinoza and Bloch, and offers an understanding of transcendental materialism in dialogue with contemporary science, especially physics, in stark opposition to the one presented here. For both, the thinking subject does not implicate an ontological trembling or pure difference within the field of being, and their respective accounts of the mind-body relationship can in no means be equated with what Žižek would perhaps be tempted to call a return to pre-modern cosmology. Moreover, considering how Žižek's own philosophy, similar in spirit to that of Zimmerman, does not possess mere implications or consequences for how we conduct empirical science, but directly engages with a broad range of disciplines such as quantum mechanics20 and cognitive science,21 by calling Žižek's philosophy a transcendental materialism I further hope to accomplish two things: to emphasize the systematic reach of Žižek's thinking and the exigence that metaphysics must also be an interlocutor with science. Ž Finally, it is worth mentioning that in Less Than Nothing Žižek also endorses Adrian Johnston's coinage of “transcendental materialism” in two passages in the last chapter on quantum mechanics, but with an important qualification that renders explicit for the first time a crucial metaphysical element of his own brand of materialism and its nuanced character. In the first instance, he claims that the key insight gained by contemporary physics is that material reality in itself does not present us with a dense field of fully constituted realities that form the ultimate building blocks of the universe, but rather with irreducibly indeterminate states lacking any substantial being and from which “hard” reality can only emerge if there Chapter 1 26 is a collapse of the wave function.22 In this sense, the micro-universe of quantum particles is strangely “less” than that of the macro-universe that constructs itself from its vicissitudes, in a way that is remarkably similar to how the Kantian subject can only construct a unified, coherent world of appearances from the inconsistent fragments of sensation. The Madness of the Symbolic In a strange logical short circuit, it would appear that not only is there no bottom-up causality at the level of experience (transcendental constitution is more real than what Kant calls “a rhapsody of perception”23), but even the most fundamental level of the universe is metaphysically more chaotic than the ordered macro-level physical world that science classically described. It is as if all reality is transcendentally constitutive, so that the only way to break free of the correlationist circle is to push “this transcendental correlation into the Thing itself:” “[i]t is against this background that one can make out the contours of what can perhaps only be designated by the oxymoron 'transcendental materialism' (proposed by Adrian Johnston).”24 The second instance adds some clarification to the metaphysical implications of this idea by stating that the “the only true consistent 'transcendental materialism' which is possible after the Kantian transcendental idealism” is one that risks the following difficult wager: “[w]hat if we posit that 'Things-in-themselves' emerge against the background of the Void of Nothingness, the way this Void is conceived in quantum physics, as not just a negative void, but the portent of all possible reality?”25 Although Žižek here takes transcendental materialism in a different direction than Johnston by introducing it to make a metaphysical point concerning the absolute rather than a more broadly ontological one concerning the subject, part of my project in what follows will be to show that his metaphysics of the void is not only completely compatible with his older ontology of the subject, but can be seen as its organic elaboration. If Žižek's approach differs significantly from that of Grant or Zi i i b hi i diff h h hi l d hi If Žižek's approach differs significantly from that of Grant or Zimmerman, it is because his game is different, even though this leads him to cover much of the same thematic domains—and hence it is interesting to label all three as “transcendental materialists” for the same reasons as it is to refer to Kant, Fichte, Hegel, and Schelling as “German Idealists” insofar as subsuming such irreconcilably different thinkers under a single category reveals a dynamic, pulsating movement, a battleground of theoretical The Madness of the Symbolic 27 positions around a shared set of problems rather than a shared doctrine, and thus something living. 1.2 The Lacanian Subject and the Irreducible Ambiguity of the Real As the fundamental presupposition of Žižek's philosophy, the Lacanian subject is to be radically distinguished from the philosophical subject of modernity. Although the former exhibits many traits that link it to early transcendental philosophy (it grounds the symbolic structures that constitute phenomenal reality through a free idealization) it is in direct opposition to the self-conscious transparency of the Cartesian cogito, the self-legislation of the Kantian rational agent, or the Hegelian account of free personality. For Lacan, the freedom of the I as witnessed in phenomenological self-experience is an illusion: completely determined by cultural and linguistic influences, the ego is an object and is constantly trying to construct a fantasmatic narcissistic space within which it can (falsely) perceive itself as a centre of self-effectuating action. Although this does not prevent the existence of human freedom for either Lacan or Žižek, it means that freedom itself gets largely displaced from consciousness into the unconscious, in a move formally similar to the middle-late Schelling of the Freiheitsschrift and Weltalter, but with an important twist. The subject is not an energetic, productive will that precedes the constitution of phenomenal reality, but is in one of its most important modalities nothing but an impersonal abyss that, uncannily, renders possible a minimal consistency of self. In this sense, the self is infinitely split at its core: when one looks inside oneself one not only finds an “extimacy,” material coming from elsewhere, where one should find one's innermost core, but if one looks long enough one only finds a void staring back. The Lacanian-Žižekian subject has no intrinsic content because it is pure form: the entire “plenitude” of cultural and psychological experience only emerges as a kind of defence formation against this primordial nothingness of the subject as an attempt to fill in its constitutive “gap” with false positive substantiality. But because this “gap” can never be filled in, it is ultimately repressed due to its traumatic, personality-shattering quality, so that we find traces of its disavowed knowledge in the slips and slides of speech, symbolic inconsistencies and non-coincidences in writing, the images of fantasies and dreams, and other phenomena. This creates two levels to any given (personal, ethical, political, or even philosophical) discourse: its surface movement, grounded in the As the fundamental presupposition of Žižek's philosophy, the Lacanian subject is to be radically distinguished from the philosophical subject of modernity. The Madness of the Symbolic What so strongly distinguishes Žižek's form of transcendental materialism from others is that, finding a fundamental structural identity between Lacanian psychoanalysis and post-Kantian idealism, he tries to illustrate the uncanny identity that exists between the psychoanalytical subject haunted by the Todestrieb as its constitutive basis and the unconscious, disavowed Grundlogik of German Idealism, with the conviction that a psychoanalytical dialogue between the two could open up a radically new possibility for metaphysics. By falling upon premonitions of the psychoanalytical experience in concepts such as Kantian unruliness, the Hegelian “night of the world,” and the Schellingian notion of the Grund, Žižek psychoanalytically reinterprets the late German Idealist attempts to give a metaphysical vision of reality compatible with the ontological emergence of the pure I in order to make us not only rethink what is at stake in the tradition of modern philosophy, a truth repressed and haunting its very history, but more primordially what is revealed with the advent of subjectivity as such: the notion of ontological catastrophe, the auto-disruption of reality into a painful not-all, at the origin of experience, with all the metaphysical implications that entails for our understanding of world in itself. Although Žižek's interpretations are heterodox, he believes that he is justified in singling out and radicalizing these premonitions, which often only appear in textual margins and often officially lack the theoretical primacy that Žižek bestows upon them, by means of the methodological application of various psychoanalytical techniques to the texts of German Idealism, these enabling him to plunge into and reveal the non-coincidences internally plaguing their symbolic space and thus retroactively restructuring their surface appearance in a manner similar to the analyst-analysand relation in therapy. We must traverse the fantasy of tradition if we are to arrive at its truth—this is Žižek's provocative claim and one that we will explore throughout most of this book. Thus, if we are to gain a preliminary sense of the driving forces of Žižek's reactualization of German Idealism and the systematicity of its method in spite of its apparent self-serving selectiveness, we must briefly pass through the Lacanian subject. 28 Chapter 1 28 Chapter 1 28 The Madness of the Symbolic The Madness of the Symbolic 29 narcissistic, self-deceiving orbit of the ego, and its “latent,” underlying truth, which shows itself negatively within the holes and inconsistencies of the former and can only be brought forth après-coup. In therapy, the task of the analyst is to make the analysand encounter and appropriate the second, the Real that afflicts the analysand often to a degree of painful agony, thereby forcing them to realign the symbolic structures underlying their personality so that the latter are more in tune with that which they reject, given that this (unconscious) act of recoil and exclusion has begun to obstruct their life. After all, the repressed always returns. To explain this complex, Lacanian psychoanalysis categorizes experience in terms of the three registers of the Imaginary, the Symbolic, and the Real. All three exist in dialectical simultaneity, so that they all depend upon and interpenetrate one another. Lacan uses a Borromean knot to illustrate this level of mutual co-existence, the point of which is to preclude the possibility of arguing for the primacy of one register over the other, as it is unclear if either can have logical priority insofar as the cutting off or isolating of one destroys the whole. The Imaginary is roughly equivalent to phenomenological experience and perception but is also related to the cogito and its “narcissistic” fantasy of existential self-mastery. It is identified with a necessary moment of misrecognition and irremovable untruth in one's everyday being and knowledge of self, world, and others, for it projects completion where there is lack. The Symbolic constitutes the logical fabric of language and the laws of culture that transcend and are anterior to the concretely existing personal subject. It therefore precedes the imaginary orbit of experience insofar as the individual phenomenological constitution of objects in a strong sense presupposes language. 1.2 The Lacanian Subject and the Irreducible Ambiguity of the Real Although the former exhibits many traits that link it to early transcendental philosophy (it grounds the symbolic structures that constitute phenomenal reality through a free idealization) it is in direct opposition to the self-conscious transparency of the Cartesian cogito, the self-legislation of the Kantian rational agent, or the Hegelian account of free personality. For Lacan, the freedom of the I as witnessed in phenomenological self-experience is an illusion: completely determined by cultural and linguistic influences, the ego is an object and is constantly trying to construct a fantasmatic narcissistic space within which it can (falsely) perceive itself as a centre of self-effectuating action. Although this does not prevent the existence of human freedom for either Lacan or Žižek, it means that freedom itself gets largely displaced from consciousness into the unconscious, in a move formally similar to the middle-late Schelling of the Freiheitsschrift and Weltalter, but with an important twist. The subject is not an energetic, productive will that precedes the constitution of phenomenal reality, but is in one of its most important modalities nothing but an impersonal abyss that, uncannily, renders possible a minimal consistency of self. In this sense, the self is infinitely split at its core: when one looks inside oneself one not only finds an “extimacy,” material coming from elsewhere, where one should find one's innermost core, but if one looks long enough one only finds a void staring back. The Lacanian-Žižekian subject has no intrinsic content because it is pure form: the entire “plenitude” of cultural and psychological experience only emerges as a kind of defence formation against this primordial nothingness of the subject as an attempt to fill in its constitutive “gap” with false positive substantiality. But because this “gap” can never be filled in, it is ultimately repressed due to its traumatic, personality-shattering quality, so that we find traces of its disavowed knowledge in the slips and slides of speech, symbolic inconsistencies and non-coincidences in writing, the images of fantasies and dreams, and other phenomena. This creates two levels to any given (personal, ethical, political, or even philosophical) discourse: its surface movement, grounded in the The Madness of the Symbolic As a self-enclosed structural system capable of reproducing and propagating itself, the Symbolic displays an irreducible autonomy that displaces the role of nature in understanding human psychology and cultural phenomena because it is able to articulate itself in utter isolation from it: the “incessant sliding of the signified under the signifier,”26 the solipsistic dance of language always in step with itself, means that the essential link between signifier and transcendent, extra-linguistic signified has been violently ruptured and that the mere chains of signifiers relating to themselves are capable of producing/ constructing meaning by themselves in an ontological void. In its simplest Chapter 1 30 form, the Real is that which does not fall under either the Imaginary or the Symbolic, whereby its upsurge is associated with experiences of breakdown and inconsistency not only of the transcendental unity at the basis of phenomenological experience, but even of language or culture itself. Lacan and Žižek therefore use a plethora of adjectives to describe it, which attempt to capture this element of irrevocable logical and existential rupture: “traumatic,” “monstrous,” “horrifying,” and “impossible,” to name a few. attempt to capture this element of irrevocable logical and existential rupture: “traumatic,” “monstrous,” “horrifying,” and “impossible,” to name a few. Yet there is an irreducible ambiguity in Lacan's definition of the Real, which serves as the starting point for Žižek's own philosophical endeavour, for it is precisely in trying to resolve this ambiguity that his metaphysical project gets off the ground. The Real elicits two potentially incompatible interpretative possibilities, and we often see Lacan oscillating back and forth between them. In its first guise, the Real is the excluded Other of the Imaginary and Symbolic, which only truly “comes to be” when the subject constitutes itself. In this sense, the Real is not only dependent upon the symbolic matrix of language and the orbit of phenomenological experience but also only shows itself negatively through their immanent obstruction. This Real-as-lack is distinctly Hegelian: it corresponds to concepts such as “tarrying with the negative” and the suffering that consciousness undergoes when it runs up against non-coincidence, paradox, and limitation in social and political action or scientific thought about the world. The Madness of the Symbolic 31 the idealizing process of human meaning makes it impossible to reach. As something that overreaches the idealizing, linguistic activity of the subject, in this modality the Real, corresponding to the Schellingian concept of the indivisible remainder (der nie aufgehende Rest), that pre-experiential darkness that can never be brought into light of consciousness yet upon which all consciousness rests, is the Real-as-excess. But such a free ciphering activity simultaneously creates the condition of the possibility of its own breakdown insofar as it will not always be capable of idealizing the Real in a way that enables its own autonomous, smooth functioning.28 These “kinks” in the Symbolic correspond with the Real-as-lack or Real2, something that cannot be integrated because it presents itself as intrinsically and paradoxically non-relational, as an inassimilable kernel within the self-referential matrix of the symbolic relations within which it emerged. The problem is as follows: Is the Real1 a necessary, illusory construct of the Symbolic designed to give a fantasmatically fabricated sense of “positivity” to a world that exists beyond its grasp (rendering it a mere secondary effect of a solipsistically self-contained structuralist metapsychology)? Or is it, more primordially, the pre-subjective, ontological basis of the Symbolic, to which we have access despite the apparent impossibility of reaching the pure Real through the differential system of language (thus showing the obscure origins of the Real2 in an ontology)? If the second is possible, what does this mean in terms of Lacan's declaration of the logical equivalence and interpenetration of the registers? The Madness of the Symbolic It has absolutely no positive content in itself even though, as an internal limit within a given symbolic space, it may effectuate an overhauling of the latter's structure and possibilities as the subject attempts to overcome its deadlock so that it is potentially productive in its very trauma. In its second guise, we could also understand the Real as the pre-subjective life of pure jouissance from which the human infant exiles itself by becoming a linguistic subject, yet upon which the Imaginary and the Symbolic logically depend, even if they only relate to it negatively through its primordial foreclosure from experience and language as the founding gesture of subjectivity itself. Bruce Fink refers to this as the Real1 because it is the necessary posit of the Symbolic despite the fact that it is inaccessible from within the latter, whose ciphering activity doesn't merely “reconstitute” objective reality by meditating it like a camera obscura. Given that this pre-subjective Real must be said to be without lack (only with language can we speak of absence and presence),27 The Madness of the Symbolic 1.3 From Logico-Dialectical Simultaneity to the Ontogenesis of the Subject Even if all three registers exist in a logico-dialectical simultaneity, within the development of Lacanian psychoanalysis we see a gradual shift in emphasis in the thematization of the registers largely due to internal reasons. Lacan's early work is largely an attempt to come to terms with the mirror stage and its implications for understanding psychogenesis. In the mirror stage, which happens around the age of six months, there is a recognition of an immanent blockage in nature that tears apart the organic unity of the body. The human infant lacks motor coordination—its self-experience is infinitely fragmented and lacking in any internal unity. Lacan's provocative Chapter 1 Chapter 1 32 thesis is that the only way out of this biological short circuit is a vel, a misrecognition of the primordial helplessness of the human organism in the “specular image”29 of its mirror self in which the child finds a mesmerizing and captivating lie of false mastery into which it libidinally invests itself. Already at this early stage we see why Lacan is so critical of the modern conception of subjectivity and rationality. The result of the mirror stage is a reorganization of the fragmented being of the child through a virtual and therefore illusionary schema as the self becomes alienated from its real chaotic being. Yet Lacan comes to see that the imaginary beginnings of psychogenesis are themselves necessarily grounded in the Symbolic: the only reason why the child becomes tantalized by his image is because their parents provoke the response. “Look, it's you!” In this sense, the entire genesis of the self is preceded by a carving out of a space for the child within the symbolic universe of familial relations even before the child was born. After this “linguistic turn,” Lacan turns all of his attention to the nature of the Symbolic and seems, in many respects, largely to leave the Imaginary behind. Inspired by the work of Levi-Strauss, who argued that “[s]tructural linguistics will certainly play the same renovating role with respect to the social sciences that nuclear physics, for example, has played for the physical sciences,”30 Lacan then begins to apply the methodology of Saussure's structuralism to psychoanalysis, accomplishing this feat largely by a retour to Freud. 1.3 From Logico-Dialectical Simultaneity to the Ontogenesis of the Subject What Lacan finds so intriguing is that, despite all of Freud's attempts to ground the unconscious within a natural vitalism of the body or biological movement of instincts, his texts themselves orbit around the analyses of images and language. Lacan's fundamental thesis is that, retroactively, we can see that Freud already had an implicit idea of the importance of linguistics for understanding the unconscious but was unable fully to articulate this fundamental insight because he lacked the appropriate methodology. This in turn creates a fundamental and irremovable non- coincidence within Freud's texts as they oscillate between purely structural analyses of language and obscure vitalistic biologism. Consequently, Lacan argues that if we read Freud against Freud, structural linguistics can give psychoanalysis the scientific rigor that it needs by systematizing the logic of the unconscious, which is the origin of Lacan's famous saying “the The Madness of the Symbolic 33 unconscious is structured like a language.” Linked to this linguistic turn are his critiques of ego-psychology as an attempt to strengthen the ego and post- Freudian attempts to biologize the unconscious. For Lacan, the unconscious is, strictly speaking, an irreducibly linguistic phenomenon: it only emerges after or alongside the advent of language, in the split between the subject of enunciation and the enunciating subject. It has nothing to do with deep- lying personality structures determining how the ego relates to the external world or instinctual energetics. Although this suggests an obvious superseding of the Imaginary by the Symbolic, commentators such as Richard Boothby and Alexander Leupin warn against this, arguing that Lacan is much more complex and subtle than he may initially appear. Lacan never backs away from the claim that all registers mutually depend upon one another in order to have any efficacy at all. Even if the self-generating matrix of language and culture historically precedes and conditions the possibility of any concretely existing person, its differential network of meaning is only possible through an originary phenomenological perception of signifiers.31 The colonization of the body by the transcendentally alienating structures of the Symbolic requires the activity of the Imaginary so that the various phonetic differences that allow signs to be intelligible in contradistinction from one another can be established in the first place. 1.3 From Logico-Dialectical Simultaneity to the Ontogenesis of the Subject Moreover, the late Lacan's topological formalizations of the psyche, as already mentioned, proclaim a strict equivalency between them, so that “the symbolic order's supremacy appears as an aporia, an ethical decision that logic does not support.”32 But how can Žižek then, as a Lacanian philosopher of the Real, justify his own theoretical preference for it over the other registers where Lacan's texts seem to contradict such an approach? Although Žižek's work holds an uncertain relation to Lacanian orthodoxy, it would be wrong to claim that this troublesome problematic is just a direct consequence of Žižek's own reading of Lacanian psychoanalysis. By the very act of embarking on a metaphysics of the Real Žižek does seem to imply that we must find a way to overstep Lacan's attempt to conserve the equivalency of the registers if psychoanalysis is to find a sufficient theoretical grounding, even if doing so means that we risk making the entire psychoanalytical edifice he constructed collapse. But Žižek's own Chapter 1 34 thinking is not as radical a rupture with Lacan as it may appear, for the late Lacan too ruminates about the understanding of nature necessarily implied by his theory and therefore himself gestures towards the possibility of a metaphysics of the Real consistent with it.33 Even as early as the seventeenth seminar, we can find Lacan proclaiming that one of the logical implications of the psychoanalytical experience is that substance is not-all (that is, nature does not present us with a spherical totality—un tout, une sphère).34 Yet if the Real is only lack, and the essential link between signifier and transcendent, extra-linguistic signified has been cut, how can Lacan even legitimately make such statements? How can he philosophically justify such a “direct touching” of the Real given the epistemological solipsism intrinsic to the cybernetic ciphering of the Symbolic? 6. Lacan, “Presentation on Psychic Causality,” in Écrits, trans. Bruce Fink (New York: W. W. Norton, 2006), p. 176/144. 5. See Freud, “The Loss of Reality in Neurosis and Psychosis” (1924), SE, XIX, especially pp. 185–87. 4. Žižek, The Parallax View (Cambridge: MIT Press, 2009), p. 231. 1.3 From Logico-Dialectical Simultaneity to the Ontogenesis of the Subject Žižek’s wager is that one can develop a metaphysics of the Real that not only does not jeopardize the equivalency of the registers, but even explains their emergence, thereby implying that to grasp the essence of psychoanalysis and draw out its philosophical implications we need to do two things: (i) metapsychologically explicate the ontogenesis of the subject in terms of a materialism compatible with the founding insights of a radical idealism or, in other words, explain the relation of the Real1 of the apparent excess of the ontological to the linguistic to the Real2 of symbolic or notional lack, since this is the great question left unanswered by Lacan and upon which his entire theoretical apparatus ultimately depends; (ii) instead of focusing on Lacan's relationship to nineteenth- and twentieth-century psychology, French structuralism, the Prague school of linguistics, or existentialism, we should return to German Idealism, since it is only in a direct dialogue with this tradition that we can find a way out of the impasses of Lacanian psychoanalysis. But to develop such a metaphysics, we must leave psychoanalysis and venture into German Idealism. Notes The Madness of the Symbolic 35 The Madness of the Symbolic 3 The Madness of the Symbolic 35 7. Žižek, Less Than Nothing, p. 77. 8. Ibid., p. 197. 9. Žižek, The Parallax View, p. 4. 10. For Johnston’s justification of “transcendental materialism,” see Žižek’s Ontology: A Transcendental Materialistic Theory of Subjectivity (Evanston: Northwestern University Press, 2000), pp. 273–74. 11. Fichte, Fichte: Early Philosophic Writings, trans. Daniel Breazeale (Ithaca: Cornell University Press, 1988), p. 147. 12. See Johnston, Žižek’s Ontology, pp. 80–92. 12. See Johnston, Žižek’s Ontology, pp. 80–92. 13. Fichte, Science of Knowledge, ed. and trans. Peter Heath and John Lachs (Cambridge: University of Cambridge Press, 1982), p. 262. 14. Žižek, The Parallax View, p. 214. 15. Žižek, The Ticklish Subject: The Absent Centre of Political Ideology (New York: Verso, 2000), pp. 33–41. 16. Žižek, The Indivisible Remainder: On Schelling and Related Matters (New York: Verso, 2007), p. 8. 17. Grant, Philosophies of Nature after Schelling (New York: Continuum, 2006). 18. Zimmerman, Die Rekonstruktion von Raum, Zeit und Materie. Moderne Implikationen Schellingscher Naturphilosophie (Frankfurt am Main: Lang, 1 19. Zimmerman, System des transzendentalen Materialismus (Paderborn: Mentis, 2004). 20. See Žižek, chapter 3 (“Quantum Physics with Lacan”) of The Indivisible Remainder, pp. 189–236; and chapter 14 (“The Ontology of Quantum Physics”) of Less Than Nothing, pp. 905–62. 21. See part 2 (“The Solar Parallax: The Unbearable Lightness of Being No One”) of The Parallax View, pp. 145–250; and interlude 6 (“Cognitivism and the Loop of Self-Positing) of Less Than Nothing, pp. 715–37. 22. Žižek, Less Than Nothing, p. 724. 23. Kant, The Critique of Pure Reason, trans. Paul Guyer and Allen W. Wood (Cambridge: Cambridge University Press, 1998), A 156/B195. 24. Žižek, Less Than Nothing, p. 906. 25. Ibid., p. 935. 25. Ibid., p. 935. 26. Lacan, “The Instance of the Letter in the Unconscious, or Reason since Freud,” in Écrits, p. 502/419. 27. One of many possible quotes: “By definition, the Real is full.” Lacan, Le séminaire, Livre IV: La relation d’objet et les structures freudiennes, 1956–1957, ed. Jacques-Alain Miller (Paris: Seuil, 1994), p. 218. I capitalize “the Real.” 28. See Fink, chapter 3 (“The Creative Function of the Word: The Symbolic and the Real”) of The Lacanian Subject: Between Language and Jouissance (New York: Princeton University Press, 1995), pp. 24–31. 29. Notes Lacan, “The Mirror Stage as Formative of the I Function as Revealed in Psychoanalytical Experience,” in Écrits, p. 94/76. 36 Chapter 1 36 Chapter 1 36 Chapter 1 30. Lévi-Strauss, “Structural Analysis in Linguistics and in Anthropology,” in Structural Anthropology, trans. Claire Jacobson and Brooke Grundfest Schoepf (New York: Basic Books, 1963), p. 33. 31. See Boothby, Freud as Philosopher: Metapsychology after Lacan (New York: Routledge, 2001), pp. 86–94. 32. See Leupin, Lacan Today: Psychoanalysis, Science and Religion (New York: Other Press, 2004), p. 27. 33. See chapters 4 and 5. 34. See Lacan, Le séminaire, Livre XVII: L’envers de la psychoanalyse, 1969–1970, ed. Jacques-Alain Miller (Paris: Seuil, 1991), p. 36. Chapter 2 Grasping the Vanishing Mediator Between the Real and the Ideal Žižek and the Unconscious Truth of German Idealism Chapter 2 Grasping the Vanishing Mediator Between the Real and the Ideal Žižek and the Unconscious Truth of German Idealism Chapter 2 Grasping the Vanishing Mediator Between the Real and the Ideal Žižek and the Unconscious Truth of German Idealism Seeing a structural homology between the contemporary concerns of psychoanalysis and those of late German Idealism's response to Kant, Žižek turns to the latter to find the resources he needs to give an account of the ontogenesis of the psychoanalytical subject. However, to do so he not only has to go against mainstream interpretations of this tradition, but also has to do great damage to the founding texts themselves. Outlining the methodology behind Žižek's reactualization, what we will see is that the only reason Žižek can apply psychoanalytical tools to restructure its symbolic space and thereby develop his own philosophy is that this tradition itself is haunted by a spectral history of an encounter with an underlying trauma: namely, the subject as the vanishing mediator between the Real and the Ideal, which both Hegel and Schelling primordially reveal in their own manner, but ultimately recoil from and disavow. In this regard, German Idealism presents us with an unconscious Grundlogik that we can only now, with the aid of Freud and especially Lacan, reconstruct, thus giving us a profoundly new and controversial view of its internal development and theoretical preoccupations. 38 Chapter 2 38 2.1 The Methodology of a Psychoanalytical Dialogue: Or, Lacan with Hegel and Hegel with Lacan What is amazing about this psychoanalytical dialogue, however, is the heterodox readings of German Idealism and Lacanian psychoanalysis it produces. To many critics, Žižek simply shows no concern for textual faithfulness or the history of ideas in his readings of Kant, Hegel, Schelling, and Lacan. His methodological approach appears, if anything, to function through a deliberate misunderstanding or liberal reconstruction that purposefully overlooks key conceptual distinctions that challenge his own philosophical outlook. Although there is some superficial truth in these critiques—one must admit that Žižek focuses on often marginal selections from texts and raises them to a level of logical priority that they do not have in the original—one of Žižek's rare comments on his own methodology is very helpful for dispelling confusion on how he proceeds: Hegel didn't know what he was doing. You have to interpret him. Let me give you a metaphoric formula. You know the term Deleuze uses for reading philosophers—anal interpretation, buggering them. Deleuze says that, in contrast to other interpreters, he anally penetrates the philosopher, because it's immaculate conception. You produce a monster. I'm trying to do what Deleuze forgot to do—to bugger Hegel, with Lacan [chuckles] so that you get monstrous Hegel, which is, for me, precisely the underlying radical dimension of subjectivity which then, I think, was missed by Heidegger. But again, the basic idea being this mutual reading, this mutual buggering [chuckles] of this focal point, radical negativity and so on, of German Idealism with the very fundamental (Germans have this nice term, grundeswig35) insight of psychoanalysis.36 Even if Žižek describes his own philosophy as an act of textual violence, almost of rape (it is also worth mentioning that the word “bugger” originates the old French bougre, meaning heretic, and acquires its colloquial sense from heresy being associated with deviate, outlawed sexual practices), this quote reveals a hidden methodological presupposition that guides all Grasping the Vanishing Mediator Between the Real and the Ideal Grasping the Vanishing Mediator Between the Real and the Ideal 39 of Žižek's interpretative work. The comparison of his own philosophy to that of Deleuze is of crucial importance and is not to be downplayed. It demonstrates that, even if Žižek is intentionally going against surface textual movements in his reading, he does not understand his own philosophy as in any sense arbitrary, a deliberate misunderstanding of the philosophers with whom he is engaging, or even as exhibiting any disregard for the tradition. Žižek recognizes that he is not doing traditional history of philosophy or any kind of philologico-exegetical interpretation, but is, instead, attempting to do something that is productive of new concepts by revealing their disavowed insights. But this generative activity of concept-creation can bring forth something unexpected, unsettling, even traumatic—we may produce monsters. Žižek is not directly interested in what the texts of the German Idealist tradition “have to say,” that is, their intended meaning, because this level of their discourse—like most discourses that fail to reflect upon the psychoanalytical conditions within which a discourse as such can take place—usually operates largely on the level of the Imaginary and its illusionary fantasy and can thus, perhaps even at most crucial junctures of conceptual argumentation, display a psychoanalytical superficiality. What therefore concerns him are hitherto unrealized textual potentialities, premonitions of which we can see in “marginal” comments or in various structures that often obstruct the general flow of a given philosophical system and consequently can be said to protrude out of its symbolic universe, negatively contorting it from the inside. Yet it is only by means of a thorough familiarity with this system and its surface affirmations that one can arrive at such unearthed possibilities and “reactualize” them. The analyst must, after all, listen to the analysand, even if, especially in its Lacanian mode, it often appears as if they are unconcerned or ignoring your needs and demands (for the goal of therapy is not the adaptation of the ego but rather a confrontation with unconscious, often traumatic truth). Žižek's own methodological approach to the history of philosophy, however, drastically differs from that of Deleuze insofar as it takes as its starting point the fundamental Lacanian claim that we can never say what we mean because there is an irremovable gap between the imaginary moi (“me” as the subject of self-conscious awareness who expresses the intention to say Chapter 2 That is what Lacan means when he says that “interpretation hits the cause”: it hits that around which the analysand is revolving without being able to “put it into words.”38 Keeping this in mind, we can easily see that Žižek's reading of German Idealism, by following its unconscious Grundlogik, is an attempt to psychoanalytically construct what the tradition in fact tries to say but cannot, by revealing what has been repressed in the first-level propositional Grasping the Vanishing Mediator Between the Real and the Ideal 41 affirmations of its texts. In other words, to borrow a Lacanian phrase, it tries to hit their cause. Although one can, of course, take issue with this methodology, one must admit that critics who take issue with Žižek's “selective” reading or “obvious misinterpretations” often just miss the point: they fail to see the underlying systematicity evident in his engagement with the tradition or understand why the conceptual moments he draws upon are so important. In the spirit of Hegel and Schelling, who, in the uncertain aftermath of Kant, sought to articulate a philosophical system that could guarantee the latter's intuition of the irreducible autonomy of the subject,39 Žižek like Lacan before him is convinced that there is something genuinely real and insurmountable in the experience of human freedom. Starting from psychoanalysis' insight into the constitutive disharmony between mind and body as a key to understanding its true essence, Žižek returns to German Idealism to see how the notion is conceptually refracted there. But in this respect Žižek's method is not a textual reconstruction of their arguments through an act of classico-philological retrieval nor is it an attempt to polish them up by using new resources at our disposal. What he notices is something primordially at odds with their surface content, which rather than rendering the texts of German Idealism logically inconsistent is more profoundly indicative of some kind of internal unconscious struggle, a struggle that is philosophically revealing and whose exploration—now made possible by the clinical tools of psychoanalysis—promises to unearth new ways of approaching its fundamental concepts. Chapter 2 Chapter 2 40 something, but somehow says something different and unintended) and the symbolic je (the elusive I as the subject that one deduces from what has in fact been said). Not only is language something that exerts control over us more than we have power over it (as an ego, rather than existing as a speaking linguistic subject, we are in a strong sense spoken), but the surface content of our own words often belies a greater (consciously) disavowed (but unconsciously known) truth, a truth that is not “hidden” in some deep, elusive place, but is so obvious that we often do not see it: “[t]he psychoanalyst is not an explorer of unknown continents or of grand depths, but a linguist: he learns to decode the writing that is already there, under his eyes, open to the look of everyone.”37 In the slips and slides of discourse, in seemingly meaningless hints and gestures, we catch a glimpse of the Real as that which cannot be said directly in discourse but around which it eternally moves, acting as a black hole drawing in everything towards it, or what Lacan refers to as its “cause” or “truth”: Lacan's theory of interpretation is based, to some extent, on a formulation similar to that of the caput mortuum: an analysand speaking in the analytic setting is often unable to say, formulate, to come out with certain things; certain words, expressions, or thoughts are unavailable to him or her at a particular moment and he or she is forced to keep circling around them, beating around the bush, as it were, never enunciating what he or she senses to be at issue. The analysand's discourse traces a contour around that which it hovers about, circles, and skirts. Those words or thoughts may become accessible to the analysand in time, in the course of analysis, but they may also be introduced by the analyst in the form of an interpretation. Chapter 2 Finding premonitions of Todestrieb protruding out of the imaginary-symbolic space of its discourse, Žižek sees a disavowed truth lurking “beneath” the tradition's own attempts to think substance as subject by means of a dialectical interconnecting of mind and body, a disavowed truth that reduces the latter to a mere reaction formation, a defence from the true horror of subjectivity finally brought to the fore by Kant's account of transcendental freedom. After all, if German Idealism's breakthrough is the freedom of the subject, according to Žižek we must also recognize that “our experience of freedom is properly traumatic.”40 Consequently Žižek gives us resources to think how, if we attentively follow the tradition's conceptual movements, we can see traces of an operative logic ephemerally emerging from time to time at crucial moments only to be Chapter 2 42 covered up by preceding steps, so that our task is to reconstruct retroactively this hidden Grundlogik constantly interfering with what the tradition takes itself to be, if we are to come to terms with the disturbing metaphysics of freedom demanded by its founding intuitions. However, the methodology of such a psychoanalytical reconstruction of philosophical trauma around which an entire tradition circulates as its unconscious cause or truth displays an important paradox that deserves to be highlighted, which we can elucidate by means of Žižek's discussion of the legend of Eppur si muove: The legend has it that, in 1633, Galileo Galilei muttered, “Eppur si muove” (“And yet it moves”), after recanting before the Inquisition his theory that the Earth moves around the Sun[. ...] There is no contemporary evidence that he did in fact mutter this phrase, but today the phrase is used to indicate that although someone who possesses true knowledge is forced to renounce it, this does not stop it from being true. But what makes this phrase so interesting is that it can also be used in the exact opposite sense, to assert a “deeper” symbolic truth about something which is not true—like the “Eppur si muove” story itself, which may well be false as a historical fact about Galileo's life, but is true as a designation of Galileo's subjective position [... B]eyond the truth of reality, there is the reality of the fiction.41 Likewise, the alternative history of German Idealism that I develop in the following in a strong sense did not happen as a historical fact. Chapter 2 Little if any evidence points towards it, and often the philological references that I have gathered can be easily opposed by numerous counter-examples. However, this is not the point: although this account of German Idealism's failed encounter with its inner truth is in many ways nothing but a fiction, this does not prevent it from directly expressing the subjective position that each of its representatives holds (even if it falsely capitulates their conscious interpretation of what they are doing) in a manner that other philosophical methodologies cannot, for what concerns us are traces of the Real in imaginary-symbolic constructs and their logic. After all, “when truth is too traumatic to be confronted directly, it can only be accepted in the guise of a fiction.”42 Grasping the Vanishing Mediator Between the Real and the Ideal 43 44 Chapter 2 Than Nothing.45 The effect of this comment is twofold: not only does it strike those immersed within “conventional,” textbook accounts of the history of philosophy as essentially wrong (which, it must be said, are losing credibility as Fichte and Schelling research has finally gained an autonomy of its own), but it also highlights a fundamental difficulty at the core of Žižek's reading of German Idealism and by consequence his own philosophy, insofar as his parallax ontology is perceived as a reactualization or continuation of this movement. This problem is only amplified when one engages with Žižek's major works on German Idealism. In both The Ticklish Subject and The Parallax View, for example, there are praises of Schelling as the greatest philosopher of the pre-symbolic Real, of the nature of the impossible X, the je ne sais quoi, which precedes consciousness as the most central theme in post-Kantian philosophy and whose problematic uncannily reappears in the wake of the Lacanian subject. Žižek even goes as far as to say that Schelling was “the first to formulate this task”46 and the philosopher who “gave the most detailed account of this X in his notion of the Ground of Existence,”47 which is why his philosophy and not that of Hegel is “at the origins of dialectical materialism.”48 Given that Žižek in Less Than Nothing describes “the key question” of philosophy as that of “how thought is possible in a universe of matter,” so that we should focus our efforts on “the very rise of representation or appearing out of the flat stupidity of being” if we are to avoid “the very rise of representation or appearing out of the flat stupidity of being” if we are to avoid “a regression to a 'naive' ontology of spheres or levels,”49 the issue of whether this project is most radically accomplished by Schelling or Hegel is more than a matter of intra-textual consistency or classico-philological accuracy, but touches the very heart of what Žižek takes to be the program of speculative philosophy. On account of this ambiguity, Žižek's own project of describing this process of the auto-disruption of the Real displays an undeniable oscillation between the characterization of this project as a Schellingian or a Hegelian one, even though he claims outright that Schelling is the culmination of this line of thought. 2.2 A Metaphysics of the Real: An Hegelian or a Schellingian Project? Central to Žižek's metaphysics of the Real as a psychoanalytical reactualization of German Idealism is his stark emphasis on the indispensable significance of Hegel. In various places, Žižek characterizes his own project as strictly Hegelian because, like Hegel, the enigma that occupies him is the possibility of appearance itself: that is, how the realm of phenomenal reality could emerge from the self-actualization of substance. Hegel is said to be the first to understand this question in terms of inscribing the subject into the absolute: a subject that, in the Kantian aftermath, is understood as intrinsically irreducible to the immanent ontological field that brought it forth, and that can freely mediate the latter for itself through its own idealizing activity. In this sense, Hegel's metaphysics could structurally supply the missing principle of a Lacanian metapsychology. Through Hegel, Žižek believes he can show that subjectivity is not illusory by situating idealism into the heart of materialism as an irruptive event, the claim being that premonitions of this can be seen throughout Hegel's philosophy. Apparently arguing by means of resources found in the latter for a self-splitting or of the noumenal within the dark pre-history of subjectivity, Žižek tries to show how the only consistent way to explain why there is experience is to posit an ontological catastrophe at the basis of the subject. Consequently, it would appear that Žižek makes the following his axiomatic first principle thanks to a direct confrontation with Hegel: Freedom is not a raw, brute fact, but an expression of the caustic collapse of material being, a brisure in the heart of Real, which is synonymous with the subject itself; “it designates [...] the primordial Big Bang, the violent self-contrast by means of which the balance and inner peace of the Void of which mystics speak are perturbed, thrown out of joint.”43 But the overtly Hegelian nature of Žižek's project does not go without saying. In one essay, Žižek makes a claim that to many readers must appear out of place insofar as he gives it no qualification: “Hegel's 'overcoming' of Schelling is a case in itself: Schelling's reaction to Hegel's idealist dialectic was so strong and profound that more and more it is counted as the next (and concluding) step in the inner development of German Idealism.”44 What is more, this claim is repeated almost verbatim in Less Grasping the Vanishing Mediator Between the Real and the Ideal 45 could) in such a metaphysics, in terms of either our understanding of ontology (the universal), natural sciences (the particular), or politics (the singular), the trinity that constitutes the conceptual fold of The Parallax View, his first theoretical magnum opus. In other respects, however, it appears that Žižek may have recently changed or distanced himself from his earlier interpretation of Schelling, perhaps precisely for these reasons. Although Žižek, despite his constant insistence that his fundamental question is a “properly Hegelian one: how does appearance itself emerge from the interlay of the Real?,”50 oscillates in his early major theoretical works The Ticklish Subject and The Parallax View in his descriptions of who most adequately answers this question, in his new masterpiece Less Than Nothing this oscillation suddenly stops. While this project is again and again characterized as Hegelian,51 the former glory once bestowed upon Schelling as the great thinker of the “phenomenalization of being”52 is not just downplayed, but apparently completely forgotten, with Žižek even going so far as to say that thinking the emergence of appearance is “what at his most radical Hegel does,”53 thus contradicting himself. However, despite appearances to the contrary, the ambiguity does not thereby disappear. Not only does Žižek claim that “[t]he minimal definition of materialism hinges of the admission of a gap between what Schelling called Existence and the Ground of Existence,”54 but the ambiguity of the Hegel-Schelling relationship in fact gets radicalized in the middle of the second chapter on Hegel, when Žižek describes the latter's dialectic as “the science of the gap between the Old and the New,” and then without explanation jumps into a two-and-a-half page discussion of Schelling's theosophic odyssey of the emergence of “the cosmos (of fully constituted reality, ruled by logos) out of the proto-cosmic pre-ontological chaos.”55 Although to many readers this may appear as just another “typical” Žižekian digression with no inner logic, the argument that I develop in this book attempts to show that it is anything but that. Grasping the Vanishing Mediator Between the Real and the Ideal Insofar as Žižek himself claims that the only way to avoid mystification is not to abandon the project of the Weltalter but rather “to reformulate it so as to avoid the mystification of the theosophic mytho-poetic narrative,”56 it appears that the proximity of his reading of Hegel and Schelling, when coupled with this brief and rare methodological elaboration, points to the core of his heterodox reading of could) in such a metaphysics, in terms of either our understanding of ontology (the universal), natural sciences (the particular), or politics (the singular), the trinity that constitutes the conceptual fold of The Parallax View, his first theoretical magnum opus. In other respects, however, it appears that Žižek may have recently changed or distanced himself from his earlier interpretation of Schelling, perhaps precisely for these reasons. Although Žižek, despite his constant insistence that his fundamental question is a “properly Hegelian one: how does appearance itself emerge from the interlay of the Real?,”50 oscillates in his early major theoretical works The Ticklish Subject and The Parallax View in his descriptions of who most adequately answers this question, in his new masterpiece Less Than Nothing this oscillation suddenly stops. While this project is again and again characterized as Hegelian,51 the former glory once bestowed upon Schelling as the great thinker of the “phenomenalization of being”52 is not just downplayed, but apparently completely forgotten, with Žižek even going so far as to say that thinking the emergence of appearance is “what at his most radical Hegel does,”53 thus contradicting himself. However, despite appearances to the contrary, the ambiguity does not thereby disappear. Not only does Žižek claim that “[t]he minimal definition of materialism hinges of the admission of a gap between what Schelling called Existence and the Ground of Existence,”54 but the ambiguity of the Hegel-Schelling relationship in fact gets radicalized in the middle of the second chapter on Hegel, when Žižek describes the latter's dialectic as “the science of the gap between the Old and the New,” and then without explanation jumps into a two-and-a-half page discussion of Schelling's theosophic odyssey of the emergence of “the cosmos (of fully constituted reality, ruled by logos) out of the proto-cosmic pre-ontological chaos.”55 Although to many readers this may appear as just another “typical” Žižekian digression with no inner logic, the argument that I develop in this book attempts to show that it is anything but that. 44 Chapter 2 Not only does it potentially suggest that Žižek tries to disavow too strong a relationship between a Lacanian-inspired metaphysics of the Real and Schelling, but it also shows that he might downplay the role that Schellingian notions play (or undermine possible roles that others Grasping the Vanishing Mediator Between the Real and the Ideal Grasping the Vanishing Mediator Between the Real and the Ideal Insofar as Žižek himself claims that the only way to avoid mystification is not to abandon the project of the Weltalter but rather “to reformulate it so as to avoid the mystification of the theosophic mytho-poetic narrative,”56 it appears that the proximity of his reading of Hegel and Schelling, when coupled with this brief and rare methodological elaboration, points to the core of his heterodox reading of Chapter 2 Grasping the Vanishing Mediator Between the Real and the Ideal 47 process of the dialectic, the third term is already the second, understood as a negativity or internal limit inscribed in the first, insofar as it has merely taken over, usurped, the primary position from which the movement began by asserting itself as such. In terms of substance and subject, this means that “this very reversal is the very definition of subject: ‘subject' is the name for the principle of Selfhood which subordinates to itself the substantial Whole whose particular moment it originally was.”61 Nothing at the level of content changes: it expresses a purely formal change at the level of structure due to the tension generated by the contorting presence of negativity. The dialectical movement that goes (i) immediacy → (ii) negation → (iii) negation of negation is superior because there is no genuine return movement to the first, although it all takes place in a single, logically immanent field. The beginning and end do not overlap because something irreducibly different emerges within the first moment (negativity now being made foundational to identity), namely, to take the example that concerns us here, an “out of joint” spirit that has a degree of notional self-reflexivity in opposition to its ontogenetic ground, the whole within which it gave birth to itself. There is no need to posit a state of “originary health” of which the devouring restlessness of the negative cannot be predicated (absolute indifference as that which stands in the difference between Grund and existence and thus is equally in one as in the other) in order to explain the dialectical movement of reality and its relation to the absolute. As we shall see, Žižek's criticism of Schelling gets more complicated because it does not universally apply to the entirety of Schelling’s work, since for Žižek the latter is not characterized by an organic unity or continuity, but rather by a series of irreconcilable ruptures. He draws a distinction between a Schelling1 of the period of quasi-Spinozism (the philosophy of indifference), a Schelling2 of the radical ontology of freedom as seen in the second draft of the Weltalter and the Freiheitsschrift, and a Schelling3 of the philosophy of mythology and revelation, which he qualifies—very violently—as a return to pre-modern essentialism. Chapter 2 46 being purely Hegelian or purely Schellingian, Žižek’s philosophy thus proves to be a hybrid of the two, an attempt—in some as yet unidentified way—to use Hegel to formalize Schelling's thinking, “saving” it from its theosophic commitments, and to use Schelling to radicalize Hegel, allowing him to draw out implicit but disavowed moves in his own philosophy and thus in German Idealism as a whole. But why does this ambiguity exist in the first place? Immediately after his remarkable and provocative reading of Schelling in the first chapter of The Indivisible Remainder, Žižek goes on to argue for the supremacy of Hegelian dialectics over Schellingian metaphysics. For him, although there are premonitions of a radical breakthrough in works such as the Freiheitsschrift and the Weltalter,57 Schelling remains philosophically inferior to his great rival because Grund and existence ultimately remain distinct from one another only by being founded within absolute indifference, which itself “is not a product of opposites, nor are they contained in it implicite; rather it is a being of its own, separated from all oppositions, on which all oppositions are broken, which is nothing other than their very non-being, and which therefore has no predicate except predicatelessness.”58 Indeed, Schelling claims that “from this neither-nor, or from this indifference, duality [...] immediately breaks forth, and without indifference, i.e., without an unground, there would be no twofoldness of the principles.”59 For Žižek, however, this means that “the [Schellingian] Absolute is primarily the 'absolute indifference' providing the neutral medium for the coexistence of the polar opposites” of the Real and the Ideal60 and, as such, is at odds with the potential latent in his middle-late philosophy, a potential Žižek sees in its articulation of the eruptive logic of Grund and its relationship to existence at the basis of subjectivity. In this regard, Hegel provides a superior logic in which there is no need for a principle of meditation exterior to Grund and existence as two reflective pairs that determine themselves through their internal dynamic, which in turn allows us to internally restructure Schelling's own descriptions of the interrelation of the two. In Hegel the very category of “and” changes—it becomes, in essence, tautological, thereby enabling us to identify Grund and existence instead of rendering them mere opposites or dual principles founded in something external to their own inner movement. In the logical Grasping the Vanishing Mediator Between the Real and the Ideal 47 For Žižek, what distinguishes the middle-late period of Schelling's thinking is, strictly speaking, the articulation of an ontogenetic emergence of self- positing freedom in a manner remarkably similar to the Hegelian dialectical movement from abstract immediacy to notional self-reflexivity, which goes Chapter 2 48 against the surface-level theosophic inclinations of the texts. It protrudes out of them and makes them non-coincidental with themselves in such a way that we can psychoanalytically reactualize their fundamental movement by “tarrying with the negative”—that is, by encountering the Real within them. What this means is that even if the self-operative logic of the Grund contains premonitions of a radical transcendental materialism, Schelling is at the same time the father of “New Age obscurantism.”62 Expressing a reliance upon and indebtedness to Schelling would, in essence, open up a possible connection between Žižek’s own thinking and everything he denies—the non-Freudian unconscious (in its Bergsonian, Jungian, Deleuzian, and other forms), “pre-modern” cosmology, Romantic theories of nature, theosophy and even its pop-culture descendant, New Age spirituality. In this sense, the very act of placing the logic of the Grund at the heart of the psychoanalytic subject appears to risk “destabilizing” the primacy of the Lacanian mode of the unconscious insofar as it opens up the possibility of interpreting it through a different account of one's relation to the pre-subjective life of the Real; covering up the pivotal importance of Schelling would seem to be nothing but a certain ideological act on Žižek's part. But here two remarks must be made. First, although it is perhaps a bit underhanded on Žižek's behalf to downplay the importance of Schelling for the development of his transcendental materialism, it must be added that this obfuscation is consistent with his overall interpretation of Schelling, for he rejects most of the latter's philosophy and needs recourse to Hegel even to reactualize the logic of the Grund that he sees as its breakthrough. But second, and more importantly, such an explanation does not solve the problem of the Hegel- Schelling relation insofar as this emphatic shift is simultaneously ambiguous, given the fact that Žižek does not distinguish which Schelling he is arguing against or justify how he is able to read the second draft of the Weltalter as an ephemeral breakthrough. In this regard, by characterizing his own philosophy again and again as against the surface-level theosophic inclinations of the texts. Grasping the Vanishing Mediator Between the Real and the Ideal 47 It protrudes out of them and makes them non-coincidental with themselves in such a way that we can psychoanalytically reactualize their fundamental movement by “tarrying with the negative”—that is, by encountering the Real within them. What this means is that even if the self-operative logic of the Grund contains premonitions of a radical transcendental materialism, Schelling is at the same time the father of “New Age obscurantism.”62 Expressing a reliance upon and indebtedness to Schelling would, in essence, open up a possible connection between Žižek’s own thinking and everything he denies—the non-Freudian unconscious (in its Bergsonian, Jungian, Deleuzian, and other forms), “pre-modern” cosmology, Romantic theories of nature, theosophy and even its pop-culture descendant, New Age spirituality. In this sense, the very act of placing the logic of the Grund at the heart of the psychoanalytic subject appears to risk “destabilizing” the primacy of the Lacanian mode of the unconscious insofar as it opens up the possibility of interpreting it through a different account of one's relation to the pre-subjective life of the Real; covering up the pivotal importance of Schelling would seem to be nothing but a certain ideological act on Žižek's part. But here two remarks must be made. First, although it is perhaps a bit underhanded on Žižek's behalf to downplay the importance of Schelling for the development of his transcendental materialism, it must be added that this obfuscation is consistent with his overall interpretation of Schelling, for he rejects most of the latter's philosophy and needs recourse to Hegel even to reactualize the logic of the Grund that he sees as its breakthrough. But second, and more importantly, such an explanation does not solve the problem of the Hegel- Schelling relation insofar as this emphatic shift is simultaneously ambiguous, given the fact that Žižek does not distinguish which Schelling he is arguing against or justify how he is able to read the second draft of the Weltalter as an ephemeral breakthrough. In this regard, by characterizing his own philosophy again and again as a Hegelian, but never a Schellingian project Žižek belies his overt reliance on texts such as Schelling's Freiheitsschrift and the Weltalter for his project. Grasping the Vanishing Mediator Between the Real and the Ideal 47 What I propose, therefore, is to read Žižek against Žižek not only to demonstrate the implicit, complex intertwining of Schellingian ontology and Hegelian logic throughout his thinking, wherein I perceive the nuance Grasping the Vanishing Mediator Between the Real and the Ideal 49 of his theoretical philosophy, but also to show how by unravelling this ambiguity we can find new resources to explore the obscure origins of the psychoanalytical subject and the fundamental structures and essence of the world within which it emerges. Yet if embarking upon a reconstruction of the Hegel-Schelling relation within Žižek's work can achieve this, it is only because Žižek sees his own work as an attempt to culminate the Grundlogik of German Idealism itself, which gestures towards the hypothesis that the intra-textual ambiguity of the Hegel-Schelling relation within his thinking is perhaps less expressive of an internal inconsistency in his philosophy than of a fundamental problem inherent in the self-unfolding of that tradition itself in its endeavour to overcome radical idealism; so that, if by a dialectical reversal we turn an epistemological limit into a positive ontological condition, apparent textual confusion into a real feature of the movement itself in its historical development, the ambiguity will prove itself in a second moment to be a side effect of a sustained methodological engagement with the fundamental insights of German Idealism which thus deserves to be investigated in its own right. As we will see, this ambiguity is in fact due to a single traumatic kernel of truth that both Hegel and Schelling, attempting to ground the Kantian subject, bring to the fore in different ways, but ultimately have to repress or force into the unconscious, a fact that therefore requires us to use a mode of inquiry that goes beyond philologico-exegetical interpretation if we are to penetrate it: The notion of Schelling's Grundoperation—the “vanishing mediator” between the two poles (the Real and the Ideal, B and A)—opens up the possibility of establishing a connection with Hegelian dialectics: the founding gesture “repressed” by the formal envelope of the “panlogicist” Hegel is the same as the gesture which is “repressed” by the formal envelope of the “obscurantist” Schelling, yet which simultaneously serves as its unacknowledged ground. [...] Does the gesture of “vanishing mediation” not point, therefore, towards what, following some German interpreters, one could call the Grundoperation des Deutschen Idealismus, the fundamental, elementary operation of German Idealism? 35. Neither this word nor a comparable one appears to actually exist in modern or old German. A simple Google search is enough to be suspicious: it only comes up with this quote. Grasping the Vanishing Mediator Between the Real and the Ideal 51 the fore and deals explicitly with the critical difficulties posed by attempting to give an account of the emergence of the Ideal out of a Real that has been, from within the former, always already lost. The psychoanalytical fusion of Schelling and Hegel implicit in his thinking—a fusion which points towards a certain unconscious struggle inherent to German Idealism itself—either reveals or resolves a potentially fatal ambivalence in the conception of the Real, an ambivalence that risks jeopardizing not only Žižek's project, but also the development of any form of metaphysics from within a radical idealism. According to Lacan, the Symbolic exhibits what may be called a form of ontological solipsism insofar as it is an irreducibly self-referential matrix of signifiers whose relation to an external, extra-ideal world has been utterly cut off. Yet to give a theoretical grounding of our inability to access objective reality we must describe its dark pre-history in the orgasm of forces that is the pre-symbolic Real. Is the Real that which precedes and exceeds consciousness, or a pure lack that only presents itself through the breakdowns of the Symbolic? Can these two modalities be reconciled within a metaphysics? If so, can a hybridization of Hegel and Schelling help us conceive of the emergence of the Symbolic out of the Real from within the Symbolic by overcoming the opposition between materialism and idealism? But before we answer these questions, we should step back and ask: why would we even want to proclaim such a Cartesian dualism of mind and body, nature and spirit, the Real and the Symbolic, especially since such a form of subjectivity has been criticized for so long in the contemporary intellectual scene as no longer sustainable? What are its theoretical advantages? 36. Žižek, “Liberation Hurts: An Interview with Slavoj Žižek (with Eric Dean Rasmussen).” Retrieved Feb. 23, 2010, from http://www.electronicbookreview. com/thread/endconstruction/desublimation. 50 Chapter 2 It is our endeavour to articulate clearly the Grundoperation of German Idealism which necessitates reference to Lacan; that is to say, our premiss is that the “royal road” to this Grundoperation involves reading German Idealism through the prism of Lacanian psychoanalytical theory.63 In this manner, to raise the question of whether Žižek's project is Schellingian or Hegelian is misleading, for what interests Žižek is not Kant, Schelling, or Hegel as particular historical thinkers per se, but a psychoanalytically retrievable unconscious truth self-unfolding throughout their works, a truth inaugurated by the Cartesian cogito but from which the entire tradition of modern philosophy has been repelled because of its horror, but to which we can now have access thanks to Freud's and Lacan's groundbreaking work on the original trauma of the human subject. In short, my wager is that Žižek forces us to consider that what makes German Idealism so singular in the history of philosophy is that it is haunted by an unconscious history that is nothing other than one of the sustained encounters with the ontological catastrophe at the very basis of human subjectivity in human thinking, this concept thus presenting itself as a Lacanian cause or truth around which the surface structures of its great epics circulate as their repressed traumatic Real and whose movement demand being systematically reconstructed retroactively. But the ambiguity of the Hegel-Schelling relationship has another advantage: it not only allows us to construct an original and perhaps controversial understanding of German Idealism and its legacy by articulating its unconscious history, a dynamic spectral history of repression and the return of the repressed, but it also allows us to evaluate the philosophical significance of Žižek's work by situating him directly within its concerns. Žižek is able to rely upon Hegel and Schelling to explicate the origins of subjectivity and their metaphysical implications because he sees them as embarking upon a metaphysics from within a self-grounding transcendental framework, whereby German Idealism and the psychoanalytical tradition in its Lacanian legacy become uncannily close due to their underlying epistemologies and the need to make sense of them ontologically. In this respect, unpacking the ambiguity of the Hegel- Schelling relation is of profound methodological importance, for it brings to Grasping the Vanishing Mediator Between the Real and the Ideal 37. Lacan, “Clefs pour la psychoanalyse (entretien avec Madeleine Chapsal).” Retrieved May 16, 2010, from http://www.ecole-lacanienne.net/docu- ments/1957-05-31.doc. Notes 37. Lacan, “Clefs pour la psychoanalyse (entretien avec Madeleine Chapsal).” Retrieved May 16, 2010, from http://www.ecole-lacanienne.net/docu- ments/1957-05-31.doc. Chapter 3 Žižek's reactualization of German Idealism represents an avid attempt to revitalize Cartesian transcendental subjectivity. In the face of a contemporary discourse that attacks from all sides the very possibility of a non-material I that stands in its own autonomous register, Žižek's philosophy aims to articulate the theoretical currency still implicit within the groundbreaking intuition that heralds modernity. But what warrants this repetition/resurgence of such an apparently “outmoded” idea? What kind of explanatory efficacy could it still exhibit in a theoretical field that believes itself to have largely overcome its shortcomings? By exposing the various theoretical holes revealed in the contemporary intellectual scene by Lacanian psychoanalysis, Žižek rethinks the modern materialism-idealism debate by showing how movements such as those in current phenomenology of the body, postmodernism, and neurobiology are philosophically insufficient and thus force us to reconceptualize transcendentalism radically. insufficient and thus force us to reconceptualize transcendentalism radically. 52 Chapter 2 38. Fink, The Lacanian Subject, p. 28. 38. Fink, The Lacanian Subject, p. 28. 39. Žižek, Less Than Nothing, p. 266. 39. Žižek, Less Than Nothing, p. 266. 40. Ibid., p. 265. 40. Ibid., p. 265. 41. Ibid., pp. 3–4. 41. Ibid., pp. 3–4. 42. Ibid., p. 24. 42. Ibid., p. 24. 43. Žižek, The Ticklish Subject, p. 31. 44. Žižek, “Fichte’s Laughter,” in Mythology, Madness and Laughter: Subjectivity in German Idealism (New York: Continuum, 2009), p. 122. 45. Žižek, Less Than Nothing, p. 137. 45. Žižek, Less Than Nothing, p. 137. 46. Žižek, The Parallax View, p. 166. 46. Žižek, The Parallax View, p. 166. 47. Žižek, The Ticklish Subject, p. 55. 47. Žižek, The Ticklish Subject, p. 55. 48. Žižek, The Indivisible Remainder, p. 11. 48. Žižek, The Indivisible Remainder, p. 11. 49. Žižek, Less Than Nothing, p. 905. 50. Žižek, The Parallax View, p. 106. 50. Žižek, The Parallax View, p. 106. 51. See, for instance, Žižek, Less Than Nothing, pp. 13f. and 642ff. 52. See Žižek, The Indivisible Remainder, p. 14; and Žižek, The Abyss of Freedom, in The Abyss of Freedom/Ages of the World (Cambridge: MIT Press, 2008), p. 15. 53. Žižek, Less Than Nothing, p. 239. 53. Žižek, Less Than Nothing, p. 239. 54. Ibid., p. 912. 54. Ibid., p. 912. 55. Ibid., p. 273. 55. Ibid., p. 273. 56. Ibid. 56. Ibid. 57. Žižek, The Indivisible Remainder, p. 8. 58. Schelling, Philosophical Investigations into the Essence of Human Freedom and Other Related Matters, trans. Priscilla Hayden-Roy, in Philosophy of German Idealism, ed. Ernst Behler (New York: Continuum, 2003) (hereafter Freiheitsschrift), p. 276. 59. Ibid., p. 278. 59. Ibid., p. 278. 60. Žižek, The Indivisible Remainder, p. 105. 60. Žižek, The Indivisible Remainder, p. 105. 61. Ibid., p. 106. 61. Ibid., p. 106. 62. Žižek, The Abyss of Freedom, p. 4. 62. Žižek, The Abyss of Freedom, p. 4. 63. Žižek, The Indivisible Remainder, p. 92. 3.1 Postmodernism and an Uncanny Defence of Transcendentalism Although phenomenologists like Merleau-Ponty and more recently Michel Henry and Jean-Luc Marion argue for the primordial unity of consciousness and the lived body or the self's immanent auto-determination from the unfolding of givenness of the flesh (which indicates the disappearance 54 Chapter 3 54 of a radical subject-object distinction by the interpenetration of both in embodiment), Žižek makes the claim that such descriptions are intrinsically lacking insofar as they fail to take account of the experience of the monstrous and the traumatic irrevocably tied to the essence of human being. One cannot merely replace classical subjectivity with a more organic theory of experience that intertwines consciousness with a phenomenological auto-affection of the flesh. The rift between the spiritual and the material uncovered by the I destabilizes the very possibility of such a primordial union or identity: if we take transcendental reflexivity seriously we see, according to Žižek, a pure self-positing that tears itself away from any kind of immersion within the field of corporeal activity. The very possibility of the psychoanalytical experience as that which reveals a split between the energetic dynamics of the body and subjective, experiential reality presupposes an irreducibly antagonistic interrelation, a discordant dichotomy, between mind and body, which proclaims that the human subject must in some sense “protrude” out of its carnal materiality and be understood on its own terms. The claim is that the modern subject not only paves the way for the psychoanalytical discourse of the unconscious, but also sets up the possibility of a genuine encounter with what it means to be a human subject existing with an intrinsic relation to a bodily substrate that paradoxically presents itself as Other. We cannot just think the self that comes after the subject in a way that does away with the latter, for transcendental subjectivity brings to our attention a primordial truth of what it means to be a free human being that, in many ways, we have yet to come to terms with: that of a structural conflict between mind and body which divides our being into two incommensurate spheres. However, the implications of Žižek's reactualization of German Idealism are not just limited to the phenomenology of the body. Psychoanalysis and the Enigma of Transcendental Subjectivity 55 55 sociopolitical activity. The unceasing play of cultural difference, the non- finite proliferation of identities and discourses, can only be adequately understood through the transcendental framework offered by Cartesian subjectivity because, as that which prevents human activity from being explicable through solely natural or biological grounds, it supplies the formal-universal structure through which such change is rendered possible. The cogito is necessary for deciphering not only human embodiment, but also human historicity insofar as it gives us a clue to what kind of ontological event could have led to the emergence of something like a symbolic universe in contrast to the “cyclical” movement of nature. Here we see another advantage of the cogito that transcends its theoretico-explicative currency, or more strongly, coincides with it: as the transcendental condition of the possibility of historical contingency, the pure I always stands above all fixed, particular sociopolitical constellations and thus presents the always possible basis of ideological critique and political revolution. sociopolitical activity. The unceasing play of cultural difference, the non- finite proliferation of identities and discourses, can only be adequately understood through the transcendental framework offered by Cartesian subjectivity because, as that which prevents human activity from being explicable through solely natural or biological grounds, it supplies the formal-universal structure through which such change is rendered possible. The cogito is necessary for deciphering not only human embodiment, but also human historicity insofar as it gives us a clue to what kind of ontological event could have led to the emergence of something like a symbolic universe in contrast to the “cyclical” movement of nature. Here we see another advantage of the cogito that transcends its theoretico-explicative currency, or more strongly, coincides with it: as the transcendental condition of the possibility of historical contingency, the pure I always stands above all fixed, particular sociopolitical constellations and thus presents the always possible basis of ideological critique and political revolution. But perhaps the greatest threat to subjectivity comes from contemporary cognitive science. In light of decades of groundbreaking research in neurobiology, there is a growing tendency to turn towards scientific models of explanation to explain away the uniqueness of human subjectivity. 3.1 Postmodernism and an Uncanny Defence of Transcendentalism Arguing against postmodern theorists like Derrida and Foucault, who claim that the subject itself is merely an empty, accidental construction that arises out of the flux of historical experience, Žižek contends that by forgetting the ontological schism between mind and body that enables the self to be determined according to linguistic and political forces in the first place, they lose sight of the very formal structure of the I that is required even to speak of the endless temporal variations of selfhood within the contingent upsurge of 56 Chapter 3 Žižek's argument here is twofold. Firstly, founding itself upon classical biology, contemporary cognitive science presupposes that every organism is a self-contained system in harmony with itself seeking homeostasis and self-preservation, which prevents it from coming to terms with the psychoanalytical concept of Todestrieb. Representing a malfunction in biology, whereby a person no more strives for pleasure and satisfaction, for the minimal possible level of distress and affliction, but rather for pain and even self-destruction, psychoanalysis identifies this apparently negative moment of short circuit within the biological machine with one of the defining traits of human subjectivity and thus of culture itself.64 Instead of being a mere haphazard disorder or a contingent feature of a sick mind, Todestrieb comes to represent a necessary feature of the singularity of our being: the condition of the possibility of psychopathological self- destruction is ultimately linked to our very freedom because the two are structurally homologous. Secondly, what Žižek adds to this argument for the supremacy of psychoanalysis over reductionist biology is the following insight: if there were nothing but the self-contained, deterministic system of the neuronal interface of the brain, then why is there (self-)experience at all?65 Why is there not just blind existence, a mere mechanism that auto- develops according to its own laws? Why does the nonconscious trembling of brute matter in its dynamic pulsations need to be aware of itself? Since the category of subjective experience is superfluous, unnecessary, to the materialism displayed by science, the mere fact of experience proclaims that neurobiological activity is not-all—that there is a gap, a series of interstices, which arise within its logical fold as a kind of unpredictable short circuit to which, perhaps, phenomenal reality arises as a response. Naming the place of this rupture the subject itself, Žižek's own work on cognitive science consequently tries to underline the inherent difficulty that the discipline has (for this very reason) to explain the emergence of consciousness, insofar as it points to a limit-situation within which the discourse itself breaks down. Neither the phenomenology of the body, postmodernism, or cognitive science can just dispel the “myth of subjectivity” because all three of them Neither the phenomenology of the body, postmodernism, or cognitive science can just dispel the “myth of subjectivity” because all three of them, in distinct ways, need it to account for the very subject matter they take as their own. Psychoanalysis and the Enigma of Transcendental Subjectivity 55 Researchers are not just constantly downplaying the role of systematic self-observation and autonomous discourses that deny the supremacy of experimental science; rather, what is increasingly coming into question is the infinite array of material offered by self-consciousness and the meaning of philosophical investigations into its culture and politics as structurally free from biological concerns. Instead of having recourse to first-person experience as it shows itself to us in the irreducibility of its complex dynamics as the site of personality (phenomenology), or the labyrinthine network of the symbolic universe of discourses informing our sense of self and other (postmodernism), they are able to explain the entire range of emotional and social characteristics through the nonconscious, asubjective pulsation of brute matter, the mere non-personal movement of neurochemico-electrical activity wherein the I becomes an epiphenomenal illusion created by a closed biological system of response mechanisms pre- determined by genetic code. 56 Chapter 3 If the phenomenology of the body is to understand the very field of the (self-)appearing of phenomena to consciousness as embodied, then it Psychoanalysis and the Enigma of Transcendental Subjectivity 57 has to come to terms with the breakthrough of transcendental reflexivity as a form of pure self-positing that institutes a subject-object schism rendering the very body that we live in Other to ourselves: “the subject (Self) is [...] immaterial: its One-ness, its self-identity, is not reducible to its material support. I am precisely not my body: the Self can only arise against the background of the death of its substantial being, of what it 'objectively' is.”66 Postmodernism needs to presuppose the pure I as something over and above the contingent field of non-finite cultural difference that it sets up even to talk about the complex network of discourses irreducible to naturalistic influences. Lastly, cognitive science cannot discard the subject if it is to explain the very possibility of how a gap in (material) being could emerge so that there is the basic distance from self that is necessary for the phenomenalization of reality. If we are to understand the true nature of the human being, Žižek's contention is that we must reread transcendental philosophy through the psychoanalytical category of Todestrieb, for both seem to cover the same theoretical set of problems in an uncanny manner; this unholy marriage of German Idealism and psychoanalysis aims to reconfigure the contemporary intellectual scene by offering a comprehensive system that is able to respond to the intrinsic limitations of all three disciplines. However, although Žižek's retrieval of subjectivity thus superficially appears to be an attempt to assert the unshakeable supreme position of the human subject against the various de-centerings that it has undergone in the twentieth century, this is an overt vel. Whereas most defenders of the transcendental ego return to Descartes, Kant, or Hegel to regain and unpack their conviction in the self-grounding ipseity at the core of human activity in face of its dissipation within flux of radical historicism, the all- engulfing force of structuralism, or the brute determination of mechanical nature, what intrigues Žižek are the various ways in which the cogito fails at its own task and the implications of this failure for our understanding of self and world. 56 Chapter 3 Instead of presenting an intuition of a self-positing substantiality that persists behind all representational content of thought while being co-given, co-equal, and ultimately fully coinciding with my own personal self, what we truly find in the thinkers of subjectivity according to Žižek is the affirmation of a negatively charged void that holds together the Chapter 3 58 sedimentations of personality through its sheer self-reflexive nothingness. Whether this be Descartes' inability to establish the positive characteristics of the I that necessarily thinks,67 Kant's failure to penetrate into the kernel of the subject an sich that makes experience possible,68 or Hegel's early account of the night of the world,69 what is obvious to Žižek is that the impervious, vacuous core of subjectivity is simultaneously its condition of possibility and impossibility. Consequently, what interests Žižek are two things: (i) the traces of this abyssal nature of subjectivity that can be found in modern philosophy, which immanently problematizes all attempts to ground experience in the intuition of a self-positing substantial core of existential familiarity and transparency; and (ii) how this grounding insight into transcendental reflexivity and the deadlocks it generates are schematized under the textual surface in different modalities by Kant and his followers, yet in such a way that they display in them a hidden logic implicitly working itself out throughout the entire tradition that can be retroactively constructed and used today in order to combat a range of theoretical problems that plague us. 3.2 Idealism and its Shadow: Materialism in the Cracks Taking the inevitable failure of self-grounding idealism to fully posit itself as a guide, Žižek's reading of German Idealism focuses on what he perceives as an ambiguous relationship in its texts: between such a self-grounding idealism, and the spectral presence of what appears as a form of materialism within its ideal-synthetic fold that must somehow be posited as theoretically more fundamental and primordial après-coup. The agonizing tension within ideality as it tries to constitute the world of experience suggests that, although a non-mediated, purely material reality has been forever lost due to the idealizing activity rendered possible by the subject, we can still, through the very limitations, blockages, and obstructions of this activity, negatively catch a glimpse of it, the very inconsistency of thought guaranteeing that we gain knowledge of reality “outside” our representations. The figure of Fichte is of utmost importance in this context for comprehending the conceptual contours of Žižek's position. Instead of being a contradictory, deficient concept within the internal trajectory of German Idealism that automatically reveals Fichte's inconsequentiality and impotence as a thinker, the concept of Anstoß manifests the truth of the materialism-idealism relation expressed within the tradition, albeit in a manner that he does not explicitly thematize. Taking the inevitable failure of self-grounding idealism to fully posit itself as a guide, Žižek's reading of German Idealism focuses on what he perceives as an ambiguous relationship in its texts: between such a self-grounding idealism, and the spectral presence of what appears as a form of materialism within its ideal-synthetic fold that must somehow be posited as theoretically more fundamental and primordial après-coup. The agonizing tension within ideality as it tries to constitute the world of experience suggests that, although a non-mediated, purely material reality has been forever lost due to the idealizing activity rendered possible by the subject, we can still, through the very limitations, blockages, and obstructions of this activity, negatively catch a glimpse of it, the very inconsistency of thought guaranteeing that we gain knowledge of reality “outside” our representations. The figure of Fichte is of utmost importance in this context for comprehending the conceptual contours of Žižek's position. 3.2 Idealism and its Shadow: Materialism in the Cracks However, to return to German Idealism and its “rampant” transcendentalist inclinations with the intention of retrieving a conception of the subject able to respond to perceived dead ends in contemporary intellectual discourse presents an immediate problem. In order to articulate an account of experience that can combine psychoanalysis and modern subjectivity in a manner capable of combating the phenomenology of the body, postmodern discourse theory, and cognitive science, Žižek must fight against the anti- materialistic tendencies evident in the German Idealist tradition itself. Žižek's fundamental thesis is—perhaps counter-intuitively—that it is the latter's very descriptions of the non-material, purely spiritual I that point the way. The nothingness of the cogito reveals more than a form of ontological dualism, the horrifying split nature of subjectivity that forever haunts psychic life: not only is the cogito unable to find an existential or ontological interiority at the heart of one's own most personal being— being a mere formal void that guarantees the self-sameness of identity through phenomenal time, the I is an empty set—but the very gesture of a Psychoanalysis and the Enigma of Transcendental Subjectivity 59 self-grounding idealism that it sets into play shows itself to be structurally incomplete and riddled with holes. In modernity's irreversible establishment of the primary function of the cogito (as a synthesizing activity that idealizes our contact with the world in such a way that we lose contact with real entities non-mediated by mind) Žižek argues that we immediately come face to face with the internal limitation and contradiction of idealism as such, insofar as absolute mediation is not possible for the subject. Instead of exhibiting a smooth transcendentally constituted fabric of experience freely brought forth by the subject on its Godlike throne, idealism generates in and of itself irremovable and unpredictable moments of blockage and obstruction that infringe upon the subject's totalizing claims for autocratic autonomy and a complete, undisturbed idealization of experience. In other words, every attempt of idealism to affirm itself as “all,” to set the stage for the transcendental constitution of phenomenal reality as an almighty, full-blown “hallucination,” fails: within idealism there is always something uncannily more than ideal, an extimacy that corrodes it from the inside and cannot be explained from within its free idealization of the world. 3.2 Idealism and its Shadow: Materialism in the Cracks Instead of being a contradictory, deficient concept within the internal trajectory of German Idealism that automatically reveals Fichte's inconsequentiality and impotence as a thinker, the concept of Anstoß manifests the truth of the materialism-idealism relation expressed within the tradition, albeit in a manner that he does not explicitly thematize. Chapter 3 Žižek's own theoretical position is therefore a rather precarious one. It contends that it is only from within the deadlock and limitations of a full- In Fichtean subjective idealism, the I is the axiomatic starting point of philosophy. Even the not-I, the entire sphere of everything that presents itself within consciousness as “extra-subjective” content, is freely posited and brought forth by the self-grounding activity of the absolute subject, in effect robbing it of any alterity that it would possess in itself as a material, non-mediated being with its own interiority that thrusts itself upon consciousness. In other words, the not-I does not affect the I through a form of causality. Yet because of the double meaning of Anstoß as a hindrance and impetus, as something that obstructs and incites our activity, one cannot just say that the Anstoß is a mere obstacle created by the subject so that it can have the necessary degree of resistance needed in order concretely to assert and actualize its own freedom. Although the not-I depends upon the pure I for its meaning, the former of itself cannot be reduced to the idealizing activity set up by the latter: it is not “like the games the proverbial perverted ascetic saint plays with himself by way of inventing ever new temptations and then, by overcoming them, confirming his strength.” For if this were so, “it would present a case of the hollow playing of the subject with itself, and we would never reach the level of objective reality.”70 There is therefore an element of speculative truth in Fichte that Žižek wants to bring to the fore: even if the circle of idealism is a self-enclosed, free creation rendered possible by the subject, due to the painful curvature of this space we must nevertheless speak of a negative materialism in Fichte's thinking.71 Here, we encounter a paradoxical coincidence of internal-external limitation and inside-outside: “Anstoß thus designates the moment of the 'run-in,' the hazardous knock, the encounter of the Real in the midst of the ideality of the absolute I: there is no subject without Anstoß, without the collision with an element of irreducible facticity and contingency—'the I is supposed to encounter within itself something foreign,”72 some inassimilable body that sticks in its throat like a bone. Chapter 3 60 In Fichtean subjective idealism, the I is the axiomatic starting point of philosophy. Even the not-I, the entire sphere of everything that presents itself within consciousness as “extra-subjective” content, is freely posited and brought forth by the self-grounding activity of the absolute subject, in effect robbing it of any alterity that it would possess in itself as a material, non-mediated being with its own interiority that thrusts itself upon consciousness. In other words, the not-I does not affect the I through a form of causality. Yet because of the double meaning of Anstoß as a hindrance and impetus, as something that obstructs and incites our activity, one cannot just say that the Anstoß is a mere obstacle created by the subject so that it can have the necessary degree of resistance needed in order concretely to assert and actualize its own freedom. Although the not-I depends upon the pure I for its meaning, the former of itself cannot be reduced to the idealizing activity set up by the latter: it is not “like the games the proverbial perverted ascetic saint plays with himself by way of inventing ever new temptations and then, by overcoming them, confirming his strength.” For if this were so, “it would present a case of the hollow playing of the subject with itself, and we would never reach the level of objective reality.”70 There is therefore an element of speculative truth in Fichte that Žižek wants to bring to the fore: even if the circle of idealism is a self-enclosed, free creation rendered possible by the subject, due to the painful curvature of this space we must nevertheless speak of a negative materialism in Fichte's thinking.71 Here, we encounter a paradoxical coincidence of internal-external limitation and inside-outside: “Anstoß thus designates the moment of the 'run-in,' the hazardous knock, the encounter of the Real in the midst of the ideality of the absolute I: there is no subject without Anstoß, without the collision with an element of irreducible facticity and contingency—'the I is supposed to encounter within itself something foreign,”72 some inassimilable body that sticks in its throat like a bone. According to Žižek, this truth is then later radicalized by Hegel's and Schelling's own response to Fichte's thinking insofar as they realize it gives us the necessary resources we need to develop a metaphysics while never leaving the transcendental idealist framework. Psychoanalysis and the Enigma of Transcendental Subjectivity 61 61 to matter—here we must think of Žižek's philosophy as a turning inside- out of Jacobi's challenge that idealism must become a speculative egoism by following it to its logical conclusion: “[t]he transcendental idealist [...] must have the courage to assert the strongest idealism that has ever been taught, and not even to fear the charge of speculative egoism”73—that we can develop a truly speculative materialism up to the task of explaining the emergence of experience, for it also suggests that only a full-blown idealism is truly capable of disclosing crucial facts about the nature of materiality. However, such a materialism rests upon the apparently paradoxical claim that it is the very auto-collapse of idealism that is epistemologically capable of overcoming itself and describing the world in itself. As Adrian Johnston says, “materialism [...] formulates itself vis-à-vis the deadlocks internal to radical transcendental idealism. On this account, materialism is philosophically tenable solely as the spectral inverse of idealism, accompanying it as the shadow cast by idealism's insurmountable incompleteness.”74 But the stark consequences of this thesis must be brought to the fore: it claims that all forms of radical idealism always already contain traces of materialism in their blind spots, even if they do not recognize this. Yet how are we to grasp this overlapping of the Real and the Ideal, this contradictory mode of inclusion/exclusion, internal/external, presence/ absence, from within a logical space that by its very essence proclaims to exclude it? How can this impossible feat of a materialism within the cracks of idealism be accomplished? Chapter 3 According to Žižek, this truth is then later radicalized by Hegel's and Schelling's own response to Fichte's thinking insofar as they realize it gives us the necessary resources we need to develop a metaphysics while never leaving the transcendental idealist framework. Žižek's own theoretical position is therefore a rather precarious one. It contends that it is only from within the deadlock and limitations of a full- Žižek's own theoretical position is therefore a rather precarious one. It contends that it is only from within the deadlock and limitations of a full- blown idealism asserting the ultimate irreducibility of mind and subjectivity Chapter 3 62 more, the very nature of idealizing activity must tell us something about the nature of the world as it is in itself and this in two ways: firstly, we cannot just understand our own inability to grasp the Real as a mere limitation due to the finitude of human cognition, but must see this inability as revelatory of an ontological state of affairs—the subject is, after all, an event in the world, such that our entrapment in the solipsistic circuitry of ideality is always already minimally revelatory of that which we are searching for once we make a parallax shift in perspective. Secondly, the very inconsistency of our notional apparatus points to the fact that there can be no absolute constructionism, which appears to epistemologically justify the possibility of using the internal limitations and obstructions of ideality to overcome ideality's very self-enclosure. The goal of Žižek's materialism is to seek to understand the precise philosophical status of the free ideal constitution of the world and how and why this autonomous register could collapse. In other words, its defining feature is that, although it strives to maintain the ontological significance of the irreconcilable difference between materialism and idealism, it searches for a way to overcome this absolute opposition at the epistemological level so that the conflict between them does not infringe upon knowledge. If the self-grounding gesture of idealism expresses a constitutive opposition to materiality, and its failure is indicative of the phantom-like presence of the latter in the former, then this disharmonious non-relation must reveal a method of explaining it. The underlying question guiding Žižek's ontology is therefore the following: What do the internal obstructions in idealization and the fact of ideality itself tell us about the mind-body relation: that is, our connection to the vital movement of being? Whereas there have been for some time in the contemporary intellectual scene two options for understanding the relationship of consciousness and world—their dynamic interconnectivity and unity in phenomenological accounts of the lived body or the outright rejection of the importance of lived first-person experience as a mere epiphenomenal effect due to the mechanical movement of nature or the structures guiding discourse, both of which comprise a disavowal of the primordial self-reflexive ipseity of the subject—Žižek opts for a third. 3.3 A Metaphysics of the Disjunctive “and” By zoning in on the limitations of idealization, the experiences of internal resistance within its own self-enclosed phenomenal space (experiences that reveal a difficult truth concerning the impotence of self-positing idealist freedom), Žižek tries to construct his own metaphysics. Only able to sustain itself from within the cracks of transcendental synthesis, his parallax ontology functions within the impossible in-between of spectral materialism and full-blown subjective idealism. But Žižek does not just claim that the latter is wholly untenable because its internal tension proclaims that there is necessarily an extimate, spectral presence of the Real within the Ideal haunting it due to its impotence to hallucinate its own world. What is Chapter 3 What is noticeable in all of these approaches is not only an inability to grasp the true insight unveiled by a transcendental freedom, but also the latter's direct Psychoanalysis and the Enigma of Transcendental Subjectivity 63 Psychoanalysis and the Enigma of Transcendental Subjectivity 63 consequence: namely, the experience of idealistic disintegration and failure, the two types of experiences being so crucial in Lacanian psychoanalysis for our understanding of psychopathologies and psychogenesis that for Žižek they demand the rehabilitation of the ontologically schismatic duality opened up by Cartesian subjectivity. These are not merely revelatory of the structure of an individual psyche lost in disarray, but rather reveal a basic metaphysical truth of subjectivity by presenting a world where “the mind and body are, so to speak, negatively related—oppositional discord is, obviously, a form of relation.”75 Pointing towards the subject being more than the matter it inhabits, insofar as the symbolic structures constituting psychic life display a quasi-absolute degree of freedom from purely naturalistic activity, the psychoanalytical experience proclaims that these two zones must resist, must be in perpetual conflict with, one another, so that the structure of psychoanalytical subjectivity is brought close to an archaic form of modern dualism while calling for a radical reconfiguration of the latter's split between mind and body. Accordingly, Žižek's attempt to think materialism and idealism requires a far-reaching remodulation of the logical conjunctive between the two into a form of psychotic non-relation, insofar as transcendentalism implies a kind of negative space isolated unto itself and alienated from external reality, an isolation that is simultaneously the logical structure of normal and pathological subjective reality. But what exactly is this disjunctive “and”? Žižek's answer is unequivocal: the place of non-coincidence between mind and body, the break or rupture between these two zones of independent activity, is nothing but the subject itself, where the subject is transformed from a mere transcendental- epistemological construct grounded through concerns in a theory of knowledge into some kind of self-positing negativity in material being, a bone in substance forever holding apart materialism and idealism. Psychoanalysis and the Enigma of Transcendental Subjectivity 63 The lacuna in contemporary thinking is therefore immense: by failing to take into account the experience of psychopathology whose possibility coincides with that of freedom (as that which institutes an infinite conflict between mind and body) its representatives fail to see the terrifying truth intrinsic to our very relation to the world: something that proclaims the need for a new paradoxical form of materialism developed within idealism to be able to consequence: namely, the experience of idealistic disintegration and failure, the two types of experiences being so crucial in Lacanian psychoanalysis for our understanding of psychopathologies and psychogenesis that for Žižek they demand the rehabilitation of the ontologically schismatic duality opened up by Cartesian subjectivity. These are not merely revelatory of the structure of an individual psyche lost in disarray, but rather reveal a basic metaphysical truth of subjectivity by presenting a world where “the mind and body are, so to speak, negatively related—oppositional discord is, obviously, a form of relation.”75 Pointing towards the subject being more than the matter it inhabits, insofar as the symbolic structures constituting psychic life display a quasi-absolute degree of freedom from purely naturalistic activity, the psychoanalytical experience proclaims that these two zones must resist, must be in perpetual conflict with, one another, so that the structure of psychoanalytical subjectivity is brought close to an archaic form of modern dualism while calling for a radical reconfiguration of the latter's split between mind and body. Accordingly, Žižek's attempt to think materialism and idealism requires a far-reaching remodulation of the logical conjunctive between the two into a form of psychotic non-relation, insofar as transcendentalism implies a kind of negative space isolated unto itself and alienated from external reality, an isolation that is simultaneously the logical structure of normal and pathological subjective reality. But what exactly is this disjunctive “and”? Žižek's answer is unequivocal: the place of non-coincidence between mind and body, the break or rupture between these two zones of independent activity, is nothing but the subject itself, where the subject is transformed from a mere transcendental- epistemological construct grounded through concerns in a theory of knowledge into some kind of self-positing negativity in material being, a bone in substance forever holding apart materialism and idealism. Psychoanalysis and the Enigma of Transcendental Subjectivity 63 The lacuna in contemporary thinking is therefore immense: by failing to take into account the experience of psychopathology whose possibility coincides with that of freedom (as that which institutes an infinite conflict between mind and body) its representatives fail to see the terrifying truth intrinsic to our very relation to the world: something that proclaims the need for a new paradoxical form of materialism developed within idealism to be able to combat phenomenological accounts of embodiment, postmodern theories Psychoanalysis and the Enigma of Transcendental Subjectivity 65 fundamental problematic guiding Žižek is consequently the following: what is the relationship between (i) this abyssal void of the I; (ii) the pre-symbolic, material X; and (iii) the mediating activity of conscious ideality? What are the “meta-transcendental” conditions of possibility for the emergence of the subject that could explain the systematic discord that persists between mind and body? Can the psychoanalytical notion of the Todestrieb as a form of biological malfunction and its avatars in the tradition tell us something about the extra-ideal state of affairs logically preceding experience? But in order to grasp the stakes at hand in this endeavour, we must first understand the problem of nature bequeathed by Lacan and how it sets up the coordinates of his own procedure. Chapter 3 64 of discourse, and cognitive science, a materialism that would be at the same time a metaphysics of the disjunctive “and.” Since it is Lacanian psychoanalysis that most strongly brings forth this disjunctive “and” as an irreducible element of human experience, Žižek's controversial thesis is not merely that Lacan brings to the fore something previously underdeveloped or neglected in other theories of subjectivity. Rather, it is much stronger—he claims that Lacan constitutes a true breakthrough in the history of thought that irreversibly restructures the field within which we can do philosophy, in a gesture that rivals that of Kant. Yet the great downfall of Lacan is that he is only interested in the systematization of the breakdowns and structural inconsistencies that define psychoanalytic experience, the antagonistic interaction of the three registers as the condition of the (im)possibility of phenomenal reality, and largely leaves unanswered the ontological question of the subject. Realizing that the Lacanian subject is lacking any explicit account of its genesis—which leads many commentators to argue that Lacan is a transcendental idealist or linguistic phenomenalist—Žižek is driven back to German Idealism to do away with this theoretical deficiency. But if German Idealism, alongside Lacanian psychoanalysis, is able to help us elucidate the paradoxical “and” negatively linking together materialism and idealism, it is because there must be something homologous in their attempts to think the subject that would allow them to be creatively read through one another. By drawing out various passages that link the basis of subjectivity in late modern philosophy to fundamental concepts of psychoanalysis, Žižek gives flesh to this disjunctive “and” by showing how the very fact of the existence of the field of ideality illustrates the necessity of the existence of the former, a fact from which many recoil. In this respect, if the Symbolic cannot assert itself as all, it is not only because of the ever-present pressure exerted from the inside upon its constitution of phenomenal reality, but also because the very “and” that precedes and sets the stage for the autonomous universe of meaning prevents the latter from being able to posit itself as a self-sustaining, total positivity. Idealization always collapses upon itself, and it is due to its self-foundering that we are led to see that there is a disharmonious relationship between mind and body that requires a new materialism to complement its internal limitations. The 73. Jacobi, “Idealism and Realism,” in Kant’s Early Critics: The Empiricist Critique of the Theoretical Philosophy, ed. Brigitte Sassen (Cambridge: Cambridge University Press, 2000), p. 175. Notes 64. Lacan, “A Theoretical Induction to the Functions of Psychoanalysis in Criminology,” in Écrits, p. 127/104. 65. Žižek, The Parallax View, p. 168. 66. Žižek, Less Than Nothing, p. 905. 67. As Žižek points out, as soon as Descartes makes the breakthrough to the cogito, he is forced to conclude: “[b]ut I do not yet understand sufficiently what I am—I, who now necessarily exists.” What follows is a discussion that prevents the confla- tion of the cogito with any specific experiential content—in short, the recognition of its intrinsic vacuous nature. However, Descartes is unable to hold onto this groundbreaking insight and quickly ends up reifying the subject into a res cogitans. See Meditations on First Philosophy, trans. Donald A. Cross (Indianapolis: Hackett Publishing, 1993), pp. 18ff.; and Žižek, “Cartesian Subject versus Cartesian Theater,” in Cogito and the Unconscious, ed. Slavoj Žižek (Durham: Duke University Press, 1998), pp. 259–61. 68. On Žižek’s reading, not only is any intimate self-relation in Kant precluded in- sofar as the self qua phenomenal is denied any experience of its “true being,” but subjectivity is reduced to a positive void: the ground of self-reflexive consciousness becomes a mysterious “this I or he or it (the thing) that thinks” that strangely guarantees the condition of possibility of experience. See Kant, The Critique of Pure Reason, A346/B404; and Žižek, Tarrying with the Negative: Kant, Hegel, and the Critique of Ideology (Durham: Duke University Press, 2003), pp. 12–18. 69. This concept will be explored in detail in chapter 6. 70. Žižek, The Abyss of Freedom, p. 45. 71. See Johnson, Žižek’s Ontology, pp. 16–20. 72. Žižek, The Abyss of Freedom, p. 45. 74. Johnson, Žižek’s Ontology, p. 19. 75. Ibid., p. 56. This is Johnston’s thesis about the nuance of Lacanian-Žižekian subjectivity. Chapter 4 The Problem of Nature in the Lacanian Subject The Obscure Origins of the Symbolic Chapter 4 The Problem of Nature in the Lacanian Subject The Obscure Origins of the Symbolic In this chapter I will lay out various theoretical problems plaguing Lacan that serve as the starting point for Žižek's own endeavour. Beginning with a precursory analysis of Žižek's identification of the Cartesian and psychoanalytical subjects, I move on to a discussion of the intrinsic limitations of Lacan's psychoanalytical methodology for understanding the ontologico-foundational basis of subjectivity. By focusing on the self- grounding, non-natural function of images and words, especially the transcendental reflexivity underlying both, Lacanian psychoanalysis has a tendency to perceive them as an external parasite that derails the substantial unity of the body. But this tendency renders problematic any account of how images and words could successfully implant themselves, thus leaving the Symbolic itself potentially without theoretical justification. Seeing an uncanny structural homology here between post-Kantian idealism and the legacy bestowed upon us by Lacan, Žižek returns to Hegel and Schelling with the conviction that a combination of psychoanalysis with late German Idealist metaphysics could help us articulate the paradoxical emergence of subjectivity. 68 Chapter 4 4.1 An Uncanny Pair: The Cartesian and Psychoanalytical Subjects Lacan's and Žižek's situating of the psychoanalytical discourse within the trajectory of modern transcendental philosophy could appear surprising. Following the works of Freud, it is clear that the father of psychoanalysis understands the psychoanalytical subject as intrinsically anti-Cartesian. Whereas the cogito conjures forth the idea of an untouchable conscious freedom, of complete self-mastery and perfect self-transparency as guaranteed by our capacities of reason, ratiocination, and deliberation, the Freudian unconscious does not merely haphazardly obstruct existential self-familiarity like some kind of accidental feature of human being, but rather puts all conscious activity in the clutches of a mysterious, obscure Other that by definition resists all ideational (re)presentation. The goal of modern philosophy as the articulation of a sound foundation for systematic philosophy through the free mediation/idealization of the world by means of the rational, self-grounding activity of consciousness is by definition foreclosed, for the very process of (re)constituting reality by the I rests upon a structurally non-representational process, a process that constantly risks interfering with the subject's synthetic mediation of self and world. It is no longer the subjective I that “thinks,” but the impersonal, non- subjective Es (the id): the “it” thinks in the same way that “it” rains. So how can “the subject of the unconscious [be] none other than that of the Cartesian cogito”?76 Although this may be true it is evident that, despite the qualitative remodulation that subjectivity undergoes in Freudian psychoanalysis, especially in terms of the nature and scope of conscious personality, there is nevertheless a series of homologous structures at the heart of both the psychoanalytical and modern Cartesian subjects. Most fundamentally, both rely on the splitting of being into two separate zones, the mental and the material, and the inability to reduce one to the other. Even if Freud's later theoretical works exhibit a propensity to reduce psychic processes to the vital force of biological activity, this very attempt self-destructs (as Lacan makes clear) in the face of the brute autonomy of images and words in his analyses over and against their subsumption within the pulsating activity of nerves or the movement of instincts. There always remains a chasm The Problem of Nature in the Lacanian Subject 69 69 separating human psychological life from the mere flux of corporeal or instinctual energetics, because ideality and materiality are operationally different registers in terms of their inner articulation or logical makeup. 70 Chapter 4 In Žižek's view, the theoretical advantage of Cartesianism and psychoanalysis is their defence of the profound ontological difference between the registers of objective material corporeality and (inter)subjective cultural and psychic experience. Difference is just another word for an irresolvable two-way resistance that constitutes a battleground of conflicting, oppositional systems and thereby sets the very stage for the experiential reality that we find ourselves thrown into. To speak in Freudian parlance, one cannot understand consciousness merely in terms of the id-forces that comprise the bodily substrate of the libidinal-material basis of human existence. Although they may serve as the ontologico-foundational basis for all psychic and ultimately phenomenological life, they are philosophically inadequate to explain the full essence of personality and its pathologies insofar as the latter are constituted through the antagonistic interaction of forces that emerge within the tripartite field of id, ego, and superego. None of these alone would be able to articulate what it is to be a human subject, but must be implicated within an intricate psychological trinitarian logic if we are to hope to come to terms with ourselves and our world of experience. Even though the ego and superego arise out of the pre-subjective activity of the id—the id immanently generates the ego out of itself as a way to cope with its own organic insufficiency and primordial Hilflosigkeit to deal with the demands of its external environment—they are not dependent upon it for their being, their very vitality, each constituting a self-subsisting system enjoying a level of genuine autonomy and irreducibility to the other. This very structure of independence is also hinted at in the breakthrough that is the Cartesian subject, which argues that although we could attempt to understand the ideality of spirit by recourse to the mechanics of nature, this attempt would ultimately collapse upon itself because something essential would be lost. It is not that the mechanical externality that forms the causal determinacy of the body's extensive field as the obscure ground of mind and its sensations is a mere underdetermination of mental activity. 70 Chapter 4 Descartes’ claim is much stronger: it is ultimately incapable of helping us establish the nature of subjectivity, even if some kind of paradoxical non-relation must nevertheless be said to hold between the two; the cogito's founding gesture exhibits an irreducible self-grounding that rejects mechanical externality in all of its modes (or however it may be reworked by In Žižek's view, the theoretical advantage of Cartesianism and psychoanalysis is their defence of the profound ontological difference between the registers of objective material corporeality and (inter)subjective cultural and psychic experience. Difference is just another word for an irresolvable two-way resistance that constitutes a battleground of conflicting, oppositional systems and thereby sets the very stage for the experiential reality that we find ourselves thrown into. To speak in Freudian parlance, one cannot understand consciousness merely in terms of the id-forces that comprise the bodily substrate of the libidinal-material basis of human existence. Although they may serve as the ontologico-foundational basis for all psychic and ultimately phenomenological life, they are philosophically inadequate to explain the full essence of personality and its pathologies insofar as the latter are constituted through the antagonistic interaction of forces that emerge within the tripartite field of id, ego, and superego. None of these alone would be able to articulate what it is to be a human subject, but must be implicated within an intricate psychological trinitarian logic if we are to hope to come to terms with ourselves and our world of experience. Even though the ego and superego arise out of the pre-subjective activity of the id—the id immanently generates the ego out of itself as a way to cope with its own organic insufficiency and primordial Hilflosigkeit to deal with the demands of its external environment—they are not dependent upon it for their being, their very vitality, each constituting a self-subsisting system enjoying a level of genuine autonomy and irreducibility to the other. This very structure of independence is also hinted at in the breakthrough that is the Cartesian subject, which argues that although we could attempt to understand the ideality of spirit by recourse to the mechanics of nature, this attempt would ultimately collapse upon itself because something essential would be lost. It is not that the mechanical externality that forms the causal determinacy of the body's extensive field as the obscure ground of mind and its sensations is a mere underdetermination of mental activity. 4.1 An Uncanny Pair: The Cartesian and Psychoanalytical Subjects For Žižek, it is not a question of the actual doctrine espoused by Descartes with respect to his own doctrine of the cogito and the essence of consciousness it entails, but rather of various theoretical potentialities and possibilities exhibited by the very structure inherent in his concept of subjectivity, which come to the fore in the aftermath of psychoanalysis. Consequently, what Žižek perceives in the Cartesian subject and its various reworkings is the possibility of extracting theoretical resources from it that could be used to help explicate the enigma of human freedom and the essence of personality as revealed in the psychoanalytical experience of the conflictual (non-)relation that holds between mind and body. In Cartesianism and psychoanalysis, we do not come across a mere conceptually regulative difference between the arena of human subjective activity and the raw processes of brute material nature, which would enable both to enjoy a strong degree of epistemic autonomy insofar as both could self-actualize themselves in terms of a standalone language game with the appearance of being intrinsically incommensurate to one another despite being on the ontological level fundamentally one. Žižek is unwilling to reduce this difference to the level of useful “working distinctions” that have the status of an “as if,” for consciousness refuses to be completely naturalized. In a similar vein, in contradistinction to a theory of monism according to which both are merely seen as attributes of a single, overarching whole (however it may be conceived) and whereby both would offer differing expressions of an ultimately identical self-same reality, Žižek wants to bestow ontological heft upon the difference. The claim is that any account of subjectivity that attempts to reduce conscious experience to the register of a pure material or substance caught within self-sustaining laws, whether it be the neuronal Real, vitalistic energetics, or nature as all, not only does not take heed of the radical metaphysical implications of the psychoanalytical experience, but is also by consequence unable to propound a satisfactory account of what it is to be a human and our relation to the world. 70 Chapter 4 Descartes’ claim is much stronger: it is ultimately incapable of helping us establish the nature of subjectivity, even if some kind of paradoxical non-relation must nevertheless be said to hold between the two; the cogito's founding gesture exhibits an irreducible self-grounding that rejects The Problem of Nature in the Lacanian Subject 71 reinterpreting it as nature, vitalistic energetics, the neuronal Real, etc.) in a process that not only makes it distinctively non-natural in terms of its inner structuration, but also thereby guarantees its capacity for transcendental self-determination and its incommensurability with other logical registers. The question is how we can explain this ontological difference opened up by Cartesianism with the aid of psychoanalysis, and vice versa. 4.2 Lacan, antiphusis, and the Parasite of Images and Words In order to expand upon this problematic, two comments can be made that will enable us to uncover the conceptual limitations inherent to the psychoanalytical theory of subjectivity and that allow us in turn to draw out its similarities with the central conceptual problems plaguing post- Kantian German Idealism as bestowed upon it by its Cartesian heritage. First, for psychoanalysis, the language that we use mediates our very experience of the biological constitution of the body in such a way that there is a radical degree of separation between the idealization of the givenness of the flesh and its objective, extra-subjective existence in itself. Freud speaks of people who suffer from paralysis of a certain part of their body, although scientifically speaking the paralysis does not coincide with where the nerve anatomically ends, and when this is pointed out the pain ceases. Here it is the common, everyday belief of where a nerve ends and begins that structures the symptom. Thus, understood as a complex cultural phenomenon and not just grammatical structures of syntax and morphology, language shows itself as an irreducible element of experience and points to a discordant relationship between mind and body. Second, as arising from the facticity of cultural, historical experience, language appears to emerge out of a symbolic interpersonal network of meaning that operates according to its own intrinsic and self-unfolding logical matrix. Even if it has a basis in our physiology (vocal folds) and neurology (language centres in the brain), words themselves appear to exist in an autonomous world of their own that, in many ways, shows itself as a kind of external parasitic attachment to the biological unity of the body. Taken together, these two points assert more than the fact that both registers, the Symbolic and the corpo-Real, exist in disunion: the former even appears to manifest itself as a “psychotic” withdrawal from the latter into some kind 72 Chapter 4 of self-enclosed sphere. What is obvious from this is not only the intrinsic Cartesian structure of the psychoanalytical subject—here the deadlock of ontological dualism is amplified, brought to its extreme, by the advocation of a constitutive dissonance—but that the latter's philosophical legitimacy depends upon the precise articulation of the delicate relation between these two mutually incommensurate spheres. The Problem of Nature in the Lacanian Subject 73 73 the Husserlian transcendental ego grounds itself and the fabric of the world of its experience in a moment of absolute freedom that establishes an insurmountable rupture between reflexive self-consciousness and the causal mechanics of nature, within Lacanian psychoanalysis we encounter the same logical move, but merely displaced. In the latter it is not a subject in any traditional sense, but rather the self-generating play of signifiers within the web of the Symbolic that expresses the impossibility of articulating a comprehensive, satisfactory psychological theory of the self only by recourse to the pulsations of neurological activity or biological instincts. There are two principal reasons for this. First, the Symbolic constitutes the possibility of the orbit of the Imaginary within phenomenological experience in such a way that it autonomously mediates all contact with the outside world, thus diminishing the latter's importance; second, as a consequence, it represents the predominance of non-natural (= irreducible to the ebb and flow of the material-objective world) influences in the explanation of the essence of psychic life. It is in this precise manner that the ciphering activity of the Symbolic can be said to obey its own self-grounding transcendental logic and be that which sustains the primordiality of the real-ideal divide: “subjectivity has no relationship to the Real, but rather to a syntax which is engendered by the signifying mark.”77 Accordingly, one can now understand why Lacan defines the object of psychoanalysis in “The Direction of the Treatment and the Principles of its Power” as “antiphusis.”78 With this terminological choice he is drawing our attention to the fact that what analysis deals with is not something natural in any sense of the nature investigated by science or understood by our common sense. Analysis operates at a level that is, by definition, incommensurate with natural laws. The effect of this designation is much greater than it may initially appear, for the human subject is characterized as “non-natural” not only insofar as the structural fabric forming the base of conscious experience enjoys a freedom over and above, or an ontological heft irreducible to, the flux of nature, but also because it transcendentally alienates us from the immediacy of the corpo-Real of our body, the ground of our objective substantial being, as the very condition of our possibility of what we call experience. 4.2 Lacan, antiphusis, and the Parasite of Images and Words Although these zones are infinitely conflictual and never to be harmonized, they must nevertheless be somehow related if the system is to be held together. The Cartesian problem of the mind-body relation is consequently inverted: the issue at hand is not how both can be in unison with one another through some kind of occasionalism, but how the conflictual gap that separates them emerged in the first place so that there can never be a harmony or overlapping of each sphere. Both Freud and Lacan spend the majority of their careers elaborating this field of disparate spheres whose interaction generates and constitutes personality. Rereading Freud through contemporary developments in linguistics and structuralism and even drawing upon cybernetics, Lacan attempts to reconfigure our understanding of the self in what he believes to be a more scientific manner than Freud could by excavating the strict logical structures constituting the condition of the possibility of the psychoanalytical subject: that is, the irreducibly non-natural basis of experience and the mind-body discord it reveals. There is something intrinsically transcendental about the project, even if the end result is apparently quite removed from the Kantian analysis of consciousness in the Critique of Pure Reason, Fichte's Wissenschaftslehre, or later attempts such as Husserl's transcendental idealism. Any project that advocates the autonomy of subjectivity over materiality and a form of self-grounding idealization or symbolization of reality must at some level seem so, for the question of the condition of the possibility of experiential reality does not pose itself in more “realist-inclined” philosophies insofar as experience is here seen as just the effect or another instantiation of a large set of dynamic relations between objects in the world. As a consequence, fighting against Freud's attempt to biologize/naturalize the unconscious by highlighting that one can never escape the ultimate primacy of images and of words in psychoanalysis, Lacan relentlessly maintains what he perceives as the Cartesian essence of subjectivity. Just as the Kantian, the Fichtean, and even to a certain extent The Problem of Nature in the Lacanian Subject 7 The Problem of Nature in the Lacanian Subject 73 Images and words not only precede us historically in terms of the temporal genesis of our identities (we are often named before Chapter 4 74 we are conceived: a space is always already carved up for us in the symbolic universe before we can be said to exist in the Real) but also structure the logico-formal space that makes personality possible in such a way as to prevent us from having a genuine access to the brute reality of our own “intimate” being as it is an sich. After the I is spoken, that is, after self- reflexive subjectivity emerges, an experience of the Real is only possible through the ideational (re)presenting capacity of a symbolic network of signifiers, thus becoming impossible: a mysterious, ever-elusive X. As soon as we enter language, the Real as a time of “fullness,” “immediacy,” and “complete immersion,” if ever there was one, is subsequently lost forever, leaving us trapped within the fragmented, ontologically solipsistic world opened up through the Symbolic-Imaginary matrix. Insofar as the symbolic network simultaneously constitutes the underlying structural support of experience and mediates all our contact with an external, subject-independent reality, Lacanian psychoanalysis appears to be a completely self-grounding transcendental idealism in the spirit of Kant. Whatever the human subject is, it is not natural in the same sense as other objects within phenomenal reality, even those that appear to have some kind of spontaneous connection with that which they find around them. Indeed, according to Lacan even animals have a kind of knowledge in the Real79 that is missing in us due to the Symbolic. Although language is intimately linked with “our” freedom—and “our” madness, we must never forget, the two being structurally identical—nevertheless it thus appears as some kind of alien, foreign presence that cannot be understood in terms of the categories of the world because it has no analogue within it. But then the question arises: where do these transcendental structures come from? How and why do they irredeemably separate the human subject from the vital ebb and flow of nature? The Problem of Nature in the Lacanian Subject 73 As a science of the symbolic network that constitutes the nature of personality and self-conscious experience, if psychoanalysis is unable to articulate the origins of these non-natural structures and the process of their genesis, it itself rests groundless insofar as the object of its inquiry remains unable to explain its own theoretical, ontogenetic basis: that is, how it itself is possible as a discourse. As Fichte, Schelling, and Hegel looked upon Kant's critical philosophy, they themselves were taken by a similar problem concerning the heritage The Problem of Nature in the Lacanian Subject 75 bestowed upon them. They asked themselves how the transcendental structures of subjectivity could be explained in a more comprehensive manner, how and why they emerged in the first place, and why they were as such necessary for the very possibility of experience. They held the brute givenness of the categories of understanding to be philosophically unacceptable and took it upon themselves to explore the Achilles' heel of the entire critical system. Their guiding intuition was that by undertaking an explanation of their genesis they would not only save the Kantian breakthrough, but also delve into the wider implications of the subject as propounded by it. Similarly, there is a brute givenness in Lacan's account of the transcendentality of the symbolic matrix because he never gives an account of its origins. When one reads his early and middle works, one is consequently struck by the impression that images and words are a purely external force that attacks the raw unity of the body and devastates it from the outside, thereby creating a denaturalized human subject out of a preexisting substantial whole. There is a movement from uncontaminated instinctual being within the positive order of the Real—the oneness of the body, the immediacy of an organic nature—to a being that speaks, to words as intruders that radically split the subject, destroying its immersion in objective being. The network of the Symbolic comes from an infinite elsewhere ravaging the unity of substance and precluding the possibility of a biologically closed libidinal economy as an inborn, innate movement of energy defined by materially articulated, instinctual schemata. The Problem of Nature in the Lacanian Subject 73 Whereas in the animal kingdom this schema directs bodily energy towards various objects that it needs within its immediate environment through a kind of immanent causal push, language eternally obstructs the dialectic of need and satisfaction, fullness and lack, by instituting an ontological going-haywire in being. Preventing the flow of the search for homeostatic constancy, the subject is thrown into the endless deferral of desire and structurally loses the possibility of attaining its object. Instead of predetermined, biological goals, we are left with the objet petit a: [T]he standard Lacanian theme in the 1950s and 1960s was h bl i i b h i l i [T]he standard Lacanian theme in the 1950s and 1960s was the unsurmountable opposition between the animal universe of imaginary captivity, of the balanced mirror-relationship between Innenwelt and Aussenwelt, and the human universe 76 Chapter 4 76 Chapter 4 of symbolic negativity, imbalance. Lacan thereby fully participates in the line of thought that begins with Hegel, according to which man is “nature sick unto death,” a being forever marked by traumatic misplacement, thrown “out of joint,” lacking its proper place, in contrast to an animal which always fits into its environment, that is to say, is immediately “grown into” it. Symptomatic here is Lacan's “mechanistic” metaphorics: an almost celebratory characterizing of the symbolic order as an automaton that follows its path, totally impervious to human emotions and needs—language is a parasitic entity that battens on the human animal, throwing his or her life rhythm off balance, derailing it, subordinating it to its own brutally imposed circuit.80 Within this advocacy of a split between the cyclical, balanced world of nature and the derailed being of man, the following theoretical concerns immediately emerge: how does the symbolic-imaginary matrix give rise to itself through a transcendental act of self-positing? How is it able to take control of and infiltrate the body, apparently lacerating its objective unity? If the symbolic order is really an external force, an alien blow, that in some way tears apart or obstructs the body's knowledge in the Real, how is it able to sustain itself in face of the corpo-Real of the biological organism? To put it another way, why is it not “rejected by this economy in a manner analogous to failed organ transplants”?81 The problem of the psychoanalytical constitution of human experience opens up unto the extra- psychological/meta-transcendental conditions or state of affairs that must have preceded the emergence of the subject if we are to give a complete account of the psyche as such. Psychoanalysis must explain its own beginnings in a lost ontological time in a move similar to how the categories of the understanding must not be taken as mere elementary facts liberated from the theoretical exigency of explaining their genesis. Just as the history of the reception of Kantian idealism has shown that it must open up onto a comprehensive metaphysics that is inclusive of it, so too we must try to develop a metaphysics in the wake of psychoanalysis. The Problem of Nature in the Lacanian Subject 77 4.3 German Idealism, Psychoanalysis, and the Quest for the Birthplace of the Transcendental Yet after the 1950s and 1960s, we see within Lacanian psychoanalysis a developing preoccupation with the ontologico-foundational basis of the subject. After the period of Lacan's “The Direction of the Treatment and the Principles of its Power” (1958), which defines the object of psychoanalysis as “antiphusis,” there is a profound shift in the meaning of this concept that is highly significant for the development of a metaphysics capable of explicating and thus justifying the psychoanalytical subject. Whereas the early to middle Lacan focuses on the derailing capacity of images that set the stage for words as that which parasitizes the corpo-Real of the body, which makes the human subject radically non-natural by preventing its immersion in the autopoietic, self-regulating unity of positive organic being by creating an alienating self-distance within it, we encounter in the late Lacan a reconceptualization of the problematic. His new emphasis on the Real proclaims that one must radicalize this notion of self-distance. The object of psychoanalysis and by consequence the human subject are not antiphysical because images and words are an infinite Other to nature, an external alterity that arises ex nihilo only to penetrate into its secret chamber like a vandal and deface its sacred inviolability. The incommensurability lies elsewhere: images and language cannot be the cause of the denaturalization of the human subject; there must be something in nature itself that immanently moves it toward denaturalization. In short, nature itself is antiphusis, self-sabotaging, self-lacerating. Although this represents a fundamental change in the meaning of the category of the non-natural, it only serves as a beginning. Insofar as the psychoanalytical experience is engulfed by images and words, which means that it is predominately ridden by non-biological influences, the only way to philosophically ground the primacy of psychoanalysis is to elaborate an underlying ontology that could explain how the subject could have emerged out a biological field that it presents itself as irreducible to. The theoretical merit of the Lacanian project is dependent upon the articulation of the workings of substantial reality that renders possible the birth of a more-than-material subjectivity out of its material Grund (to borrow an expression from Adrian Johnston), a paradoxical ontologization of the 78 Chapter 4 8 Chapter 4 8 Chapter 4 If our generation of The Problem of Nature in the Lacanian Subject 79 79 experience proves itself to be unable to posit itself as an absolute and unbridled hallucination of objects, then we must encounter some kind of paradoxical limit to our autonomous ciphering and constitution of phenomenal reality from within; the impasses obstructing the self-grounding idealization of the world demonstrate that, although we are forever stuck within ideality, we are not simply prisoners of the completely solipsistic sphere of the self-referential, masturbatory play of thought within thought and that a metaphysics of the Real, an account of the noumenal, appears to be theoretically possible. The inassimilable kernel of the Real within our notional, symbolic code points to the paradoxical negative coinciding of inside with outside, the Real and the Ideal, within thinking: the cracks of ideality cast an abyssal shadow that opens up onto the materiality of being, albeit only as refracted through the impossibilities of the Ideal, in such a way that tarrying with the latter offers a way to develop idealism into a science of the Real. Idealism has been overcome because the materialism-idealism distinction has become intra-discursive, that is, internal to idealism itself. In this sense, we should not understand Žižek's proclamation of Hegel's “transcendentalism” to say that Hegel, like Kant, remains at the level of a pure subjectivism: due to the constitutive movement of notional tension intrinsic to the concept of dialectics, not only is idealism prevented from being subjective, but, more strongly, “[t]he opposition between idealistic and realistic philosophy is therefore without meaning,” which is why idealism can be said to be absolute.83 Embracing the paradox of the critical system, its breakthrough articulation of the irreducible idealizing activity of consciousness, the mediative structures that freely transcendentally constitute the fabric of experience, Hegel's philosophy is a daring attempt to rethink the possibility of a metaphysics in the wake of the Kantian revolution, to carve a space between idealistic ontological solipsism and a speculative materialism within the former. It is only within such a space that the question: “What is the transcendental subject's relation to nature?” or, in Lacanian parlance, “What is the Symbolic's relation to the pre-symbolic Real?” can be asked. 8 Chapter 4 78 transcendental structures that constitute and found experience by separating themselves from and effacing their ontological foundation. It is exactly this problematic that thematically binds together the theoretical concerns of the psychoanalytical and German Idealist traditions, for according to the latter the only way to save the breakthrough of the Kantian critical system is to find a way, from within the self-founding idealism that it advocates, to reach out into the extra-subjective Real, or to speak in its parlance, the field of noumena, and ground it within a system that does not represent a return to previous modes of thinking (naïve dogmatic realism) but rather somehow manages to reach the Real through the Ideal despite the self-enclosed, self- subsistent ideal activity of consciousness. But how? The very trap in which we find ourselves proves to hold the way out: We can now see in what sense Hegel's logic remains “transcendental” in the strict Kantian sense—that is, in what sense its notional network is not merely formal, but constitutive of reality itself, whose categorical structure it describes. Hegel's Logic is the inherent tension in the status of every determinate/limited category: each concept is simultaneously necessary (i.e. indispensable if we are to conceive reality, its underlying ontological structure) and impossible (i.e. self-refuting, inconsistent: the moment we fully and consequently “apply” it to reality, it disintegrates and/or turns into its opposite). This notional tension/contradiction is simultaneously the ultimate spiritus movens of “reality” itself: far from signalling the failure of our thought to grasp reality, the inherent inconsistency of our notional apparatus is the ultimate proof that our thought is not merely a logical game we play but is able to reach reality itself, expressing its inherent structuring principle.82 Looking back at the history of the inner development of modern transcendental philosophy, Žižek sees a structural homology to his own task of searching for the ontogenetic condition of the possibility of the psychoanalytical subject, a question left unanswered by Freud and Lacan, in the tradition's attempt to find a way to construct a metaphysics from within the internal deadlock of transcendental idealism. 8 Chapter 4 Žižek's retrieval of the cogito and his reactualization of German Žižek's retrieval of the cogito and his reactualization of German Idealism are therefore irrevocably caught up in his own attempt to develop a metaphysics of the Real able to explicate the obscure grounds of the Chapter 4 80 psychoanalytical subject. Attempting to excavate the ontological edifice implied by the latter, his more overtly theoretical works pass through modern to late modern philosophy by means of an intuition of a basic structural parallelism binding together such seemingly different traditions. Although the starting point is always psychoanalysis—“psychoanalysis is ultimately a tool to reactualize, to render actual for today's time, the legacy of German Idealism”84—it is clear that Žižek does not understand his work as an intrusion or unmethodological tearing apart of the tradition according to extrinsic philosophical principles he is imposing upon it. In fact, for him, the psychoanalytical subject is not merely a direct consequence of German Idealism's grounding insights—there is actually something intrinsically identical in these traditions, so much so that it seems that the only way to fully realize the former is to supplement it with the latter and vice versa, insofar as they are both really dealing with the same central issues that each fail in their own way to adequately thematize. Describing his own project, Žižek says: If you were to ask me at gunpoint, like Hollywood producers who are too stupid to read books and say, “give me the punchline,” and were to demand, “Three sentences. What are you really trying to do?” I would say, Screw ideology. Screw movie analyses. What really interests me is the following insight: if you look at the very core of psychoanalytic theory, of which even Freud was not aware, it's properly read death drive—this idea of beyond the pleasure principle, self- sabotaging, etc.—the only way to read this properly is to read it against the background of the notion of subjectivity as self-relating negativity in German Idealism. 8 Chapter 4 That is to say, I just take literally Lacan's indication that the subject of psychoanalysis is the Cartesian cogito—of course, I would add, as reread by Kant, Schelling, and Hegel.85 By zoning in on this basic identity, Žižek's philosophy doesn't lose methodological rigor, as if it made him blind to other, more broad-reaching concerns in the tradition, thereby reducing his thinking to a mere haphazard picking out of various conceptual structures and ideas that are useful for his own project to the neglect of others. Rather, it enables him to show The Problem of Nature in the Lacanian Subject 8 The Problem of Nature in the Lacanian Subject 81 Žižek's answer Chapter 4 82 not only questions our conventional reading of this movement, but more fundamentally challenges our very conception of ourselves and the world. not only questions our conventional reading of this movement, but more fundamentally challenges our very conception of ourselves and the world. The Problem of Nature in the Lacanian Subject 81 81 how there is an unconscious, disavowed Grundlogik that repeats and self- unfolds throughout modern transcendental philosophy and finally comes out into the open with the advent of psychoanalysis, which shows itself as the culmination of this entire lineage. Because Žižek perceives some kind of self-developing truth inherent within the tradition that binds it together with psychoanalysis, his reading is pushed on by a sense of fidelity to the movement as such so that his return to German Idealism resembles Fichte's, Hegel's, and Schelling's return to Kant86 just as much as Lacan's return to Freud: what is at stake is never merely textual faithfulness, but a hidden kernel of truth that has been simultaneously opened up and obscured by the tradition itself. The effect here is twofold: while psychoanalysis allows us to reconstruct retroactively the Grundlogik inherent in the German Idealist account of subjectivity by bringing to the fore the relevance of previously underemphasized concepts and textual moves, this reconstruction of its fundamental concepts also allows us to elaborate our understanding of the psychoanalytical subject. It is not merely that psychoanalysis reveals a hidden unconscious logic at work throughout tradition; the backward retroactive glance is simultaneously a forward-looking task that tries to pave the way for the new. Žižek's investigation into the history of philosophy is never a mere activity of philological exegesis, the retrieval of the “primordial” meaning of a text, but a creative generation of new concepts that, paradoxically, must be said to have been already present in the now lost factical past, yet to have existed there in the paradoxical temporality of a futur antérieur (“I will have been”) whose contours are now first visible in the après-coup reconstruction of said past. A repetition is always the establishment of a difference, so that the legitimacy of Žižek's reading rests not so much upon his apparent “lack” of faithfulness to the words or the spirit of the great German philosophers, but how this temporal intersection of philosophico-psychoanalytical interpretation, the space within which Žižek effectuates his reactualization, functions and realizes itself. But what is the true breakthrough of the German Idealist and psychoanalytic traditions and why can only a psychoanalytical dialogue between the two enable us to see the conceptual impetus underlying the former's unconscious Grundlogik and also to come to terms with the ontology of the subject? Notes 76. Žižek, Less Than Nothing, p. 632. 77. See Lacan, “Seminar on ‘The Purloined Letter,’” in Écrits, p. 51/38. I capitalize “the Real” for consistency. 78. Lacan, “The Direction of the Treatment and the Principles of Its Power,” in Écrits, p. 615/514. 79. Knowledge in the Real (savoir dans le réel) refers to the fact that “it is as if there is a knowledge of the laws of nature directly inscribed into the Real of natural objects and processes—for instance, a stone ‘knows’ what laws of gravity to obey when it is falling.” Žižek, How to Read Lacan (New York: W. W. Norton, 2007), pp. 74–75. 80. Žižek, The Indivisible Remainder, p. 218. 81. Johnston, Žižek’s Ontology, p. 271. 81. Johnston, Žižek’s Ontology, p. 271. 82. Žižek, The Ticklish Subject, pp. 98–99. 83. Hegel, The Science of Logic, trans. George di Giovanni (Cambridge: Cambridge University Press, 2010), p. 124. 84. Žižek, “Liberation Hurts.” 85. Ibid. 86. “The Kantian philosophy needed to have its spirit distinguished from its letter.” Hegel, The Difference between Fichte’s and Schelling’s System of Philosophy, trans. H. S. Harris and Walter Cerf (Albany: SUNY Press, 1977), p. 79. Fichte’s says the same thing: Kant should be “studied, not as the Kantians without exception have studied him (holding on to the literal text [...]), but rather on the basis of what he actually says, raising oneself to what he does not say but which he must assume in order to be able to say what he does.” Fichte, The Science of Knowing: Fichte’s 1804 Lectures on the Wissenschaftslehre, trans. Walter E. Wright (Albany: SUNY Press, 2005), p. 30. II Nature Torn Apart Nature Torn Apart Chapter 5 Kant, Todestrieb, and Beyond the Pleasure Principle The Unruly Basis of Transcendental Freedom Chapter 5 Kant, Todestrieb, and Beyond the Pleasure Principle The Unruly Basis of Transcendental Freedom In this chapter I begin an examination of Žižek's metaphysics by showing how the thematic intersection between German Idealism and psychoanalysis allows him to develop a highly original approach to the transcendental. Beginning with a brief discussion of Adrian Johnston's summary of Lacan's passing remarks on nature in largely unavailable seminars stemming from the late period, I then precede to analyze how these remarks lay the foundation for Žižek's own radical rethinking of the Symbolic-Real relationship. Trying to comprehend the emergence of the Symbolic, Žižek attempts to demonstrate that the very fact of its existence must be revelatory of some ontological process that set the stage for its immanent genesis out of the Real as an extimate Other, even if, in turn, it makes the very Real from which it arose an impossible concept. Perceiving the key for understanding this paradoxical point of discordant (non-) relation between the pre-subjective Real and the transcendental matrix of the Symbolic in the Todestrieb, and following Lacan's claim that “Kant's practical philosophy [is] the starting point of the lineage culminating in Freud's invention of psychoanalysis,”87 Žižek reactualizes the legacy of German Idealism in order to articulate his own parallax ontology through an intuition of a fundamental identity between Kantian transcendentalism and psychoanalysis as established in the former's concept of the primordial unruliness and diabolical evil at the core of subjectivity. 86 Chapter 5 86 5.1 From the Rottenness of Nature... Although Lacan lacks an explicitly developed philosophy of nature as a complement to his structuralist metapsychology, throughout his career we see a growing interest in and appreciation for the underlying ontology implied by his theory of the subject. Adrian Johnston succinctly describes the theoretical situation plaguing the late Lacan in his shift from the primacy of the Symbolic to that of the Real, a problematic of utmost importance for understanding the deficiency in Lacanian psychoanalysis that guides Žižek's own project: A psychoanalytically influenced theory of the subject that fails to furnish a basic delineation of human nature as the precondition for the genesis of subjectivity is groundless, incapable of explaining a foundational dimension of its object of inquiry. In the later seminars of the 1970s, a series of somewhat cryptic remarks testify to Lacan's awareness of the need to redefine nature itself in order to account for why human nature is predisposed to being thoroughly altered by the denaturalizing mediation of socio-symbolic structures. In both the twenty-first and twenty-fourth seminars, Lacan contends that nature is far from being entirely natural. However, this isn't just a slightly reworked reiteration of his earlier remarks from the 1950s about humanity's denaturalized nature. Rather than grounding his assertions here by invoking the externally imposed intrusion of images and signifiers as the ultimate cause of the denaturalization involved in subjectification, Lacan takes the additional step of pointing to something within nature itself that inclines it in the direction of its own effacement.88 What is missing in Lacan, however, is a fully worked out account of the consequences of this shift, a detailed investigation into the paradoxical ground of the subject intrinsic to the very gesture of psychoanalysis. Yet he makes a crucial advance by suggesting that the Real is not to be, despite the fact that we can only posit its existence from within the differential network Kant, Todestrieb, and Beyond the Pleasure Principle 87 Kant, Todestrieb, and Beyond the Pleasure Principle 87 of signifiers, merely taken as that which must be said to logically precede the emergence of the linguistic subject, but also as that which renders the latter in a certain sense possible by virtue of a self-destructive tendency always already within it that opens up the space for its infinite loss to self through the colonizing activity of images and words. We encounter a metaphysical thesis: subjectivity does not come on the scene as a scar inflicted upon an otherwise harmonious nature, as a disturbance of its symphonized order by means of a haphazard intrusion into its sphere of non-natural influences that produce an accidental zone of ontological non-coincidence. The psychoanalytical experience is, rather, revelatory of something much more primordial: namely, that nature itself must be always already antiphusis, self-sabotaging, self- lacerating, and responsible for its own demise in the human being's denaturalized essence. But why? Lacan provides only a few hints. At one point, he identifies “liberty” (liberté) with “the non-existence of the sexual relationship,” which, in light of the above, can be understood as indicating that the freedom enjoyed by the autonomous subject is made possible by the lack of an integrated organic foundation as the grounding basis of the subject's being. Similarly, several years later, Lacan speaks of nature as not all that natural due to being internally plagued by “rottenness” (pourriture), by a decay or defect out of which culture (as antiphusis) bubbles forth (bouillonner). Viewed thus, human nature is naturally destined for denaturalization. Put differently, more-than-material subjectivity immanently arises out of the dysfunctionality of a libidinal-material ground.89 Yet it would perhaps be erroneous to say that this theoretical awareness is limited to the late Lacan's move from the primacy of the Symbolic towards that of the Real. Kant, Todestrieb, and Beyond the Pleasure Principle 87 As early as 1949 in his work on the Imaginary and the mirror stage, Lacan had already said that “these reflections lead me to recognize in the spatial capture manifested by the mirror stage, the effect in man, even prior to this social dialectic, of an organic inadequacy of his natural reality—assuming we can give some meaning to the word 'nature,'” an inadequacy that points to “a certain dehiscence at the very heart of the organism, a primordial Discord betrayed by the signs of malaise and motor Chapter 5 88 uncoordination of the neonatal months”90 which is, in fact, responsible for the alteration we see in our relationship to nature in comparison with other animals insofar as it represents “the shattering of the Innenwelt and Umwelt circle”91 and thus functions as the true “intersection of nature and culture.”92 Here it is noteworthy to mention that the French word stade does not completely map onto the English stage. Although it does correspond in one of its principal meanings to the latter (a distinct stage in a process of evolution: les stades de la vie) it also means stadium (a terrain or area where something takes place) and thus signifies a primordial scene constituting the foundation or arena within/through which an activity unfolds. Consequently, Lacan's thesis is that the mirror stage can never be subsumed in a later phase of development, forcing us to conclude that, even in the early period, it must necessarily refer to some kind of self-effacing force immanent within nature that gives rise to and simultaneously sustains the ontogenetic condition of the possibility of the paradoxical emergence of a more-than-material subject. This force thereby institutes the infinitely denaturalizing process of flirtation with images and symbolic castration, so that organic discord in the motor coordination of the body is not a mere failure of the biological system but also a “positive” support that persists in its very non-naturalness even after the Imaginary and the Symbolic have taken hold as their dark origin. If the quasi-experience of dismemberment is to be taken as originary, as that which incites the libidinal investment of the captivating mirror picture the human infant sees of itself as the beginning of psychogenesis by letting itself be alienated by the Otherness of images and words, then nature here must also be seen—at least in the case of human being—as a festering, half-living corpse. Kant, Todestrieb, and Beyond the Pleasure Principle 87 The shift of emphasis in the late seminars is already contained within the founding texts of Lacanian psychoanalysis, which not only suggests their central thematic unity, despite the stark differences that they may exhibit, but more strongly a historical unfolding that follows an internal development conforming to the model of the Hegelian movement from the in-itself to the for-itself. But we should avoid looking at the various hints and suggestions in the i But we should avoid looking at the various hints and suggestions in the late Lacan that gesture towards the character of the material edifice upon which the subject rests as a mere immanent elaboration of the implications of his previously laid out position, given that there are important changes Kant, Todestrieb, and Beyond the Pleasure Principle 89 Kant, Todestrieb, and Beyond the Pleasure Principle 89 of position in the development of his thinking. What is important is not the unity or disunity of Lacan, but rather the radicality and nuance inherent in his thinking of the subject as brought to the fore when we focus on this very specific constellation of problems hovering around the obscure relation between nature and the essence of human being, a constellation that proposes a frightening metaphysical conception of the world, albeit only implicitly. Synonymous with the irrevocable organic inadequacy of its biological prematurity at birth, and functioning as such as the basis for full-fledged subjectivity, the primordial Hilflosigkeit of the human infant already points toward a vision of the world that exceeds the constraints of psychoanalysis as a mere investigation into psychogenesis and its pathologies as to be dealt with in the psychiatric setting. Driven by its own concerns, psychoanalysis—indeed, perhaps like any strong theory of subjectivity— offers a metaphysics, or at least must become a metaphysics, since we can never safely isolate the subject under investigation from the greater scheme of ontology within which it is inscribed as a thing, process, or event. The subject is. What Lacan proclaims about its modality of “being” is that subjectivity can no longer be perceived as unnatural in the sense of an external-parasitical invasion into the vital movement of nature through the alienating effects of flirtatious images and castrating words, which somehow spoil or disfigure its pure unity by disrupting the smooth functioning of its immanent laws. The necessary theoretical posit of an originary rottenness of nature contends that there was never a realm of innocence, a pre-symbolic whole whose peaceful in-itself precluded division, a self-pervasive oneness whose unbroken energetic flow was then interrupted through the advent of language, which would be said not only to forever ideally fragment and lacerate it through artificial categories, but more disconcertingly to upset the very positivity of its movement, the cyclical repetition of things in the Real of nature, by short-circuiting the body's self-determining laws striving after homeostatic balance by giving rise to desire. No: it is not that it is only here that we see a snag, a breakdown, in the natural flow of things. Lacan's claim is much stronger: nature, in some sense, was never completely natural (it is in this spirit that we should interpret Lacan's hesitation in “The Mirror 90 Chapter 5 For Žižek, it is precisely this intuition of a necessary moment of negativity, which simultaneously rots nature from the inside out and gestures towards its constitutive weakness, that allows us to come to terms with the Symbolic-Real relation in Lacanian psychoanalysis. It alludes to the necessity of a metaphysics of the Real to explicate what the subject truly is and sketches its contours. Yet due to his conceptual reworking of Lacan, Žižek is led to part ways with and challenge many conventional ways of understanding this relation. Bruce Fink, for example, says: So too, Lacan's Real is without zones, subdivision, localized highs and lows, or gaps and plenitudes: the Real is a sort of unrent, undifferentiated, fabric, woven in such a way as to be full everywhere, there being no space between the threads that are its “stuff.” It is a sort of smooth, seamless surface or space which applies as much to a child's body as to the whole universe. The division of the Real into separate zones, distinct features, and contrasting structures is a result of the symbolic order, which, in a manner of speaking, cuts into the smooth facade of the Real, creating divisions, gaps, and distinguishable entities and laying the Real to rest, that is, drawing or sucking it into the symbols used to describe it, and thereby annihilating it.94 For Žižek, if we are to understand how language emerges out of/within the Real, this pre-symbolic, pre-logical Real sans fissure does not go far enough and must be argued against for two reasons, even if traces of it can be found in Lacan. First, it is a necessary posit created by the Symbolic at the moment of its free self-instituting, just as the transcendental subject posits the notion of a pure noumenon as a consequence of its (re)constitution of phenomenal reality. In this sense, the idea of the extra-subjective Real as an undifferentiated “mass” exhibiting no absence and negativity, just like the noumenon, risks being a mere fantasy of some kind of positive state of ontological completion outside of symbolization and idealization, which psychoanalytical experience (the mind-body discord) disproves. The Real prior to language may not possess linguistic and conceptual determination into a system of strict symbolic differences, but it cannot be said to be a substantial reality fully existing unto itself in such a way that language Kant, Todestrieb, and Beyond the Pleasure Principle 91 Kant, Todestrieb, and Beyond the Pleasure Principle 91 “pierces” its smoothness by “cutting into it” it like a flesh wound, which such presentations of the problematic appear to imply. This means that the above reading (represented by Lacanians such as Fink) is not false, but must be qualified. The Real sans fissure and the noumenon represent a compensation for the impossibility of an intimate experience of the Real within the Symbolic by claiming that, outside the reach of this synthetic (re)constitution of reality, it can still be said to persist in a state lacking contradiction and antagonism. It safeguards us from the realization that the Real itself is morcelé: it does not merely get itself into traps, producing monsters that disrupt the flow of knowledge in the Real by making the latter howl under ontological pain (chaotic states such as black holes, wherein the laws of physics seem to break down, or states in which animals, misreading meteorological conditions, perceive warm days in winter as the beginning of spring and act accordingly, “not only rendering themselves vulnerable to later onslaughts of cold, but also perturbing the entire rhythm of natural reproduction”95) but is always already riddled with internal differences, in such a manner that symbolic categories, due to a certain kind of “family resemblance,” cannot be said to be some kind of lacerating agent that first cuts up the stuff of the world into a system of divisions. Speaking of quantum mechanics (which Žižek is interested in precisely because it gives us resources that prevent us from having recourse to “a 'naive' ontology of spheres or levels”96 and challenges our understanding of nature and culture/ the Real and the Symbolic), he says: According to [our] “spontaneous ideology,” nature stands for the primacy of actuality over potentiality, its domain is the domain of the pure positivity of being where there are no lacks (gaps) in the strict symbolic sense; if, however, we take the ontological consequences of quantum physics seriously, then we have to suppose that the symbolic order pre-exists in a “wild” form, albeit in what Schelling would have called a lower potency.97 Žižek outlines four precise ways in which the symbolic order unexpectedly “pre-exists” in the Real as according to quantum mechanics, which deserve to be paraphrased in full in this context: 92 Chapter 5 92 Chapter 5 1. Possibility as such possesses actuality, that is, has effects. Just like parental authority imposes itself all the time despite the fact that it is normally only virtually and not actually present, understanding a particle's trajectory at the quantum level presupposes that we already know its possible trajectories within its wave function, which have a “being” of their own. What is more, the actualization of one of these latter does not do away with the rest: similar to the case of parental authority, as various phenomena of guilty conscience arising from an act or thought that no parental authority (or their stand-in) could ever find out or demonstrate, “what might have happened continues to echo in what actually happens as its virtual background.”98 2. Both possess knowledge in the Real. As the now (in)famous double- slit experiment testifies, if we observe a particle to see through which slit it will pass, it will always behave as a particle, but if we do not observe it, it will always behave as a wave; it is as if the particle knows when it is and when it is not been watched by scientists. We display similar behaviour in the Symbolic—often, for instance, when others project certain roles on us, we act appropriately, being aware of the projection and assuming it. 3. Each exhibits the phenomenon of registration. In the symbolic universe of meaning, an event only truly occurs when the surrounding “external” environment takes note of or registers it, that is, if it can leave a trace. In order to explain the phenomenon of the collapse of the wave function, physicists must also resort to such metaphors: even at the quantum level, an event only “fully actualizes itself only through its symbolic registration, its inscription into a symbolic network, which is external to it.”99 For this reason, particles can pop into and out of existence, just as long as the universe does not notice—like it is possible to cheat a banking system if one does not violate normal functioning. 4. Both exhibit an irreducible openness. In the Symbolic, there is always a delay between an event and its symbolic registration. The rise of a new master signifier that rewrites the entire logical field 4. Both exhibit an irreducible openness. In the Symbolic, there is always a delay between an event and its symbolic registration. Kant, Todestrieb, and Beyond the Pleasure Principle 93 within which it occurs is not a substantial, fully constituted and self-unfolding process that was determined from the get-go, like a plant growing from a seed: it was not until the precise moment when it fully actualizes itself (when it inscribes itself into its surroundings as a master signifier) that it comes to be that which it retroactively always already was, thus rewriting its own entire past. Similarly, in the double-slit experiment, when a particle is observed, it “will not only (now) behave as a particle, its past will also retroactively become (‘will have been’) that of a particle,”100 so that beforehand it could be said to have only existed as a form of proto-reality. within which it occurs is not a substantial, fully constituted and self-unfolding process that was determined from the get-go, like a plant growing from a seed: it was not until the precise moment when it fully actualizes itself (when it inscribes itself into its surroundings as a master signifier) that it comes to be that which it retroactively always already was, thus rewriting its own entire past. Similarly, in the double-slit experiment, when a particle is observed, it “will not only (now) behave as a particle, its past will also retroactively become (‘will have been’) that of a particle,”100 so that beforehand it could be said to have only existed as a form of proto-reality. Secondly, if we try to understand the pre-logical Real as in itself an undifferentiated “mass” or ontologically complete, we just cannot comprehend the possibility of the emergence of the Symbolic in the first place. If something like the human subject is to emerge, then nature must be self-divided, wrought with tension-ridden zones of inner laceration, for otherwise we cannot account for its ontogenesis in any adequate manner, insofar as the subject, intrinsically exhibiting an originary ontologico-natural discord, could not be inscribed into the world. 92 Chapter 5 The rise of a new master signifier that rewrites the entire logical field Kant, Todestrieb, and Beyond the Pleasure Principle 93 Kant, Todestrieb, and Beyond the Pleasure Principle 93 The subject and its linguistic capacities must be seen as expressive of the underlying ontological status of the Real, even if they represent within Lacanian metapsychology the loss and impossibility of such a non-mediated, pre-symbolic reality: the Symbolic is not merely some kind of extraneous, self-unfolding construct (a self-generating matrix of “meaning” that can assert itself in complete freedom from the Real as such) for the mere fact that we are entrapped within it must be revelatory of the essence of objective reality at some level since it must have given birth to it. The meaning of this is twofold: firstly, descriptions of the Symbolic-Real relation such as Fink's risk obfuscating what is for Žižek of essential importance for understanding what is at stake because they describe language as “encrusted upon the living,”101 thereby reducing language to an external reflection upon substance and rendering the task of explicating its obscure origins impossible; secondly, the apparently self-grounding idealism of Lacanian psychoanalysis—the autonomous, self-positing ciphering of the Real by the Symbolic—already points to a way of explaining its emergence, and thus of breaking free from Chapter 5 94 its correlationalist prison, by zoning in on that very feature that seems to prohibit such an inquiry: that is, its ontological solipsism now understood as an ontological event. It is necessary to explain how the Real can open itself up and give rise to a force seemingly Other to itself, to explain how images and words can “colonize” being from within, but this process forces us to include a moment of non-coincidence and antagonism with the Real. Although it may be without meaning to say that in the Real there is lack—lack only being brought forth in the reign of the Symbolic—it would nevertheless be erroneous to deduce from this that there is no negativity or difference within it. Yet how are we to understand this element of negativity in the all- pervasive fabric of the pre-symbolic Real, this self-sabotaging moment in the very heart of being? 5.2 …to a Denaturalized Monstrosity We can now begin to see why the psychoanalytical experience of the infinite dichotomy between the structures underlying personality (subjectivation, culture) and the vital flow of energy sustaining us in objective being (nature, the corpo-Real of the body) is so pivotal for Žižek. The two registers of the Symbolic and the Real may function without any degree of reciprocal interaction or mutual interconnectivity, but the simple declaration of this unstable Cartesian bipolarity actually belies a third element that subsists throughout the discord and, in fact, paradoxically ties them together in their very antagonistic dialectical (non-)relationship. Here, following another hint given by Lacan— While psychoanalysis cannot, since its experience is limited to the individual, claim to grasp the totality of any sociological object or even the whole set of forces currently operating in our society, the fact remains that it discovered in analytical experience relational tendencies that seem to play a basic role in all societies, as if the discontent in civilization [das Unbehagen in der Kultur] went so far as to lay bare the very meeting point of nature and culture.102 Kant, Todestrieb, and Beyond the Pleasure Principle 95 Self-positing autonomy, as both freedom from the laws of a closed libidinal-material economy and the relatively closed structural determinism of the symbolic law of culture, rests upon the subject as a self-relating point of infinite negativity, a positively Chapter 5 96 charged, excessive void, but it can only beget itself if there is first of all a short circuit, a breakdown, in the dynamic flow of energy constitutive of nature's rhythms as that which carves up room for its more-than-materiality. Full-fledged subjectivity is rendered possible by a devastating ontological violence and is consequently nothing but a denaturalized monstrosity (Todestrieb) logically existing above and beyond the flux of being (due to its non-coincidence with the latter) while never able to leave its immanent plane within which it is primordially inscribed as out of joint: representing the failure, the collapse, of a self-enclosed biological system based upon the homeostatic self-preservation of the organism, it is an extimate inassimilable body in nature that exhibits the double feature of inclusion/exclusion, internal/external, presence/absence so characteristic of the Real in Lacan, which simultaneously demonstrates why the pre-symbolic Real must be said to be morcelé. In other words, set up as nature turned against itself, the first dull stirrings of the human subject refer to some kind of trauma that eventually incites the growth of individual ego life and culture in such a way that the latter exhibit the structural form of a reaction formation against this dysfunctionality in being and thus can never prevent themselves from being a negative, symptomatic expression of their basis in an ontological crisis, no matter how they may try to occult this fact. The psychoanalytical experience by definition presupposes an emergent schism in the fabric of the world between substance and subject, matter and mind, the Real and the Symbolic. But how can psychoanalysis—explicitly a theory of psychogenesis and its pathologies—explain where this denaturalization comes from, since such an investigation by principal must be external to its theoretical field and methodology? charged, excessive void, but it can only beget itself if there is first of all a short circuit, a breakdown, in the dynamic flow of energy constitutive of nature's rhythms as that which carves up room for its more-than-materiality. Kant, Todestrieb, and Beyond the Pleasure Principle 95 Furthermore, is not the object of psychoanalysis precisely this gap between first and second nature—the insecure position of a human subject who, after losing his footing in the first nature, can never feel fully at case in the second: what Freud called das Unbehagen in der Kultur, the different way the subject's passage from first to second nature can go wrong (psychosis, neurosis ...)? There is thus a core that resists the subject's full reconciliation with his second nature: the Freudian name for this kernel is drive, the Hegelian name for it is “abstract negativity” (or, in more poetic terms of the young Hegel, the “night of the world”).103 The conceptual contours of this passage are much more complex than they may originally appear. Žižek is saying that central to any psychoanalytical theory of psychogenesis and psychopathology is the claim that the subject is out of joint with both the biological needs of the corpo-Real of its body (the anorexic eats nothing, the romantic is willing to die for love) and the symbolically constituted “second” nature that is created to compensate for the primordial Hilflosigkeit of human organic insufficiency. Understood in the context of seeking the obscure origins of the Symbolic in the Real, the subject, as the very gap between first nature (Real) and second nature (Symbolic), cannot be said to fit into either: it is a paradoxically self-standing space of non-relation that protrudes out of and obstructs both. Strictly speaking, the subject is neither Real nor Symbolic—it is a pure logical non-coincidence that possesses no place in either, so that the question of its upsurge within being must also go beyond a mere exploration of the breakdown of the libidinal-material fold of its biological nature as that which sets the stage for its emergence by opening up a liberating space within nature's hold. What is crucial and groundbreaking here is the outlining of the ontological edifice that grounds human subjectivity qua cogito: we see the articulation of the “site” or “juncture” from which transcendental freedom and spontaneity emerge and take told. Kant, Todestrieb, and Beyond the Pleasure Principle 97 We just have to look at how the latter's key representatives have recourse to various concepts (unruliness and diabolic evil in Kant, the night of the world in Hegel, or the Entscheidung in Schelling) that indicate a necessary disruption, breach, or violence at the very basis of the founding attributes they bestow upon the subject (self- legislative reason, the irreducibility of spirit, or freedom as the capacity for good and evil). It is exactly this paradoxical connection between real discord and ideal freedom throughout both traditions that enables Žižek to develop a new metaphysics by working in the intersections of both traditions, a metaphysics whose first conceptual contours we already see in Kant. Kant, Todestrieb, and Beyond the Pleasure Principle 97 as having two goals.105 First, because the Lacanian subject is lacking any account of its ultimate origins, Žižek turns to German Idealism to develop a transcendental materialism insofar as there exists a structural parallelism in the underlying problematic plaguing both post-Kantian idealism and contemporary psychoanalysis. Second, and more strongly, Žižek's claim is that this parallelism is more than a mere shared set of theoretical concerns hovering around the grounding of the subject. If we read Kant, Schelling, and Hegel through Freud and Lacan, we actually see that there is a fundamental identity between the psychoanalytical subject haunted by the Todestrieb as constitutive of its very existence and the unconscious Grundlogik of German Idealism. We just have to look at how the latter's key representatives have recourse to various concepts (unruliness and diabolic evil in Kant, the night of the world in Hegel, or the Entscheidung in Schelling) that indicate a necessary disruption, breach, or violence at the very basis of the founding attributes they bestow upon the subject (self- legislative reason, the irreducibility of spirit, or freedom as the capacity for good and evil). It is exactly this paradoxical connection between real discord and ideal freedom throughout both traditions that enables Žižek to develop a new metaphysics by working in the intersections of both traditions, a metaphysics whose first conceptual contours we already see in Kant. as having two goals.105 First, because the Lacanian subject is lacking any account of its ultimate origins, Žižek turns to German Idealism to develop a transcendental materialism insofar as there exists a structural parallelism in the underlying problematic plaguing both post-Kantian idealism and contemporary psychoanalysis. Second, and more strongly, Žižek's claim is that this parallelism is more than a mere shared set of theoretical concerns hovering around the grounding of the subject. If we read Kant, Schelling, and Hegel through Freud and Lacan, we actually see that there is a fundamental identity between the psychoanalytical subject haunted by the Todestrieb as constitutive of its very existence and the unconscious Grundlogik of German Idealism. Kant, Todestrieb, and Beyond the Pleasure Principle 95 Full-fledged subjectivity is rendered possible by a devastating ontological violence and is consequently nothing but a denaturalized monstrosity (Todestrieb) logically existing above and beyond the flux of being (due to its non-coincidence with the latter) while never able to leave its immanent plane within which it is primordially inscribed as out of joint: representing the failure, the collapse, of a self-enclosed biological system based upon the homeostatic self-preservation of the organism, it is an extimate inassimilable body in nature that exhibits the double feature of inclusion/exclusion, internal/external, presence/absence so characteristic of the Real in Lacan, which simultaneously demonstrates why the pre-symbolic Real must be said to be morcelé. In other words, set up as nature turned against itself, the first dull stirrings of the human subject refer to some kind of trauma that eventually incites the growth of individual ego life and culture in such a way that the latter exhibit the structural form of a reaction formation against this dysfunctionality in being and thus can never prevent themselves from being a negative, symptomatic expression of their basis in an ontological crisis, no matter how they may try to occult this fact. The psychoanalytical experience by definition presupposes an emergent schism in the fabric of the world between substance and subject, matter and mind, the Real and the Symbolic. But how can psychoanalysis—explicitly a theory of psychogenesis and its pathologies—explain where this denaturalization comes from, since such an investigation by principal must be external to its theoretical field and methodology? Using Lacan's gesture towards an originary rottenness plaguing nature as a theoretical starting point, Žižek seeks to expand this structuralist metapsychology in order to secure the means of articulating the ontogenetic possibility-conditions of the Symbolic in the Real. Seeing psychoanalysis as conceptually unable to fulfill this task, he expands its horizon by recourse to modern philosophy, seeing therein a certain homology that enables him to draw upon its resources.104 Following Lacan's claim that “Kant's practical philosophy [is] the starting point of the lineage culminating in Freud's invention of psychoanalysis,” Žižek's project could be described Kant, Todestrieb, and Beyond the Pleasure Principle 97 Chapter 5 98 nothing to do with the unconscious in the Lacanian sense; on top of that, it displays great hope in the modern Enlightenment project of establishing the self-transparency and powers of reason—albeit through reason's own self-critique—as a means of historical progress and concretizing man's perfectibility. However, even if this might be the image of Kant's practical philosophy that always comes to mind, the matter at hand is far more complicated. Kant's practical philosophy is of central importance because it is an expression and systematization of the experience of freedom in its irreducible essence, freedom understood as the self-legislative spontaneity at the core of human subjectivity, a faculty that separates us from the rest of mechanical nature insofar as we generate and obey our own laws. One must also remember that, for Kant, the Critique of Pure Reason is an attempt to make room for faith by limiting knowledge and reason, a point that directs the entirety of the critical enterprise from beginning to end by penetrating into the originary self-positing of human liberty at all costs, something that the late German Idealists were retroactively convinced revealed a decisive deficiency present in all the great thinkers in the history of philosophy. It is not the Copernican revolution—indeed, Schelling's106 and Hegel's107 projects are founded upon an attempt to escape its consequences—which forces us to rethink the very possibility of philosophy, but rather Kant's account of freedom.108 This is also true of Žižek: “Kantian practical reason provides a glimpse into the abyss of freedom beyond (or beneath) the constraints of traditional metaphysical ontology.”109 It is all a matter of how one understands the Kantian breakthrough. Even if it is true that it is a response to specific epistemological and scientific concerns that emerge out of modern philosophy (the [im]possibility of universal and necessary knowledge, the nature of the correlation of our ideas to reality in the genesis of concepts, etc.) it is clear that Kant's revolution cannot merely be reduced to his innovative way of rethinking the question of the subject-object relation, for the radical reflexivity of the cogito that imposes upon us the task of reconceptualizing what it is for an object to be present in the field of experience is also at the basis of what it means to be a practical subject. 5.3 Kant, Unruliness, and the Cry of the Newborn Lacan's claim that the beginning of psychoanalysis is in Kant's practical writings appears, at first, counter-intuitive. Especially given Lacan's structuralist bent, one would perhaps expect the clearest elaboration of the subject to be found in Kant's philosophical treatises on the mediating structures of consciousness. What do we see by delving into his practical philosophy except an attempt to found the ethical in the self-legislative spontaneity of human reason through articulating the self-imposing impetus of the categorical imperative and a listing of the a priori duties that automatically follow from its law? How could such a cold, machine-like way of determining the legitimacy of existential action be the immediate origin of psychoanalysis? Although we do encounter traces here of the irrevocably split nature of subjectivity in the tension between reason and sensible inclination, Kant's practical philosophy appears to have absolutely Chapter 5 Kant, Todestrieb, and Beyond the Pleasure Principle 99 Kant, Todestrieb, and Beyond the Pleasure Principle 99 99 Kant appears to be making the self-legislative freedom we witness in concrete, existential situations the very basis of theoretical philosophy— instead of a passive thinking subject that receives external reality as a kind of inert receptacle, a mere spectator, we have a reflecting subject that spontaneously and freely generates the very fabric of its own experience into a continuous, unitary whole through an activity of synthetic integration in a way similar to how it gives itself its own laws. The theoretical subject that is unearthed in transcendental apperception is ultimately identical with the practical subject of self-legislative freedom—or in other words, one cannot speak of one without the other, because they form a dialectical whole: if one wants to plunge into the labyrinthine depths of subjectivity, one should not read the Critique of Pure Reason in isolation from later works such as Lectures on Pedagogy and Religion within the Limits of Reason Alone. In a similar vein, Žižek locates the true Kantian breakthrough in Kant’s practical thinking on subjectivity instead of his epistemologico- transcendental destruction of metaphysics insofar as it is in the former that we most directly see the abyss of freedom at the obscure origin of subjectivity. But what is so primordial in the former that could bestow upon Kant's pedagogical writings such a privilege for understanding the radicality of critical philosophy, while also enabling us to shed light on the basis of the Cartesian-psychoanalytical subject? Finding numerous textual traces of the death-drive understood as a self-sabotaging tendency in nature as logically prior to subjectivity in both Kant, Fichte, Schelling, and Hegel, what interests Žižek in Kant's pedagogical writings is how they set the stage for what he claims to be the unconscious Grundlogik of German Idealism: that is, its founding intuition of the passage from nature to culture centred around a disturbing moment of irreducible negativity inscribed within the palpitating heart of being, which suggests that what first appears as a mere homology in conceptual structure between psychoanalysis and German Idealism is in fact a strict identity. Chapter 5 The movement goes both ways: if we read the Critique of Pure Reason through the later ethical, pedagogical, and religious writings, Kant, Todestrieb, and Beyond the Pleasure Principle 99 What they bring to the fore is a thematization of the subject as some kind of disjunctive “and”: The key point is thus that the passage from “nature” to “culture” is not direct, that one cannot account for it within a continuous evolutionary narrative: something has to intervene between the two, a kind of “vanishing mediator,” which 100 Chapter 5 Chapter 5 is neither nature nor culture—this In-between is silently presupposed in all evolutionary narratives. We are not idealists: this In-between is not the spark of logos magically conferred on Homo sapiens, enabling them to form his supplementary virtual symbolic surroundings, but precisely something that, although it is also no longer nature, is not yet logos, and has to be “repressed” by logos—the Freudian name for this In-between, of course, is the death drive. Kant, Todestrieb, and Beyond the Pleasure Principle 99 Speaking of this In-between, it is interesting to note how philosophical narratives of the “birth of man” are always compelled to presuppose such a moment of human (pre)history when (what will become) man is no longer a mere animal and simultaneously not a “being of language,” bound by symbolic Law; a moment of thoroughly “perverted,” “denaturalized,” “derailed” nature which is not yet culture.110 According to Žižek, within Kant this “in-between” finds its expression in the necessity to discipline the excessive “unruliness” (Wildheit) of human nature, the “wild, unconstrained propensity to insist stubbornly on one's own will, cost what it may.”111 What is to be emphasized here is the drastic nature of this claim: if we do not tame this primordial rawness (Rohigkeit) that presents itself as the zero-level of human spontaneity, not only do we fail to become full-fledged, fully adjusted subjects in the sociopolitical field of the world, but our freedom even threatens to devour itself in its frenzy in such a way that we become failed subjects: we are not born as humans, but rather become human—or, as Kant says, “[m]an only becomes man by education,”112 which leads him to contend that “with education is involved the great secret of the perfection of human nature”113 insofar as it is only through the principles offered by this act of disciplining, a means of schematizing our originary unruliness, that a “second nature”114 can emerge as a response to the grounding ontological dilemma of human being, “nature ha[ving] placed no instinct in [man] for that purpose.”115 Even if the dysfunction of nature, this ontological abortion that is a monstrous, uncontainable excess of life, can in a second dialectical moment serve as a “positive” foundation or support, it can also collapse upon itself like a dying the dysfunction of nature, this ontological abortion that is a monstrous, uncontainable excess of life, can in a second dialectical moment serve as a “positive” foundation or support, it can also collapse upon itself like a dying star. Yet this “unruliness” cannot be equated with the brute reality of animal “positive” foundation or support, it can also collapse upon itself like a dying star. Kant, Todestrieb, and Beyond the Pleasure Principle 99 Yet this “unruliness” cannot be equated with the brute reality of animal Kant, Todestrieb, and Beyond the Pleasure Principle 101 existence—even if it exists within nature, it is strictly speaking something non-natural: The love of freedom is naturally so strong in man, that when once he has grown accustomed to freedom, he will sacrifice everything for its sake [...]. Owing to his natural love of freedom, it is necessary that man should have his natural roughness smoothed down; with animals, their instinct renders this unnecessary.116 For Žižek, this extract shows that the enigma of the emergence of subjectivity in German Idealism cannot be reduced to a mere dichotomy between nature and culture, as if in order to conform to the symbolic law of our own making we must first tame the blind, egotistical pleasure-seeking principles of our animal nature. The self-creative, logically autonomous milieu of culture is only possible through a prior, infinitely uncontainable freedom that acts as the “vanishing mediator” between brute animal reality and structured human intersubjective existence. The passage to culture does not consist in a sublimation of animalistic needs, but rather in a disciplining or symbolic re-articulation of a monstrous and logically irreducible unruliness that marks the essence of the human being, a disciplining that, when it succeeds (the possibility of neurosis always lurks in the air), simultaneously functions as that which once and for all separates us from nature by causing this denaturalized Grund (our ontogenetic “origins”) to withdraw from the scene. It is worth pointing out that Žižek strangely overlooks an important passage on the first page of the transcript we have of Kant's Lectures on Pedagogy, the very text that he makes use of at such a crucial point in his argument, which actually further supports his own Lacanian-inspired and ontologically oriented rereading of the vision of practical freedom it offers. In a perhaps unexpected move, Kant defines the human neonate as non- natural, claiming that, if an animal came to the world crying as a human does, it would merely attract attention to itself as potential prey, thus establishing that there is something off, primordially non-advantageous from a biological point of view, about the obscure ontogenetic beginnings of human subjectivity. Kant, Todestrieb, and Beyond the Pleasure Principle 99 In this context, we only need to cite a passage from the 02 Chapter 5 Anthropology from a Pragmatic Point of View to show how explicitly Lacanian Kant's point is: The cry of a newborn child is not the sound of distress but rather indignation and furious anger; not because something hurts him, but because something annoys him: presumably because he wants to move above and his inability to do so feels like a fetter through which his freedom is taken from him.—What could nature's intention be here in letting the child come into the world with loud cries which, in the crude state of nature, are extremely dangerous for himself and his mother? For a wolf or even a pig would thereby be lured to eat the child, if the mother is absent or exhausted from childbirth. However, no animal except the human being (as he is now) will loudly announce his existence at the moment of birth [...]. One must therefore assume that in the first epoch of nature with respect to this class of animals (namely, in the time of crudity), this crying of the child at birth did not yet exist; and then only later in a second epoch set in, when both parents had already reached the level of culture necessary for domestic life; without our knowing how, or through what contributing causes, nature brought about such a development.117 Not only does Kant relate the dark unruliness that sets the stage for full- fledged human freedom to some kind of ontologically disjointed state of natural being, but he more radically links the cry of the newborn to the infinite dis-coordination of the corpo-Real of the human neonate, its feeling of utter dismemberment so central to the mirror stage in Lacan, and even suggests that this direct expression of painful negativity immanent in the fold of material being is fundamental to the passage from nature to culture. These passages could be further drawn out by supplementing them with a number of possible citations from Religion within the Limits of Reason Alone, the first book in which Kant attempts to deal with the insurmountable propensity to evil that lies at the core of human subjectivity and ethical action. Kant, Todestrieb, and Beyond the Pleasure Principle 99 It argues two major points: (i) “the ground of this evil cannot be placed, as is so commonly done, in man's sensuous nature” and (ii) “neither can the ground of this evil be placed in a corruption of the morally Kant, Todestrieb, and Beyond the Pleasure Principle 103 legislative reason.”118 As such, this diabolic evil can neither be linked to the corpo-Real of the body and its pleasure-seeking tendencies or to the self's being within the law of a criminal or coercive symbolic order, but must itself be a foundational, constitutive part of the subject insofar as, being an infinitely self-asserting activity irreducible to and incommensurate with either zone, it articulates this same paradoxical structure of the in-between. Protruding out of nature and culture and failing to be understood except by its own self-positing, self-determining logic, the diabolic evil at the basis of freedom can only appear as an uncontrollable urge threatening to devour everything, even itself, in its self-destructive forward thrust. But here we see one crucial difference: whereas before we were at the strict level of ontogenesis (the “origins” of transcendental subjectivity) we now encounter full-fledged speaking subjects acting in a world (the ambiguity of freedom). Linking this consuming fire at the core of subjectivity to the perverse truth hidden in the Cartesian gap between mind and body, nature and culture, we already see in Kant the outlines of a radical materialist ontology, for it is as if in the movement from the former to the latter we see unruliness positing or owning itself in its own attempt at self-domestication, the structural consequences of which have profound metaphysical consequences for our understanding of reality as a substantial whole or totality. Since the ontogenetic origins of the subject are linked to a denaturalized unruliness and a full-fledged subject only comes on the scene when the latter posits itself as such in this endeavour of schematizing itself, which in turn institutes a pure difference in being by forever sustaining the gap it attempts to fill in, subjectivity exhibits an insurmountable propensity to evil because its very founding gesture is structurally evil. It is a radical, egotistical “No!” that reverses the order of the world according to its own self-assertion and cannot be undone without undoing itself. 104 Chapter 5 Kant's breakthrough is the following: if human subjectivity is to be truly self-legislative, at its zero-level there can be no formal distinction between a good and evil free act insofar as both are self-chosen in a non-disciplinable frenzy that has no definitive (real or symbolic) status and knows no influence exterior to its own self-asserting, self-positing activity. In itself, freedom is indifferent to both: there is no intrinsic difference between a will that wills evil and a will that wills good in terms of the pure act itself insofar as both are merely following their own self-given causality, an unconstrained self- legislation that tears apart the very fabric of nature in its self-imposition.119 A will that wills evil is not merely giving itself over to the animalistic impulses of the body, nor is it expressing its ignorance or even corruption of the symbolic fold of cultural laws; it is merely forcefully upholding its own diabolical evil for the sake of it, basking in its own self-grounding tyranny, even when it has complete knowledge of its nature and repercussions. A will that wills evil is not merely giving itself over to the animalistic impulses of the body, nor is it expressing its ignorance or even corruption of the symbolic fold of cultural laws; it is merely forcefully upholding its own diabolical evil for the sake of it, basking in its own self-grounding tyranny, even when it has complete knowledge of its nature and repercussions. What is so difficult to come to terms with in the theoretical positing of this state of unruliness, and the ambiguity at the core of the self-positing of the subject, is its proclamation that the good itself is only possible through the gentrification or taming of evil. In order for good to be truly good, it too must present itself as a non-deducible act that breaks free from any order within which it could be contained by refusing all inscription within a field of heterogeneous, external forces that could impose itself upon the absolute originarity of its uncompromising self-assertion. Kant, Todestrieb, and Beyond the Pleasure Principle 99 What is more, not only did Kant identify the intrinsic break from the order of substantial being that sets the stage for the cogito's very autonomy and sketch this movement, he also already saw the paradox at the heart of subjectivity. That is to say, he implicitly understood that we can only grasp the latter as a pure act non- deducible from the obscure libidinal-material grounding that renders it possible (after all, for Kant evil is intrinsically enigmatic insofar as once as it 104 Chapter 5 The Yes of union depends upon the No of separation, but the process of converting the latter into the former can never be complete, since this would reduce freedom to a mere moment of cultural law as a kind of quasi-natural immersion within a pre-given logistics, thereby robbing human spontaneity of its untouchable autonomy in face of everything else that may be said to have influence upon it. Posited as that which transcendentally precedes and even conditions the possibility of its (logico-symbolic) articulation, at the very heart of the good there paradoxically lies evil as its extimate Other and menacing ground, threatening at any moment to erupt and disturb its smooth surface. Evil and good are not infinitely different and opposed, but merely two logical modalities of freedom, a freedom that in and of itself knows no law except its own uncontainable upsurge. If subjectivity is evil and the subject is an Kant, Todestrieb, and Beyond the Pleasure Principle 105 Kant, Todestrieb, and Beyond the Pleasure Principle 105 event in the world, then the world must not be understood as not-all, for the latter is not incapable of subsuming the former within its smooth touch. It is precisely for this reason that Žižek, along with Lacan, believes that Kant's practical philosophy is the beginning of the lineage that culminates in psychoanalysis, the latter being understood as the second great revolution in philosophy that inflicts upon us the task of radically reconfiguring how we view ourselves and our relation to the world. Not only can we already therein see its traits principaux being thematized in an implicit way, but paradoxically one could even say that it in any uncanny manner develops them in different directions from Freud or Lacan so that bringing psychoanalysis and the German Idealist tradition together promises to produce something new. Just as Kant asserts an ultimate identity between the theoretical and practical ego, Žižek argues for the interpenetration of modern transcendental philosophy and psychoanalysis through the Kantian notion of the original “unruliness” of human nature. Todestrieb becomes a synonym for the transcendental “I,” the cogito, by giving expression to the pre-subjective conditions of the possibility of freedom as some kind of non-masterable excess in nature that must be “tamed” if full-fledged subjectivity is to come on the scene. Exposing an activity uncontainable within positive being, it alludes to an ontologically self-violent “wildness” that serves as the ontogenetically constitutive basis of the subject's self- positing, in such a way that makes the late Lacan's passing remarks over the ontology of the psychoanalytical subject (“the rottenness of nature”) come strikingly close to those of Kant (“[w]hat could nature's intention be here [...] ?”). In Kant, however, just as in Lacan, the actual status of the subject remains ambivalent and theoretically undetermined, even if there are various suggestions littered throughout their texts that programmatically outline how to proceed if one were to develop a materialism of transcendental freedom, despite the insurmountable problem that such gestures surpass the very constraints imposed upon the epistemology that they develop. The exact same set of theoretical questions is brought to the fore by both thinkers but remains unsolved: why does transcendental freedom develop? What is its exact relation to the “unruliness,” synonymous with the excess of life presented by the Todestrieb, at the core of our being that appears to logically precede it? Kant, Todestrieb, and Beyond the Pleasure Principle 105 Insofar as transcendental spontaneity is just as related The exact same set of theoretical questions is brought to the fore by both thinkers but remains unsolved: why does transcendental freedom develop? What is its exact relation to the “unruliness,” synonymous with the excess of life presented by the Todestrieb, at the core of our being that appears to logically precede it? Insofar as transcendental spontaneity is just as related Chapter 5 106 to the synthetic powers of the imagination as it is to self-legislative reason in Kant, what role does the former play in this picture (or, in a Lacanian parlance, how can the Symbolic emerge as that which [re]constitutes the fold of experience)? It is only in Kant's successors that such concerns will be addressed in a more explicit manner, and it is through a psychoanalytical reactualization of their thinking that the transcendental materialist ontology at the heart of Kant's breakthrough can finally come to light. Notes 87. Žižek, The Ticklish Subject, p. 48. 88. Johnston, Žižek’s Ontology, p. 272. Here Johnston is referring to Le Séminaire de Jacques Lacan, Livre XXI: Les non-dupes errent, 1973–1974 (unpublished type- script), pp. 2, 21, and 74; and Le Séminaire de Jacques Lacan, Livre XXIII: Le sinthome, 1975–1976, ed. Jacques-Alain Miller (Paris: Éditions du Seuil, 2005), pp. 5, 17, and 77 (untranslated). 89. Ibid., p. 273. Following Johnston’s bibliography, the first quote comes from Le Séminaire de Jacques Lacan, Livre XVIII: D’un discours qui ne serait pas du semblant, 1971 (unpublished typescript), pp. 2, 17, and 71, while the second seminar he makes reference to is (as in the previous footnote) Le Séminaire de Jacques Lacan, Livre XXIV: L’insu que sait de l’une bévue s’aile à mourre, 1976–1977, ed. Jacques- Alain Miller, in Ornicar? 12-18, pp. 5, 17, and 77. 90. Lacan, “The Mirror Stage as Formative of the I Function,” Écrits, p. 96/77. 90. Lacan, The Mirror Stage as Formative of the I Function, Écrits, p. 96/77. 91. Ibid., p. 97/78. 91. Ibid., p. 97/78. 91. Ibid., p. 97/78. 92. Ibid., p 100/80. 93. Ibid., p. 96/77. 94. Fink, The Lacanian Subject, p. 24. I capitalize “the Real” for consistency. 95. Žižek, Living in the End Times (New York: Verso, 2011), pp. 350–51. 96. Žižek, Less Than Nothing, p. 905. 96. Žižek, Less Than Nothing, p. 905. 97. Ibid., p. 921. 97. Ibid., p. 921. 98. Ibid., p. 920. 99. Ibid. 99. Ibid. 100. Ibid., p. 921. 100. Ibid., p. 921. 101. Fink, The Lacanian Subject, p. 12. 102. Lacan, “A Theoretical Induction to the Functions of Psychoanalysis in Criminology,” Écrits, p. 127/104. 103. Žižek, The Ticklish Subject, pp. 81–82. 103. Žižek, The Ticklish Subject, pp. 81–82. 104. Žižek, “Liberation Hurts.” 104. Žižek, “Liberation Hurts.” Kant, Todestrieb, and Beyond the Pleasure Principle 107 Kant, Todestrieb, and Beyond the Pleasure Principle 107 105. Žižek, The Ticklish Subject, p. 48. 106. The founding gesture of Schelling’s entire project begins with an attempt to surmount the Copernican revolution. Only the modalities of his response differ: whether it be through an attempt to give a genesis of the categories from the I as the first principle, a daring naturephilosophy, or eventually by a theosophic mytho-poetics of creation, one thing is clear—we must go “beyond simple rep- resentation.” Einleitung in die Philosophie, ed. Walter E. Ehrhardt (Stuttgard-Bad Connstatt: Frommann-Holzboog, 1989). 106. The founding gesture of Schelling’s entire project begins with an attempt to surmount the Copernican revolution. Only the modalities of his response differ: whether it be through an attempt to give a genesis of the categories from the I as the first principle, a daring naturephilosophy, or eventually by a theosophic mytho-poetics of creation, one thing is clear—we must go “beyond simple rep- resentation.” Einleitung in die Philosophie, ed. Walter E. Ehrhardt (Stuttgard-Bad Connstatt: Frommann-Holzboog, 1989). 107. Although people are wont to speak about the tight relationship between Kant and Hegel, one must point out that in the Lesser Logic Hegel badmouths Kant by by throwing upon him the ultimate insult one could give to a systematic philosopher (except, of course, that their absolute is a dead dog)—namely, that he was just too lazy to think through what he says and therefore completely misses the point: “[i]n deal- ing with this highest Idea [teleological causality], however, the laziness of thought, as we may call it, finds in the ‘ought’ an all too easy way out.” The Encyclopedia Logic (with the Zusätze), trans. T. F. Geraets, W. A. Suchting, and H. S. Harris (Indianapolis: Hackett Publishing, 1991), p. 101 (§55A). 108. See chapter 6. 109. Žižek, The Ticklish Subject, p. 48. 110. Ibid., p. 36. 111. Ibid. 112. Taken from Kant, Kant on Education, trans. Annette Churton (Boston: D. C. Heath, 1900), p. 6; for a more easily findable edition, see Lectures on Pedagogy, trans. Robert B. Louden, Anthropology, History, and Education, ed. Günter Zöller and Robert B. Louden (Cambridge: Cambridge University Press, 2007), p. 439. 113. Ibid., pp. 7 and 439. 113. Ibid., pp. 7 and 439. 114. Ibid., pp. 9 and 440. 115. Ibid., pp. 13 and 442. 115. Ibid., pp. 13 and 442. 116. Ibid., pp. 4–5 and 438; quoted by Žižek, The Ticklish Subject, p. 36. 117. Chapter 6 This chapter comprises a Žižekian-inspired interpretation of the philosophical movement from Kant to Hegel by focusing on Kant's thematization of freedom and how it radically reconfigures the possibility of metaphysical inquiry. In the aftermath of critical philosophy, what is clear is that any philosophy unable to think system and the irreducibility of the human subject is to be rejected. By following certain premonitions within Kant's pedagogical writings that appear to link transcendental spontaneity to the psychoanalytical concept of Todestrieb, Žižek gives us resources to read Hegelian Absolute Idealism against standard interpretations by claiming that Hegel's attempt to think substance as subject implies the ontogenetic emergence of freedom through a self-sundering of being, the immanent advent of a devastating ontological non-coincidence, which forces upon us the necessity of a new kind of metaphysics: a metaphysics of the not-all. This enables us not only to rethink the Kant-Hegel relation in a provocative manner, but also to explain how Žižek is able to draw upon post-Kantian idealism to lay the foundation for the logic of his own transcendental materialism. Kant, Todestrieb, and Beyond the Pleasure Principle 107 Anthropology from a Pragmatic Point of View, trans. Robert B. Louden, Anthropology, History, and Education, p. 423. First set of italics is my own. 118. Kant, Religion within the Limits of Reason Alone, trans. T. M. Greene and H. H. Hudson (New York: Harper & Row, 1960), p. 30. 119. Žižek & Daly, Conversations with Žižek (Cambridge: Polity, 2004), p. 124. From Transcendental Philosophy to Substance as Subject 109 From Transcendental Philosophy to Substance as Subject 109 109 penetrating account of the essence of human freedom.120 Although much of what he says concerning freedom is already laid out in Descartes' thinking on the cogito, it was Kant who gave it a full, profound expression. For Žižek, this means that it is here that the principal intuitions that heralded forth modernity—the ontologically shattering schism between the thinking mind and extended substance and subjectivity's irreducible reflexivity as that which institutes this very split—are radicalized and find an overpowering degree of theoretical articulation. After Kant, there is just no going back, for this would be to give up on what it means to be an infinitely self-standing, autonomous subject, to turn one's back on one's own freedom, whose apparently indemonstrable existence has been proven once and for all.121 Any system that regresses into a “primitive,” “pre-critical” way of philosophizing is in effect merely recoiling from the difficulty that is the burden of freedom, “our experience of freedom [being] properly traumatic, even for Kant himself.”122 Herein lies the fundamental undecidability intrinsic to the Kantian breakthrough: not only is the freedom of human subjectivity liberating, but it is also (and perhaps more originally) monstrous, insofar as we are infinitely given over to it and therefore responsible for it, yet can only comprehend it according to the frenzy that is its own self-positing essence. In the wake of the Kantian system, there is only “the uncanny abyss of freedom without any guarantee in the Order of Being”:123 in Kantian ethics, the true tension is not between the subject's idea that he is acting only for the sake of duty, and the hidden fact that there was actually some pathological motivation at work (vulgar psychoanalysis); the true tension is exactly the opposite one: the free act in its abyss is unbearable, traumatic, so that when we accomplish an act out of freedom, in order to be able to bear it, we experience it as conditioned by some pathological motivation. Here I am tempted to bring in the key Kantian concept of schematization: a free act cannot be schematized, integrated into our experience; so, in order to schematize it, we have to “pathologize it.”124 Immediately following the birth of transcendental idealism, however, there is an overall ambiguity as to how to proceed. 6.1 Fichte and the Frailty of Freedom Within the trajectory of modern philosophy, the inheritors of the legacy of the critical system all agree that it is with Kant that we see the first truly From Transcendental Philosophy to Substance as Subject 109 Although there is some general consensus concerning the various ways that the critical system is by itself 110 Chapter 6 110 Chapter 6 110 incomplete, internal discord quickly arises. Leaving aside Reinhold's and Maimon's own responses to its perceived insufficiencies, Fichte, Schelling, and Hegel, despite all agreeing that (i) the categories of the understanding are dogmatically asserted (they lack a genetic deduction grounding their necessity and universality) and ultimately too static (there is no clear articulation of their systematic interconnectivity) and (ii) concepts such as the thing-in-itself in their Kantian mode are internally contradictory or at least theoretically unnecessary, each offer different strategies to think through the deadlock of Kant's legacy to retain its grounding insights. The early Fichte of the Jena period—the only Fichte who had a significant impact on the internal development of German Idealism— proceeds by removing the extra-subjective alterity of the thing-in-itself, often referred to as the residual trace of a materialism in Kant, by reducing its status to a mere generated effect of subject's purely autonomous activity that knows no outside. Taking as his starting point the immanent field of absolute actuation presented by the subject's radical freedom, whose self- manifestedness is revealed in the unavoidable transcendental reduction of any given fact of consciousness to its activity and most primordially demonstrated by intellectual intuition, and which is best exemplified in the infinite thetic judgement I am,125 subjectivity, as uncontainable freedom, cannot be trumped. Groundless, nothing can get beyond it: it is the ultimate, self-explanatory condition of experience. Yet, although a wondrous fountainhead of activity, the theoretical and practical unconditional beginning that is the I is paradoxically lacking. Not only is its essence (an essence that is its very act of existing) indistinguishable from nothingness insofar as it knows no bounds; more disconcertingly, it is also immensely fragile and immediately threatened by a not-I that risks destroying its very omnipotence from within its own sphere. In the face of the “not-I,” which is transcendentally simultaneous with the “I,” freedom cannot be simply restricted to the undetermined nothingness of the I am, but it must upsurge in an attempt through our very actions and finitude to overcome that which opposes it. From Transcendental Philosophy to Substance as Subject 111 From Transcendental Philosophy to Substance as Subject 111 as the pure act of freedom tries to posit itself as such, it runs up against the impossibility of asserting itself as an autarchic all. In this conflict-ridden battle, the not-I proves to be more than a pure alterity wreaking havoc on the subject's freedom. Instead, it becomes a determined other, an Anstoß (an obstacle and impetus). Through encountering this Anstoß there can be a continual overcoming of the menacing not-I and a never-ending perfecting of the I’s own savage freedom by attaining an ever greater degree of concrete autonomy. In this sense, consciousness emerges from the shock of the not-I on the immanent field of actuation that is the unconscious I in its pure freedom and the latter's defiant cry and refusal to submit when confronted with the possibility of its own demise. The victory of this freedom is never ontologically guaranteed, but can only ever be won again and again in the onslaught of time, whose basic structure is described by the total theoretico- practical syntheses of divisibility opened by the third principle, thus making all principles simultaneous in experience. In this picture there is nothing outside of the pure immanence of the I as freedom and the dynamics of its subjectification as necessitated by the opposition it encounters to its raw, unconditional power. Due to this internal opposition, the I is divided between the absolute I (which is less an egological pole than a faceless, even inhuman activity that only warrants the title of I because it is always spoken of in relation to persons amongst whom it incarnates itself, thus making it in its very essence cryptic126) and the finite (which in turn is divided into passive and active aspects wherein the absolute totality of reality as expressed by the I is never completely annihilated). There is no need for a hard, impenetrable remainder left over from the transcendental constitution of reality: that which threatens to destroy the subject if not gentrified through the syntheses of divisibility, the not-I, is a mere negative magnitude, even if its contorting effects upon the subject can never be predicated and could potentially upsurge as traumatic events. We do not need to go beyond this logical role of an internal pressure proclaiming the possible implosion of the I as freedom to explain experience. From Transcendental Philosophy to Substance as Subject 109 In order to truly sustain its theorectio-practical firstness, to assert its own primordiality against the not-I that “desires” its annihilation, the subject must bring forth the freedom that is its own capacity for absolute actuation in the natural and sociopolitical field of the world, since as soon 112 Chapter 6 to underline the paradoxical simultaneity of radical spontaneity and fragility in the free constitution of phenomenal reality as it continually comes upon an alien presence within its very intimacy: namely, the irresolvable contradiction that exists in subjectivity between the groundlessness of consciousness as a radically self-grounding idealist freedom and the necessary realist contingency that continually jeopardizes it. For Fichte, the removal of an extra-subjective reality is a necessity imposed upon us not just by Kant's radical articulation of freedom, but more broadly by idealism as such. From within the latter, there can be no coherent assertion of a self-subsisting thing-in-itself which, existing outside of the closed idealist circle of phenomenality in an infinite elsewhere, causes our representations. This would be to transgress the epistemological constraints imposed upon us by the very confines of the idealization of the world; any assertion of the thing-in-itself would constitute a return to the worst kind of dogmatism. The Anstoß is therefore not merely the true limit of idealism, but contains the necessary resources to synthesize idealism and realism into a greater unity:127 instead of proclaiming that all our idealizations are first caused by a foreign intruder pushing in upon subjectivity, it says that if there are immanent obstructions from within the mediating activity of the Ideal, if phenomenal experience is plagued by internal inconsistencies, we can legitimately say that these knots negatively point to some extra-subjective, inassimilable body in subjectivity itself forcing it partially to negate itself and transfer its power to that which is extimately Other in order to save itself from complete collapse.128 Giving the immanent intruder reality is a way to appropriate it: in short, to idealize it. In this way, one of the major tasks of Fichte’s 1794 Wissenschaftslehre is to draw our attention to the radicality of this conditional: if we take idealism seriously then we can without difficulty account for realism, for the realist character of our everyday experience can be entirely explained by the very movement of the Ideal itself. Idealism does not infringe upon the freedom of the objective world, since it is clear that the latter can in some way make itself known through the very obstructions of our idealizations of it. From Transcendental Philosophy to Substance as Subject 111 Thus, to say that the thing-in-itself becomes a theoretical posit of the subject is to say that its irreducibility to conscious, finite subjectivity and thus its apparently extra-subjective status have been deduced from the self-manifestedness offered by subjectivity as a first principle: its function is 112 Chapter 6 112 Chapter 6 112 Chapter 6 The epistemic priority of the Ideal is not to be equated with the unknowability of the world, since its autonomous operational self-closure is precisely that which enables us to have a world at all. From Transcendental Philosophy to Substance as Subject 113 114 Chapter 6 From within this originary psychosis at the heart of subjectivity, there is no escape “except” accepting it and taking the path it opens up to its end by the self-conscious creation of fabulous, imaginative identities (criticism) that can be used Subjectification is nothing more than a spinning in the positively charged void of freedom. In the latter's aftermath, we become irrevocably lost in a series of dream-like images, a rhapsody of sociopolitical phantasmagoria, which give a transcendental structure to the fabric of our experience and thus to our ethical striving, thus even making our own self just one image amongst others, but without having any basis in the world at large. If we take Freud's definition of psychosis as a loss of the causal impact of the world upon the self—a “loosening of the relationship to reality”130—due to a radical withdrawal into primary narcissism, whereby object cathexes are libidinally cut off and the primary process slowly takes over psychic reality, we could venture the claim that the founding gesture of the Fichtean subject is a form of psychosis (the I “posits itself absolutely, and is thereby complete in itself and closed to any impression from without”131), justified in the name of autonomy. Here, just as in the illusionary, image-filled world of the psychotic, the “objective” world is reduced to a mere haphazard obstruction to a self-unfolding and self-sustaining tale creative of its own experiential reality (which for Fichte, “absolutely creates itself [...] in a genesis out of nothing”132), a mere haphazard obstruction that is to be overcome and integrated within the tale if the latter is to sustain its very consistency. What is more, in the same manner that the psychotic must continually refuse new perceptions so as to “autocratically” construct an external and internal world that pleases the id's wishful impulses,133 the Fichtean subject, struggling to subsist as a pure I due to the contemporaneous emergence of the not-I and its violence with its self-positing, strives to rid itself of all influence of the not-I in the syntheses of divisibility and thereby actualize the sphere of absolute self-positing at the empirical level. In this respect, its ideal is a full-blown psychosis, an “alloplastic”134 creation of its own experience, which, though technically impossible (without the influence of the not-I, the I would lack determination), is a structural tendency in all experience, the paradoxical basis of practical freedom. 114 Chapter 6 imagine, of my being, my power, my purposes), thought is the dream of this dream.129 Subjectification is nothing more than a spinning in the positively charged void of freedom. In the latter's aftermath, we become irrevocably lost in a series of dream-like images, a rhapsody of sociopolitical phantasmagoria, which give a transcendental structure to the fabric of our experience and thus to our ethical striving, thus even making our own self just one image amongst others, but without having any basis in the world at large. If we take Freud's definition of psychosis as a loss of the causal impact of the world upon the self—a “loosening of the relationship to reality”130—due to a radical withdrawal into primary narcissism, whereby object cathexes are libidinally cut off and the primary process slowly takes over psychic reality, we could venture the claim that the founding gesture of the Fichtean subject is a form of psychosis (the I “posits itself absolutely, and is thereby complete in itself and closed to any impression from without”131), justified in the name of autonomy. Here, just as in the illusionary, image-filled world of the psychotic, the “objective” world is reduced to a mere haphazard obstruction to a self-unfolding and self-sustaining tale creative of its own experiential reality (which for Fichte, “absolutely creates itself [...] in a genesis out of nothing”132), a mere haphazard obstruction that is to be overcome and integrated within the tale if the latter is to sustain its very consistency. What is more, in the same manner that the psychotic must continually refuse new perceptions so as to “autocratically” construct an external and internal world that pleases the id's wishful impulses,133 the Fichtean subject, struggling to subsist as a pure I due to the contemporaneous emergence of the not-I and its violence with its self-positing, strives to rid itself of all influence of the not-I in the syntheses of divisibility and thereby actualize the sphere of absolute self-positing at the empirical level. In this respect, its ideal is a full-blown psychosis, an “alloplastic”134 creation of its own experience, which, though technically impossible (without the influence of the not-I, the I would lack determination), is a structural tendency in all experience, the paradoxical basis of practical freedom. From Transcendental Philosophy to Substance as Subject 113 For Fichte, however, the most decisive consequence to draw from the removal of the thing-in-itself is not a rethinking of the realism-idealism debate. He never takes an interest in the resources that this new way of philosophizing would offer for developing a new form of spectral materialism. The reason is simple: taking the unbridled freedom of the I as his starting point, Fichte agrees with Kant's prohibition on searching for the ontological origins of the subject because, even if it seemingly becomes methodologically possible in Fichte, he argues that it is a futile project, the Ideal being self-explanatory without recourse to the Real. That the subject has no need for the thing-in-itself signifies that the subject is characterized by a constitutive groundlessness: transcendental spontaneity demonstrates that it is totally engulfed within a self-unfolding practical world of its own making, so that what is of primordial importance is never an extra-subjective reality, but the unconditioned freedom of our concrete activity. Fichte does not need to embark upon a metaphysical archaeology of the subject, instead focusing all of his attention on the dynamic inherent in the process of subjectification itself, which inflicts upon us a frightening realization ridden with stark ontological and political implications. Not only are all our actions—and thereby the identities and the collectives that we construct through them—irreducible in themselves insofar as they have no foothold in objective reality, but they become, as it were, mere parts of the free play of an infinite imagination so engrossed by its own self-composing stories that it almost lacks the power to know its self-narrating fiction as a fiction: There is no being. I myself do not know at all and don't exist. There are images: they are all that exists and they know about themselves in the manner of images—images which drift by without there being anything by which they drift; images which hang together through images; images which do not represent anything, without meaning and purpose. I myself am one of these images. No, I am not even that, but only a distorted image of these images. All reality is transformed into a fabulous dream, without there being any life the dream is about, without there being a mind which dreams; a dream which hangs together in a dream of itself. Intuition is the dream; thought (the source of all being and all reality which I 114 Chapter 6 114 Chapter 6 From within this originary psychosis at the heart of subjectivity, there is no escape “except” accepting it and taking the path it opens up to its end by the self-conscious creation of fabulous, imaginative identities (criticism) that can be used From Transcendental Philosophy to Substance as Subject 115 From Transcendental Philosophy to Substance as Subject 115 If the human being is an animal, then he is an utterly incomplete animal, and for that very reason he is not an animal.137 What is striking about this quote is not only that Fichte, like Kant before him, draws attention to the utter helplessness of the neonate as an indication 116 Chapter 6 116 Chapter 6 116 of some kind of ontological indeterminacy within nature as that which sets the stage for subjectivity and freedom, an indeterminacy that is non- natural (“nature withdraws her hand”), but also that he is forced to do so from within the confines of a self-grounding idealism. To account for the very transcendental unity of experience, there is a point during the deduction of its laws that something meta-transcendental must be posited whose very existence appears to be responsible for the subject's groundlessness. That there is no possible explication of the leap from not-I to I appears now shaken, for a certain relation between the two has been established despite the fact that such a relation would seem to jeopardize the I's self- standingness. What is at stake is not that Fichte creates an unacceptable subjective idealism within which knowledge of the world is lost and even precluded through being constructed, but that his philosophy ultimately lacks the resources to tie together an ontology of nature with the spontaneity of the I, though such an impossible link has been immanently deduced. Although Fichte too outlines a transcendental materialist ontology of the subject, he, like Kant before him, cannot answer the question “How does appearance itself emerge from the Real?,” even if he stumbles upon the solution. If Fichte and Kant are right in these intuitions in the same way that Lacan could be said to be right in his late musings on the status of nature in light of the psychoanalytical subject, the task facing Hegel, Schelling, and Žižek is thus remarkably similar: how can being and thinking, the Real and the Symbolic, be reconciled to one another, for surely the latter must exist in the former? From Transcendental Philosophy to Substance as Subject 115 to give the subject the resources it needs to actualize itself through the formation of an absolutely free identity liberated from all external causality (dogmatism). What this means is that “the human being is originally nothing:”135 it is free to strive, and should strive, to absolutely create itself according to its own transcendental self-positing/primary narcissism. For both Schelling and Hegel, however, this complete removal of the problematic of the grounding of the subject is not satisfying on two accounts. First, although Fichte's notion of the Anstoß in the 1794 Wissenschaftslehre does allow us to develop something like a spectral materialism, Fichte does not use his own real-idealism/ideal-realism to investigate the obscure origins of the subject, rejecting any transcendental materialist account of the emergence of the I out of the not-I as contrary to reason.136 Transcendentalism must remain purely immanent—we need not investigate its ontological origins, since these will never suffice. But this does not necessarily mean we cannot embark on such an inquiry (a “pre-history” of the Ideal). Second, Fichte himself, despite his remarks concerning its impossibility, like Kant cannot refrain from commenting on the paradoxical ground of the subject's freedom, as if to say that the matter at hand cannot be limited to the transcendental structures at the heart of the self-unfolding conceptual-imaginative fabric underlying subjectification, but must also include the birthplace of the I as causa sui: Every animal, a few hours after its birth, moves and seeks nourishment at the breast of its mother [...]. To be sure, the human being has a plant-like instinct, but he has no animal instinct at all in the meaning given here. He needs the freely given assistance of other human beings, and without it would die shortly after birth. When the human offspring has left its mother's body, nature withdraws her hand from it and cuts it loose, so to speak [...]. For it is precisely nature's abandonment of him that proves that the human being, as such, neither is nor should be nature's pupil. From Transcendental Philosophy to Substance as Subject 117 of its one-sided countermove.141 But what, then, exactly drives Hegel to part ways from Schelling and develop his own way of balancing idealism (radically self-positing freedom) with a philosophy of nature (a system of the world)? And why is this juncture so important for understanding Žižek's own metaphysics of the disjunctive “and”? To answer this question, we must first make a brief detour through the development of modern philosophy. From Transcendental Philosophy to Substance as Subject 115 For the early Schelling, what is necessary is a theoretical project that supplements the ontologically solipsistic Fichtean subject with an account of its emergence out of an unconsciously creative nature, which would implicate the interpenetration and ultimate identity of the Real and the Ideal, so that such a problem is shown to be ultimately moot.138 Interestingly, though he is initially satisfied with Schelling's response to the deadlock of Fichtean idealism,139 Hegel later breaks from what Schelling himself refers to as a “real” or “objective idealism,”140 claiming that by attempting to solve the internal contradictions of Fichte's idealism, Schelling unknowingly becomes its inverted opposite, a mere reactionary form spurred on by the inconsistencies of the former that ultimately fails because From Transcendental Philosophy to Substance as Subject 117 118 Chapter 6 are Hegel and Schelling attempting to demonstrate how it is still possible to do metaphysics within the very breakthrough of critical philosophy without denying any of what they consider to be its necessary/essential presuppositions, but also, and most importantly, why it is necessary to do so: are Hegel and Schelling attempting to demonstrate how it is still possible to do metaphysics within the very breakthrough of critical philosophy without denying any of what they consider to be its necessary/essential presuppositions, but also, and most importantly, why it is necessary to do so: Here, however, a gap between Kant and his followers occurs: for Kant, freedom is an “irrational” fact of reason, it is simply and inexplicably given, something like an umbilical cord inexplicably rooting our experience in the unknown noumenal reality, not the First Principle out of which one can develop a systematic notion of reality, while the Idealists from Fichte onwards cross this limit and endeavour to provide a systematic account of freedom itself. [... F]or the Idealists [this is] just an indication that Kant was not yet ready to pursue his project to the end, to draw all the consequences from his breakthrough. For the Idealists, Kant got stuck half-way.144 Although this may mean a vigorous rethinking of transcendental spontaneity and imagination, noumena and the status of nature, ultimately neither Hegel nor Schelling wants to give up on Kant's descriptions of freedom in order to substitute transcendental idealism with just another classico-metaphysical system. Yet for this endeavour to be successful, they realize that they need to fight against the Critique of Pure Reason's own prohibition of pure ontological inquiry; they deny one of the major claims of critical philosophy in the hope of saving it from itself. This becomes clear when we realize that for both Hegel and Schelling, Spinoza is the very emblem of a philosopher. His rationalist system is admirable not only for its depth and beauty, but for its self-consistency, clarity, and comprehensiveness. Case of Spinoza A common critique of Schelling and Hegel as they attempt to think through the problems bequeathed by Kantian idealism is that they ignore the basic breakthrough of the Critique of Pure Reason: that is, the recognition of the insurmountable finitude of human reason and its inability to grasp the absolute structure of reality. In face of its debilitation of a priori enquiry by pure reason, they return to a metaphysical thinking that has already been debunked. According to the canonical reading, while Schelling talks of the interrelated poles of subject and object in his attempt to balance transcendental idealism with a naturephilosophy, and then of their ultimate identity in the absolute as indifference by developing an account of absolute reason, Hegel supposedly attempts to show that the logical structures that the thinking subject uses to constitute the world transcendentally are actually one with its ontological, that is, its extra-conscious structure. What Hegel does is dialectically “fix up” Schelling by revealing that reality in all its facets (mind and matter, nature and subject) is merely an expression of the rational self-actualization of the absolute as a logical self-unfolding totality, which does away with the Schellingian indifference as an indeterminate void lacking genuine philosophical content, “the night in which, as the saying goes, all cows are black.”142 Žižek, however, refuses this interpretation, calling it “the standard cliché according to which German Idealism pleads the 'pan-logicist' reduction of all reality to the product of the self-mediation of the Notion.”143 Žižek gives us material that allows us to shed new light on the movement from Kant to Hegel by showing that this conventional reading of German Idealism levels off the daring character of the post-Kantian gesture by making it look like just another classical metaphysical system. Not only 118 Chapter 6 From Transcendental Philosophy to Substance as Subject 119 investigation. They reject this for two reasons: first, it seems arbitrary, and, second, it threatens to destabilize the very Kantian edifice. Although both Schelling and Hegel believe that metaphysical knowledge of an objective reality is still possible, they are at the same time unwilling to abandon idealism insofar as they are not willing to back down from its articulation of freedom, which leads them to see both questions as intimately intertwined and refuse to separate them as Kant does in order to make room for faith and human autonomy. Schelling, for instance, bemoans the fact that “idealism,” whose founding intuition he identifies in the Freiheitsschrift with the principle of freedom, “is not a work of reason,” and that consequently “the supposedly sad honor of being a system of reason remains only for pantheism and Spinozism.”146 Even if Kant's systematization of freedom is of irreducible importance for the late German Idealists, it must be stressed that of itself it remains theoretically negative in a strong sense: largely remaining at the level of formality, it never really manages to get off the ground and provide a thoroughly developed basis for itself.147 We must provide the missing link between system (Spinozism, absolute determinism) and idealism (Kantianism, the true philosophy of freedom) by showing that freedom itself is a metaphysical possibility. But what does it mean for system if freedom is inscribed within it? investigation. They reject this for two reasons: first, it seems arbitrary, and, second, it threatens to destabilize the very Kantian edifice. Although both Schelling and Hegel believe that metaphysical knowledge of an objective reality is still possible, they are at the same time unwilling to abandon idealism insofar as they are not willing to back down from its articulation of freedom, which leads them to see both questions as intimately intertwined and refuse to separate them as Kant does in order to make room for faith and human autonomy. 118 Chapter 6 In terms of an expression of metaphysics, each looks up to him and sees something fundamentally askew in Kant's preclusion of its possibility—or, as Hegel says “[i]t is therefore worthy of note that thought must begin by placing itself at the standpoint of Spinozism; to be a follower of Spinoza is the essential commencement of all Philosophy.”145 Not only did Kant not show trust in the capacities of human reason, more problematically his critical system was lacking any foundation insofar as it left the origins of the transcendental subject, the very object of its thematic, a mystery and even went so far as to put a ban on their From Transcendental Philosophy to Substance as Subject 119 From Transcendental Philosophy to Substance as Subject 119 Schelling, for instance, bemoans the fact that “idealism,” whose founding intuition he identifies in the Freiheitsschrift with the principle of freedom, “is not a work of reason,” and that consequently “the supposedly sad honor of being a system of reason remains only for pantheism and Spinozism.”146 Even if Kant's systematization of freedom is of irreducible importance for the late German Idealists, it must be stressed that of itself it remains theoretically negative in a strong sense: largely remaining at the level of formality, it never really manages to get off the ground and provide a thoroughly developed basis for itself.147 We must provide the missing link between system (Spinozism, absolute determinism) and idealism (Kantianism, the true philosophy of freedom) by showing that freedom itself is a metaphysical possibility. But what does it mean for system if freedom is inscribed within it? Amongst other things, Spinozistic metaphysics represents an avid attempt to rethink the ontological splitting of mind and matter by reconceiving the very notion of substance so that the two categories no longer represent a schismatic split but are subsumable under a single, unified substrate of which they are merely different attributes, all the while preserving as much as possible the basic intuitions of Cartesianism. Mind and matter, the brute material Real of the universe and the self-reflexive powers of ideal spirit, are merely differing “perspectives” on the same, unchanging substance,148 a kind of epistemic parallax shift between two of the countless logical modalities of an all-pervasive “weave” that encompasses all things within its vital ebb and flow. Although they succeed in manifesting the infinite power of substance through different refraction mediums irreducible to each other, they ultimately must be said to interpenetrate one another insofar as they articulate the same content: there is no possible rupturing chasm between them, but only an untouchable oneness.149 But 120 Chapter 6 120 Chapter 6 Chapter 6 120 this oneness is not some kind of undifferentiated, static whole, an abyssal metaphysical void that devours all difference within its cruel awesomeness, like Chronos who eats his own offspring, but rather a pure power or force that is capable of expressing itself in an infinite variety of ways,150 and indeed is only one insofar as it does so. If “nothing exists except substance and its modes,”151 it is because the two constitute one another in an immanent pulsating field teeming, overflowing, with immanent life. From Transcendental Philosophy to Substance as Subject 119 Substance is a raw, pulsating activity, an unconstrained upsurge of a dynamic, quivering freedom that is at the very core of the flux that is perceived reality and circulates through its most minute and insignificant features: “God's power whereby he and all things are and act, is his very essence.”152 Substance exists absolutely as a harmonious play of forces even in its most seeming conflict, tension, and struggle. It is a wondrous ballet of cosmic energy whose dance is something peaceful and inspiring, sometimes macabre and dreadful, for us mortals who without choice are engaged in its spectacle. Although this may appear to preclude human liberty, it actually proclaims that human beings are in a certain sense free insofar as they directly participate in the self-actualizing movement of substance (God, nature) as causa sui. In Spinozism, however, there is a crucial modification of the underlying logic of modern subjectivity already seen in Descartes: the intuition of irreducible subjective freedom is said to arise out of a misrecognition of our fundamentally determined character as egos. By locating freedom within the kernel of my being, my will, I am merely misperceiving its notion, for real spontaneity lies in the impersonal self- writing symphony of the universe, the self-creative flow of life and difference (“we are in God's power as clay in the hands of the potter”153), in which I also play a constitutive part insofar as its energy expresses itself through me, animates me, constitutes me, so that any radical distinction disappears. The cause is fully in its effect, for it itself is immanent. Spinoza's account of human freedom, instead of being a pure cancellation of the significance of concrete human ethical striving by its submission to the total system of the world in its self-imposing oneness, is an attempt to show its greater truth, meaning, and role by its inclusion in the intimate life of God or nature: in short, its function within the autonomous and infinitely inventive self- actualization of substance. It is precisely for this reason that metaphysics From Transcendental Philosophy to Substance as Subject 121 122 Chapter 6 What repels him is that freedom is “not my freedom at all but rather that of an alien force outside me:”157 “[t]o stand there cold and dead and merely to look at the change of events an inert mirror or fleeting forms—that is an unbearable existence and I disdain and deplore it. I want to love, I want to lose myself in taking an interest, I want to be glad or sad. [...] Only in love is there life; without it there is death and annihilation.”158 At this juncture, a Spinozist may argue that the German Idealist misgivings of Spinoza's naturalistic pantheism are false. Is not the Ethics itself a profound celebration of life rather than death, love rather than indifference, a text that itself can be read as a quasi-psychotherapeutic intervention that aims, with the aid of its geometrical proofs, to liberate one from the passivity of the passions and in so doing find a new manner of radically asserting the (limited, though existent) freedom of one's own conatus? As Spinoza says, the Ethics “concerns the method, or way, leading to freedom.”159 By demanding that we build a new organ of sight,160 it asks us to undergo a profound change in our relationship to self and world, to become a new species, by giving up ill-founded politico-theological ideals and conceptions of humanity (mere fictions),161 which we can only accomplish by giving into and working with the relational dynamics at the heart of what it means for to be singular essence insurmountably inter- bound with the infinity of other essences. Substance as a causal network of complete interpenetration wherein each being attains its life force reveals the immanent “potentials” of existence: that is, how it can achieve more power, more strength, more force in this or that existent. However, even if in this respect Spinoza does allow for the mind to have some power over the body and thus a certain degree of spontaneous activity (for surely the Ethics is emancipatory for the subject only because it is a work of ideas), according to the German Idealists this does not come close to articulating the irreducibility of freedom attested by Kant.162 Spinoza fails to see the true kernel of human liberty, a failure that not only makes the Spinozistic system insufficient in terms of the lived essence of freedom, but also thereby takes away life and richness from its ontology. From Transcendental Philosophy to Substance as Subject 121 122 Chapter 6 From Transcendental Philosophy to Substance as Subject 121 is an ethics: the whole endeavour of Spinozism is meant to show how, by coming to this realization step by step through clear and precise reasoning, one can liberate oneself from the bondage of one's passive emotions and see how one directly takes part in the freedom of the self-unfolding cosmos, overflowing with uncontainable energy. Showing the structure of the world forces us to act differently—logical proofs are equivalent to opening the mind's eye to the dynamics of substance as totality.154 For the late German Idealists working in the aftermath of Kant, however, this vision reduces the apparent autonomous essence of subjectivity to a mere epiphenomenon, a false appearance, of the vital flux of a more primordial life force that runs through and simultaneously is the universe, thus leaving nothing untouched and no room for a transcendence within its omnipresent pull. This “direct participation” can only be paradoxically interpreted as a passive participation, a forced enactment, which befalls us. We see this most unsettlingly in Spinoza's stone: Furthermore, conceive, if you please, that while continuing in motion the stone thinks, and knows that it is endeavouring, as far as in it lies, to continue in motion. Now this stone, since it is conscious only of its endeavour and is not at all indifferent, will think it is completely free, and that it continues in motion for no other reason than that it so wishes. This, then, is that human freedom which all men boast of possessing, and which consists solely in this, that men are conscious of their desire and unaware of the causes by which they are determined. In the same way that a baby thinks that it freely desires milk [...].155 For the late German Idealists, if this is freedom, it is a grotesque joke: instead of being liberating, it is claustrophobic, for our infinite strivings are reduced to a mere puppet show for an impersonal God sive nature whose power drowns any hope we may have for true self-standing independence in its might. Whatever freedom is here, it is distant from us: “[i]n the mind there is no absolute, or free, will. The mind is determined to this or that volition by a cause, which is likewise determined by another cause, and this again by another, and so ad infinitum.”156 It is without a doubt Fichte who most succinctly expresses the German Idealist disdain at this picture. From Transcendental Philosophy to Substance as Subject 123 The error of his system lies by no means in the positing of things in God, but rather in that there are things in the abstract concept of the world's being, instead of infinite substance itself, which in fact is also a thing for him. Thus his arguments against freedom are entirely deterministic, and in nowise pantheistic. He treats the will, too, as a thing, and then naturally proves that it must be determined in its every action by another thing, which, in turn, is determined by yet another thing, etc., into infinity. Hence the lifelessness of his system: the mindlessness of its form, the impoverishment of its concepts and expressions, the unyielding acerbity of its definitions [...]. One could view Spinozism in its rigidity as Pygmalion's statue: it needed to be given a soul by the warm breath of love.163 Hegel makes a similar point in his Science of Logic: Of course, substance is the absolute unity of thought and being or extension; it therefore contains thought itself, but only in its unity with extension, that is to say, not as separating itself from extension and hence, in general, not as determining and informing, nor as a movement of return that begins from itself. For this reason, on the one hand substance lacks the principle of personality—a defect that has especially aroused indignation against Spinoza’s system.164 Hegel makes a similar point in his Science of Logic: Of course, substance is the absolute unity of thought and being or extension; it therefore contains thought itself, but only in its unity with extension, that is to say, not as separating itself from extension and hence, in general, not as determining and informing, nor as a movement of return that begins from itself. For this reason, on the one hand substance lacks the principle of personality—a defect that has especially aroused indignation against Spinoza’s system.164 Humans are not mere passive players in the self-unfolding drama of the universe, but constitutive writers of it, dominating it from the inside out, to such an extent that they even present a challenge to the autonomy of substance. In the face of man, the very fabric of substance appears lacerated, for it encounters a transcendent Other within its heart of hearts that makes it non-coincident to itself, infringes upon its oneness and thereby renders it not-all. 122 Chapter 6 Without inscribing the difference announced by subjectivity into substance, we miss something essential about the life of the absolute, a critique raised by Schelling in his Freiheitsschrift: From Transcendental Philosophy to Substance as Subject 123 From Transcendental Philosophy to Substance as Subject 123 According to Žižek, what both Hegel and the middle-late Schelling find unsatisfactory about Spinoza is that he is unable to articulate the ontogenetic condition of the possibility of the emergence of a freely existing transcendent(al) subjectivity out of the purely immanent plane of being and its implications for understanding the metaphysical nature of reality. The problem is that freedom—in its very specific articulation in German Idealism—is 124 Chapter 6 Chapter 6 124 not compatible with substance qua devouring totality. How, then, are we to think substance and subject/system and freedom if we are to retain the spontaneity first brought to light, albeit for the most part formally, by Kant? For Žižek, the answer is clear: “[t]he passage from the Spinozan One qua the neutral medium/container of its modes [to] the One's inherent gap is the very passage from Substance to Subject.”165 But the recognition of this gap has to be intrinsically traumatic, terrifying—it demonstrates a radical shift in our understanding of the world as some sort of harmonious cosmos that holds itself together in its infinite rational majesty to a world that, lacking totalizing order, must be predicated upon disruption and upheaval. If such an intuition did arise in the history of German Idealism, we would expect to see a series of psychoanalytical defence mechanisms against a conscious acknowledgment of its truth, which in turn obstruct its texts. It is a direct confrontation with this Real of the tradition that will enable Žižek to bring forth the true metaphysical horror of subjectivity that he thinks Descartes had already glimpsed and that has been haunting philosophy like a spectre ever since. It is this specific spin on the dialectical “union” of system and freedom that is of utmost importance for understanding Žižek's psychoanalytical reactualization of German Idealism. It has two functions. First, it demonstrates the heterodox character of Žižek’s appropriation of the tradition insofar as he proclaims that its real truth has always been this disjunctive, parallax relationship between the two terms.166 It clearly shows us how and why Žižek challenges our normal preconceptions of German Idealism's internal historico-theoretical concerns and puts us in a position to evaluate Žižek's own readings more clearly. From Transcendental Philosophy to Substance as Subject 125 From Transcendental Philosophy to Substance as Subject 125 of the Imaginary that often tells itself lies to cover up its dirty spots) and various symbolically non-integrated elements visibly existing in its fold (the ephemeral flickering of a traumatic Real interrupting its normal discourse pointing to its repressed truth), whose integration would demand a radical reconfiguration of its own self-perception (in the spirit of Wo Es war, soll ich werden). Žižek seeks to understand what the role played by this disavowed knowledge could teach us about this crucial turning point in the history of philosophy, the nature of subjectivity, and, ultimately, the very ontological structure of the world we live in. From Transcendental Philosophy to Substance as Subject 123 Second, it demonstrates that Žižek's own specific take on its unconscious Grundlogik is grounded in an extremely coherent reading of the stakes at play in post-Kantian philosophy, even if these have been drastically reformatted along the way according to a perceived and unrealized textual possibility. This makes reading Žižek a strange experience because there is an irreconcilable tension between Žižek's account of German Idealism and what its representatives on the surface appear to accomplish with their systems. Yet through this productive clash, Žižek is trying to create a space of “therapeutic” interplay between German Idealism's surface content (what it takes itself to be in the narcissistic orbit 6.3 The Suffocating Deficiency of a Naturphilosophie The immediate problem facing the late German Idealists is that the Kantian affirmation of transcendental freedom must be grounded in an ontological edifice that can rival Spinozism, for otherwise a Spinozist could argue that freedom is merely the misrecognition of the absolutely free self-unfolding oneness of substance, of man's subsumption within the positive order of being driven by a divinely energized and productive nature as its immanent cause. While both the early Schelling and Hegel offer their own solutions, Hegel remains unsatisfied with the results of his onetime colleague and friend, both in terms of the former's naturephilosophical response to the early Fichte and his quasi-Spinozistic attempts to ground transcendental subjectivity and creative nature in a point of absolute indifference that is neither subjectivity nor nature, but possesses both equally within it. Both projects seem to miss the mark, but why this would lead to a break between Hegel and Schelling is not clear. As hinted by Žižek's vision of Hegel and the middle-late Schelling, Hegel must have implicitly recognized here that Schelling fails to grasp the true radicality of Kantian freedom and its implications by adhering too much to a unitary view of the absolute, the seamless oneness of all that is. Consequently, Hegel tries to save the breakthrough of the critical system by thinking substance as subject, by thinking how the positive order of being ex-ists (existere in the sense of stepping or standing out) in the mode of subjectivity, instead of merely tying two apparently different yet complementary areas together into a precarious, “dead” harmony in indifference, wherein all qualitative difference between Chapter 6 126 subjectivity and objectivity becomes secondary, unimportant, and ultimately lost. As Hegel himself says in his Philosophy of Nature: The cause of [Naturphilosophie's] aberration lay in the fundamental error of first defining the Absolute as the absolute indifference of subjective and objective being, and then supposing that all determination is a merely quantitative difference. The truth is rather, that the soul of absolute form, which is the concept and living reality, is solely qualitative self- sublating differentiation, the dialectic of absolute antithesis. One may think, in so far as one is not aware of this genuinely infinite negativity, that one is unable to hold fast to the absolute identity of life, without converting the moment of difference into a simply external moment of reflection. 6.3 The Suffocating Deficiency of a Naturphilosophie This is of course the case with Spinoza, whose attributes and modes occur in an external understanding; life must then completely lack the leaping point of selfhood, the principle of autonomous movement, of internal self-diremption.167 This citation clearly shows the task Hegel believes must be accomplished: a full actualization of the primordial insight underlying the cogito by instituting the I and the schism it evokes into the very immanent activity of the absolute. For if we follow Fichte's intuition that the subject emerges in being “by absolute spontaneity alone,” that is, “not through a transition, but by means of a leap,”168 in the wake of the subject the life of substance must be said to undergo a process of internal self-diremption. Accordingly, Hegel's project is an attempt to ontologize the Kantian framework by exploring conditions of the possibility of the emergence of the subject out of a ground that remains Other to it (its “pre-history”), insofar as its self-positing must be identical to a liberating self-caused separation from substance that leaves the latter bleeding in its ontological fold. Hegel's goal is to balance Spinoza and Kant by creating a metaphysical system that renders possible rather than precludes freedom in such a way that his “transcendentalism” reaches far beyond the conditions of theoretical knowing or practical action and directly opens up to a metaphysics of the ontological rupture that presents itself as the extra-subjective condition of human autonomy. From Transcendental Philosophy to Substance as Subject 127 From Transcendental Philosophy to Substance as Subject 127 The issue is to explain how a true freely existing subject can arise from within the internal mechanics of substance. Žižek's heterodox and challenging claim is that the only way to do this is by taking the split announced by Cartesian subjectivity and pushing it to its limits by inscribing the non-coincidence of mind with matter into the very heart of being. If human freedom is irreducibly self-reflexive and self-legislative it cannot be understood in terms of the basal energetic pulsating of the absolute. Reading the Hegelian response to Schelling remodulated through psychoanalysis, Žižek suggests that what provokes the movement from transcendental philosophy to Hegelian substance as subject is how Spinozism and the Kantian articulation of freedom reciprocally expose each other's intrinsic limitations, which simultaneously force us to rethink our understanding of ourselves and our relation to the world. While the latter lacks a metaphysics, the former misses the irrevocable (ontological) disturbance of/in nature at the basis of the cogito, which signals that human spontaneity cannot be contained in the positive order of being, something also missed in Schelling's own early Naturphilosophie. For Žižek, the true breakthrough of Kantian idealism, made explicit for the first time in Hegel and in the middle-late Schelling and then most acutely in psychoanalysis, is the proclamation of transcendental freedom as linked to Todestrieb, an excess of being that breaks from all externally given laws, dirempts being from the inside out, and thereby produces a tension-stricken not-all bursting at the seams from inner turmoil. Because of the value Žižek accords to the psychoanalytical experience of the discord between mind and body, he arrives here at a conditional: If freedom exists, substance cannot be all. Substance's auto-disruption is the first-level condition of the possibility of the subject. Although this is perhaps a tenuous claim to make within the context of post-Kantian idealism (it exhibits an abundance of other ways of understanding the substance-subject relation), we should be wary of dismissing Žižek for purely “historico-contextual” reasons. He himself is more interested in another possibility of understanding German Idealism he sees hinted at behind the scenes of its texts. The fact that his reading is not a mere line-by-line commentary is no reason to proclaim that it is outright wrong. As a Lacanian, Žižek does not share the presuppositions that would 128 Chapter 6 128 Chapter 6 128 Chapter 6 128 methodologies and constraints of truth for evaluating his interpretation would, in fact, merely do to Žižek what his critics accuse him of doing. In this respect, their critiques are a performative contradiction. What intrigues Žižek in Hegel's articulation of the subject as self- relating negativity is its connection with the Kantian pedagogical concept of “unruliness” and “diabolic evil.” Insofar as self-relating negativity indicates that human subjectivity is non-natural, it shows that, if we follow the internal course of German Idealism, many of Hegel's concepts such as the “night of the world” or “the activity of dissolution [which] is the power and work of Understanding”169 appear to be nothing other than an elaboration of the subject's origins within an ontological disruption from the closed circuitry of nature's homeostatic laws as already alluded to but not fully developed by Kant. Žižek proclaims that we normally overlook something crucial in Hegel, for what Žižek's Hegel adds to the Kantian notion of the transcendental constitution of experience and rational self-determination is a gesture towards their dark commencement, a glimpse into how the spectral pandemonium of the pre-logical Real we see in “unruliness” precedes and makes possible the autonomy of the cogito and its originary “diabolic evil,” thus drawing a more explicit link between notions whose interdependence Kant merely suggested: The human being is this night, this empty nothing, that contains everything in its simplicity—an unending wealth of many representations, images, of which none belongs to him—or which are not present. This night, the interior of nature, that exists here—pure self—in phantasmagorical representations, is night all around it, in which here shoots a bloody head—there another white ghastly apparition, suddenly here before it, and just so disappears. One catches sight of this night when one looks human beings in the eye—into a night that becomes awful.170 Prior to the self-legislative laws of practical reason and the synthesis of transcendental imagination constituting the unity of phenomenal reality, we must posit some kind of ontological going-haywire that represents a savage tearing apart of the flow of vital being (“here shoots a bloody head—there another white ghastly apparition”) as that which opens up their logical From Transcendental Philosophy to Substance as Subject 129 As a result, the unruliness of the human organism is nothing other than another logical modality of transcendental imagination, its ontological zero-level as a disruption in/of the circuitry of 130 Chapter 6 0 Chapter 6 130 nature's laws that demands its recombination, a recombination that can only be done in a non-natural (virtual) register; and reading this insight in light of Kant's pedagogical writings on the necessity of disciplining unruliness for the emergence of culture, we can thus further say that the epigenesis of the categories173 as that which bestows upon experience its form cannot be limited to the logico-scientific structure that the latter assumes, but must also extend to the sociopolitical code underlying culture itself. The out- of-control freedom of the subject is intimately linked with the power of imagination (Einbildungskraft) precisely because it is only by means of the latter that the subject is capable of producing images (Bilder or schemata) by which it can give structure to this ghastly state of chaos in the Real, a quasi-phenomenal field lacking subject-object articulation since, although the subject itself has emerged in a primordial sense, as of yet there are no conceptual structures and no symbolic network necessary to mould reality into an integrated, smooth fabric (a process of transcendental Bildung, a schematization of the night of the world). The paradox lies in the following: we can only explain the order of experiential reality in its multifarious modes by presupposing an originary, impenetrable pandemonium that we can only glimpse, but never know (it being always already “overcome” as soon as experience has come on the scene), a pandemonium that logically precedes and ontologically renders possible the consistency of full-fledged subjectivity and psychic life. The I itself as an irreducible core of transcendental reflexivity can only emerge out of this chaos, this macabre seizure of forms that represents ontological mayhem/madness at its finest, which in turn signals that, at its zero-level, the subject is nature in the mode of auto-denaturalization. Hegel's horrifying ontologization of transcendental imagination in the night of the world, however, goes a step further. The night of the world does not merely indicate a radical breakdown in the flux of materiality in the Real; it is also the beginning of an infinite withdrawal of being into itself that cuts all ties with the outside world through obeying its own self-given law. From Transcendental Philosophy to Substance as Subject 129 From Transcendental Philosophy to Substance as Subject 129 possibility. If practical reason and transcendental imagination go hand in hand, it is because both are a response to subjectivity itself, a radicalization of this denaturalizing tendency, to a nature whose fold has been disrupted and thus demands re-articulation (“a night that becomes awful”): The pre-synthetic Real, its pure, not-yet-fashioned “multitude” not yet synthesized by a minimum of transcendental imagination, is, stricto sensu, impossible: a level that must be retroactively presupposed, but can never actually be encountered. Our (Hegelian) point, however, is that this mythical/impossible starting point, the presupposition of imagination, is already the product, the result of, the imagination's disruptive activity. In short, the mythic, inaccessible zero-level of pure multitude not yet affected/ fashioned by imagination is nothing but pure imagination itself, imagination at its most violent, as the activity of disrupting the continuity of the inertia of the pre-symbolic “natural” Real. This pre-synthetic “multitude” is what Hegel describes as the “night of the world,” as the “unruliness” of the subject's abyssal freedom which violently explodes reality into a dispersed floating of membra disjecta.171 This chaotic aggregate of ghastly forms and shapes making up the quasi- phenomenological self-experience of the night of the world is the pure expression of the unruliness/biological short circuitry of the human organism, the German Idealist variation upon Lacan's mirror stage.172 As in the latter, this moment is not to be taken in isolation, but to be supplemented with what it ontogenetically makes possible, namely, the ideal-symbolical realm of ordered experience. What we see here is quasi- phenomenological because, in actuality, there is no fully developed I that stands in relationship to an alterity over and against which it can stand (rather than there being well-defined Gegen-stände, there is nothing but a fragmentary field lacking coherence). This I can only emerge après coup after a free act of synthesis of this initial state of chaotic dispersion, that is, its ideal-symbolic re-articulation. From Transcendental Philosophy to Substance as Subject 131 Just as Kant demonstrates in Religion within the Bounds of Reason Alone, the basis of subjectivity for Hegel is inherently ambiguous, insupportably undecidable: not only does it necessarily bring our own understanding of freedom dangerously close to that of evil, where the difference between them threatens to dissipate, but more disconcertingly it points to the ancestral past of the subject as entangled in some kind of unfathomable ontological crime.175 Whereas early post-Kantian philosophy, namely that of Reinhold and the early Fichte, sought to unify the critical project through elementary philosophy and the absolute self-positing of the I, respectively, Hegel came across something disquieting in his own endeavour to escape its impasses: theoretical and practical reason coincide in a chasm in being that testifies to an unspeakable extra-subjective violence. What both Kant's pedagogic writings and Hegel's night of the world reveal is that in order for idealization/ symbolization to occur as a purely autonomous activity, there must be something in nature that leaves it lacerated, wounded, bleeding, this being the only way that a zone freed from its hegemony could arise. In other words, even if we take the breakthrough of Kantian idealism to be the priority of the symbolic-conceptual categories whereby the subject engenders experience rather than its account of freedom (a move that also assures that culture is always of our own making because not subject to natural determination) even on this reading, this is the dark horizon within which ideality and, by implication, the sociopolitical as a logically autonomous milieu can operate: the death of nature in us. In this respect, if the major insight of German Idealism and psychoanalysis is that “the passage from 'nature' to 'culture' is not direct,”176 then one of its major implications is that the Symbolic is not only a “kingdom within a kingdom” (imperium in imperio), but more radically one that has won its way to ontological sovereignty through a destructive, murdering force, thus establishing the avid anti-Spinozism of both traditions.177 The pure I inaugurated by transcendental philosophy exposes a bone The pure I inaugurated by transcendental philosophy exposes a bone in the throat of substance, a snag in natural causality, proclaiming that the absolute as nature or the physical universe cannot be all. From Transcendental Philosophy to Substance as Subject 129 Consequently, if this ontogenetic mayhem/madness is that which renders possible the absolute spontaneity of the I, then it shares an uncanny structural identity with Hegel's definition of evil in the Jenaer Realphilosophie as an “internal reality, absolute certainty of itself, the pure night of being-for-itself.”174 From Transcendental Philosophy to Substance as Subject 131 132 Chapter 6 constitutes the zero-level of human freedom represents an ontological catastrophe, a catastrophe that is synonymous with the subject itself: “it designates [...] the primordial Big Bang, the violent self-contrast by means of which the balance and inner peace of the Void of which mystics speak are perturbed, thrown out of joint.”178 In this sense, the Žižekian appropriation of the Hegelian attempt to think substance as subject readdresses what really differentiates Hegel from Schelling the Naturphilosoph and sets the stage for his own philosophical career. Although the early Hegel does support Schelling's project to develop a rich teleological account of nature (as various sketches in the Jena period demonstrate), he must be said to have come to some kind of recognition of the inherent limitations of Schelling's endeavour. It is this vital hemorrhaging of nature that, preventing the ideality of the subject from being subsumed within the self-actualizing of the absolute, pushes Hegel away from Schelling: before the transcendental (re)constitution of reality by the I can occur, an internal ontological short circuit, a metaphysical breakdown of substance, must be posited, which not only renders impossible a complete immersion of subjectivity within a natural evolutionary narrative, but declares the ultimate discontinuity between nature and spirit instead of their peaceful identity and mutual interpretation in nature as “the unconscious poetry of spirit.”179 From Transcendental Philosophy to Substance as Subject 131 What must be taken from this is one of the key principles of Žižek's ontology: freedom is not a raw, brute fact, but depends upon the caustic collapse of the vital fold of being, a brisure in the heart of the Real; the chaotic aggregate of ghastly forms that 132 Chapter 6 From Transcendental Philosophy to Substance as Subject 133 subject, therefore, has no positive ontological substrate, so that in Hegel the German Idealist attempt to supply a foundation for freedom strangely backfires on itself. But in its very failure, this attempt stumbles upon a great truth: the zero-level of human freedom is a blockage, an obstruction in the mechanics of nature, for the ontological status of the transcendental expresses its dependence upon a self-destructive negativity in substance itself. The Žižekian thesis is that without the articulation of this paradoxical site of excessive negativity (Todestrieb) as emerging immanently within being, all metaphysical accounts of human subjectivity and freedom risk their reductionist-monistic cancelation—otherwise we cannot explain the leap constitutive of the latter without risking their subsumption within an evolutionary transition. If we are to defend the fact of freedom, we must assume it and go all the way, which leads us to a single coherent conclusion: “either subjectivity is an illusion, or reality itself (not only epistemologically) is not-All.”181 subject, therefore, has no positive ontological substrate, so that in Hegel the German Idealist attempt to supply a foundation for freedom strangely backfires on itself. But in its very failure, this attempt stumbles upon a great truth: the zero-level of human freedom is a blockage, an obstruction in the mechanics of nature, for the ontological status of the transcendental expresses its dependence upon a self-destructive negativity in substance itself. The Žižekian thesis is that without the articulation of this paradoxical site of excessive negativity (Todestrieb) as emerging immanently within being, all metaphysical accounts of human subjectivity and freedom risk their reductionist-monistic cancelation—otherwise we cannot explain the leap constitutive of the latter without risking their subsumption within an evolutionary transition. If we are to defend the fact of freedom, we must assume it and go all the way, which leads us to a single coherent conclusion: “either subjectivity is an illusion, or reality itself (not only epistemologically) is not-All.”181 Here we see again how Žižek's reactualization fights against textbook accounts of German Idealism. If Hegel is to be a philosopher of freedom, his metaphysical articulation of the absolute must pass through the Kantian critical system, which means it cannot just be a simple retour to Spinozistic metaphysics; we must witness some kind of brisure within the absolute that disrupts the blind, immanent movement of natural laws and thereby enables the possibility of a freely existing subject. 6.4 The Night of the World/A Monism Bursting at Its Seams Hegel makes a crucial step towards elaborating the paradoxical ground of the subject by demonstrating that it is this going-haywire, this dysfunctioning of substance that makes subjectivity incommensurable with material being and renders possible freedom in the truly “idealist” sense of the word. It is Hegel's account of the advent of the I in nature that is the first to truly explicate the eruption of the subject as an extimacy that cannot afterwards be resubsumed within the oneness of the self-integrating unity of the absolute. There is no smooth union, no ultimate self-penetrating identity within the fabric of all-pervasive being, for we encounter an infinite breakdown, an internal tension, that causes irrevocable havoc in the life of the absolute: “Subject designates the 'imperfection' of Substance, the inherent gap, self-deferral, distance-from-itself, which forever prevents Substance from fully realizing itself, from becoming 'fully itself.'”180 The From Transcendental Philosophy to Substance as Subject 133 From Transcendental Philosophy to Substance as Subject 133 Although this is a rather controversial claim to make in the context of German Idealism, Žižek believes it allows us to reconceptualize its attempt to think substance as subject in such a way that it does not fall into conventional platitudes that smother its theoretical potential: platitudes such as comprehending the absolute as a cosmic subject-like agency that remains constant through all determinate change, preceding and (sub/con)suming the freedom of concrete existence by autonomously actualizing itself from a safe distance from the flux and contingency of reality, thereby guaranteeing the movement of history.182 The subject only emerges from an accidental blockage, an irrevocable moment of self-division, which means that substance and subject are in themselves dirempt and caught in finitude: “Substance is Subject” means that the split which separates “Substance is Subject” means that the split which separates Subject from Substance, from the inaccessible In-itself beyond Subject from Substance, from the inaccessible In-itself beyond 134 Chapter 6 Chapter 6 phenomenal reality, is inherent to the Substance itself. [...] The point is not that Substance (the ultimate foundation of all entities, the Absolute) is not a pre-subjective Ground but a subject, an agent of self-differentiation, which posits its otherness and then reappropriates it, and so on: “Subject” stands for the non-substantial agency of phenomenalization, appearance, “illusion,” split, finitude, Understanding, and so on, and to conceive Substance as Subject means precisely that split, phenomenalization, and so forth, are inherent to the life of the Absolute itself.183 Not only does the transcendental reconstitution of reality depend upon a prior upsurge of pure ontological violence, but the very phenomenalization of experience is co-incidental with this rupturing, the not-all, of substance. In other words, subject is substance in its mode of self-alienation: the transcendental reconstitution of reality is nothing but an immanent attempt by the absolute as nature to overcome its split by filling in the gap opened up in the core of its vital being, whereby ego development and concrete experience are reduced to a reaction formation, an attempt to suture a wound. Substance is not subject (as a proclamation of identity): substance becomes subject (in a moment of trauma). The very fact that there is experience demonstrates this for Žižek, for there is no intrinsic reason for experience within the “smooth” self-articulation of substance. From Transcendental Philosophy to Substance as Subject 135 critique of Propositions 12 and 13 of Part I of the Ethics, which argue for the absolute indivisibility and untouchable oneness of substance, but in such a way that we do not fall into the logical conundrum outlined in Propositions 2 through 6, whose goal is to show the impossibility of two substances having nothing in common being able to have an effect upon one another or to produce one another, all the while paradoxically without violating Propositions 14 and 15, which argue that all things must be within one substance. What idealism reveals to us is that freedom expresses something operatively new that has emerged within being and is irreducible to its ebb and flow, yet that does not exist outside of it, a transcendence in immanence that indicates some kind of split within substance itself infringing upon the logical hegemony of its infinite power by means of its internal breakdown. The subject “is not a new name for the One which grounds all, but the name for the inner impossibility or self-blockage of the One.”185 In man, negativity, having attained notional self-reflexivity, shows itself to be foundational to identity rather than a mere subcategory of positivity of the absolute used in the determination of finite things, so that it possesses its own monstrous logic that colonizes the immanent field within which it has emerged from a place that can only appear, from within that field, as an infinite elsewhere. However, what do we see here except the beginnings of a systematic ontologization of Lacanian metapsychology, centred in the mirror stage as a primordial organic disharmony indicative of a disjunctive “and” inscribed within being? Doesn't Žižek imply here that the only possible way to answer Miller's famous question: “Lacan, what's your ontology?” is by a passage through German Idealism, because of the distinctly “psychoanalytical” themes omnipresent in the latter once we psychoanalytically reconstruct its Grundlogik? Yet one other thing should be clear at this juncture. Although Žižek's Yet one other thing should be clear at this juncture. Although Žižek's Hegel glimpses into the ontologico-foundational basis of human spontaneity preceding the transcendental constitution of experiential reality, he is unable to account for two things. First, the immanent generation of this negativity within the material flux of substance that sets the stage for subjectivity by enacting the first lacerations upon substance. How does the vital flow of being itself rupture? From Transcendental Philosophy to Substance as Subject 135 critique of Propositions 12 and 13 of Part I of the Ethics, which argue for the absolute indivisibility and untouchable oneness of substance, but in such a way that we do not fall into the logical conundrum outlined in Propositions 2 through 6, whose goal is to show the impossibility of two substances having nothing in common being able to have an effect upon one another or to produce one another, all the while paradoxically without violating Propositions 14 and 15, which argue that all things must be within one substance. What idealism reveals to us is that freedom expresses something operatively new that has emerged within being and is irreducible to its ebb and flow, yet that does not exist outside of it, a transcendence in immanence that indicates some kind of split within substance itself infringing upon the logical hegemony of its infinite power by means of its internal breakdown. The subject “is not a new name for the One which grounds all, but the name for the inner impossibility or self-blockage of the One.”185 In man, negativity, having attained notional self-reflexivity, shows itself to be foundational to identity rather than a mere subcategory of positivity of the absolute used in the determination of finite things, so that it possesses its own monstrous logic that colonizes the immanent field within which it has emerged from a place that can only appear, from within that field, as an infinite elsewhere. However, what do we see here except the beginnings of a systematic ontologization of Lacanian metapsychology, centred in the mirror stage as a primordial organic disharmony indicative of a disjunctive “and” inscribed within being? Doesn't Žižek imply here that the only possible way to answer Miller's famous question: “Lacan, what's your ontology?” is by a passage through German Idealism, because of the distinctly “psychoanalytical” themes omnipresent in the latter once we psychoanalytically reconstruct its Grundlogik? From Transcendental Philosophy to Substance as Subject 133 Not only does the transcendental reconstitution of reality depend upon a prior upsurge of pure ontological violence, but the very phenomenalization of experience is co-incidental with this rupturing, the not-all, of substance. In other words, subject is substance in its mode of self-alienation: the j f f transcendental reconstitution of reality is nothing but an immanent attempt by the absolute as nature to overcome its split by filling in the gap opened up in the core of its vital being, whereby ego development and concrete experience are reduced to a reaction formation, an attempt to suture a wound. Substance is not subject (as a proclamation of identity): substance becomes subject (in a moment of trauma). The very fact that there is experience demonstrates this for Žižek, for there is no intrinsic reason for experience within the “smooth” self-articulation of substance. But here we should make another addition. When we say that the subject is a transcendent Other created from within the immanence of being, we must be careful. It is not that substance creates or produces another substance, in which case the enigma concerning how one could explain the genesis of one out of the other would remain unsolved. Rather, the subject is nothing but the void of substance, the minimal difference of the absolute to itself,184 which creates a metaphysical vacuum that is able to have devastating effects in the fabric of the Real of nature from which it emerged as soon as it develops the capacity to paradoxically relate to itself (movement from “unruliness” to “diabolic evil,” from the tearing apart of the flow of vital being to its radicalization in “the night of the world”). In this sense we should understand late German Idealism, and by consequence Žižek's own metaphysics, as attempts to think system and freedom as a From Transcendental Philosophy to Substance as Subject 135 136 Chapter 6 the Real, thus making it barred, nothing but a series of membra disjecta? Second, the absolute spontaneity of the very founding gesture of subjectivity that depends on nothing but itself for its own self-positing, a self-positing that presents itself as a fiat that of itself institutes a pure difference in being: the problem for us is how we are to conceive of the founding gesture of subjectivity, the “passive violence,” the negative act of (not yet imagination, but) abstraction, self-withdrawal into the “night of the world.” This “abstraction” is the abyss concealed by the ontological synthesis: by the transcendental imagination constitutive of reality—as such, it is the point of the mysterious emergence of transcendental “spontaneity.”186 To answer these questions, we will need to delve into Žižek's oft-neglected work on Schelling, for it is Schelling who delved most profoundly into the obscure origins of subjectivity. From Transcendental Philosophy to Substance as Subject 135 How does this extimate core germinate within the Real and incite a violent explosion that forever precludes the ontological life of 120. In the Freiheitsschrift, for instance, Schelling says that it is idealism that “we have to thank for the first perfect concept of freedom” and then situates the true Kantian breakthrough in its revolutionary articulation. See pp. 231ff. Hegel also praises Kant’s theory of freedom. See Encyclopedia Logic, p. 101 (§54Z). The same applies to Žižek: “No wonder Kant is the philosopher of freedom: with him, the deadlock of freedom emerges.” Žižek, The Parallax View, p. 9. But all this was stressed by Kant himself, who declares that freedom “constitutes the keystone of the whole architecture of the system of pure reason and even of speculative reason.” Critique of Practical Reason, in Practical Philosophy, trans. and ed. Mary J. Gregor (Cambridge: Cambridge University Press, 1999) p. 139. Notes 120. In the Freiheitsschrift, for instance, Schelling says that it is idealism that “we have to thank for the first perfect concept of freedom” and then situates the true Kantian breakthrough in its revolutionary articulation. See pp. 231ff. Hegel also praises Kant’s theory of freedom. See Encyclopedia Logic, p. 101 (§54Z). The same applies to Žižek: “No wonder Kant is the philosopher of freedom: with him, the deadlock of freedom emerges.” Žižek, The Parallax View, p. 9. But all this was stressed by Kant himself, who declares that freedom “constitutes the keystone of the whole architecture of the system of pure reason and even of speculative reason.” Critique of Practical Reason, in Practical Philosophy, trans. and ed. Mary J. Gregor (Cambridge: Cambridge University Press, 1999) p. 139. 121. In this sense, both Hegel and Schelling are following Fichte: “I live in a new world since I read the Critique of Practical Reason; it destroyed theses that I thought were irrefutable, proved things I thought indemonstrable, like the concept of abso- lute freedom.” Johann Gottlieb Fichte, Gesamtausgabe der Bayerischen Akademie der Wissenschaften, Division III: Briefe, vol. I, ed. R. Lauth, Hans Jacob, and H. Gliwitzky (Frommann: Stuttgart-Bad Cannstatt, 1964–), no. 63, p. 168. 122. Žižek, Less Than Nothing, p. 265. 123. Žižek, The Parallax View, p. 93. 124. Ibid., p. 92. 125. Fichte, Science of Knowledge, p. 114. 126. Maldiney, Penser l’homme et la folie (Paris: Gillon, 2007), p. 114. 127. Fichte, Science of Knowledge, p. 247. 127. Fichte, Science of Knowledge, p. 247. 128. Ibid., p. 152. 128. Ibid., p. 152. 129. Fichte, The Vocation of Man, trans. Peter Preuss (Indianapolis: Hackett Publishing, 1987), p. 64. 130. Freud, “The Loss of Reality in Neurosis and Psychosis,” SE, XIX, p. 183. 131. Fichte, The Science of Knowledge, p. 243. 131. Fichte, The Science of Knowledge, p. 243. 132. Fichte, Wissenschaftslehre 1805, ed. H. Gliwitzky (Hamburg: Felix Meiner, 1984), pp. 127–28. 133. Freud, “Neurosis and Psychosis,” SE, XIX, p. 151. 134. Freud, “The Loss of Reality in Neurosis and Psychosis,” SE, XIX, p. 185. 135. Fichte, Foundations of Natural Right, ed. Frederick Neuhouser, trans. Michael Baur (Cambridge: Cambridge University Press, 2000), p. 74. 136. Fichte, Fichte: Early Philosophic Writings, p. 147. 137. Fichte, Foundations of Natural Right, p. 76. Also compare p. 74. 138. See Schelling, System of Transcendental Idealism, trans. Peter Heath (Charlottesville: University Press of Virginia, 2001), p. 232. 139. Notes See Hegel, The Difference between Fichte’s and Schelling’s System of Philosophy, pp. 143 and 168. 140. Schelling, Darstellung meines Systems der Philosophie, Schellings sämmtliche Werke, ed. K. F. A. Schelling, division I, vol. 4 (Stuttgart and Augsburg: J. G. Cotta’scher Verlag, 1856–1861), p. 109. 141. In his “mature period,” Hegel declares Schelling as merely Fichte’s successor, ironically going against the spirit of his early work on the insurmountable differ- ence between them. Cf. Lectures on the History of Philosophy: Medieval and Modern Philosophy, trans. E. S. Haldane and Frances H. Simson (London: University of Nebraska Press, 1995), p. 529. 142. Hegel, The Phenomenology of Spirit, trans. A. V. Miller (London: Oxford University Press, 1977), p. 9. 143. Žižek, The Ticklish Subject, p. 55. 144. Žižek, Less Than Nothing, p. 266. 145. Hegel, Lectures on the History of Philosophy: Medieval and Modern Philosophy, p. 257. 146. Schelling, Freiheitsschrift, p. 231. 146. Schelling, Freiheitsschrift, p. 231. 147. Ibid., p. 232. 147. Ibid., p. 232. 148. Spinoza, Ethics and Selected Letters, ed. Seymour Feldman and trans. Samuel Shirley (Indianapolis: Hackett Publishing, 1982), II p7 (p. 66). 149. Ibid., I p13c (p. 39). 149. Ibid., I p13c (p. 39). 150. Ibid., I p16 (p. 43). 150. Ibid., I p16 (p. 43). 151. Ibid., I p15 (p. 40) and I ax1 (p. 31). 151. Ibid., I p15 (p. 40) and I ax1 (p. 31). 152. Ibid., I p34 (p. 56). 152. Ibid., I p34 (p. 56). 152. Ibid., I p34 (p. 56). 153. Ibid., Letter 78 to Henry Oldenburg (p. 254). 154. Ibid., V p23s (p. 216). 154. Ibid., V p23s (p. 216). 138 Chapter 6 155. Ibid., Letter 58 to G. H. Schuller (p. 250). Compare with I p24 (p. 49), I p26 and 27 (p. 50), and I p33 (p. 54). 156. Ibid., II p48 (p. 95). 156. Ibid., II p48 (p. 95). 157. Fichte, The Vocation of Man, pp. 21–22. 157. Fichte, The Vocation of Man, pp. 21–22. 158. Ibid., p. 24. 158. Ibid., p. 24. 159. Spinoza, The Ethics, p. V pref (p. 203). 160. Ibid., V p23s (p. 216). 160. Ibid., V p23s (p. 216). 161. Ibid., I app (pp. 57–62). 161. Ibid., I app (pp. 57–62). 162. Schelling, Freiheitsschrift, p. 227. 163. Ibid., pp. 230ff. 163. Ibid., pp. 230ff. 164. Hegel, The Science of Logic, p. 472. 165. Žižek, The Parallax View, p. 42. 166. From Transcendental Philosophy to Substance as Subject 139 184. Žižek, Less Than Nothing, p. 905. 186. Žižek, The Ticklish Subject, p. 61. 185. Ibid., p. 380. 184. Žižek, Less Than Nothing, p. 905. Notes See Žižek, The Abyss of Freedom, pp. 11–14. 167. Hegel, Hegel’s Philosophy of Nature, vol. 3, pp. 142–43 (§359A). Translation modified. 168. Fichte, Science of Knowledge, p. 262. 169. See Hegel, Phenomenology of Spirit, pp. 18–19. 170. See Hegel, Frühe politische Systeme (Frankfurt am Main: Ullstein, 1974), p. 204; quoted in Donald Philip Verene, Hegel’s Recollection: A Study of Images in the Phenomenology of Spirit (Albany: SUNY Press, 1985), pp. 7–8, whom Žižek takes it from. For a discussion, see The Abyss of Freedom, pp. 4–14; The Ticklish Subject, pp. 26–48; and The Parallax View, pp. 43–45. 171. Žižek, The Ticklish Subject, p. 33. 172. Kant knew this very well. See Kant, Anthropology from a Pragmatic Point of View, p. 423. 173. Kant, The Critique of Pure Reason, B167. 173. Kant, The Critique of Pure Reason, B167. 174. Hegel, Frühe politische Systeme, p. 262. 174. Hegel, Frühe politische Systeme, p. 262. 175. Žižek and Daly, Conversations with Žižek, p. 126. 176. Žižek, The Ticklish Subject, p. 36. 176. Žižek, The Ticklish Subject, p. 36. 177. Compare Spinoza, The Ethics, III pre (p. 103). 177. Compare Spinoza, The Ethics, III pre (p. 103). 178. Žižek, The Ticklish Subject, p. 31. 178. Žižek, The Ticklish Subject, p. 31. 179. Schelling, System of Transcendental Idealism, p. 12. 180. Žižek, The Abyss of Freedom, p. 7. 180. Žižek, The Abyss of Freedom, p. 7. 181. Žižek, The Parallax View, p. 168; Less Than Zero, p. 725; and Žižek & Woodard, “Interview,” The Speculative Turn: Continental Materialism and Realism, ed. Bryant, Levi, Nick Srnicek, and Graham Harman (Melbourne: re.press, 2011), p. 407. My italics. 182. See Žižek, Less Than Nothing, pp. 234–35 and 286–92. 183. Žižek, The Ticklish Subject, pp. 88–89. 183. Žižek, The Ticklish Subject, pp. 88–89. 185. Ibid., p. 380. 186. Žižek, The Ticklish Subject, p. 61. Chapter 7 The Logic of Transcendental Materialism Schelling and the Spectral Other Side of German Idealism Žižek needs to go beyond Hegel to articulate a crucial dialectical moment of transcendental materialism: the emergence of an ontological violence within being that prohibits the indivisibility of the absolute as an infinitely powerful, self-sustaining whole and creates room for irreducible freedom and ideality. To demonstrate this, I will outline the inherent limitations in Hegel's attempt to think substance as subject by focusing on Žižek's criticisms of the mature Hegelian Encyclopedia, thereby showing the theoretical hole simultaneously opened up and concealed by Hegel. Spurred on by the perceived threat of Absolute Idealism to human freedom, what we will see is how Schelling's investigation of the abyssal origins of subjectivity presents us with a passionate attempt to rethink the grounding of the subject and its role in being so that the former is never reducible to the latter. In this regard not only can Schelling's “materialist” response to Hegel be seen as the culminating, concluding step in what Žižek takes to be the unconscious Grundlogik of German Idealism, but it puts us face to face with the spectral Other Side of that tradition which it previously recoiled from to varying degrees: the insurmountable tension between the Real and the Ideal, which imposes a new conception of a never-to-be-reconciled quadruple dialectics with stark consequences for our understanding of nature, human historicity, and the absolute. The Logic of Transcendental Materialism 141 142 Chapter 7 upon Schelling remains largely behind the scenes potentially demonstrates some kind of error, inconsistency, or sleight of hand. Not only does Žižek fail to give any systematic argumentation for the superiority of Schelling over Hegel in terms of the obscure origins of the I out of its pre-subjective Grund, he also levels off the differences between the two insofar as he appears, as will become clear, to read them reciprocally through one other. Here I am thinking specifically of his endeavour in The Parallax View to show that, “far from posing an irreducible obstacle to dialectics, the notion of the parallax gap provides the key which enables us to discern its subversive core. To theorize this parallax gap properly is the necessary first step in the rehabilitation of the philosophy of dialectical materialism.”188 But this quote is suspicious: remarking that Schelling was “the first to formulate the post- idealist motifs of finitude, contingency and temporality,”189 and is even thus “at the origins of dialectical materialism,”190 it is clear that Žižek associates Schelling with the beginning of this tradition rather than Hegel; Schelling occupies an “immediate” place, acting “as a kind of 'vanishing mediator' between the Idealism of the Absolute and the post-Hegelian universe of finitude-temporality-contingency, [such] that his thought—for a brief moment, as it were in a flash—renders visible something that was invisible and withdrew into invisibility thereafter.”191 It is Schelling, not Hegel, who supplies us with the premonitions of a new radical way of philosophizing, a new dialectics. Moreover, the very idea of an insurmountable internal limit (a gap) as constitutive of (onto)‍logical movement—the necessity of positing the non-coincidence and tension of its moments to one another in order for it to function—has a more manifest affinity with Schelling's middle-late philosophy, which, developed as a response to the absoluteness of the Hegelian self-mediating Notion, bases itself on the idea of the indivisible remainder, der nie aufgehende Rest, as an irremovable snag in every system that guarantees its very vitality. 7.1 The Hegelian Recoil, The Schellingian Breakthrough In order to explicate how substance auto-disrupts into a chaotic series of membra disjecta, the ontological zero-level preceding the synthetic modality of transcendental imagination, we have to move outside Žižek's reading of Hegel and confront his works on Schelling. The night of the world is merely a profile portrait of the disarray and pandemonium that pave the way for the transcendental constitution of reality into a (relatively) unified fabric of experience. It does not of itself explain the originary moment of withdrawal from organic immersion in the positive order rendered possible by Todestrieb as the obscure birthplace of an irreducible more-than-material subjectivity; comprehending this event requires us to first plunge headfirst into the immanent pulsation of the vital ebb and flow of being itself if we are to see how it sets the stage for the latter—a project that Žižek explicitly says is most acutely developed in Schelling: Kant was the first to detect this crack in the ontological edifice of reality: if (what we experience as) “objective reality” is not simply given “out there,” waiting to be perceived by the subject, but an artificial composite constituted through the subject's active participation—that is, through the act of transcendental synthesis—then the question crops up sooner or later: what is the status of the uncanny X that precedes the transcendentally constituted reality? F. W. J. Schelling gave the most detailed account of this X in his notion of the Ground of Existence—of that which “in God Himself is not yet God”: the “divine madness,” the obscure pre-ontological domain of “drives,” the pre-logical Real that forever remains the elusive Ground of Reason that can never be grasped “as such,” merely glimpsed in the very gesture of its withdrawal.187 This, however, creates an internal problem for Žižek's work insofar as he describes his own project time and time again as Hegelian and never as Schellingian. If it is Schelling who is the philosopher who most fully describes the material ontogenetic conditions for the emergence of the subject rather than Hegel and thus more predominantly influences Žižek's metapsychology and ontology, then the fact that his own overt reliance 142 Chapter 7 142 Chapter 7 142 Chapter 7 Žižek in many ways appears to be interpreting Hegel retroactively through Schelling, which would explain why the irreducibility of the parallax between moments in contradistinction to their organic interpenetration in the notional structure of any given phenomenon is the “perverse” truth of Hegelian dialectics.192 What is more, in a key passage in Less Than Nothing that describes dialectics as “the science of the gap between the Old and the New,” Žižek abruptly and without The Logic of Transcendental Materialism 143 The Logic of Transcendental Materialism 143 explication jumps into a discussion of the middle-late Schelling, going so far as to say that to avoid mystification properly we should not abandon his project from this period of his thinking, but rather “reformulate it so as to avoid the mystification of the theosophic mytho-poetic narrative,”193 in such a way that it appears that the proximity of his reading of Hegel and Schelling when coupled with this brief and rare methodological explanation points to the core of his heterodox reading of German Idealism and thus his philosophy. This suggests that the core of Žižek's philosophy is a hybridism of Schellingianism and Hegelianism, so that exploring this intersection puts us face to face with the theoretico-ontological stakes underlying his entire project. Yet the following question imposes itself: at what point is Žižek's own metaphysical archaeology of the subject Schellingian or Hegelian? The very posing of this question is relatively misleading within the context of Žižek's reactualization of German Idealism, considering that what interests him is not Kant, Schelling, or Hegel as particular historical thinkers whose respective philosophies often display insurmountable differences to one another, not to mention incompatible concerns (to such a degree that one could even question whether German Idealism constitutes a coherent tradition with a single logical nucleus). What he finds alluring is a psychoanalytical truth self-unfolding throughout their works, a truth inaugurated by the Cartesian cogito and ultimately culminating in Freud and Lacan, but that they are unable to articulate fully due to its traumatic nature, “our experience of freedom” being, after all, “properly traumatic.”194 However, even if what intrigues Žižek is the disavowed Grundlogik implicitly driving the movement as an unconscious formation—something that appears in a flash only to withdraw again into the abyss from which it came—we can nevertheless demonstrate a certain dominating influence of Schelling by showing how the latter fills in a theoretical void opened up by Hegel and thereby radicalizes the founding insight of German Idealism. According to a Žižekian narrative of the untold history of German Idealism, the essence of that impossible X that eternally precedes the birth of consciousness remains underdeveloped in the Hegelian attempt to think substance as subject. The Logic of Transcendental Materialism 143 Although the Hegel of the night of the world hints at the disturbing metaphysical paradoxes that arise out of the ontologization of 144 Chapter 7 144 transcendental imagination and its concomitant concept of freedom, in The Ticklish Subject Žižek expresses outright dissatisfaction with Hegel's most systematic undertaking to inscribe the subject within a dialectics of nature as propounded in the various versions of The Encyclopedia of the Philosophical Sciences. Since this book is written after The Indivisible Remainder, it would appear that Žižek's critical engagement with Hegel's mature system and its account of the passage to culture is based on the presuppositions that guide his own transcendental materialism as worked out in this vehement work on Schelling published only three years earlier. In other words, his admitted disapproval is an implicit proof of the prioritization given to Schellingian ontology for the theorization of the parallax as a metaphysics of the disjunctive “and.” Pointing to an ambiguity persisting within the movement from self- contained Notion to nature and then to spirit in his mature system, Žižek suggests that Hegel was unable to bring into conceptual fullness the earth- shattering realization that he was on the verge of.195 What is left aside is, strictly speaking, the night of the world that Hegel's earlier Realphilosophie had uncovered. In the Encyclopedia, it is uncertain how this radical negativity, this moment of irremediable ontological breakdown haunted by sanguinary spirits, truly fits in. Instead of the precarious, never-to-be- complete “reconciliation” between nature and finite spirit brought forth by the subject, culture itself becomes a closed circuit, a complete return of the Idea to itself out of its infinite self-outsidedness in nature, a move that, by rendering culture a self-sufficient, self-contained all, completely does away with the unruliness that is the zero-level of freedom as revealed in psychoanalytical experience and potentially jeopardizes the irreducibility of the practico-concrete in Kant and made explicit for the first time in Fichte. The Logic of Transcendental Materialism 145 the snag in the machine of the Symbolic, loses all currency: we would paradoxically lose the very condition of free spiritual activity, for a complete sublation of nature into culture would herald the destruction of culture as a process of building an artificial, second nature where one is missing. Losing the obstacle causes us, in turn, to lose the goal; dialectics needs its own inner impediment to get off the ground. Corresponding to the reality/Real distinction in Lacanian psychoanalysis, we may thus venture that there are two forms of Hegelian dialectics: either we have the perfect dialectical triad of the mature system (Logic → Nature → Spirit) or a non-closed tetrad that signals the self-collapse of dialectical logic itself as seen in the Realphilosophie (Logic → Nature → finite Spirit → objective/naturalized Spirit).197 The triad is, strictly speaking, not merely inconsistent with Hegel's—and ultimately Kant's—true earlier insight, but self-defeating: it robs dialectics of its own energy, energy that can only be mobilized due to the structural impossibility of completing the task it sets out to complete. At the most basic level, culture can never utterly sublate the excessive kernel of human being and simply make it a moment of the self- meditation of the Notion as it seeks to actualize itself: there must always be a minimal, insurmountable distance between the unruliness of human nature, the withdrawal into the nocturnal Innenwelt of the world that is the founding gesture of subjectivity, and the symbolic, cultural network that attempts to form and discipline this non-natural violence into a new order after subjectivity has posited itself as such. The two can never overlap within an all-pervasive totality, insofar as for Žižek this overlapping would not merely level out the singularity that marks the subject (namely, the fact that it cannot be fully explained by either material or cultural determinations) but also in the same breath radically preclude the condition of the possibility of human freedom and the exploration of its larger metaphysical implications. The difference between traditional accounts of Absolute Idealism and the quadruple dialectic of the Realphilosophie thus enables us to demonstrate the logic Žižek wants to defend both in terms of his own transcendental materialism and the unconscious Grundlogik of modern philosophy that he psychoanalytically constructs. The Logic of Transcendental Materialism 143 The issue is that the Idea is nothing other than this very act of its own returning to itself, nothing but the attempt at reconciliation, so that not only is this very movement generative of that to which the movement returns, but more drastically the self-alienation of the Idea is a condition of its returning to itself.196 If the ontological zone wherein the fabric of the world is torn apart (“here shoots a bloody head—there another ghostly apparition”) disappears, the claim is stronger than simply the subject as the irreconcilable in-between of nature and culture, the bone in the throat of substance and The Logic of Transcendental Materialism 145 Whereas the former articulates itself according to a series of upward-moving spirals wherein each new turn completely encompasses the previous one in an act of subsumption (so 146 Chapter 7 146 Chapter 7 The Logic of Transcendental Materialism 147 self-development of the absolute by instituting a new age of the world that can never be reconciled with that which came before, in a moment of ontological triumph. As a consequence, if we inscribe culture into the fabric of the universe according to the second model of dialectics, we are forced to conclude that the absolute is open, precarious, and necessarily incomplete, for the symbolic universe is not only constitutively out of joint with nature, but as the always doomed attempt to reconcile itself with the latter, is constantly forced to reinvent itself. The process of subjectification (culture) emerges out of the ontological chasm opened up by the pure I and holds a position of infinite difference with respect to nature insofar as it operates within a zone of logical non- coincidence that has been carved out from within the laws of the latter. Instead of a self-enclosed spiral or circle of circles, we see an immanent “break” that prevents the next dialectical phase of self-appropriation from occurring and by means of which another level of autonomous activity irreducible to the first can take hold. The image is of two cones—one ontologically positive, the other immersed in a virtual zone of nonbeing— linked together by a black hole that is the pure I, the night of the world, whereby nature and culture self-actualize in isolation to one another, but are nevertheless negatively tied together by the abyssal void of subjectivity—that which “protrudes” out of both as an impossible in-between non-explicable in either. The subject stands for the bone in the throat of substance that prevents it from being a devouring all following its own immanent laws (nature's non-coincidence to self) just as much as it stands for that snag in the cultural machine (the non-all of the symbolic Other) that can never be filled in or completely overcome, and that thus constitutes the impetus for all subjectification as a series of reaction formations and the infinite proliferation of the forms it can take on due to its necessary failure of covering up, schematizing, the primordial trauma. In this regard, transcendental materialism presents a radically different view of dialectics as Absolute Idealism, going so far as to claim that what the latter misses is that it is, at best, a mere compensation in fantasy for the traumatic truth of the former. 146 Chapter 7 With culture, we see that nature had immanently produced an eruptive, shattering transcendence (the subject) that bursts The Logic of Transcendental Materialism 147 146 Chapter 7 that we encounter a completely self-enclosed, organic oneness that slowly articulates itself in increasing complexity) the very self-unfolding operation of the later precludes the possibility of such a self-totalizing activity. While Absolute Idealism itself does move forward on the basis of fundamental non-coincidence or immanent contradiction (there is conflict internal to the system), it is always productive of new, evermore comprehensive unity, but in such a manner that its innate teleological push towards greater order and self-coherence is able to project an upper limit. Since it knows no radical inner impediment, the ever-expanding series of upward spirals predicts a point in history when the Idea would attain perfection by returning to itself and in turn overcome its prior self-alienation, that is to say, when nature and culture would become reconciled in a moment of ontological jubilation. Here, understood as the self-development of the structure of the world, the absolute is seen as an immanent processual movement from self-externality to absolute self-mediation: by making itself into the Idea, nature (as a realm of pure contingency) would have succeeded by coming to a complete grasp of itself in the freedom of thought, whereby the end of the movement would see itself in the beginning, thus closing the circle of circles. Within transcendental materialism, however, the passage from nature to culture does not reveal a struggle of notional transmutation as culture endeavours to rid itself of its basis in nature in the onslaught of history with the promise of completion, but rather reveals a standstill in the heart of being that cannot be brought into a higher moment of truth of free spirit that would bring the circle of circles to an end: the ebb and flow of substance ontogenetically incites the birth of a freely existing subject only through a self-sabotaging, self-destructive movement that defies perfect reconciliation, because this unruliness inheres in all culturally achieved unity and disrupts it from within. Conflict, though here too internal to the system, articulates at this juncture of the passage from nature to culture an irrevocable place of rupture, devastation, or laceration in the absolute, which points to a dialectical residue that can never become a vehicle of internal growth of the structure of the world, yet that simultaneously sustains culture as the very attempt to overcome it. 148 Chapter 7 with the Freiheitsshcrift or the Weltalter, this demonstrates the manifest Schellingian character of Žižek's criticism of Hegel, while at the same time locating the germ of the former's logic of the Grund within Hegel's early Realphilosophie: But dependence does not annul autonomy or even freedom. It does not determine essence, but merely says that the dependent, whatever it might be, can only be as a consequent of that upon which it is dependent; it does not say what it is, and what it is not. Each organic individual, as something which has become, has its being only through another, and to this existent it is dependent in terms of becoming, but not at all in terms of being. It is not incongruous, says Leibniz, that he who is God is at the same time begotten, or vice versa; as it is no more a contradiction to say that he who is the son of a man is himself a man.198 The Logic of Transcendental Materialism 147 Here we have a rich account of the emergence of two zones of activity wherein, although the second is dependent upon the first that constitutes its genetic ground, it remains entirely free. To anyone familiar 148 Chapter 7 48 Chapter 7 The Logic of Transcendental Materialism 149 The Logic of Transcendental Materialism 149 that got it wrong: it is not Schelling who is the misfit, but rather Hegel, for it is he who turns away from the abyss brought forth by the idealist account of freedom after gazing too deeply into its traumatic core. After the Realphilosophie, something prevents Hegel, holds him back—there is a recoil, a hesitation. What is so compelling for Žižek about works such as the Weltalter is not their anti-Hegelian character, but their ability to penetrate into the breakthrough heralded by modern philosophy and to bring it to a new, higher level. It is Schelling who gives the complete articulation of its underlying but disavowed core insofar as it is he who most fully outlines the principles of a quadruple dialectical logic, whereas Hegel, going against his own initial tendencies, falls back into a triad at a crucial moment and loses sight of the “deontologized being” of the subject. What thus characterizes the passionate fury of the middle-late period is its embrace of the paradoxes surrounding subjectivity. This is what makes Schelling that which is in Hegel more than Hegel himself, the extimate core deeply entrenched within the body and soul of Hegel's philosophy that he could not own, as if Schelling were the real spectre haunting and destabilizing his mature system. Consequently Schelling, more than anyone else, is the culmination of German Idealism: it is he who most passionately tarries with its Real, for, in their “very failure, [the Weltalter drafts] are arguably the acme of German Idealism and, simultaneously, a breakthrough into an unknown domain whose contours became discernible only in the aftermath of German Idealism”;200 “Hegel's 'overcoming' of Schelling is a case in itself: Schelling's reaction to Hegel's idealist dialectic was so strong and profound that more and more it is counted as the next (and concluding) step in the inner development of German Idealism.”201 In the context of the Žižekian reactualization of German Idealism, the fundamental assertion to be made is that to understand the movement towards the middle-late period in Schelling we must at some level say that Schelling himself came to realize the deficiencies of his previous philosophical endeavours, perhaps either through a rethinking of the Kantian critical system or by being spurred on by Hegel. 7.2 The Weltalter and the Systematization of Freedom One of the most interesting aspects of Žižek's reactualization of German Idealism is its claim that the middle-late Schelling's “departure” from the throes of reason and “descent” into theosophical obscurantism does not demonstrate a break from modern rationality as inaugurated by Descartes' search for a self-evident Archimedean starting point for all philosophy (famously developed further in Kant's transcendental conditions of the possibility of knowledge, and ultimately epitomized by Hegel's self- mediating Notion).199 On the contrary, Žižek's reactualization states that Schelling actually makes explicit for the first time the perverse, unconscious truth that remains hidden throughout the entire tradition, but only appears ephemerally through the distortion of its imaginary-symbolic universe: his attempt to present a system that would be able to combat the perceived threat posed by Hegel's horrifying “pan-logicism” presents a radicalization, a completion, of modernity's fundamental insight into the paradoxical origins of subjectivity, an insight that Hegel himself was unable to follow through in his own endeavour to solve the impasse bequeathed by Kant's critical philosophy. In this sense, Žižek implies that it is philosophical orthodoxy The Logic of Transcendental Materialism 149 Although, for instance, in the Naturphilosophie Schelling is also interested in the dark side of nature, the project forecloses the possibility of Todestrieb as an 150 Chapter 7 150 Chapter 7 Chapter 7 emergent and infinitely disruptive force in being. In this sense, Žižek's rejection of the early Schellingian attempts to balance transcendental idealism with materialism follows the same line of argument as his denial of the theoretical weight of contemporary evolutionary models of self- reflexive consciousness,202 for both share the same fault: the ultimate identification of mind and matter instead of the articulation of their ultimate irreconcilability to one another. From the standpoint of the immanent laws of the pre-symbolic Real (as avowed by Spinozistic monism or reductionistic materialism), Žižek's claim is that the Ideal cannot be explained either on the basis of a teleological or purely naturalistic emergence. The Ideal explodes from within the vital throes of positive being,203 rather than just being one specific (albeit complex) mode of physical organization, for it names an alienating distance to self, a non-coincident split that literally short-circuits the world. Rather than inhering in matter as its implicit structure, mind can only emerge within the void of this ontological scar, thus making it impossible to reconcile with matter. Written in the aftermath of the birth of the Hegelian system with the publication of the Phenomenology of Spirit in 1807, which contains a famous explicit criticism of the Schellingian philosophy of absolute indifference as an attempt to balance the two poles of realism and idealism,204 the Freiheitsschrift (1809) and the Weltalter (1810–1815) radically restructure the problematic that had occupied Schelling's philosophical career. The Logic of Transcendental Materialism 149 But what complicates the issue at hand is the fundamental ambiguity of the Hegel-Schelling relationship in Žižek's own thinking, which is brought to an extreme at this crucial juncture: while one could say that much of the young Schelling's work is an attempt to rethink the subject's relation to the noumenal thing-in-itself that haunts its representations or to explicate the androgynous complementarity of the ideal-real poles, the middle- late Schelling's problematic, on Žižek's reading, is the one he erroneously attributes to Hegel: “the true problem is not how to reach the Real when we are confined to the interplay of the (inconsistent) multitude of appearances, but, more radically, the properly Hegelian one: how does appearance itself emerge from the interplay of the Real?”205 Although Žižek does oscillate between calling this problematic Hegelian and Schellingian, what should be clear is that it is more accurately Schellingian, insofar as it is the latter— The Logic of Transcendental Materialism 151 The Logic of Transcendental Materialism 151 according to Žižek's own words—who most fully develops the quadruple logic of the passage from the pre-logical Real into the Symbolic as the unconscious Grundlogik of German Idealism, whereas Hegel recoiled at the most crucial moment. Interpreting the Weltalter through this theoretical framework, Žižek is then able to interpret its ontology as an attempt to articulate a transcendental materialism capable of grounding the psychoanalytico- Cartesian subject by thematizating the vanishing mediator between the Real and the Symbolic. He can do this, perhaps surprisingly, because first and foremost the Weltalter manuscripts present themselves as a theosophic exploration of creation. Perceiving Hegelian logic as a purely conceptual artifice that suffocates the freedom under the self-articulating necessity of the Notion, Schelling puts his philosophical prowess to use to give his own account of the emergence of temporality and finitude that could rival the dialectics of his great adversary. His basic thesis is that, although Hegelian logic can express notional necessity (what something ideally is) it ultimately fails to grasp the fact of any being, the thatness of its existence, especially if that being has its primordial basis in the brute, raw reality of freedom, something that forever eludes the self-mediation of conceptuality.206 For Schelling, however, this failure of pan-logistic dialectics in the face of a freely deciding being (the emergence of the subject) does not amount to a mere admittance of the intrinsic limitations of knowledge and human reason. It must be distinguished from a merely negative constraint upon philosophizing because this dialectical dead end we come across in explicating freedom does not arise due to the limited synthesizing activity of the subject and the finite conditions of the possibility of knowledge, but rather through an (onto)logically disruptive and yet productive metaphysical activity: to say that the fact of a specific being that possesses freedom as its essential predicate cannot be conceptualized according to an a priori dialectics is to point to the uncontainable act constituting its very self-positing, which is therefore capable of continually heralding forth the new and tearing apart any given causal matrix in which it finds itself,207 thereby making itself only graspable après-coup in the wake of its own self-instituting revelation in the world. The Logic of Transcendental Materialism 151 The inability to conceptualize the advent of eruptive subjectivity in substance through 152 Chapter 7 152 Chapter 7 pure reason transcends mere epistemological constraints and reaches out unto the ontological: it is not merely that we must be agnostic concerning the existence of a totalizing principle that holds being together, but more disconcertingly, freedom proclaims that there is not any. The subject is an unpredictable event in being that rewrites what we consider to be possible. Thus, in trying to systematize freedom in a way to escape the perceived threat of Hegelian Absolute Idealism, Schelling reaches a contradiction, a contradiction that paradoxically becomes the very vitality of his system itself, insofar as it declares that the totality of being must be understood in terms of a constitutive yet conflict-ridden relation with an immanent Other: “[w]ere the first nature in harmony with itself, it would remain so. It would be constantly One and would never become Two. It would be an eternal rigidity without progress.”208 Both the Freiheitsschrift and the Weltalter give expression to the necessary snag in the dialectical machine, the primordial, unruly excess of the Real over the Ideal that prevents any system of thinking from being self-enclosed unto itself and in the same breath guarantees the dynamic character of the latter by making it inclusive of freedom as an irrepressible, self-rupturing event at its very core. In order to situate ourselves more firmly within the dialectical nuance of the Weltalter and show how, in relation to Žižek's ontology, Schelling holds a position of theoretical primacy over Hegel, we can use the problem of evil as an entry point, since it is perhaps in their respective theories thereof that they most strongly distance themselves from one other. Whereas for the mature Hegel evil becomes a mere sublated moment in the self-developmen of the good, a necessary phase for its establishment, for Schelling evil remains at its very core irrational and illogical. By definition it cannot be sublated as a moment within a higher dialectical standpoint because it is, at its primordial basis, the effect of an irreducible act of will. There is something spontaneous about evil that forever eludes conceptualization, something insurmountable about the wildness of a soul that insists on that which it wants and will sacrifice whatever it can to achieve it. The Logic of Transcendental Materialism 153 articulation of freedom as the capacity for good and evil: freedom in itself must rest intrinsically incomprehensible, that which cannot be dialectically mediated, which means that its pure self-positing can only resemble madness insofar as it precedes and makes possible any articulation of a table of values that could be used to comprehend it. It of itself knows no order, no rationality—if it did, it would be explicable in terms of the principle of sufficient reason and at risk of being thrown into a subordinate position within a greater self-articulating whole of which it is a mere functional part. There is therefore something always essentially impenetrable in every good and evil act done out of freedom, something always irreducible and violent in each act of self-positing: without this intuition, we lose the breakthrough of the critical system as revealed most poignantly in Kant's pedagogical writings and succumb to another form of determinism (dogmatism) that cancels out the primordial meaning of autonomy within a logic of overarching and self-unfolding reason. Insofar as the act itself is concerned, both the modalities of good and evil as expressions of freedom are formally identical; they involve an act logically distinct at its zero-level from any set of values—or in other words, an act done without any guarantee and without any external determination or influence. More radically, this testifies that evil is itself at the core of every good act, that evil is actually more primordial than the good. In order for an act to be truly good and authentically free at the same time, it must “pass” through evil, discipline it, and use it as the tamed Grund for its own expansive power—any Yes (adherence to rationally determined ethical principles) must first be a No (an egoistic self-assertion) if it is to be utterly self-determined and not just a blind following of laws: “the day lies concealed in the night, albeit overwhelmed by the night; likewise the night in the day, albeit kept down by the day, although it can establish itself as soon as the repressive potency disappears. The Logic of Transcendental Materialism 151 Evil has something crazed and frantic about it: it is the capacity to say “No!” with the full knowledge of the implications of one's action. As soon as evil is understood, it fails to be evil; it becomes, rather, misguided good in the Platonic sense that no one does wrong willingly. Hence Schelling's The Logic of Transcendental Materialism 4 Chapter 7 co-related concept of the original “unnatural” unruliness of the human organism, so that these original insights become an intrinsic part of his own logic of the Grund as the indivisible remainder, the “incomprehensible basis of reality,” which is missing in mature Hegelian dialectics. As a full- fledged ontologization of Kant's declaration of the radical spontaneity at the basis of human practical activity,211 Schelling's philosophical impulse initiated by the Freiheitsschrift is an attempt to develop a system wherein freedom is irreducible to notional necessity, for “[o]nly he who has tasted of freedom can sense the desire to make everything its analogue, to spread it throughout the whole universe.”212 What intrigues him is the fact that there is an insurmountable enigmatic blind spot at the core of every action, every decision, a blind spot that not only presents the truth, mystery, and potential horror of human freedom, but more primordially reveals a deep hole that has been carved out in the flesh of being, making it tremble from within, for the unpredictable has emerged in its core. It is this strong conviction in freedom that leads Schelling into the abyssal labyrinths of self-exploration that constitute the conceptual fabric of the Weltalter, in the same way the intuition of freedom made Kant limit knowledge in order to make room for faith and embark down the path where he would eventually articulate the necessity of diabolic evil and unruliness in his pedagogical writings after years of original investigation into the essence of self-legislative practical reason. For Žižek, it is not an accident that Schelling's own project in the Weltalter ends up radicalizing Hegel's descriptions of the night of the world or Kant's account of unruliness, which in turn proves that his response to Hegel is the concluding step of German Idealism: all are driven by an attempt to give a philosophically adequate bedrock to freedom,213 with Schelling merely following its intuition right to the metaphysical conclusions it forces upon us in a way other representatives in the tradition were unable to do. What distinguishes Schelling is that he, propelled by an immense energy to battle against what he perceived as the threat posed by Hegelian dialectics on human freedom, had the strength to go further than the others in the symbolization of the unconscious Grundlogik inherent in the tradition. The Logic of Transcendental Materialism Hence, good lies concealed in evil, albeit made unrecognizable by evil; likewise evil in good, albeit mastered by the good and brought to inactivity.”209 In this respect, a priori dialectics must fail: if all birth is a birth from darkness to light, there is always something in the emergence of rational order that remains impervious to the latter.210 From this it becomes clear that the Schellingian concept of freedom From this it becomes clear that the Schellingian concept of freedom is an explicit rethinking of the Kantian notion of diabolic evil and its 4 Chapter 7 154 The Logic of Transcendental Materialism 155 than of humanity, it is because it is a work that delves into the ontological implications of the freedom revealed by idealism. 4 Chapter 7 If hi b t k k i ll d Phil phi l I i i i H co-related concept of the original “unnatural” unruliness of the human organism, so that these original insights become an intrinsic part of his own logic of the Grund as the indivisible remainder, the “incomprehensible basis of reality,” which is missing in mature Hegelian dialectics. As a full- fledged ontologization of Kant's declaration of the radical spontaneity at the basis of human practical activity,211 Schelling's philosophical impulse initiated by the Freiheitsschrift is an attempt to develop a system wherein freedom is irreducible to notional necessity, for “[o]nly he who has tasted of freedom can sense the desire to make everything its analogue, to spread it throughout the whole universe.”212 What intrigues him is the fact that there The Logic of Transcendental Materialism 7.3 The Problem of the Beginning Itself: Schelling's Uncanny Response to Idealism Just as in the Freiheitsschrift, and using the operative logic that it had already programmatically developed as a guide, Schelling in the Weltalter embarks upon a specific form of introspective analysis with the aim of developing a theosophy, the founding intuition of which is that the same process underlying the birth of human subjectivity is fundamentally structurally identical to God's creation of the world (as exhibited in the alchemical principle “so above, so below”).214 In another vein, the idea is that psychological experience is in some sense directly revelatory of the absolute drama of divine being in all its vicissitudes, even if it must pass through the meditating filters of self-reflexive consciousness: the experience of the relationship of dependence and autonomy that holds between one's pre- subjective, material Grund and free personality is primordially disclosive of an ontological event that is a symbol of God's relation to the finite created world. In this way, the theosophic odyssey of the birth of God out of that which in God is not God himself, is irrevocably intertwined with a parallel investigation into the ontogenesis of subjectivity out of a nature that presents itself as Other to and irreconcilable with its free self-standingness. Since the methodological starting point is similar to the psychoanalytical experience of disharmony between mind and body as the obscure basis of freedom (which hints at the vanishing mediator between them), Žižek is led to discard the entire theosophic scope of the work as ultimately accidental to its “true” philosophical core, so that Schelling's narration of the painful process of the self-begetting of God and the decision of divine creation presents itself as a mere “metapsychological work in the strict Freudian sense of the term.”215 Whether or not Žižek himself is justified in completely removing the theosophic scope from Schelling's argument, one must at the very least admit that Žižek's wager follows the spirit of Schelling's middle-late philosophy to the letter, for Schelling himself declares in the Freiheitsschrift to “have established the first clear concept of personality.”216 156 Chapter 7 What makes Žižek's appropriation of Schelling at times so provocative and compelling is his profound ability to penetrate into the fine details of the conceptual structures that make up the operative logic of the Freiheitsschrift and the Weltalter in a way no one else has. What interests him is how Schelling advances the descriptions in the tradition of the status of the elusive X, which simultaneously haunts transcendentally constituted reality, precedes it, and appears to set the stage for its condition of possibility. What makes Žižek s appropriation of Schelling at times so provocative and compelling is his profound ability to penetrate into the fine details of the conceptual structures that make up the operative logic of the Freiheitsschrift and the Weltalter in a way no one else has. What interests him is how Schelling advances the descriptions in the tradition of the status of the elusive X, which simultaneously haunts transcendentally constituted reality, precedes it, and appears to set the stage for its condition of possibility. These three conceptual aspects of this je ne sais quoi map directly unto the three modalities of the Real: (i) the Real as a “kink” in the Symbolic, which pressurizes phenomenal reality; (ii) the Real as pre-symbolic “immediacy” that is lost through the advent of language; and (iii) the barred Real (R⁄ ) now understood as an auto-disruptive substance (N ≠ N) whose self-laceration creates the necessary room within which the transcendental constitution of reality through the Symbolic-Imaginary matrices underlying self-experience can take place, thus drawing attention to the interconnection of each aspect. Prior to these middle-late works, as we have seen, our relation to this mysterious X had already been partially “schematized” by a list of concepts (from Kantian transcendental freedom, diabolic evil, and unruliness to the Hegelian accounts of the night of the world and substance as subject). However, for Žižek, it is only with Schelling's own additions that we move away from the paradoxes of the ideal representation of the extra-subjective world or from a mere haphazard glimpse into the self-effacing ontological catastrophe that precedes the very possibility of free idealization. 156 Chapter 7 With him, we completely plunge into the auto-disruptive logic of the pre-symbolic Real at the basis of subjectivity so that the unconscious Grundlogik plaguing the German Idealist tradition, which had already from time to time appeared only to fall back into the darkness from which it came, finally comes clearly into light, becoming now minimally subjectified, as it were. The major difficulty, however, is how to articulate a philosophical system that can synthesize the various aspects of the Real together into a stable whole insofar as the very ontological space whose exploration would enable it retreats the very moment that conscious experience begins and is only visible in its very gesture of self-withdrawal.217 It is no accident that the problem that haunts the entirety of the middle-late Schelling of the Freiheitsschrift and the Weltalter is as Žižek emphasizes: The Logic of Transcendental Materialism 157 the problem of the Beginning itself, the crucial problem of German Idealism—suffice it to recall Hegel's detailed elaboration of this problem and all its implications in his Science of Logic. Schelling's “materialist” contribution is best epitomized by his fundamental thesis according to which, to put it bluntly, the true Beginning is not at the beginning: there is something that precedes the Beginning itself—a rotary motion whose vicious cycle is broken, in a gesture analogous to the cutting of the Gordian knot, by the Beginning proper, that is, the primordial act of decision. The beginning of all beginnings, the beginning kat' exohen—“the mother of all beginnings” as one would say today—is, of course, the “in the beginning was the Word” from the Gospel according to St John: prior to it, there was nothing, that is, the void of divine eternity. According to Schelling, however, “eternity” is not a nondescript mass—a lot of things take place in it. Prior to the Word there is the chaotic-psychotic universe of blind drives, their rotary motion, their undifferentiated pulsating; and the Beginning occurs when the Word is pronounced which “represses,” rejects into the eternal Past, this self-enclosed circuit of drives. 156 Chapter 7 In short, at the Beginning proper stands a resolution, an act of decision which, by differentiating between past and present, resolves the preceding unbearable tension of the rotary motion of drives: the true Beginning is the passage from the “closed” rotary motion to “open” progress, from drive to desire—or, in Lacanian terms, from the Real to the Symbolic.218 the problem of the Beginning itself, the crucial problem of German Idealism—suffice it to recall Hegel's detailed elaboration of this problem and all its implications in his Science of Logic. Schelling's “materialist” contribution is best epitomized by his fundamental thesis according to which, to put it bluntly, the true Beginning is not at the beginning: there is something that precedes the Beginning itself—a rotary motion whose vicious cycle is broken, in a gesture analogous to the cutting of the Gordian knot, by the Beginning proper, that is, the primordial act of decision. The beginning of all beginnings, the beginning kat' exohen—“the mother of all beginnings” as one would say today—is, of course, the “in the beginning was the Word” from the Gospel according to St John: prior to it, there was nothing, that is, the void of divine eternity. According to Schelling, however, “eternity” is not a nondescript mass—a lot of things take place in it. Prior to the Word there is the chaotic-psychotic universe of blind drives, their rotary motion, their undifferentiated pulsating; and the Beginning occurs when the Word is pronounced which “represses,” rejects into the eternal Past, this self-enclosed circuit of drives. In short, at the Beginning proper stands a resolution, an act of decision which, by differentiating between past and present, resolves the preceding unbearable tension of the rotary motion of drives: the true Beginning is the passage from the “closed” rotary motion to “open” progress, from drive to desire—or, in Lacanian terms, from the Real to the Symbolic.218 What Žižek refers to as “Schelling-in-itself: the 'Orgasm of Forces'”219 is the remarkable capacity Schelling's philosophy possesses of being able to descend into the immanent driving forces governing the extra- subjective, material Real, the elusive, obscure phase of darkness that precedes and sets the stage for the birth of the light that is the openness of self-reflexive consciousness. 156 Chapter 7 But what fascinates Žižek is the depth of his materialist response to Hegel, which still remains immersed in the fabric of transcendental idealism, a response that is “at the origins 158 Chapter 7 158 Chapter 7 of dialectical materialism.”220 This is why Žižek describes Schelling as a vanishing mediator between classical philosophy and the contemporary discourse of finitude: Schelling stands in a position of irreconcilable contradiction between the two, a tension that Žižek takes upon himself to further develop insofar as Schelling, according to him, is unable to endure his own breakthrough and recoils.221 If, onto the ground/existence distinction propounded in the Freiheitsschrift and systematically laid out in the Weltalter, we superimpose the real(ity)/ideal(ity) distinction operative within modern philosophy from Descartes onward, we perceive a nuance in the ontologization/grounding of Cartesian subjectivity: this split announced between mind and matter, which makes them non-reconcilable to one another, occurs “within” or “on the side of” the material Real through an ontologico-metaphysical deadlock, a schismatic rupture. The standard debate between idealism (ideality precedes and structurally makes possible the positive order of physical being and is thus the insurmountable metaphysical zero-level as in Platonism and textbook Hegelianism, or constitutes completely self-grounding and self-justifying transcendental conditions or normative values that make discourse possible as in Kantianism or much linguistic philosophy) and materialism (there is nothing but the ebb and flow of brute matter, the rest being reducible to an epiphenomenal production of nature's self-enclosed laws, as in the Greek atomists, conventional cognitive science, and logical positivism) is thus stood on its head in Schelling: idealism posits an ideal Event which cannot be accounted for in terms of its material (pre)conditions, while the materialist wager is that we can get “behind” the event and explore how Event explodes out of the gap in/of the order of Being. 156 Chapter 7 The first to formulate this task was Schelling, who, in his Weltalter fragments, outlined the dark territory of the “prehistory of Logos,” of what had to occur in pre-ontological proto- reality so that the openness of Logos and temporality could take place.222 This quote expresses Schelling as one of the most crucial figures (if not the most crucial figure) in the history of dialectical materialism for Žižek, and thus establishes once and for all the pivotal role Schelling's ontology plays in his own philosophical development, despite his own characterizations The Logic of Transcendental Materialism 159 of his project as Hegelian. So how, then, do the vicissitudes of pure, raw materiality open up unto the irreducible event of the Ideal and the transcendentally constituted reality of phenomenological (self-)experience? 7.4 Grund and Existence: The Pulsating Heart of Nature and the Upward Spiral of Human Temporality The reason for this is not that nature, instead of being characterized by a smooth teleological development, is plagued by unpredictable catastrophism, irruptive disharmonies of widespread murder and extinction that cover up their own traces, so that the history of the world cannot be subsumable under a single, all-pervasive trajectory that, preceding from simple to more complex organizations in the unimaginable passage of aeons and aeons, would crown man as the summit of an unconscious yearning for the Word, that is, the structured logos of the symbolic world (contra the surface structure of Schelling's thinking225). This would effectively prevent nature from being motivated by an unknowing search for the light of self- consciousness from within the darkness of its raw, productive potency,226 but the reason lies elsewhere. There are, of course, differing stages, periods, and epochs in nature—and ones that have been lost forever, never to be recovered—due to varying levels of dynamic evolution and growth within its immanent activity, even if there is always unforeseeable violence risking to wreak havoc. Yet when we look around, we cannot find the subject within the system of nature. It just does not fit, a fact that has stark metaphysical implications: the progressive, transformative time of nature presents itself as radically Other to the distinctiveness of human (spiritual) temporality because it does not display the same intensity of uncontrollable dialectical self-sublation. In nature, beginning and end for the most part coincide: in the darkness of the soil, the seed gives birth to the plant, which, after reaching the life-giving light of the radiant sun, finally dies, leaving behind its fruit and thereby returns to itself, only to burst forth again in an eternal recurrence of the same. Change can only transpire in the span of incomprehensible ages: unmeasurably dilatory, dialectical movement is here “enchained” within the unbreakable spurious infinity of endless circularity and does not display the same frantic upward-moving spiral of human temporality, the never repetitious onslaught of history, where beginning and end exist in a productive non-coincidence that is the very vital force of its unimaginably fast paced metamorphosis and unending creation of nuance.227 A new era of culture displaces and supersedes the previous one, going in an unfathomable number of conflicting directions at once, so that self-transmutation that leads to man, so that he is ultimately included within it. 7.4 Grund and Existence: The Pulsating Heart of Nature and the Upward Spiral of Human Temporality Schellingian nature is more than a mere symbol or paradoxical representation of the eternal Past that precedes consciousness: it is that elusive, impossible X, that je ne sais quoi in the modality of the pre-symbolic Real prior to conceptual-linguistic mediation. Yet when we move from the world of human meaning and into the circuitry of nature's vital ebb and flow, we see that “[e]verything that surrounds us points back to a past of incredibly grandeur. The oldest formations of the earth bear such a foreign aspect that we are hardly in a position to form a concept of their time or origin or of the forces that were then at work,”223 in such a way that the task of philosophy becomes to reconstruct this ancestral trajectory of the immemorial into a system of times: We find the greatest part of [its formations] collapsed in ruins, witnesses to a savage devastation. More tranquil eras followed, but they were interrupted by storms as well, and lie buried with their creations beneath those of a new era. In a series from time immemorial, each era has always obscured its predecessor, so that it hardly betrays any sign of an origin; an abundance of strata—the work of thousands of years— must be stripped away to come at last to the foundation, to the ground.224 It is at this very juncture in the Weltalter that Žižek invites us to risk a daring thesis. If we draw our attention to its operative logic, we see that Schelling's system of times does something more radical than displace the primordiality of the human subject by illustrating its subsumption within the tenebrous pulsating heart of nature's productive potency, whose own self-unfolding takes place in the abyssal dregs of an immemorial time that threatens to engulf they who look into it. The crucial observation to be made here is that nature does not have a history that evolves by means of an activity of internal 160 Chapter 7 160 Chapter 7 160 Chapter 7 self-transmutation that leads to man, so that he is ultimately included within it. The Logic of Transcendental Materialism 1 The Logic of Transcendental Materialism 161 always out of place in the inaccessible residue that is the past. The end, the result, is a qualitative break rewiring the plethora of human thoughts, expressions, and emotions, and even the past itself,228 thus setting up a new beginning to be surmounted in its own right. As Žižek succinctly puts it, “[h]ere we encounter the key feature of the Symbolic: the fundamental 'openness' it introduces into a closed order of reality,”229 in such a manner that saying nature yearns for humanity in its depths as the solution to its enigma230 merely covers up the fact that humanity is an irreconcilable break from it. But if the subject is a break from the system of times that constitutes nature and its catastrophism by bringing forth a new age of self-unfolding activity, this does not mean that we are liberated from all dismay by being brought into the luminous sphere of holy spirit. Instead, bound by the erratic and excessive life of freedom that overflows itself, we face our own non- natural catastrophes. We also encounter traces of this paradoxical upward spiralling and uncontrollable linear time in Kant and Hegel, which establish its integral place within founding intuitions of the tradition. Kant's account of unruliness as the ontogenetic starting point of transcendental freedom not only establishes the non-natural basis of human sociopolitical activity—not only is the human being “the only creature that must be educated,” but due to his unruliness, man “has no instinct” and is therefore separated from the vital throes of nature231—but, what is more, it demands to be disciplined if it is not to devour the subject in its frenzy. Due to this exigence of discipline, there is an intrinsic link between what Kant calls moral education and the historical destiny of man that institutes a new form of temporality driven forward through a productive non-coincidence at the core of what it is to be man. Although presenting itself as an excessive energy whose domestication/ schematization will enable us to attain terrestrial perfection, the very ground of our progress is in actuality its own inevitable obstacle: the meta- transcendental condition of subjectivity, while it opens up a distinctly human sphere within which progress is possible, at the same time tarnishes it with the inevitability of eventual collapse, misfire, failure. 7.4 Grund and Existence: The Pulsating Heart of Nature and the Upward Spiral of Human Temporality The reason for this is not that nature, instead of being characterized by a smooth teleological development, is plagued by unpredictable catastrophism, irruptive disharmonies of widespread murder and extinction that cover up their own traces, so that the history of the world cannot be subsumable under a single, all-pervasive trajectory that, preceding from simple to more complex organizations in the unimaginable passage of aeons and aeons, would crown man as the summit of an unconscious yearning for the Word, that is, the structured logos of the symbolic world (contra the surface structure of Schelling's thinking225). This would effectively prevent nature from being motivated by an unknowing search for the light of self- consciousness from within the darkness of its raw, productive potency,226 but the reason lies elsewhere. There are, of course, differing stages, periods, and epochs in nature—and ones that have been lost forever, never to be recovered—due to varying levels of dynamic evolution and growth within its immanent activity, even if there is always unforeseeable violence risking to wreak havoc. Yet when we look around, we cannot find the subject within the system of nature. It just does not fit, a fact that has stark metaphysical implications: the progressive, transformative time of nature presents itself as radically Other to the distinctiveness of human (spiritual) temporality because it does not display the same intensity of uncontrollable dialectical self-sublation. In nature, beginning and end for the most part coincide: in the darkness of the soil, the seed gives birth to the plant, which, after reaching the life-giving light of the radiant sun, finally dies, leaving behind its fruit and thereby returns to itself, only to burst forth again in an eternal recurrence of the same. Change can only transpire in the span of incomprehensible ages: unmeasurably dilatory, dialectical movement is here “enchained” within the unbreakable spurious infinity of endless circularity and does not display the same frantic upward-moving spiral of human temporality, the never repetitious onslaught of history, where beginning and end exist in a productive non-coincidence that is the very vital force of its unimaginably fast paced metamorphosis and unending creation of nuance.227 A new era of culture displaces and supersedes the previous one, going in an unfathomable number of conflicting directions at once, so that no return to the beginning is even possible: the beginning, the origin, is 162 Chapter 7 being, so that man is forever spurred on in the course of history to reinvent himself because of the insurmountable ontological violence preceding all acts of subjectification: “[t]he human being must therefore be educated to the good; but he who is to educate him is on the other hand a human being who still lies in the crudity of nature and who is now supposed to bring about what he himself needs. Hence the continuous deviation from his destiny with the always repeated returns to it.”232 But the consequence of this is that man is nothing more than this perpetual deviation from his destiny, that “man only becomes man”233 by continually (re)creating his own identity by means of education through the construction of a second nature within the forward onslaught of history, its fragile movement of contraction and expansion, history itself thus circulating around an ideal sociopolitical point that it posits by its very activity as necessary to it but which it can never reach, for beginning and end can never coincide as in the realm of nature. In other words, the impossibility of reaching our destiny is that which constitutes its very possibility, that which gives us a destiny in the first place. Due to this impossibility, we have historical time. This idea of a sharp distinction between the natural and the spiritual in terms of time is also taken up by the mature Hegel, even if, on Žižek's reading, he misses the radicality of the vanishing mediator—the night of the world—that enacts the passage from the former to the latter. There are two sides to the story. First, Hegel's starting point in the philosophy of nature is nature as “the Idea in the form of otherness.”234 This means that nature cannot exhibit the characteristics Hegel associates with the Idea, such as development qua self-unfolding activity, whose image we see in the growth of plants from seeds, wherein we encounter a purposive causality guiding all change and movement. “The abstract universality of nature's self- outsidedness”235 demonstrates that nature lacks any inner structuration that would enable it to realize itself freely according to a pre- or self-given telos. In this sense, there is no teleological activity intrinsic to nature: its zero-level is a deterministic mechanics ruled by the contingency of its relations that are always external to themselves. The Logic of Transcendental Materialism 1 But this is not a mere proclamation of defeatism, resignation, and forfeit: the very impossibility of our task is the impetus for action, that which provokes an infinite plurality of new ways of “purifying” the insupportable surplus of our 162 Chapter 7 162 Chapter 7 The Logic of Transcendental Materialism 163 a meaningless giving birth to and destruction of its own offspring.236 Second, at the level of organic life, although nature has sublated this prior staleness and intrinsic lack of purposiveness within its ebb and flow, it nevertheless exhibits a kind of claustrophobic immanence wherein the freedom distinctive of spiritual temporality is foreclosed by the eternal repetition of sameness within nature's cycle of life and death, fullness and lack, for there is a suffocating coincidence between the birth of one individual and the death of the other. Even though there is teleological self-unfolding explicit within organic living being—there is a universal genus that concretizes itself by constituting itself within the series of particulars that it generates and that generates it in turn, a complex unity that sustains itself within difference throughout the dispersion of time—there is no real difference and thus no history possible, even if we are allowed to speak of gradual sedimentations of change over the course of living being's activity. To put it crudely (borrowing one of Hegel's favourite animal examples) in nature we see nothing but one damn parrot after another,237 which ultimately makes nature uninteresting for him: nature remains immensely poor with regard to its notional reflexivity, for in contrast with historical existence, it is not capable of the faster-than-light transformations constitutive of the latter's essence. The Logic of Transcendental Materialism 163 a meaningless giving birth to and destruction of its own offspring.236 Second, at the level of organic life, although nature has sublated this prior staleness and intrinsic lack of purposiveness within its ebb and flow, it nevertheless exhibits a kind of claustrophobic immanence wherein the freedom distinctive of spiritual temporality is foreclosed by the eternal repetition of sameness within nature's cycle of life and death, fullness and lack, for there is a suffocating coincidence between the birth of one individual and the death of the other. Even though there is teleological self-unfolding explicit within organic living being—there is a universal genus that concretizes itself by constituting itself within the series of particulars that it generates and that generates it in turn, a complex unity that sustains itself within difference throughout the dispersion of time—there is no real difference and thus no history possible, even if we are allowed to speak of gradual sedimentations of change over the course of living being's activity. To put it crudely (borrowing one of Hegel's favourite animal examples) in nature we see nothing but one damn parrot after another,237 which ultimately makes nature uninteresting for him: nature remains immensely poor with regard to its notional reflexivity, for in contrast with historical existence, it is not capable of the faster-than-light transformations constitutive of the latter's essence. Hegel refers to this deficiency as the “impotence of nature,”238 insofar as it is incapable of the power of self-relating negativity and therefore displays a spurious infinity.239 Consequently, we must assert the following “distinction between the spiritual and the natural worlds: that, whilst the latter continues simply to return into itself, there is certainly a progression taking place in the former as well.”240 As such, it is thus only with the rise of human spirit that we see contradiction posited as such, and with it, the possibility for real metamorphosis: “[s]pirit is posited as contradiction existing for itself, for there is an objective contradiction between the Idea in its infinite freedom and in the form of singularity, which occurs in nature only as an implicit contradiction, or as a contradiction which has being for us in that otherness appears in the Idea as a stable form.”241 In German Idealism, a human subject is not merely born and then, by dying, proliferating his progeny in an endless repetition of the same. 162 Chapter 7 Exclusively determined by the conditions that engulf them, the bodies emerging here do not display any capacity for self- reproduction, but merely stumble against one another due to empirical laws, whereby nature presents itself as a dead husk and time is nothing other than y g p g In this sense, there is no teleological activity intrinsic to nature: its zero-level is a deterministic mechanics ruled by the contingency of its relations that are always external to themselves. Exclusively determined by the conditions that engulf them, the bodies emerging here do not display any capacity for self- reproduction, but merely stumble against one another due to empirical laws, whereby nature presents itself as a dead husk and time is nothing other than The Logic of Transcendental Materialism 163 The Logic of Transcendental Materialism 163 Strictly speaking, nature is nonconscious.243 In it, we only encounter a pulsation of matter, an annular rotary movement of contraction and expansion that follows its own automatic rhythm—what we see knows no pure upsurge of the irrevocable forward march of time, no dynamic linear temporality, as first witnessed in the human symbolic universe, even if its constitutive openness already appears in a “wild” form at lower levels of being. Nature eternally repeats itself in an infinite, relatively self-enclosed cycle of life and death, day and night, fullness and lack, wherein change sediments excruciatingly slowly over inscrutable eons and eons through a sluggishly self-developing, self-growing activity. Outside of it, there is nothing—everything is caught within an agonizing deadlock insofar as there is no room for completely free movement, for there is nothing but a symphonized flow of energy within the indivisibility of nature that is at the same time, from our perspective, a “blind” oscillation because our singularity is there lost. When one looks into nature as that impossible X, that je ne sais quoi, which sustains our life as subjects, one is almost forced to collapse: in face of the all-encompassing immanent laws of substance, one is pushed into an infinitely claustrophobic space. For Žižek we get a sense of this all-devouring, all-consuming force when we look inside the body and specifically the skull—“the realization that, when we look behind the face into the skull, we find nothing; 'there’s no one at home' there, just piles of grey matter—it is difﬁcult to tarry with this gap between meaning and the pure Real.”244 This raw flow of biochemical and electrical energy is so “terrifying” for two reasons. First, it is faceless, personless—it has absolutely difference representing an irreconcilable rupture with the autopoiesis of nature, a self-legislative spontaneity that defines itself in direct opposition to its self-organizing totality, even if it must rely upon it as a dark, inaccessible ground. Human history begins with a cutting off of immemorial natural history, a tearing itself away from the natural cycle of life and death. Although our biology falters and brings us to our end, we die as men, not as mere creatures immersed in the world of substantial being. The Logic of Transcendental Materialism 163 Hegel refers to this deficiency as the “impotence of nature,”238 insofar as it is incapable of the power of self-relating negativity and therefore displays a spurious infinity.239 Consequently, we must assert the following “distinction between the spiritual and the natural worlds: that, whilst the latter continues simply to return into itself, there is certainly a progression taking place in the former as well.”240 As such, it is thus only with the rise of human spirit that we see contradiction posited as such, and with it, the possibility for real metamorphosis: “[s]pirit is posited as contradiction existing for itself, for there is an objective contradiction between the Idea in its infinite freedom and in the form of singularity, which occurs in nature only as an implicit contradiction, or as a contradiction which has being for us in that otherness appears in the Idea as a stable form.”241 In German Idealism, a human subject is not merely born and then, b d i lif ti hi i dl titi f th In German Idealism, a human subject is not merely born and then, by dying, proliferating his progeny in an endless repetition of the same. During man's slow march towards his oblivion, a frenzy of naturally uncontainable and unfathomable activity articulating itself emerges, a Chapter 7 164 difference representing an irreconcilable rupture with the autopoiesis of nature, a self-legislative spontaneity that defines itself in direct opposition to its self-organizing totality, even if it must rely upon it as a dark, inaccessible ground. Human history begins with a cutting off of immemorial natural history, a tearing itself away from the natural cycle of life and death. Although our biology falters and brings us to our end, we die as men, not as mere creatures immersed in the world of substantial being. Schelling testifies to this insight, and advances it further than other representatives of the tradition (despite the fact he simultaneously recoils from, represses, the radicality of his own thematization of it242) insofar as, when we examine the genealogy of natural history in the Weltalter, we realize that nature is not the unconscious proper. The Logic of Transcendental Materialism 165 nothing to do with either the orbit of phenomenal experience or the human universe of meaning. There is no indication of any genuine human quality: we are only confronted with anonymous, dull palpitations, which resemble the industrial buzzing of automatic machinery, a machinery that may amaze us with its complexity and dynamism (the plasticity of the neuronal network) but that nevertheless exists as a matrix of closed circuitry locked within its own self- enclosed, self-sustaining movement, a movement that is not only greater than us but also thereby appears to “threaten” our very existence as free subjects at every step. Second, the passage from the pure, senseless Real of nature in its mechanism to the absolute spontaneity of the I—the rupturing advent of a dialectical leap—is stricto sensu inexplicable, for given our inability to locate the full-fledged human subject in nature, there is always a moment of arbitrariness and fiat. nothing to do with either the orbit of phenomenal experience or the human universe of meaning. There is no indication of any genuine human quality: we are only confronted with anonymous, dull palpitations, which resemble the industrial buzzing of automatic machinery, a machinery that may amaze us with its complexity and dynamism (the plasticity of the neuronal network) but that nevertheless exists as a matrix of closed circuitry locked within its own self- enclosed, self-sustaining movement, a movement that is not only greater than us but also thereby appears to “threaten” our very existence as free subjects at every step. Second, the passage from the pure, senseless Real of nature in its mechanism to the absolute spontaneity of the I—the rupturing advent of a dialectical leap—is stricto sensu inexplicable, for given our inability to locate the full-fledged human subject in nature, there is always a moment of arbitrariness and fiat. The Logic of Transcendental Materialism 163 Schelling testifies to this insight, and advances it further than other representatives of the tradition (despite the fact he simultaneously recoils from, represses, the radicality of his own thematization of it242) insofar as, when we examine the genealogy of natural history in the Weltalter, we realize that nature is not the unconscious proper. Strictly speaking, nature is nonconscious.243 In it, we only encounter a pulsation of matter, an annular rotary movement of contraction and expansion that follows its own automatic rhythm—what we see knows no pure upsurge of the irrevocable forward march of time, no dynamic linear temporality, as first witnessed in the human symbolic universe, even if its constitutive openness already appears in a “wild” form at lower levels of being. Nature eternally repeats itself in an infinite, relatively self-enclosed cycle of life and death, day and night, fullness and lack, wherein change sediments excruciatingly slowly over inscrutable eons and eons through a sluggishly self-developing, self-growing activity. Outside of it, there is nothing—everything is caught within an agonizing deadlock insofar as there is no room for completely free movement, for there is nothing but a symphonized flow of energy within the indivisibility of nature that is at the same time, from our perspective, a “blind” oscillation because our singularity is there lost. When one looks into nature as that impossible X, that je ne sais quoi, which sustains our life as subjects, one is almost forced to collapse: in face of the all-encompassing immanent laws of substance, one is pushed into an infinitely claustrophobic space. For Žižek we get a sense of this all-devouring, all-consuming force when we look inside the body and specifically the skull—“the realization that, when we look behind the face into the skull, we find nothing; 'there’s no one at home' there, just piles of grey matter—it is difﬁcult to tarry with this gap between meaning and the pure Real.”244 This raw flow of biochemical and electrical energy is so “terrifying” for two reasons First it is faceless personless—it has absolutely The Logic of Transcendental Materialism The Logic of Transcendental Materialism In the contemporary scientific scene, however, these menacing dimensions of the writhing, pulsating material of the Grund and its irreconcilable tension with free existence are constantly being brought into a new power, because neuroscience puts the very gap itself in question: the neuronal Grund, as a seething, all-devouring force, comes closer and closer to annihilating the distance from nature necessary for the autonomous transcendental constitution of reality, insofar as the fact of experience here risks being reduced to a mere epiphenomenon of a complex biological interface that uses the I as a system of representation to mediate itself to the world. As Žižek reiterates time and time again, “there are two options here: either subjectivity is an illusion, or reality itself (not only epistemologically) is not-Al.”245 That being said, Žižek's reactualization of Schelling's revamped Cartesian positing of the difference between nature (cyclical time, body, ground) and human being (dialectical temporality, mind, existence) allows him to rethink the significance of contemporary neuroscience. The divide between our world of experience and the mechanisms of the natural world does not proclaim the irrelevance of the latter for our understanding of human subjectivity in face of the pure power of scientific explanatory models, their efficacy and statistical guarantee, as perhaps various representatives of phenomenological psychiatry or even psychoanalysis would advocate. On the contrary, according to Žižek, these models adequately describe the Real of our lives with a rigorous vigor and precision 166 Chapter 7 166 Chapter 7 Chapter 7 never before imaginable by penetrating into the true ontologico-foundational basis of experience. The Logic of Transcendental Materialism Žižek criticizes attempts to respond to the threat announced by neuroscience that merely assert the irreducible character of the subject, seeing instead the only feasible way to find a solution being to “develop one approach to its extreme, radically abstracting from the other—to develop the logic of brain science, for instance, at its purest.”246 The question is how pp , y g p the logic of brain science, for instance, at its purest.”246 The question is how a parallax gap could emerge from within the self-regulated biochemical and electrical activity inside the skull, how “the 'mental' itself explodes within the neuronal through a kind of 'ontological explosion.'”247 The question and problematic here is distinctly Schellingian: what is the nature of the copula in judgement?248 Grund is existence in exactly the same way that the neuronal is the mental: the copula here does not primordially distinguish a relation of identity or pure equivalence, so that the latter is entirely subsumable under the former (ground = existence; the neuronal = the mental). It represents an activity that, through the logical self-withdrawal of its pervasiveness and primordiality, results in the production of irreducible difference (ground generates existence; the neuronal immanently gives rise to the mental), wherein each exists as opposite and therefore autonomous to one another, although the unpredictably new—a pure difference—that emerges nevertheless retains an internal thread of logical dependence upon that which gave birth to it at an originary level of theoretical investigation: that is, one of ontogenesis, whereby a productive or creative (schöpferisch) identity emerges between the irreducibly different terms.249 But one must be careful. Grund and existence are just as much “contemporaneous” logical relations as stages of historical development. Although the emergent split institutes two autonomous zones of activity—or, to speak in the parlance of the Weltalter, although the divide in being created by the irreducible spontaneity of the unconscious decision or de-scission (Entscheidung as Ent-Scheidung) sets the stage for the irreconcilability of the Present and the Past as epochs or ages in nature that forever alter it—they both exist simultaneously after the act of separation, despite the fact that the Grund also represents the dark “pre-history” of existence. The Logic of Transcendental Materialism It is in this way that the body as an independent entity existing in infinite contradistinction to mind can still follow its own laws, even if mind ultimately proves itself to be superior to its ontogenetic origins by existing in its own free register only in the a parallax gap could emerge from within the self-regulated biochemical and electrical activity inside the skull, how “the 'mental' itself explodes within the neuronal through a kind of 'ontological explosion.'”247 The question The Logic of Transcendental Materialism 167 aftermath of its hegemonizing self-positing or usurping of the primacy of body—or, to put it differently, natural cyclical time can still exist alongside, albeit in tension with, the out-of-joint dialectical temporality of spirit in the same creature. The neuronal interface can subsist in two paradoxical times, in the non-coincident two-way pull of the parallax as a multistable figure, the eternal “Past” of nonconscious material pulsation and the eternal “Present” of self-consciousness, both being “held” together in the positively charged void that is the subject as the impossible in-between (the vanishing mediator) generated within/by the negativity of being, so that the gap that sustains the subjective consistency of the universe of meaning can be maintained without denying the autonomy and power of cognitivism to describe the pure Real of biochemical and electrical activity that is the brain. The two zones are not to be confounded with one another, even though in a certain sense there is only the brute matter of the neuronal interface. Here we must recognize an implicit wordplay in Schelling: the copula in judgement (Urteil) is not merely an act of mind, a mental synthesis bringing a subject and predicate into relation with one another, but the expression of an act of primordial ontological division (Ur-Teil as ursprüngliche Teilung) exhibited by the thing in question with itself.250 The principle of identity should be able to explain eruptive breaches in the fold of being instead of being doomed to subsume everything under the dead univocity of a claustrophobic immanence: “[t]his principle does not give expression to an unity which, revolving in a circle of sameness, would be unprogressive, and thus insensitive or unalive. The Logic of Transcendental Materialism This leads us into a detailed analysis of the role of ontological catastrophe in the emergence of experience in being. expressions for a metaphysics of the disjunctive “and.” But what does this moment of the breaking of existence out of Grund, of the explosion of the Ideal out of the Real, look like? This leads us into a detailed analysis of the role of ontological catastrophe in the emergence of experience in being. 189. Žižek, The Indivisible Remainder. Taken from the book description. The Logic of Transcendental Materialism The unity of this law is immediately creative.”251 The copula in judgement is, in this sense, one of Schelling's many expressions for a metaphysics of the disjunctive “and.” But what does this moment of the breaking of existence out of Grund, of the explosion of the Ideal out of the Real, look like? This leads us into a detailed analysis of the role of ontological catastrophe in the emergence of experience in being. aftermath of its hegemonizing self-positing or usurping of the primacy of body—or, to put it differently, natural cyclical time can still exist alongside, albeit in tension with, the out-of-joint dialectical temporality of spirit in the same creature. The neuronal interface can subsist in two paradoxical times, in the non-coincident two-way pull of the parallax as a multistable figure, the eternal “Past” of nonconscious material pulsation and the eternal “Present” of self-consciousness, both being “held” together in the positively charged void that is the subject as the impossible in-between (the vanishing mediator) generated within/by the negativity of being, so that the gap that sustains the subjective consistency of the universe of meaning can be maintained without denying the autonomy and power of cognitivism to describe the pure Real of biochemical and electrical activity that is the brain. The two zones are not to be confounded with one another, even though in a certain sense there is only the brute matter of the neuronal interface. Here we must recognize an implicit wordplay in Schelling: the copula in judgement (Urteil) is not merely an act of mind, a mental synthesis bringing a subject and predicate into relation with one another, but the expression of an act of primordial ontological division (Ur-Teil as ursprüngliche Teilung) exhibited by the thing in question with itself.250 The principle of identity should be able to explain eruptive breaches in the fold of being instead of being doomed to subsume everything under the dead univocity of a claustrophobic immanence: “[t]his principle does not give expression to an unity which, revolving in a circle of sameness, would be unprogressive, and thus insensitive or unalive. The unity of this law is immediately creative.”251 The copula in judgement is, in this sense, one of Schelling's many expressions for a metaphysics of the disjunctive “and.” But what does this moment of the breaking of existence out of Grund, of the explosion of the Ideal out of the Real, look like? 188. Žižek, The Parallax View, p. 4. 187. Ibid., p. 55. 187. Ibid., p. 55. 188. Žižek, The Parallax View, p. 4. 189. Žižek, The Indivisible Remainder. Taken from the book description. 188. Žižek, The Parallax View, p. 4. 213. Žižek, Less Than Nothing, p. 266. 168 Chapter 7 190. Ibid., p. 11. 191. Ibid., p. 8. 192. The other important figure here is, of course, Lacan. Since Lacanian psycho- analysis declares that the Symbolic is never all, it is only natural that dialectical logic for Žižek would necessarily include an irremovable moment of irreconcilabil- ity. After all, for Lacan “when one gives rise to two, there is never a return. They don’t revert to making one again, even if it is a new one. The Aufhebung is one of philosophy’s pretty little dreams.” The Seminar. Book XX. Encore, On Feminine Sexuality, The Limits of Love and Knowledge, 1972–3, ed. Jacques-Alain Miller, trans. Bruce Fink (New York: Norton, 1998), p. 86. Yet insofar as Žižek’s project rests on the claim that psychoanalysis is the culmination of a lineage that begins in German Idealism, to assert that he merely reads Hegel through Lacan does not suffice, especially given the similarities between his own dialectics and his reading of Schelling. 193. Žižek, Less Than Nothing, p. 265. 194. Ibid. 194. Ibid. 195. See The Ticklish Subject, pp. 79–86. 196. Žižek, Less Than Nothing, pp. 234–35. 197. Žižek, The Ticklish Subject, p. 82. 198. Schelling, Freiheitsschrift, p. 227. 199. Žižek, Less Than Nothing, p. 922. 200. Žižek, The Abyss of Freedom, pp. 3–4. 201. Žižek, “Fichte’s Laughter,” p. 122. 202. See, for instance, The Parallax View, pp. 197–99. 203. Žižek, The Parallax View, p. 197 and 210. 203. Žižek, The Parallax View, p. 197 and 210. 204. Hegel, The Phenomenology of Spirit, p. 9. 204. Hegel, The Phenomenology of Spirit, p. 9. 205. Žižek, The Parallax View, p. 106 (repeated in Less Than Nothing, pp. 13f. and 642ff.). Contrast this with what he says of Schelling at ibid., p. 166;The Ticklish Subject, p. 55; The Indivisible Remainder, p. 14; and The Abyss of Freedom, p. 15. 206. This stays the same in the late philosophy. See The Grounding of Positive Philosophy, trans. Bruce Matthews (Albany: SUNY Press, 2007), p. 207. 207. Žižek, Less than Nothing, p. 265. 208. Schelling, The Ages of the World: Third Version (c. 1815), trans. Jason M. Wirth (Albany: SUNY Press, 2000) (hereafter Weltalter III), p. 219. 209. Ibid., p. 217. 210. Schelling, Freiheitsschrift, p. 239. 211. Ibid., p. 232. 212. Ibid. 213. Žižek, Less Than Nothing, p. 266. The Logic of Transcendental Materialism 169 214. Schelling, Ages of the World, in The Abyss of Freedom/Ages of the World, trans. Judith Norman (Ann Arbor: University of Michigan Press, 2008) (hereafter Weltalter II), p. 121. 214. Schelling, Ages of the World, in The Abyss of Freedom/Ages of the World, trans. Judith Norman (Ann Arbor: University of Michigan Press, 2008) (hereafter Weltalter II), p. 121. 215. Žižek, The Indivisible Remainder, p. 9. 215. Žižek, The Indivisible Remainder, p. 9. 216. Schelling, Freiheitsschrift, p. 281. 216. Schelling, Freiheitsschrift, p. 281. 217. Schelling, Weltalter II, p. 6. 218. The Indivisible Remainder, p. 13. 218. The Indivisible Remainder, p. 13. 219. Ibid. 219. Ibid. 220. Ibid., p. 11. 220. Ibid., p. 11. 221. Ibid., pp. 35–39. I take up this point in chapter 9. 222. Žižek, The Parallax View, p. 166. 222. Žižek, The Parallax View, p. 166. 223. Schelling, Weltalter II, p. 121. 223. Schelling, Weltalter II, p. 121. 224. Ibid. 224. Ibid. 225. Schelling, Freiheitsschrift, pp. 241–42. 225. Schelling, Freiheitsschrift, pp. 241–42. 226. Ibid., p. 239. 226. Ibid., p. 239. 227. Žižek, Less Than Nothing, p. 233. 227. Žižek, Less Than Nothing, p. 233. 228. See Žižek, The Parallax View, pp. 201–4. 228. See Žižek, The Parallax View, pp. 201–4. 229. Žižek, Less Than Nothing, p. 558. 229. Žižek, Less Than Nothing, p. 558. 230. Schelling, Freiheitsschrift, p. 239. 231. Kant, Kant on Education, p. 1; Lectures on Pedagogy, p. 437. For my discussion, see chapter 5. 232. Kant, Anthropology from a Pragmatic Point of View, p. 420. 233. Kant, Kant on Education, p. 6; Lectures on Pedagogy, p. 439. 234. Hegel, Hegel’s Philosophy of Nature, vol. 1, p. 205 (§247). 235. Ibid., p. 223 (§254). Translation modified. 235. Ibid., p. 223 (§254). Translation modified. 236. Ibid., p. 257 (§257A). 236. Ibid., p. 257 (§257A). 237. This example comes from my first Hegel teacher and philosophy mentor, Toni Stafford. 238. Hegel, The Science of Logic, p. 536. 238. Hegel, The Science of Logic, p. 536. 239. Hegel, The Encyclopedia Logic, p. 293 (§221Z). 239. Hegel, The Encyclopedia Logic, p. 293 (§221Z). 240. Ibid., pp. 302–3 (§234Z). 240. Ibid., pp. 302–3 (§234Z). 241. Hegel, Hegel’s Philosophy of Nature, vol. 1, p. 206 (§247Z). 242. Žižek, Indivisible Remainder, p. 92. 242. Žižek, Indivisible Remainder, p. 92. 242. Žižek, Indivisible Remainder, p. 92. 243. The full argument for this will be spilled out in chapter 10. 244. The Logic of Transcendental Materialism 169 Žižek, The Parallax View, p. 7. 244. Žižek, The Parallax View, p. 7. 245. Ibid., p. 168 (again in Less Than Zero, p. 725; and Žižek & Woodard, “Interview,” The Speculative Turn: Continental Materialism and Realism, p. 407). 250. Hegel draws upon the same wordplay in his Logic. Cf. The Encyclopedia Logic, p. 244 (§166A); and The Science of Logic, p. 552. 170 Chapter 7 246. Ibid., p. 175. 247. Ibid., p. 210. 248. Schelling, Freiheitsschrift, pp. 223–25. 249. Ibid., p. 227. 249. Ibid., p. 227. 250. Hegel draws upon the same wordplay in his Logic. Cf. The Encyclopedia Logic, p. 244 (§166A); and The Science of Logic, p. 552. 251. Schelling, Freiheitsschrift, p. 227. 251. Schelling, Freiheitsschrift, p. 227. Chapter 8 When the World Opens its Eyes The Traumatic Fissure of Ontological Catastrophe Chapter 8 When the World Opens its Eyes The Traumatic Fissure of Ontological Catastrophe Žižek's quadruple dialectics sets forth a conception of the absolute that is incomplete, insofar as it has split itself through a moment of primordial division into two irreconcilable zones of activity: body and mind, nature and spirit, the Real and the Symbolic. However, to explain this moment of self-sundering, Žižek must reappropriate various elements from Schellingian ontology to articulate the pre-conditions of subjectivity in the throes of being, something that the mature Hegel, recoiling from his Jenaer night of the world, fails to do. Žižek's provocative claim is that if we attentively read Schelling's account of the eruption of a freely existing subjectivity out of a nature that becomes infinitely Other, we encounter two startling insights. First, the emergence of desire in being as the ontogenetic condition of the possibility of subjectivity and phenomenalization displays a structural parallel to Schelling's own theory of disease and evil. Second, we must follow the implications of this to the end: rather than exhibiting a great triumph at the end of the odyssey of being, self-consciousness is merely the possible aftereffect of a cancerous negativity in being, an ontological catastrophe, which points to an irreversible fracturing of the very essence of the world. We will also explore the consequences of this for evolutionary theory and contemporary philosophy of mind. 172 Chapter 8 8.1 Desire, the Disease-Stricken Body of Being Following Schelling's descriptions of the eternal Past forever anterior to consciousness and language, a concern immediately arises: when we look at the elusive X of nature in the immemorial epochs of cosmological and geological time or the evolutionary strata of biological auto-development, we encounter an all-encompassing/all-consuming whole that precludes the absolute freedom of the subject. Insofar as this self-totalizing causality immanent in nature represents a relatively closed circle, how is this deterministic “deadlock” surpassed so that autonomy is possible? How exactly can the Grund/neuronal interface act in the self-effacing yet world- giving mode of existence/the mental? Although Žižek's own descriptions of the passage in The Indivisible Remainder and The Abyss of Freedom focus on the founding gesture of subjectivity as a self-instituting fiat, this is not enough. It is only one side of the story. The question is how the undifferentiated circuit of drives that constitute the pre-logical Real could paradoxically ground—give rise to or help incite—the irreducible self- positing act of decision. As Adrian Johnston argues, although this self- positing is ultimately an arbitrary, groundless act “analogous to the cutting of the Gordian knot”—which, as Žižek himself says, “can be described (narrated) only post festum, after it has already taken place, since we are dealing not with a necessary act but with a free act which could also not have happened”252—Schelling himself searches for a way to inscribe the very condition of the possibility of the act itself within the material palpitations of nature, in works that Žižek for the most part does not discuss.253 In this sense, Žižek's own account is not satisfactory because it has a tendency to present the drives as an irrevocably closed and blind system without explaining how of themselves they could short-circuit, a theoretical point that would be advantageous to his overall project. As Johnston points out, within the Schellingian ontogenetic narrative, the self-positing of the subject is first possiblized by the emergence of desire (Begierde) within being. Desire marks the first juncture of some kind of primordial blockage in the heart of blind necessity that upsets the automatic, unbridled oscillation of drives by shattering their pure immanence. This has two effects. 8.1 Desire, the Disease-Stricken Body of Being First, in psychoanalytical terms, it means that instead of a relative homeostasis as the inner telos guiding the entirety of an organism's When the World Opens its Eyes 173 biological life, we see for the first time a relative short-circuiting within the pleasure principle, an inability to find satisfaction through the mere repetition of the same constitutive of the movement of instincts. Second, in place of a smooth, determined relation to the environment wholly programmed by instincts (the coincidence of Innenwelt and Aussenwelt through a predetermined set of biological schemata that hardwire the organism into its “exterior” surroundings) we get a degree of liberation from the various sense data of perception that normally mechanically determine an organism's actions as it enters into a state of denaturalization that is contemporaneous with an act of withdrawing from its immersion in being, and thereby the first stirrings of a free creation of a world of experience. Desire in its Schellingian mode is thus an intermediary stage between nature and the violent unruliness that is the dark birthplace of the transcendental I. But what must be noted here is how desire, as the beginning of the idealization of reality, is essentially identical to the conventional definitions of psychosis as withdrawal from objective reality into self, but here at the ontological level instead of that of sociopolitically structured reality. Consequently, it is Schelling and not Hegel who most succinctly describes the ontological passage through madness insofar as it is the former and not the latter who describes how the night of the world could disrupt the world into a series of membra disjecta. In this respect, when Žižek in The Ticklish Subject and Less Than Nothing proclaims that it is Hegel who is the most radical philosopher of the abyss of madness at the core of subjectivity and the minimal paranoia at the basis of order itself,254 he appears to be completely unaware of how strongly his reading of Schelling influences his own reactualization of German Idealism. 8.1 Desire, the Disease-Stricken Body of Being On his own account, not only is the Schellingian concept of the erratic oscillation of drives that precedes fully constituted reality the expression of the abyss of madness at the core of subjectivity, which Schelling himself supplements with the emergence of desire/ontological madness in being, but at the end of the first chapter of The Indivisible Remainder Žižek discusses Schelling's great insight into the necessarily paranoiac structure of universality as such, a point made explicit for the first time by Lacan, thus establishing a link between the two.255 In this sense, in both Žižekian and Schellingian ontology we can speak of something like a spectrum of subjectivity or ideality inherent within In this sense, in both Žižekian and Schellingian ontology we can speak of something like a spectrum of subjectivity or ideality inherent within 174 Chapter 8 Chapter 8 174 nature. Following this claim literally, we must say that we come across traces of desire within other organisms to varying degrees: there is a kind of quantitative accumulation of desire (an evolutionary genesis) that may lead to a complete qualitative break with nature (the splitting act of Ent- Scheidung) but in such a way that the possibility of the latter is not logically contained within the former as a kind of hidden, self-unfolding kernel— rather, the former can only incite it, so that only after the fact can we establish a “relation” between the two. There is no guarantee for freedom in the realm of mere being. Yet, what desire shows us is that the pure Real is not completely all-consuming: it is teeming with crevices within its positive fold, interstices within its being, which present a restless negativity tearing it apart. However, these sites of negativity are to be distinguished from human subjectivity insofar as despite expressing a minimal level of liberation from nature's cycles, they are unable to completely liberate themselves from nature's biological hardwiring and thus seek a new form of non- natural (virtual) organization. In this respect, they would only resemble the primordial unruliness prior to the advent of the Symbolic insofar as they would not exhibit a violent process of utter denaturalization, although we must nevertheless speak of a tension-ridden trembling of the organic system as it begins to quiver under its own weight. 8.1 Desire, the Disease-Stricken Body of Being It is for this reason that Žižek can adopt Heidegger's claim in The Fundamental Concepts of Metaphysics that animals feel “the ‘poorness’ of their relating to the world” in such a way that we see “an infinite pain pervading the whole of living nature.”256 As Žižek points out, this shares common ground with Schelling's notion of “the veil of despondency that spreads itself over nature.”257 But we must understand this pain or despondency in nature in two separate but interrelated ways. First, one could say that in desire as the beginnings of the idealization, that is, of the world's psychotic replication of itself within itself, animals have a kind of implicit yet nonconscious “knowledge” that they are unfree because they exist in a mode of unfathomable tension between the Real (instinctual-deterministic schemata) and the Ideal (desire as a blockage), but without this distinction being posited as such, as if here the Ideal has begun to inhere/persist within the Real, disrupting it from the inside, without yet being autonomous. The Real rules supreme—and not only has full-fledged ideality not yet emerged When the World Opens its Eyes 175 175 in being, but what we encounter here may only be referred to as a form of ontological proto-ideality once ideality in its freedom has come on the scene, that is, retroactively. By consequence, the slightest tinge of desire within the biological system that is supposed to determine the animal's reaction with its environment in advance through a pre-set logistics gives it, as it were, a taste of its own possible, but unreachable freedom, the animal being liberated yet confined in its movement. It is this simultaneous nonconscious “dreamlike” premonition of subjective freedom and its ultimate ontological foreclosure that constitutes nature's despondency or melancholy (Schwermut). But it must be noted that the “despondency” or “melancholy” of nature is not a moment of mere poetical rhetoric or uncalled-for anthropomorphism. As Henri Maldiney points out, Heidegger's descriptions of living beings as plagued by a constitutive Benommenheit (captivation, dazedness) coincide with that “mode of being of the type melancolicus” elaborated by Tellenbach.258 If nature is overcome by a veil of despondency, it is because non-human life is wrought by a structure that is, for us, distinctively pathological: it is as if it is held back, stuck in its tracks, because it is incapable of a truly effective willing. 176 Chapter 8 The Žižekian night of the world emerges as the nonconscious drives of nature for the first time liberate themselves from their blind rotation in being through an immanently generated pandemonium within the corpo-Real of the body. Properly speaking, desire is an impasse within the ontological life of substance—“[s]ince there is consequently an unremitting urge to be and since it cannot be, it comes to a standstill in desire, as an unremitting striving, an eternally insatiable obsession with Being”259—that prevents substance from encompassing all, for the organism now obeys its own non-natural logic. It stands for the irremovable and impenetrable kernel of the Real qua logical paradox/internal limit that disturbs the annular circulation of drives through its own self-assertive violence. Here, the analogue with the body again proves to be useful to perceive the radicality of Žižek's reactualization of Schelling. Although the biological unity of the corpo-Real can astound us with its organic dynamism and systematic efficacy, the very awe-inspiring force of this self-organizing totality can cast a shadow over its dark underbelly, whose traumatic fact is often attested by nature's production of monstrosities, degenerative diseases, the mindless proliferation of cancerous tumors, or even the emergence of various forms of mental illness and psychosis caused by pure organic dysfunction, a fact that demonstrates how, from within the closed totality of a determinist system, a part can assert itself from within and hegemonize the organic whole, restructuring it according to its own “unruly” whim and thereby perturbing its harmonious, symphonized flow. Even if everything is logically pre-determined (the ebb and flow of matter can only follow certain paths carved out by genetics, the neuronal interface of the brain, and various different autopoietic systems), the laws that normally regulate the body can by themselves immanently generate a (bio)logical short circuit, thereby opening up a negative space within the body's corpo-Real that can assert itself and wreak havoc over its self-governing unity through a glitch in its programming. That which guarantees the placid functioning of everyday life and the health of body can suddenly turn into a ghastly apparition of true terror. 176 Chapter 8 Like an illness or disease within Schellingian ontology, desire does not stand for a positive ontological unity, but for an internal scrambling of the biological system that does not follow its supposed path within the whole and instead stubbornly asserts its own self at all costs—even its own The Žižekian night of the world emerges as the nonconscious drives of nature for the first time liberate themselves from their blind rotation in being through an immanently generated pandemonium within the corpo-Real of the body. Properly speaking, desire is an impasse within the ontological life of substance—“[s]ince there is consequently an unremitting urge to be and since it cannot be, it comes to a standstill in desire, as an unremitting striving, an eternally insatiable obsession with Being”259—that prevents substance from encompassing all, for the organism now obeys its own non-natural logic. It stands for the irremovable and impenetrable kernel of the Real qua logical paradox/internal limit that disturbs the annular circulation of drives through its own self-assertive violence. Here, the analogue with the body again proves to be useful to perceive the radicality of Žižek's reactualization of Schelling. Although the biological unity of the corpo-Real can astound us with its organic dynamism and systematic efficacy, the very awe-inspiring force of this self-organizing totality can cast a shadow over its dark underbelly, whose traumatic fact is often attested by nature's production of monstrosities, degenerative diseases, the mindless proliferation of cancerous tumors, or even the emergence of various forms of mental illness and psychosis caused by pure organic dysfunction, a fact that demonstrates how, from within the closed totality of a determinist system, a part can assert itself from within and hegemonize the organic whole, restructuring it according to its own “unruly” whim and thereby perturbing its harmonious, symphonized flow. Even if everything is logically pre-determined (the ebb and flow of matter can only follow certain paths carved out by genetics, the neuronal interface of the brain, and various different autopoietic systems), the laws that normally regulate the body can by themselves immanently generate a (bio)logical short circuit, thereby opening up a negative space within the body's corpo-Real that can assert itself and wreak havoc over its self-governing unity through a glitch in its programming. That which guarantees the placid functioning of everyday life and the health of body can suddenly turn into a ghastly apparition of true terror. 8.1 Desire, the Disease-Stricken Body of Being Second, with emergence of desire, the self-sustaining circuitry of nature becomes disturbed by the interruptive presence of an extimacy, an inassimilable Real, within its very ebb and flow, causing its relatively balanced rotation to fall into a painful deadlock, an erratic oscillation of conflictual tendencies that become more and more uncontrollable as desire increases. If the animal gets its first taste of freedom in desire, then nature in the same moment gets the first taste of the madness that awaits it if this freedom were to fully actualize itself (the night of the world). Nature's pain is the unsteady, unpredictable palpitation of its heart of hearts threatening to explode in one excessive outburst as Todestrieb begins to awaken itself, but because it has yet to self-accumulate to a great intensity, and nothing has arisen to tame this propensity, there is a bleak darkness of antagonism. What is at issue here is the state of nature that precedes and sets the stage for humanity as a response; the emphasis is not on us, but rather on ontogenetic conditions, so that the apparent anthropomorphization of nature is coincident with the dehumanization of 176 Chapter 8 Like an illness or disease within Schellingian ontology, desire does not stand for a positive ontological unity, but for an internal scrambling of the biological system that does not follow its supposed path within the When the World Opens its Eyes 177 dismal downfall by obstructing its own life source. A false unity, it simultaneously represents an ontological perversion and a metaphysical distortion: freedom is a devouring black hole, a mere disturbance, in the vital throes of being. In this context, Žižek talks of Jacques-Alain Miller's remarks on an unsettling rat experiment mentioned in one of Lacan's unpublished seminars, where it is only through a kind of neurological mutilation that a rat can be made to behave like a human. Formally, the specific character that distinguishes human freedom and separates it from the rest of the world is identical to rampant malfunction, a violent ontological disfiguring.260 As the force of desire is raised to a higher and higher degree of ideality, matter enters into a self-lacerating rage (sich selbst zereißende Wut) like a cancer-ridden, disease-stricken body howling under its own out-of-control energy.261 Desire is a self-destructive mania that tears apart the smooth fabric of the world. This is why Žižek finds Schelling's “Wagnerian” vision of God so terrifying. It depicts a being that, by means of the painful, crippling amplification of desire into Todestrieb, becomes completely denaturalized and can thus posit itself as distinct from the anonymous, faceless pulsation of substance by the carving up of a self: “[t]he horror of the rotary motion resides in the fact that it is no longer impersonal: God already exists as One, as the Subject who suffers and endures the antagonism of drives,” “a state of an endless 'pleasure in pain,' agonizing and struggling with Himself, affected by an unbearable anxiety.”262 The primordial unruliness of human nature and its coequal term diabolical evil are therefore synonymous with this grotesque excess of life that we witness in the breakdown of the corpo- Real in times of illness, with what occurs when the self-enclosed logic of nature auto-disrupts through pure dysfunction. 178 Chapter 8 Žižek. What I am currently engaged with is the paradoxical idea that, from a strict evolutionary standpoint, consciousness is a kind of mistake—a malfunction of evolution—and that out of this mistake a miracle emerged. That is to say, consciousness developed as an unintended by-product that acquired a kind of second-degree survivalist function. Basically, consciousness is not something which enables us to function better. On the contrary, I am more and more convinced that consciousness originates with something going terribly wrong—even at the most personal level. For example, when do we become aware of something, fully aware? Precisely at the point where something no longer functions properly or not in the expected way. Daly. Consciousness comes about as a result of some Real encounter? Žižek. Yes, consciousness is originally linked to this moment when “something is wrong,” or, to put it in Lacanian terms, an experience of the Real, of an impossible limit. Original awareness is impelled by a certain experience of failure and mortality—a kind of snag in the biological weave. And all the metaphysical dimensions concerning humanity, philosophical self-reflection, progress and so on emerge ultimately because of this basic traumatic fissure.264 176 Chapter 8 In this regard, the freedom of the subject—that anarchic state that precedes the birth of the Symbolic as a form of retroactive damage control—is not a positive characteristic or attribute: it is the failure of the auto-actualization of the essence of nature, its inability to contain itself within its own preset logistics, which causes an ontological catastrophe.263 As Žižek makes clear in an interview, when viewed from the standpoint of the natural world we can only even understand the peculiarity of (self-) consciousness and human intelligence by positing something going horribly wrong in its internal development: 178 Chapter 8 8.2 Malfunction, Mal-adaptation, Breakdown: Žižek and the Sciences We should pause for a moment and consider this Žižekian notion of a “snag in the biological weave” in order to draw out its full meaning and implications for our understanding of the sciences, which will simultaneously allow us to show that such an ontogenetic account of the emergence of Todestrieb is not enough to explain the emergence of subjectivity. Taken at a purely formal level, both standard accounts of evolution and Žižek's theory of the ontogenesis of the subject appear to share a fundamental presupposition. Functioning through random mishaps When the World Opens its Eyes 179 When the World Opens its Eyes 179 179 in the self-replication of genetic code producing new, unforeseeable ontological differences in living being, neo-Darwinian evolution is rendered possible by a moment of malfunction, error, the always possible upsurge of inconsistency in the “rhythmic” flow of nature. Irreversible mutations form the basal and primordial stuff from which life gains its mercurial character. Consequently, for modern-day biology, nature too is not-all insofar as there is no eternal, overarching unity in the vital energetics of substance, no all-inclusive weave with a preset structure, plan, or predictable movement: there are constant slips and slides in positive reality, glitches and functional disturbances. Evolution is nothing other than nature encountering its own real limit, which proves to be the actual driving force of material creation and change: its weakness does not prove to be a mere limitation, a deficiency, but is often even its strength, allowing its forms to endure by adaption. However, what we could call the biological movement of the negative is only able to ground this transformative activity insofar as it sets up a new “dialectic” between organism and environment. As malfunction slowly and contingently gives rise to new characteristics by the sedimentation of small changes over the sluggish march of evolutionary time, an automatic rubric of natural selection occurs: those who are, by pure chance, more “fit” for their particular niche survive and pass on the biological glitch that, paradoxically, shows itself retroactively to have been a source of force and power, making those that were not struck by accident with the given mishap of self-replication in question disadvantageous, leading them to die out. Though here the possibility of monstrosities coincides with the possibility for new natural forms, a new emergent logistic is able to subsume both within a greater system of the struggle for life and death, even if this struggle is, in and of itself, non-teleological. in the self-replication of genetic code producing new, unforeseeable ontological differences in living being, neo-Darwinian evolution is rendered possible by a moment of malfunction, error, the always possible upsurge of inconsistency in the “rhythmic” flow of nature. Irreversible mutations form the basal and primordial stuff from which life gains its mercurial character. When the World Opens its Eyes 179 Consequently, for modern-day biology, nature too is not-all insofar as there is no eternal, overarching unity in the vital energetics of substance, no all-inclusive weave with a preset structure, plan, or predictable movement: there are constant slips and slides in positive reality, glitches and functional disturbances. Evolution is nothing other than nature encountering its own real limit, which proves to be the actual driving force of material creation and change: its weakness does not prove to be a mere limitation, a deficiency, but is often even its strength, allowing its forms to endure by adaption. However, what we could call the biological movement of the negative is only able to ground this transformative activity insofar as it sets up a new “dialectic” between organism and environment. As malfunction slowly and contingently gives rise to new characteristics by the sedimentation of small changes over the sluggish march of evolutionary time, an automatic rubric of natural selection occurs: those who are, by pure chance, more “fit” for their particular niche survive and pass on the biological glitch that, paradoxically, shows itself retroactively to have been a source of force and power, making those that were not struck by accident with the given mishap of self-replication in question disadvantageous, leading them to die out. Though here the possibility of monstrosities coincides with the possibility for new natural forms, a new emergent logistic is able to subsume both within a greater system of the struggle for life and death, even if this struggle is, in and of itself, non-teleological. For Žižek, however, this naturalistic account of biological “negativity” within being at the level of the self-reproduction of organisms does not go far enough. Although he agrees with its fundamental presuppositions— nature as not-all, as driven forward by an internal yet productive inconsistency—he believes that the mechanism of neo-Darwinian evolution is unable to explain the emergence of subjectivity because it falls back into the trap of an organic theory of nature. When the World Opens its Eyes 179 Even if it does not posit an indwelling, necessary tendency for progression, a forward-moving 180 Chapter 8 0 Chapter 8 180 advancement towards more sophisticated life-forms, the “dialectic” between organism and environment is all-inclusive: the law of self-preservation and homeostasis becomes so primordial that even a mishap in reproduction becomes ultimately subsumed under a species' attempt to assert itself and stay alive, so that the moment of the negative retroactively posits itself as being always already a teleologically guided, rational development. In other words, we can always give reasons as to why this or that evolutionary feature benefited the organism as a being immersed in the natural cycle of life and death. Even if within the naturalistic viewpoint nature/substance is in a certain respect not-all—there is nothing guaranteeing its completion— it does not go far enough, for nothing transcends its grasp. There may be conflict and negativity within the system, but this in no way poses a problem to the inner algorithm of its biological “dialectics.” The problem with this theoretical position is that it fails to see how the subject, haunted by Todestrieb, demonstrates that there is no single overarching principle of explanation, that there is a “place” in nature that is, strictly speaking, non- natural and defines itself against the rhythm of life and death, fullness and lack: the negativity that opens up the space for self-consciousness cannot be, even retroactively, subsumed within an evolutionary narrative of survival. Scientific methodology cannot explain the human, for the zero-level (= unruliness) of experience not only provides no functional advantage in nature, but even disturbs the dialectic of organism and environment because it cuts the link between Innenwelt and Aussenwelt. As Žižek says, “we should bear in mind the basic anti-Darwinian lesson of psychoanalysis repeatedly emphasized by Lacan: man’s radical and fundamental dis-adaptation, mal- adaptation, to his environs,”265 a point not only argued for by Lacan, but also by Kant and Fichte at the beginning of the German Idealist tradition.266 Ž advancement towards more sophisticated life-forms, the “dialectic” between organism and environment is all-inclusive: the law of self-preservation and homeostasis becomes so primordial that even a mishap in reproduction becomes ultimately subsumed under a species' attempt to assert itself and stay alive, so that the moment of the negative retroactively posits itself as being always already a teleologically guided, rational development. When the World Opens its Eyes 179 In other words, we can always give reasons as to why this or that evolutionary feature benefited the organism as a being immersed in the natural cycle of life and death. Even if within the naturalistic viewpoint nature/substance is in a certain respect not-all—there is nothing guaranteeing its completion— it does not go far enough, for nothing transcends its grasp. There may be conflict and negativity within the system, but this in no way poses a problem to the inner algorithm of its biological “dialectics.” The problem with this theoretical position is that it fails to see how the subject, haunted by Todestrieb, demonstrates that there is no single overarching principle of explanation, that there is a “place” in nature that is, strictly speaking, non- natural and defines itself against the rhythm of life and death, fullness and lack: the negativity that opens up the space for self-consciousness cannot be, even retroactively, subsumed within an evolutionary narrative of survival. Scientific methodology cannot explain the human, for the zero-level (= unruliness) of experience not only provides no functional advantage in nature, but even disturbs the dialectic of organism and environment because it cuts the link between Innenwelt and Aussenwelt. As Žižek says, “we should bear in mind the basic anti-Darwinian lesson of psychoanalysis repeatedly emphasized by Lacan: man’s radical and fundamental dis-adaptation, mal- adaptation, to his environs,”265 a point not only argued for by Lacan, but also by Kant and Fichte at the beginning of the German Idealist tradition.266 In his own work on cognitive science, Žižek attempts to show for this very reason how a variety of cognitive scientists and neurophilosophers falter at the enigma of consciousness and thereby create a plurality of approaches at odds with one another. He divides the latter into four groups, which cover the logical possibilities of explaining consciousness in terms of the standard natural model (sadly, the details of each on the Žižekian reading must be left aside here): When the World Opens its Eyes 181 181 I. eliminative/reductive materialism: the proclamation that there is no such thing as consciousness and qualitative experience is a “naturalized” illusion (Patricia and Paul Churchland); II. the antimaterialist position: the necessity of positing a new fundamental force of nature yet to be discovered (David Chalmers); II. the antimaterialist position: the necessity of positing a new fundamental force of nature yet to be discovered (David Chalmers); III. 182 Chapter 8 182 Chapter 8 182 Chapter 8 its singular character as ideal self-reflexivity—both an attempt to show the theoretical prowess of materialism) or by tackling it head-on ([ii] declaring the irreducibility of mind or [iii] making a possible materialist explanation disappear—both an attempt to demonstrate the necessity of idealism), what each option shows, positively or negatively, is that the self-reflexivity constitutive of consciousness is a disruption of natural laws that demands a new metaphysical vision of the world. For these reasons, Žižek does not locate the moment of malfunction constitutive of the subject at the level of genetic mutation and, in a second step, he refuses to equate it completely with a breakdown of the libidinal- material ground of nature. It goes beyond a scientifically measurable “unit of change” (even if is ultimately incited by a certain series of genetic mutations) insofar as it breaks with all totalizing, homogenizing principles or laws. As a closed loop of infinite self-relating, consciousness can only be made possible by a prior psychotic withdrawal of the world into its nocturnal, ontologically solipsistic self, the primordial rupture dividing Innenwelt and Aussenwelt. It is this founding gesture that sets an internal limit to scientific models because it obstructs the “dialectic” of organism and environment: the self-organizing, instinctual schemata offered by our genetic code go haywire in the life of the organism, so that we are no longer immersed in the world through a preset logistical program, but open up a space within which we are able to freely relate to it as an Other. The point is not to search for how the raw immediacy of the brute Real of neurons and their ancestral history in immemorial time dissipates the primordiality of self-experience, but how the irreducible reflexivity that sustains consciousness itself emerges from within the brute, faceless abyss of asubjective brain matter in such a way that the former remains incommensurate with latter, even if the latter is its obscure birthplace. Relying upon John Taylor's theory of consciousness, Žižek locates this primordial act of ontological division at a very specific point: through the relation of past working memory to present input, present experience spontaneously acquires the ability to relate to itself through a detour through the (its) past, the condition of which is “bubbles” of neuronal activity in local cortical regions establishing complex feedback systems. When the World Opens its Eyes 179 the inherent inexplicability of consciousness: given the “cognitive closure” limiting knowledge, we must assert that the birthplace of conscious awareness is unknowable, even if it did arise out of materiality (Colin McGinn, Steve Pinker); IV. an “as-if” non-reductive materialism: consciousness exists, and we can use teleological language to describe it, but it has emerged entirely due to natural laws and can ultimately be completely subsumed within them (Daniel Dennett).267 IV. an “as-if” non-reductive materialism: consciousness exists, and we can use teleological language to describe it, but it has emerged entirely due to natural laws and can ultimately be completely subsumed within them (Daniel Dennett).267 What remains common to each of these approaches, according to Žižek, is the failure of a purely scientific account of consciousness. In typical psychoanalytical style, it is the places where these discourses reach an impasse, where their symbolic space is internally obstructed, that are the most revealing; paradoxically, this occurs whenever they face the essentials of the very object of their investigation. Whether it be (i) the outright dismissal of self-consciousness reflexivity as a pseudo-problem, (ii) the thesis that the only way to inscribe the singularity of consciousness within nature is to posit a new force comparable to gravity or electromagnetism, (iii) entirely giving up any possible material explanation of consciousness, or (iv) the forced attempt to understand conscious reflexivity as a purely natural phenomenon, the closed loop of infinite self-relating that is the condition of the possibility of self- consciousness is itself the impasse of each discourse, for consciousness cannot be naturalized. This leads Žižek to say: “I am therefore tempted to apply here the dialectical reversal of epistemological obstacle into positive ontological condition: what if the 'enigma of consciousness,' its inexplicable character, contains its own solution? What if all we have to do is to transpose the gap which makes consciousness (as the object of our study) 'inexplicable' into consciousness itself?”268 Either by trying to bypass the problem by fiat ([i] completely eliminating the object, “consciousness,” or [iv] stripping it of 8.3 Terror, Perplexity, and the Awakening of the World All at once and as if in a cosmic flash, through an accident of overlapping in the neuronal network, material being encounters its own internal limit amplified to the maximum and the world opens its eyes: with the rise of subjectivity it appears that an unsolvable glitch in the smooth functioning of its own inward-dwelling logic posits itself as such, enabling the world for the first time to find the distance to Situating his own philosophical project within the heritage of German Idealism, Žižek's psychoanalytical reactualization of the Schellingian eruptive logic of the Grund is an attempt to show how the subject is not external to the absolute. In other words, the gaze of the subject in Žižekian ontology must be seen as the material universe finally “gaining” the power to look upon itself through an internal reflection: “the whole domain of the representation of the world (call it mind, spirit, language, consciousness, or whatever medium you prefer) needs to be understood as an event within and of the world itself. Thought is not at all opposed to being, it is rather being's replication within itself.”271 As the dense, closed circuitry of nature dissolves under the impact of the Todestrieb, it gives way to the possibility of the “miracle”272 of human thinking and its free idealization of the world as immanent in the latter. However, this moment of parallax shift from mere being to thought must be taken for what it is. Within the ancestral genealogy of forgotten, phenomenologically inaccessible time, the system of nature slowly and contingently grows in complexity, eventually reaching its autopoietic apotheosis in the structure of the brain, which, because of its some one hundred billion neurons possessing a total of at least one hundred trillion synaptic connections, thereby displaying one hundred times more synapses than the estimated number of stars in our galaxy, is unable to hold itself together according to its own immanent, self-regulating laws. Unintended loops and gaps in its processes become visible as they writhe under the infinite pressure of their own labyrinthine intricacy, nature failing to find its own way in its own production. 182 Chapter 8 It is this direct self-instituting short-circuiting that allows for free, infinitely self-standing thought, the ease, energy and speed of mental operations, in When the World Opens its Eyes 183 contradistinction to the complex mediative channels of neuronal activity that produced the organism as a biological system, for it allows present experience to liberate itself radically from a mere attachment to input.269 In a flash, the organism stops being determined through external conditions, but creates a zone within which it can relate to itself as a self so that bit by bit this self-relation takes charge of instinctual schemata. For Žižek, this “short circuit” or “(bio)logical glitch” between various faculties within the brain—a kind of naturally inexplicable overlapping that creates the possibility of psychotic self-relation, which, just like Schelling's account of the self-begetting of God, “can be described (narrated) only post festum, after it has already taken place, since we are dealing not with a necessary act but with a free act which could also not have happened”270—as a meta- transcendental condition of consciousness is related only in a derivative or secondary sense to the “malfunction” so fundamental to the process of neo-Darwinian evolution.This is true for two reasons. First, it makes itself superior to the natural basis that paves the way for it and thereby elevates itself above it. Second, it is not predictable from the mere level of genetic mutation. In this sense, not only is it a pure self-positing that depends on nothing but itself (even if it is incited by a genetic mutation, it remains irreducible to and logically distinct from it: it is a mere neuronal mishap, a disruptive emergence in the interstices of the logical network sustained by the biochemical and electrical interface of brain matter), but this capacity for self-assertion also allows it to liberate itself from its immersion/ imprisonment in the natural world (the generated closed loop of self-relation breaks the interpenetration of Innenwelt and Aussenwelt in such a way that it cannot be retroactively subsumed within a survivalist narrative of newfound functionality, even if it does manage to acquire a second-degree survivalist function). Genetic mutation, the “dialectics” of organism and environment, and with it the breakdown of the libidinal-material ground of the organism, may be necessary for explaining the origins of subjectivity, but they are not sufficient. For full-fledged subjectivity to emerge, natural breakdown must paradoxically double itself 184 Chapter 8 8.3 Terror, Perplexity, and the Awakening of the World Situating his own philosophical project within the heritage of German Idealism, Žižek's psychoanalytical reactualization of the Schellingian eruptive logic of the Grund is an attempt to show how the subject is not external to the absolute. In other words, the gaze of the subject in Žižekian ontology must be seen as the material universe finally “gaining” the power to look upon itself through an internal reflection: “the whole domain of the representation of the world (call it mind, spirit, language, consciousness, or whatever medium you prefer) needs to be understood as an event within and of the world itself. Thought is not at all opposed to being, it is rather being's replication within itself.”271 As the dense, closed circuitry of nature dissolves under the impact of the Todestrieb, it gives way to the possibility of the “miracle”272 of human thinking and its free idealization of the world as immanent in the latter. However, this moment of parallax shift from mere being to thought must be taken for what it is. Within the ancestral genealogy of forgotten, phenomenologically inaccessible time, the system of nature slowly and contingently grows in complexity, eventually reaching its autopoietic apotheosis in the structure of the brain, which, because of its some one hundred billion neurons possessing a total of at least one hundred trillion synaptic connections, thereby displaying one hundred times more synapses than the estimated number of stars in our galaxy, is unable to hold itself together according to its own immanent, self-regulating laws. Unintended loops and gaps in its processes become visible as they writhe under the infinite pressure of their own labyrinthine intricacy, nature failing to find its own way in its own production. Substance falls into deadlock: with the emergence of the complex neuronal interface of man, it can no longer successfully posit itself as a fragile all, and it breaks into a series of membra disjecta, which opens up the space within which the self-enclosed loop of experience asserts itself out of nowhere, further intruding upon and obstructing its activity from within. 8.3 Terror, Perplexity, and the Awakening of the World Substance falls into deadlock: with the emergence of the complex neuronal interface of man, it can no longer successfully posit itself as a fragile all, and it breaks into a series of membra disjecta, which opens up the space within which the self-enclosed loop of experience asserts itself out of nowhere, further intruding upon and obstructing its activity from within. All at once and as if in a cosmic flash, through an accident of overlapping in the neuronal network, material being encounters its own internal limit amplified to the maximum and the world opens its eyes: with the rise of subjectivity it appears that an unsolvable glitch in the smooth functioning of its own inward-dwelling logic posits itself as such, enabling the world for the first time to find the distance to When the World Opens its Eyes 185 185 self necessary for its own self-phenemonalization by virtue of a shock, a blow, a violence, for which it itself is responsible. The claustrophobic immanence that drowns everything in the blind void of non-experience is finally shattered—yet when the world finally opens its eyes for the very first time in the mode of subjectivity it does not rejoice, it does not celebrate, nor does it feel a passive, grateful joy from the beauty surrounding it and bask in the bliss aroused by the wonder and amazement of the brute fact of its existence. It whimpers under its own weight, hearing its own inarticulate cry as it experiences itself in a moment of unbearable agony and catastrophic self-diremption, “a mixture of terror and perplexity” that Žižek compares to the atrocity of sexual abuse and the horrific pictures of children dying from radiation exposure in Chernobyl: Although one of today's main candidates for the figure of Evil is child sexual abuse, there is nevertheless something in the image of a hurt, vulnerable child which makes it unbearably touching: the figure of a child, between two and five years old, deeply wounded but retaining a defiant attitude, his face and poise remaining stubborn, although he is barely able to prevent an outburst of tears—is this not one of the figures of the Absolute? 8.3 Terror, Perplexity, and the Awakening of the World One thinks here about the photos of children dying from exposure to radiation after the Chernobyl accident, or—also from Ukraine—one of the photos on a child-porn website showing a really young child, no more than four years old, confronting a big ejaculating penis, face covered with fresh sperm. Although the shot probably plays on the link between the penis ejaculating sperm and the mother’s breast full of milk, the expression on the child's face is clearly a mixture of terror and perplexity: the child cannot make out what is going on.273 What the world first sees is not its own awe-striking unity and oneness, the spiritual fullness of a self-seizing, self-actualizing centre that holds everything together in an all-encompassing totality. All it discerns is the tumultuous uproar of erratic pulsation, an insurmountable, non-masterable chaos resulting from the degradation or collapse of its own positive, autopoietic activity. The self-awareness of the world, its self-experience 186 Chapter 8 But it must be insisted that this can only be accomplished at the level of the virtual: “[t]he third moment which 'resolves' the contradiction is by definition 'prothetic' (virtual, artificial, symbolic, not substantially natural).”278 When the world “perceives itself” as lost to self and infinitely When the World Opens its Eyes 187 dirempt (N ≠ N as the originary [onto]logical violence preceding and possibilizing transcendental imagination) in the mode of subjectivity, it recoils into culture as a defence mechanism to try to sublate this gap in its being, to fill in this hole in its depths and thereby posits itself as such as a full-fledged subjectivity.279 In short, the passage from darkness to light only occurs at the level of the Symbolic: in the Real, nothing changes, unruliness (our break from nature) is left untouched. It is this aspect of the intrinsic madness of culture, language, and phenomenal reality, its psychotic lack of contact with the world, that Žižek claims we forget, that we must necessarily forget, if the transcendental misrecognition of reality necessary to subjectification as a reaction formation is to be a successful “compensation.” All our discourses, all our “truths,” are nothing but the deluded ravings of the asylum unaware of their true origin within the founding gesture of subjectivity as a recoil spurred on by the brutal trauma of violently awakening up into a dismembering hemorrhaging of being, the ultimate ontological catastrophe. All the beauty of the world merely belies its true, unbearable horror: “[i]f we take into consideration the many terrible things in nature and the spiritual world and the great many other things that a benevolent hand seems to cover up from us, then we could not doubt that [the ego] sits enthroned over a world of terrors.”280 In this respect, “the true point of 'madness' [...] is not the pure excess of the ‘night of the world,' but the madness of the passage to the Symbolic itself, of imposing a symbolic order onto the chaos of the Real. 186 Chapter 8 86 Chapter 8 186 Chapter 8 in the first person as made possible by the existence of subjectivity and thus all experience as such, is necessarily preceded by this irreducible and irreversible auto-disruption that must be seen as catastrophic. The ontogenetic basis of subjectivity is the trauma of primordial loss, where nature is forever alienated from itself through complete breakdown (N ≠ N), so that we must say that the subject is paradoxically that which has survived its own death, for “the past traumatic loss of substance [...] is constitutive of the very dimension of subjectivity”274—or as Schelling himself puts it, “[p]ain is something universal and necessary in all life, the unavoidable transition point to freedom [...]. It is the path to glory.”275 But this is no mere rhetoric on behalf of Žižek: as his Schellingian-inspired argument makes clear, this psychosis-inducing auto-disruption of being is a necessary theoretical posit if free experience is to be possible instead of a blind experiential void: We cannot pass directly from nature to culture. Something goes terribly wrong in nature: nature produces an unnatural monstrosity and I claim that it is in order to cope with, to domesticate, this monstrosity that we symbolize. Taking Freud's fort/da as a model: something is primordially broken (the absence of the mother and so on) and symbolization functions as a way of living with that kind of trauma.276 In short, the ontological necessity of “madness” resides in the fact that it is not possible to pass directly from the “animal soul” immersed in its natural life-world to “normal” subjectivity dwelling in its symbolic universe—the vanishing mediator between the two is the “mad” gesture of radical withdrawal from reality that opens up the space for its symbolic reconstitution.277 The implication of this is that the Symbolic (the reconstitution of reality, the world's self-replication within itself) is nothing but an attempt to tame, to gentrify this constitutive mayhem/madness in the immanent fold of being. 188 Chapter 8 188 Chapter 8 188 Chapter 8 this being the fundamental logical moment for understanding how his own parallax ontology can bring together a materialism and a self-grounding idealism. It is not the mediating filters of language that are primordially responsible for our lack of access to extra-subjective reality due to some internal limitation—rather, it is reality itself that renders impossible its own access to itself. The Symbolic is always already revelatory of the nature of the world, albeit in an intrinsically negative manner: the prison house of language, our inability to “transcend” it and grasp reality as it is in itself,284 necessarily refers to the material processes that gave birth to the alienating linguistic-conceptual structures that permanently rob us of the immediacy of being. Psychosis is not the feature of a single isolated subject lost in disarray—it is the rite of passage of becoming a subject, a point explicitly brought to the fore by Lacan285 and that justifies rereading Schelling through Lacanian psychoanalysis. It is no accident that both Schelling's Stuttgarter Privatvorlesungen and the third draft of the Weltalter contain at a crucial point a meditation on the relationship between subjectivity and madness and the latter's irreducibility (in the former when discussing the soul's higher faculties, in the latter at the very end of the book of the Past): “[u]nderstanding, if it is to be an actual, living and active understanding, is therefore properly nothing other than a coordinated madness”;286 “[f]or in what does the intellect prove itself than in the coping with and governance and regulation of madness? [...] Without continuous solicitation of it, there would be no consciousness.”287 The difficult paradox of Žižek’s metaphysical archaeology of the subject is that it is only through this ontological catastrophe that the true “miracle”288 of human thinking can emerge, so that there is a speculative identity between the highest (that which sustains and creates the autonomous sphere of spirit in its dialectical creativity) and the lowest (the irrevocable alienation of being to itself, its schismatic auto-disruption that produces a non-natural excess). As that which thereby negatively binds together materialism and idealism, the impossible in-between that is the subject is neither Real nor Symbolic insofar as it dialectically coincides with both modalities simultaneously, yet is also outside of them, thus rendering its status as a vanishing mediator undecidable. In both, it is internally contained as external, present as absent, included as excluded. 186 Chapter 8 If madness is constitutive, then every system of meaning is minimally paranoid, 'mad.'”281 Paradoxically, the world can only become known to itself—being can only replicate itself within thought—if its medium of self-disclosure operates “with no external support of its truth,”282 without ever touching the Real.283 Accordingly, Schellingian desire must be said to be the beginning of being's withdrawal into its nocturnal, irreal self and that which opens up the space necessary for ego development as symptom formation (a defence). Following various hints and gestures in the middle-late Schelling, Žižek takes premonitions of the psychoanalytical experience of mind-body discord and brings to the fore their underlying eruptive logic. This leads him to the idea of Grund and existence as a dialectically irreconcilable pair emerging through the caustic collapse of being as it gains notional self-reflexivity, 188 Chapter 8 The subject When the World Opens its Eyes 189 189 is therefore infinitely non-coincidental and contradictory: having no place in either mind or body, it can only show itself as the caustic breakdown of substance (N ≠ N) or in the fleeting, ephemeral distortions of symbolic space between signifiers ($), but these two traces fail to grasp its essence in its purity, for the subject is more than the material collapse of being and the limits of discourse. But what is more, not only is the subject outside the Symbolic because it is minimally non-coincident with it, but insofar as it is at its very origin and must withdraw in order for the Symbolic to take hold, we need a new form of language even to discuss its genesis out of pre-logical antagonism, since not even the inconsistencies of our notional apparatus could thus aid us to explain its pure upsurge. The subject is “a non-provable presupposition, something whose existence cannot be demonstrated but only inferred through the failure of its direct demonstration.”289 As a result, the metaphysical archaeology of the subject I have been executing contains a necessary moment of speculative fabulation to fill in the unavoidable gaps of the narrative of how thought arises within the flat plane of being. Here, however, we encounter a major methodological difficulty. Here, however, we encounter a major methodological difficulty. Drawing upon Schellingian ontology to embark upon such a mytho-poetic articulation of how substance could act in the self-effacing, self-sundering mode of subjectivity presents an immediate problem to those who are familiar with the texts upon which Žižek relies, for the Freiheitsschrift and the Weltalter display the structure of a quaternity, with a principle exterior to the dynamic of Grund and existence that ties them together as androgynous opposites in a point of absolute indifference, an Ungrund, whose conceptual contours appear to have starkly different consequences for human self-consciousness and culture, namely, that humans participate in the theo-cosmogonic drama of God's search for self-manifestedness in creation, or to put it differently, in the emergence of “the cosmos (of fully constituted reality, ruled by logos) out of the proto-cosmic pre-ontological chaos.”290 According to this picture, ontological catastrophe (the subject) is less a cataclysmic breakdown than part of the inherent negativity in the intimate life of God that makes His personality truly alive. 188 Chapter 8 Only through the preclusion of a theosophic philosophy of nature as the illusion that there is “a secret, invisible, all-powerful agent who effectively 'pulls the strings' behind the visible, public Power... [an] obscene, invisible power structure 190 Chapter 8 190 Chapter 8 Chapter 8 190 Chap 190 C 190 acts the part of the 'Other of the Other' in the Lacanian sense, the part of the meta-guarantee of the consistency of the big Other (the symbolic order that regulates social life),”291 can Žižek ground his reading of the ontogenesis of the subject by identifying the act of decision—the pure self-positing of the I that separates Grund from existence for the first time—with the self-assertive violence of evil and disease. Because there is no God who needs a being like Himself in order to recognize Himself as Himself through a mirror that is His Other, the irreducible freedom of the human subject can only be seen as an ontologically narcissistic disturbance of the relatively ordered whole of nature: there can be no internal teleology guided by divine understanding and will whereby man participates in the divine drama of being's search for self-disclosure. Nature is not the primordial, first revelation of God,292 but the obscure basis of subjectivity, that against which it defines itself by a self-caused break. Rejecting the theosophic Ungrund central to Schelling's middle-late texts, Žižek sees traces of a disavowed analytic of finitude within them that, wrought with self-destructive, self-sabotaging tendencies, can paradoxically efface itself in the production of an extimate kernel of negativity, which then, in another moment, hegemonically takes over. In this sense, Žižek's own ontology—just like his work on Hegel—must be understood as an attempt to psychoanalytically construct the unconscious truth of Schelling's thinking by demonstrating how “his thought—for a brief moment, as it were in a flash—renders visible something that was invisible beforehand and withdrew into invisibility thereafter,” and how this tension constitutes the central difficulty of his theosophic epic.293 What, then, motivates and philosophically founds Žižek's violent “reactualization” of the Schellingian subject? To come to terms with this, we will have to explore the motivates and philosophically founds Žižek's violent “reactualization” of the Schellingian subject? To come to terms with this, we will have to explore the psychoanalytical conflict omnipresent in the Weltalter project. 252. Žižek, The Indivisible Remainder, pp. 22–23. 253. See Johnston, Žižek’s Ontology, pp. 80–92. 254. Žižek, The Ticklish Subject, p. 78; and Less Than Nothing, p. 331. 255. Žižek, The Indivisible Remainder, p. 76. 255. Žižek, The Indivisible Remainder, p. 76. 254. Žižek, The Ticklish Subject, p. 78; and Less Than Nothing, p. 331. When the World Opens its Eyes 191 256. Žižek, The Fragile Absolute: Or, Why Is the Christian Legacy is Worth Fighting For? (London: Verso, 2008), pp. 86–89. 257. Schelling, Freiheitsschrift, p. 271. 257. Schelling, Freiheitsschrift, p. 271. 258. Maldiney, Penser l’homme et la folie, p. 270. 259. Schelling, Weltalter III, p. 232. 259. Schelling, Weltalter III, p. 232. 260. See Žižek, The Indivisible Remainder, pp. 219–20. 260. See Žižek, The Indivisible Remainder, pp. 219–20. 261. Schelling, Weltalter III, p. 322. 261. Schelling, Weltalter III, p. 322. 262. Žižek, The Indivisible Remainder, p. 24. 262. Žižek, The Indivisible Remainder, p. 24. 263. Or, as Johnston succinctly puts it, “[t]he surplus of autonomy is made possible by the deficit of heteronomy. Freedom emerges from the dysfunctioning of determin- ism.” Žižek’s Ontology, p. 114. 264. Žižek and Daly, Conversations with Žižek, p. 59. 265. Žižek, The Parallax View, p. 231. 265. Žižek, The Parallax View, p. 231. 266. See chapters 5 and 6. 267. Žižek, The Parallax View, pp. 177–78. 267. Žižek, The Parallax View, pp. 177–78. 268. Ibid., p. 241. 268. Ibid., p. 241. 269. See ibid., pp. 210–14, for a rather interesting, detailed discussion. 270. Žižek, The Indivisible Remainder, pp. 22–23. 271. Gabriel and Žižek, “Introduction: A Plea for a Return to Post-Kantian Idealism,” in Mythology, Madness and Laughter, p. 3. 272. Žižek and Daly, Conversations with Žižek, p. 59. 273. Žižek, The Parallax View, p. 73. Strangely enough, this passage is omitted in the French translation. Cf. La Parallaxe (Paris: Fayard, 2008). 274. Žižek, Living in the End Times, p. 312. 274. Žižek, Living in the End Times, p. 312. 275. Schelling, Weltalter III, p. 335. 275. Schelling, Weltalter III, p. 335. 276. Žižek and Daly, Conversations with Žižek, pp. 64–65. 277. Žižek, The Ticklish Subject, p. 35. 277. Žižek, The Ticklish Subject, p. 35. 278. Žižek, Less Than Nothing, p. 314. 278. Žižek, Less Than Nothing, p. 314. 279. Žižek, Living in the End Times, p. 398. 280. Schelling, Weltalter III, p. 291. 280. Schelling, Weltalter III, p. 291. 281. Žižek, Less Than Nothing, p. 331. , g, p 282. Ibid., p. 77. 282. Ibid., p. 77. 283. Ibid., p. 959. 283. Ibid., p. 959. 284. Žižek, The Ticklish Subject, p. 33. 284. Žižek, The Ticklish Subject, p. 33. 285. Lacan, “Presentation on Psychic Causality,” in Écrits, p. 176/144. 286. Schelling, Stuttgart Seminars, in Idealism and the Endgame of Theory, trans. 286. Schelling, Stuttgart Seminars, in Idealism and the Endgame of Theory, trans. Thomas Pfau (Albany: SUNY Press, 1994), p. 233. When the World Opens its Eyes 191 Thomas Pfau (Albany: SUNY Press, 1994), p. 233. 192 Chapter 8 287. Schelling, Weltalter III, pp. 338–39. 288. Žižek and Daly, Conversations with Žižek, p. 59. 289. Žižek, Less Than Nothing, p. 730. 289. Žižek, Less Than Nothing, p. 730. 290. Ibid., pp. 273ff. 290. Ibid., pp. 273ff. 291. Žižek, “The Big Other Doesn’t Exist,” Journal of European Psychoanalysis, Spring- Fall 1997. Retrieved Jan. 7, 2013, from www.lacan.com/zizekother.htm. 292. Schelling, Freiheitsschrift, p. 284. 292. Schelling, Freiheitsschrift, p. 284. 293. Žižek, The Indivisible Remainder, p. 8. 9.1 Into the Void: The Frenzy of God's Self-Diremption The ambiguity of the Hegel-Schelling relationship within Žižek stems from his critique of Schellingian metaphysics. Even if Žižek's own transcendental materialism is founded upon a notion of emergent ontological catastrophe at the origin of subjectivity, a notion he largely derives from the Freiheitsschrift and the Weltalter, Žižek is quite adamant in distancing himself from these texts, even if in the same breath he praises Schelling's profound ability to penetrate into the pre-symbolic material of the Real.294 Indeed, immediately after his remarkable and provocative reading of Schelling in The Indivisible Remainder, Žižek argues for the supremacy of Hegelian dialectics. Despite the ephemeral moments of genuine breakthrough that emerge “as it were in a flash,”295 Žižek insists that Schelling remains philosophically inferior to his great rival: he ultimately fails to conceive the radicality implicit in the self-positing act of separating Grund from existence and tries to cover up the non-coincidence in being it indicates by making the two distinct from one another only by being founded within absolute indifference, which itself, as Schelling says, “is not a product of opposites, nor are they contained in it implicite; rather it is a being of its own, separated from all oppositions, on which all oppositions are broken, which is nothing other than their very non-being, and which therefore has no predicate except predicatelessness.”296 The eruptive logic of the Grund, intrinsic to the notion of ontological catastrophe, is thereby completely lost: “from this neither-nor, or from this indifference, duality [...] immediately breaks forth, and without indifference, i.e., without an unground, there would be no twofoldness of the principles,”297 in such a way that “the [Schellingian] Absolute is primarily the 'absolute indifference' providing the neutral medium for the coexistence of the polar opposites” of the Real and the Ideal.298 As such, it provides a way out of the abyss of freedom as a kind of metaphysical violence: the act of unconscious decision, the vanishing mediator that mediates between the real and ideal poles, “'repressed' by the formal envelope of the ‘obscurantist’ Schelling,” becomes relegated to a mere secondary position in a theosophic drama that subsumes it.299 The ambiguity of the Hegel-Schelling relationship within Žižek stems from his critique of Schellingian metaphysics. Chapter 9 The Abyss of Unconscious Decision Schelling's Weltalter and Psychoanalytical Horror of Substance as Subject Chapter 9 The Abyss of Unconscious Decision Schelling's Weltalter and Psychoanalytical Horror of Substance as Subject Since Schelling's own account of the Grund has theosophic tendencies in contradiction with the Lacanian subject, and by consequence with the disavowed yet formative Grundlogik of German Idealism, Žižek is only able to reactualize Schelling by “formalizing” its content. By elaborating Žižek's psychoanalytical methodology, I will show how Žižek is able to legitimate such a violent overhauling of Schelling insofar as he sees an inability to assimilate an encounter with the Real within key conceptual moments, a maddening struggle that simultaneously causes the “failure” of his middle-late philosophy and testifies to his prowess as the greatest thinker of subjectivity, thus presenting us with a nuanced and controversial reading of Schelling's philosophical development and his relation to German Idealism as a whole. What is more, reconstructing this “therapeutic space” will enable us to not only come to a greater understanding of the systematic rigour of Žižek's approach in opposition to what his critics want us to believe, but also bring us face to face with the psychoanalytical horror of the founding insight of modernity—the ontologically catastrophic nature of the subject as the vanishing mediator between the Real and the Symbolic—and the mechanisms by which even the greatest thinker of subjectivity can be seduced by fantasies as he tarries with its essence. 194 Chapter 9 The Abyss of Unconscious Decision 195 night of the world, nevertheless Hegel develops a superior logic because within it there is no need to leave the internal dynamic of Grund and existence and posit some transcendent principle. All can be done at the level of (a self-destructive, negativity-wrought) immanence. What Hegel fails to bring to full conceptual expression is how in dialectics the very category of “and” changes, along with the full range of implications this presents for any metaphysical system of the world: through the paradoxical identification of Grund and existence “and” becomes, in essence, tautological,300 which not only prevents Grund and existence from being mere opposites existing alongside one another through their foundation in something external to their own movement, but also excludes the possibility of any self-totalizing activity. In the logical process of the dialectic, the third term is the second, understood as a negativity or internal limit inscribed in the first, but only insofar as it has successfully taken over, usurped, the originary position from which the movement began by asserting itself as such—that is, the passage from the second to the third is that of an emergent extimacy within the first moment, which renders it non-coincident to itself, and thereby not-all, by owning itself and taking over its originary position to which it was once held at bay through an uprising. In terms of substance and subject, this means that “this very reversal is the very definition of subject: ‘subject’ is the name for the principle of Selfhood that subordinates to itself the substantial Whole whose particular moment it originally was.”301 Nothing at the level of content changes: it expresses a purely formal self-relationality giving birth to itself from within the radical non-coincidence of the absolute with itself, a self-begetting that resets the latter's logical hardwiring in an unpredictable way from within an unforeseeable self-posited zone of operation. The dialectical movement from (i) immediacy → (ii) negation → (iii) negation of negation is superior to Schellingian metaphysical narrative because there is no genuine return movement to the first, for everything takes place within a self-effacing, yet productive immanent field: the beginning and end do not overlap because something irreducibly different emerges within the first moment (negativity being now made foundational to identity): namely, an “out of joint” spirit that has a degree of notional self-reflexivity. 9.1 Into the Void: The Frenzy of God's Self-Diremption Even if Žižek's own transcendental materialism is founded upon a notion of emergent ontological catastrophe at the origin of subjectivity, a notion he largely derives from the Freiheitsschrift and the Weltalter, Žižek is quite adamant in distancing himself from these texts, even if in the same breath he praises Schelling's profound ability to penetrate into the pre-symbolic material of the Real.294 Indeed, immediately after his remarkable and provocative reading of Schelling in The Indivisible Remainder, Žižek argues for the supremacy of Hegelian dialectics. Despite the ephemeral moments of genuine breakthrough that emerge “as it were in a flash,”295 Žižek insists that Schelling remains philosophically inferior to his great rival: he ultimately fails to conceive the radicality implicit in the self-positing act of separating Grund from existence and tries to cover up the non-coincidence in being it indicates by making the two distinct from one another only by being founded within absolute indifference, which itself, as Schelling says, “is not a product of opposites, nor are they contained in it implicite; rather it is a being of its own, separated from all oppositions, on which all oppositions are broken, which is nothing other than their very non-being, and which therefore has no predicate except predicatelessness.”296 The eruptive logic of the Grund, intrinsic to the notion of ontological catastrophe, is thereby completely lost: “from this neither-nor, or from this indifference, duality [...] immediately breaks forth, and without indifference, i.e., without an unground, there would be no twofoldness of the principles,”297 in such a way that “the [Schellingian] Absolute is primarily the 'absolute indifference' providing the neutral medium for the coexistence of the polar opposites” of the Real and the Ideal.298 As such, it provides a way out of the abyss of freedom as a kind of metaphysical violence: the act of unconscious decision, the vanishing mediator that mediates between the real and ideal poles, “'repressed' by the formal envelope of the ‘obscurantist’ Schelling,” becomes relegated to a mere secondary position in a theosophic drama that subsumes it.299 Although Hegel's mature ontology as outlined in the Encyclopedia suffers, Ži Although Hegel's mature ontology as outlined in the Encyclopedia suffers, in Žižek's view, from a similar deficiency by articulating the complete return of the Idea to itself in a manner that attempts to get rid of the psychotic The Abyss of Unconscious Decision 195 The Abyss of Unconscious Decision 195 In other words, the tautology indicated by the category of “and” is, in fact, revelatory of a monism bursting at the seams: to say that Grund and existence are 196 Chapter 9 196 Chapter 9 Chapter 9 196 C identified is to say that Grund can only attain to existence, can only subsist in the modality of existence, insofar as it has erased itself, withdrawn from the scene. In this way it makes itself identical to the latter through the institution of its very difference from it, a difference that at some level is always a self-difference, a self-sabotaging, for there is no outside.302 We come across a “self-enclosed” immanence that has produced a transcendence that persists in its heart of hearts in the paradoxical mode of the double feature of inclusion/exclusion, internal/external, presence/absence, so characteristic of Lacan's descriptions of the Real, but in such a way that what is excluded, external, absent, has a power over that from which it has emerged. Negativity means that the indivisibility and power of substance is shattered from within, because it is colonized by its own parasites like a madman is terrorized by his own hallucinations, but with the added effect that there is no need to posit a state of “originary health” of which the devouring restlessness of the negative cannot be predicated to explain the dynamism inherent to reality. Because of the theosophic structure of the middle-late Schelling, Žižek's own appropriations of concepts such as Grund and Entscheidung in the development of his own metaphysics put him in a delicate situation. The issue at hand is further complicated by Žižek's division of Schelling's philosophy into three distinct and irreconcilable stages, which he finds reflected in the three existent drafts of the Weltalter.303 Schelling1 largely coincides with his quasi-Spinozistic philosophy of absolute indifference, where freedom is completely subsumed under the positive order of being. In the first draft this is seen in terms of the explication of freedom as a logical mode of necessity within the inner articulation of substance, its subsumption within the self-harmonizing genesis of the latter's rational structure and order. In the Schelling2 of the second draft of the Weltalter and the Freiheitsschrift, we see an interesting twist with regard to the concern with how the contraction of material being itself is made possible. The Abyss of Unconscious Decision 195 By conceiving the act of contraction as ultimately free and self-positing, here Schelling is able to think the will-to-contraction (the No) and the will-to- expansion (the Yes) as identical and therefore internal to the dynamic of freedom, which makes his thinking approach that of Hegelian dialectics and secures his position as the concluding step of the unconscious Grundlogik of The Abyss of Unconscious Decision 197 The Abyss of Unconscious Decision 197 German Idealism whose lineage culminates in the advent of psychoanalysis. For Žižek, this brief period of breakthrough was quickly left behind by the Schelling3 of the philosophy of mythology and revelation, where we see a return to a pre-modern “essentialism,” traces of which he claims are already hinted at in the third draft of the Weltalter, where Schelling posits another principle of synthesis external to the movement of contraction and expansion within which freedom and determinism are grounded as opposites. It is because of these tendencies (which explain why Žižek qualifies the revolutionary character of Schelling's philosophy by describing him as the father of “New Age obscurantism” just as much as the father of contemporary philosophy of finitude)304 that Žižek so quickly changes tone in the second chapter of The Indivisible Remainder. Yet while consistent with his overall interpretation of Schelling, this emphatic shift is simultaneously ambiguous insofar as Žižek does not distinguish which Schelling he is arguing against or justify how he is able to read the second draft of the Weltalter as an ephemeral rupture in Schelling's thought “which goes farthest in the direction of Freedom.”305 Given that the only way for freedom to exist according to Žižek is through the space opened up by the caustic collapse of being, and that both Hegel's ontology and Schelling's logic fail in their own fashion to come to terms with this insight,306 even if each encounters it in a significant manner, how exactly is Žižek able to retrieve this concept from the second draft without falling into the pitfalls of Schelling's own thinking, insofar as it is evidently against its letter and spirit even as he presents it? The Abyss of Unconscious Decision 195 How is he able to maintain that the Weltalter and the Freiheitsschrift are the most sustained confrontation in the entire tradition with the frightening origins of subjectivity and its metaphysical implications, that they reveal the most profound penetration into the “perverted” core of the cogito that lurked latent in itself since the very founding gesture of modernity? What specifically interests Žižek in the middle-late work of Schelling is its frantic and uncertain nature. For all its intense, uncontrollable passion, for all its feverish outpouring, it does not get off the ground. Schelling's masterpiece, the Weltalter, never gets finished. Although this is not an outlier in Schelling's long philosophical career—his entire corpus is ridden with rapidly written sketches of systems and allusions to publications on the 198 Chapter 9 198 Chapter 9 horizon that never appear—there is an undeniable hesitation saturating this period. The trunk of stillborn, abortive drafts of the first book of the Weltalter found in the library of Munich, of which only three remain today due to the fires that wrought havoc in the city in the aftermath of Allied bombings (but of which there were more than twelve different handwritten versions) is enough to demonstrate that this work haunted Schelling in a manner and with an intensity that others did not, as does the fact that after the 1809 Freiheitsschrift he refrained from publishing another major work, and largely withdrew from the German intellectual scene where he was once the rising star. Aside from scattered lectures—for instance, an 1827 and an 1833 course on the System der Weltalter—he would not return to the public eye with any sustained vigor until he was called to Berlin in 1841. At that time, assuming Hegel's chair ten years after the latter's untimely death, addressing an audience that had eagerly awaited him to break his silence for decades (an audience that included both Engels and Kierkegaard) and whose final reappearance was exacerbated by the uncertain fate of Hegelianism, he gave lectures on the philosophy of mythology and revelation. But the Hörsäle, first overflowing with excited, enthusiastic students of philosophy anxious to hear the words of Hegel’s old rival, were quickly abandoned in utter disappointment, leaving only a handful of devoted students, Fichte's son Immanuel Hermann von Fichte among them. But what explains this sudden change in Schelling's career, this lack of desire to publish, to engage in dialogue, to spread his philosophy over the European landscape that had eagerly welcomed it? Jason Wirth hints at something that comes to mind for anyone who knows Schelling's life story: Yet 1809 marked a turning point in Schelling's zeal to publish. Already Schelling's reputation had been injured by Hegel's unwarranted dismissal of the intellectual intuition as the “night when all cows are black.” [...] More seriously, however, Schelling's wife, Caroline, had become very ill. It is hard to read the Freedom essay, published in May 1809, with its analogy between sickness and evil (sickness is to Being as evil is to human being), without thinking of Caroline. 198 Chapter 9 In the treatise, Schelling claimed that the “veil of melancholy [Schwermut] that is spread out over all of nature is the The Abyss of Unconscious Decision 199 200 Chapter 9 After Hegel’s critique of the Schellingian absolute indifference308 in The Phenomenology of Spirit, and recognizing the strength of Hegel's dialectics, Schelling is forced to rethink the philosophical foundation of his thought and its attempt to balance transcendental idealism with an organic philosophy of nature in such a way that he can at the same time fight against the perceived threats of Hegel's philosophy. He puts his own prowess to use in articulating his account of the emergence of temporality and finitude that does not succumb to the claustrophobic subsumption of human freedom within the self-mediation of the absolute. Arguing that Hegel can only show the notional necessity of things and never their brute reality, he embarks on a theosophic exploration of the creation of the world that would not only challenge, but hopefully put a stake in the heart of the philosophy of his now adversary and former friend. Yet Schelling's middle-late philosophy is not just a theo-cosmogonic odyssey of the vicissitudes of divine being in its restless search for self-revelation. Following reason by analogy and identifying a structural parallel between God's speaking of the Word by which he becomes a full person and the birth of self-experience out of the eternal darkness that precedes it,309 Schelling's philosophy simultaneously functions on two levels, that of the theo-cosmological and that of the metapsychological, in such a way that Schelling is able to say, at the end of the Freiheitsschrift, that we “have established the first clear concept of personality.”310 It assumes that we can pass from the lowest to the highest, from the known to the unknown, by a careful, methodologically guided introspection, insofar as there is “a system of times [...] for us, of which the human system would be just a copy, a repetition within a narrower sphere.”311 What is so intriguing about Schelling's attempt to describe the emergence of finitude from eternity and human subjectivity out of nature is the conceptual restlessness evident in the existent drafts of the project, each of which oscillates around an insurmountable deadlock located in the movement from the Past to the Present: that is, how the divine and human subject could emerge from the non-experiential void that precedes them. The Abyss of Unconscious Decision 199 199 profound and indestructible melancholy of all life.” Caroline died on September 7, 1809. Schelling was devastated. In a letter written less than a month after Caroline's death, Schelling claimed that “I now need friends who are not strangers to the real seriousness of pain and who feel that the single right and happy [glücklich] state of the soul is the divine mourning [Traurigkeit] in which all earthly pain is immersed.” A year later, Schelling began work on Die Weltalter, a philosophical poem about the rotatory movement of natality and fatality, pain and joy, comedy and tragedy within God, that is, within the whole of Being, itself.307 Žižek allows us to offer a new, interesting, and controversial spin on this enigma. If we follow and reconstruct his implicit methodology, we see that he refuses the claim that Schelling's “retreat” was spurred on by the tragic death of his true love and great muse Caroline in 1809 just before the publication of the Freiheitsschrift, or for that matter even by the overwhelming influence of the Hegelian system that could also be seen as leaving him in a state of philosophical and existential paralysis. What Žižek invites us to argue is that, when one looks at the very structure of the Weltalter drafts themselves, one is confronted with an uncanny struggle of composition that reveals something primordial concerning what its texts give witness to and attempt—but ultimately fail—to bring forth. This painful unrest is not to be reduced to the mere level of personal dissatisfaction, as if there were a conscious recognition by Schelling that his own “masterpiece” could never hold its own as a rival of Hegelian logic, nor could it just boil down to some personal trauma that began to cloud Schelling's own philosophical capacities through a devouring melancholy that made the world during these years an agonizing repetition of bitter grey upon bitter grey. Instead, it provides the key to understanding both the self-deploying historico-dialectical (Lacanian) cause of German Idealism at work in Schelling's thought and ultimately dividing it into the three distinct stages as outlined by Žižek and establishing its true greatness in this fragmented second stage, whereby we can open up a legitimate space in which to reactualize it and let it come to terms with that which, by itself, it was not able to do. 200 Chapter 9 Schelling remains unable fully to explicate the meaning of what Žižek calls the “breach of symmetry,” God's contraction of finitude, and in the same vein the emergence of a free, deciding being (the subject) in a manner The Abyss of Unconscious Decision 201 The Abyss of Unconscious Decision 201 that leaves him at ease. In the first draft the abyss of freedom contracts materiality out of necessity, so that both divine and human freedom are subsumed within the self-unfolding of substance, while the second declares that the very contraction itself is an act of freedom, a self-positing, which not only renders the two opposed principles of the Yes and the No identical in the internal dynamic of freedom, but also (and more disconcertingly) results in a structural homology with sickness and evil as an unpredictable negative reversal of established ordered and wholeness from within. The third merely complicates the picture insofar as it breaks from the “Spinozistic” determinism and closed systematicity of the first and the radical philosophy of freedom with its potentially unsettling implications proclaimed by the second: by synthesizing both polar principles within a point of metaphysical simultaneity within the Godhead that is the Ungrund within which both rest as androgynous pairs, God the Creator's personal freedom is saved from being logically dependent upon a moment of cancer in divine being that upsets the joyful bliss of eternity, and in a similar move, subjectivity is no longer made possible by that which disrupts the smooth placidity of substance. These basic conceptual differences at such a foundational level of inquiry reverberate through the entire movement of the three stages in a meaningful way in each draft: 1. Not only is human freedom actually a misrecognition of blind (Spinozistic) causality, so that the movement of dialectical human temporality is an illusion, all being co-present in the self-harmonizing synchronicity of the system—here we see Schelling struggling to rearticulate his old ideas in a new format—but even the act of contracting finitude is a necessity, so it could not not have happened, deriving from the “primordial Freedom in an absolutely immediate, 'blind,' non-reflected, unaccountable way.”312 2. The fact of finitude in God or of freedom in man proclaims that synchronic substance cannot be a devouring totality, but must be an open not-all plagued with fragility and inconsistency all the way down. The Abyss of Unconscious Decision 203 The absolute yearns for self-disclosure once it has willed it, so that all things in the self-institution of finitude and negativity he stumbled across in the Potenzenlehre of the second draft and already implicit in the eruptive logic of the Grund in the Freiheitsschrift; in both there is an attempt to retreat from the notion of a barred organic whole, a metaphysics of the not-all, as forced upon us by the philosophy of freedom whose first outlines we see already in Kant, substantiating Žižek's claim that both Hegel and Schelling repress the unacknowledged truth, the Grundlogik, of German Idealism.313 By referring to the Godhead as das Überexistierende that summons the blind rotatory movement of drives, the third draft strives to save reality from the irredeemable conflict that it is faced with in the contraction of finitude: instead of the latter being absolutely self-positing as a moment in the movement from the potentiality of freedom in its abyssal eternity to its actualization, God in some strong sense now precedes the Grund of His existence, assuring His protection from the non-coincidence to self announced by the dependence of light on darkness: there is “an activity performed at a safe distance.”314 This becomes even more evident in the late philosophy of mythology, where the entire theoretical edifice becomes grounded on the distinction between the Was and the Daß of God. The transcendence of the Daß from the flux of the Potenzen not only allows for an implicit, yet self-articulating teleological movement intrinsic to the antagonisms of finitude of the latter so that there are stages that unfold throughout nature and then human history that ultimately pave the way for the final revelation of God, but it also makes any appearance of ontological disarray merely a “perceptual illusion” generated by a self-harmonizing scheme deduced by negative philosophy (in Žižek's words, this is Schelling’s regress to pre-modern essentialism). The Abyss of Unconscious Decision 201 The very self-positing of finite being in God 202 Chapter 9 not only precludes all attempts to enclose it within a totalizing whole even within the divine understanding itself, since self- positing is equated with an absolute self-assertion, but (even more devastatingly) the emergence of God as a personal being out of its contracted material Grund is uncertain, insofar as there is no guarantee that He will come to His own by speaking the Word, unlike in the first draft where it is part of divine's substance infinite self-articulation. 3. The problematic ramifications of the second position are foreclosed by making God's free existence as an entity predetermined and guaranteed in advance through the dialectical simultaneity of freedom and determinism in a higher synthetic principle. Thus the creation of the world is an absolutely contingent act following from a volitional arbitrariness expressing God's essence as love, making divine being, rather than a turmoil- ridden drama with an unforeseeable end, a self-maintaining whole. In this sense the third draft symbolizes a kind of retreat from the second; one is even tempted to say an (unconscious) attempt to save oneself from the traumatic, horrifying implications of its philosophy of freedom. The true basis of spontaneity was, as it were, too much for Schelling—he had to pull back. Not only do the existent drafts of the Weltalter give testament to an uncompromising tension, an endless circulation around an inassimilable kernel, both in terms of the relationship of God as person and consciousness to the eternal darkness of their ground, but it also appears as if Schelling's own philosophical career could only continue once it cut off, repressed, this encounter with the Real. In the turn to the Schelling3 of the philosophy of mythology (already hinted at in the third draft of the Weltalter) we can glimpse a recoil from the abyssal basis of freedom as pure self-positing unleashed in the second in the same way that the earlier Realphilosophie of Hegel is later abandoned in order to embrace a consoling triadic dialectics: just as Hegel tries to save the absolute from a diremptive wound that can never be healed, here Schelling attempts to save the essence of God and nature from the insoluble deadlock of drives eventually engendered by The Abyss of Unconscious Decision 203 The Abyss of Unconscious Decision 203 the self-institution of finitude and negativity he stumbled across in the Potenzenlehre of the second draft and already implicit in the eruptive logic of the Grund in the Freiheitsschrift; in both there is an attempt to retreat from the notion of a barred organic whole, a metaphysics of the not-all, as forced upon us by the philosophy of freedom whose first outlines we see already in Kant, substantiating Žižek's claim that both Hegel and Schelling repress the unacknowledged truth, the Grundlogik, of German Idealism.313 By referring to the Godhead as das Überexistierende that summons the blind rotatory movement of drives, the third draft strives to save reality from the irredeemable conflict that it is faced with in the contraction of finitude: instead of the latter being absolutely self-positing as a moment in the movement from the potentiality of freedom in its abyssal eternity to its actualization, God in some strong sense now precedes the Grund of His existence, assuring His protection from the non-coincidence to self announced by the dependence of light on darkness: there is “an activity performed at a safe distance.”314 This becomes even more evident in the late philosophy of mythology, where the entire theoretical edifice becomes grounded on the distinction between the Was and the Daß of God. The transcendence of the Daß from the flux of the Potenzen not only allows for an implicit, yet self-articulating teleological movement intrinsic to the antagonisms of finitude of the latter so that there are stages that unfold throughout nature and then human history that ultimately pave the way for the final revelation of God, but it also makes any appearance of ontological disarray merely a “perceptual illusion” generated by a self-harmonizing scheme deduced by negative philosophy (in Žižek's words, this is Schelling’s regress to pre-modern essentialism). Complementing negative philosophy, Schelling envisages a form of positive philosophy, one of whose primary tasks is to find and explicate these stages in nature and history: that is to say, “to function as a kind of 'transcendental empiricism,' and to 'test' the truth of rational construction in actual life,”315 thereby subsuming the restless negativity at the heart of reality as revealed specifically in the second draft into a mere secondary feature of God's plan within the spectacle of the absolute's own self-development, deflating it to a mere ephemeral phase in an overarching cosmic dance towards revelation. The Abyss of Unconscious Decision 203 Complementing negative philosophy, Schelling envisages a form of positive philosophy, one of whose primary tasks is to find and explicate these stages in nature and history: that is to say, “to function as a kind of 'transcendental empiricism,' and to 'test' the truth of rational construction in actual life,”315 thereby subsuming the restless negativity at the heart of reality as revealed specifically in the second draft into a mere secondary feature of God's plan within the spectacle of the absolute's own self-development, deflating it to a mere ephemeral phase in an overarching cosmic dance towards revelation. The absolute yearns for self-disclosure once it has willed it, so that all things in 204 Chapter 9 204 Chapter 9 Chapter 9 204 creation move towards it, even if unknowingly: here, not only is nature's unpredictable catastrophism and widespread murder and extinction316 no longer indicative of a lack of a totalizing principle, but man, denied the right to be an autonomous writer in the drama of divine being, is reduced to a mere actor or puppet who follows a pre-written script (a telos) given by the very structure of the Potenzen themselves for the joy of its author in His distant, cold kingdom of divine eternity, the non-rational Daß of God being, after all, distinct from and existing over and above the Was of God. But in the movement from Schelling2 to Schelling3 we should not just draw our attention to how in the latter there is absolutely no room for subjectivity with the very technical sense of the word that we have been using. What is much more revealing is how concerned Schelling3 is to establish God as that which guarantees the consistency and internal coherence of nature and history, previously jeopardized, for us and God Himself: there is a frantic obsession to demonstrate the existence of the Other of the Other in the Real when the inconsistencies and frailty of order have already been pushed upon us. The third stage in Schelling's thought is nothing but a reaction formation against his radical philosophy of freedom. The Abyss of Unconscious Decision 205 redemption, which would merely just institute a triadic dialectics similar to that of Hegel's mature system—it also brings Schelling's philosophy to a halt, a standstill, as it comes upon metaphysical implications revealed by the split nature of subjectivity. Only ever successfully completing the first, preliminary part of his mytho-poetic ages of the world, his description of the eternal Past, Schelling remains forever unable to proceed; it is this which causes him to fade slowly from the public eye, where he was once a star and the leader of one of the greatest intellectual movements the world has ever witnessed, because he came too close to its true horrifying core. The Abyss of Unconscious Decision 203 Leaving aside the possible theological implications of Žižek's reading of Schelling—which deserve to be evaluated in their own right—due to his outright rejection of theosophy as ultimately nothing but a mytho-poetics of the Symbolic's coming into being, what Žižek's Schelling unearths in his frenzied attempt to develop a philosophical program able to battle Hegelian Absolute Idealism is the not-all nature of the absolute. Trying to give an account of the emergence of finitude and driven on by the alchemical intuition that there is an identity between the highest and the lowest, the self-begetting of God and the self-caused birth of the subject in its freedom, he delves into the nocturnal site out of which the human self is born and experiences the terror of the failure of positive being to ground itself, its “self-lacerating rage” (sich selbst zerreißende Wut), which acts as the vanishing mediator between nature and culture by opening up the possibility for the self-assertion of freedom from within the Real and its vicissitudes. This not only by definition prevents the completion of the project of the Weltalter insofar as it precludes any possibility of the reconciliation promised by the Third Age, the Future wherein Grund and existence become reconciled in The Abyss of Unconscious Decision 205 206 Chapter 9 The “victory” that Žižek perceives in the Weltalter is in its gestures towards philosophical logic underlying the birth of consciousness and its wider metaphysical implications. The strangeness and density of the Weltalter project are not due to its out-of-jointness with the modern tradition, but due to the fact that it comes too close to its disavowed essence. The anxiety-inducing reverberations that shake one's body when one reads the descriptions of “Schelling's grandiose 'Wagnerian' vision of God in the state of a endless 'pleasure in pain,' agonizing and struggling with Himself, affected by an unbearable anxiety, the vision of a 'psychotic,' mad God”318 are so difficult because they make us approach the very kernel not just of our existence as subjects but also the nature of the world at large. This experience of reading its drafts is properly “traumatic” in the meaning Lacan gives to this term drawing upon its etymological origins in ancient Greek: they bring forth a devastating encounter by which the symbolic support of our personality is “wounded” or “pierced,” during which the coherence of our identity is torn apart as it comes upon its truth.319 The effect is twofold. i The “victory” that Žižek perceives in the Weltalter is in its gestures towards philosophical logic underlying the birth of consciousness and its wider metaphysical implications. The strangeness and density of the Weltalter project are not due to its out-of-jointness with the modern tradition, but due to the fact that it comes too close to its disavowed essence. The anxiety-inducing reverberations that shake one's body when one reads the descriptions of “Schelling's grandiose 'Wagnerian' vision of God in the state of a endless 'pleasure in pain,' agonizing and struggling with Himself, affected by an unbearable anxiety, the vision of a 'psychotic,' mad God”318 are so difficult because they make us approach the very kernel not just of our existence as subjects but also the nature of the world at large. This experience of reading its drafts is properly “traumatic” in the meaning Lacan gives to this term drawing upon its etymological origins in ancient Greek: they bring forth a devastating encounter by which the symbolic support of our personality is “wounded” or “pierced,” during which the coherence of our identity is torn apart as it comes upon its truth.319 The effect is twofold. 9.2 That Which Is in Schelling More than Schelling Himself—Žižek Schelling's failure to finish the Weltalter is not due to his lack of conceptual prowess or an earth-shattering, existential strife: it results from an encounter with the Real, an encounter that is philosophically meaningful. Because there is an inassimilable kernel within its symbolic space tearing it apart, its discourse is riddled with harrowing tension: unable to bring this encounter to its complete symbolization, Schelling's Weltalter falls to the ground and is only able to save itself through an unconscious disavowal that takes the form of an insistence on the existence of the Other of the Other. By consequence, it has to be interpreted psychoanalytically so that its truth or cause can finally be integrated into its symbolic space, and its place as the concluding step of German Idealism's formative Grundlogik can be firmly established. If this can be accomplished, then we will be permitted to say that, retroactively, Schelling's project fell to the ground not only in the sense of a purely negative collapse, but also as the condition of the possible opening of a new beginning, following the double connotation of the German zugrunde gehen. The failure of the Weltalter, the fact of its incompletion, turns out to be a triumph of human reason:317 its apparent limitations—the feverish frenzy of the work, the inconsistency brought forth by its conceptual unrest—are actually expressive of an underlying ontological and metaphysical intuition that we can catch a glimpse of through the interstices of its discourse. Žižek's claim is that if we pass through the negative determination at the heart of the work, the non-coincident in-itself we witness can become a for-itself. 206 Chapter 9 Firstly, both Schelling's own inability to finish the project, his heap of lost manuscripts, and the subsequent necessity to “invent” a solution to continue his philosophical career and its neglect in the philosophical community as a whole could thus be seen as nothing but a recoil from the Real of our being as finally explicitly brought into the open within his texts. It is thus Schelling who, in an attempt to give a death-dealing materialist response to Hegel's “pan-logicism,” most fully experiences out of all the representatives of the modern tradition the “incomprehensible basis of reality,” that mysterious X that forever haunts transcendentally constituted reality (the Real as a “kink” in the Symbolic), precedes it (the Real as pre-symbolic immediacy lost due to language), and in some modality conditions its very possibility (the Real [R⁄ ] as auto-disruptive substance [N ≠ N] whose self-laceration creates the space for free experience). In an attempt to articulate the irreducibility of freedom, Schelling plunges headfirst into the paradoxical character of subjectivity—not only into the denaturalized state of unruliness we must posit as prior to the emergence of phenomenal reality and culture, the pulsating cauldron of chaotic, heterogeneous forces that first make possible the gesture of infinite withdrawal into the nocturnal irreal self of The “victory” that Žižek perceives in the Weltalter is in its gestures towards philosophical logic underlying the birth of consciousness and its wider metaphysical implications. The strangeness and density of the Weltalter project are not due to its out-of-jointness with the modern tradition, but due to the fact that it comes too close to its disavowed essence. The anxiety-inducing reverberations that shake one's body when one reads the descriptions of “Schelling's grandiose 'Wagnerian' vision of God in the state of a endless 'pleasure in pain,' agonizing and struggling with Himself, affected by an unbearable anxiety, the vision of a 'psychotic,' mad God”318 are so difficult because they make us approach the very kernel not just of our existence as subjects but also the nature of the world at large. The Abyss of Unconscious Decision 207 act. Secondly, the originality of this narrative of German Idealism that Žižek allows us to construct is its implicit claim that the Weltalter is the text of modernity and that its incompletion, fragmentation, and unpopularity are philosophically rich in meaning, perhaps even necessary reactions to its truth. Not only does this contribute to elevating Schelling to his rightful place in history of philosophy after having long been overshadowed by Hegel, it also (and more importantly) calls us to own up to other elements of our being that may be more difficult to bear, even too difficult to bear, by one of their greatest theorists. Žižek's work on Schelling is so crucial for understanding his philosophy as a whole, not only because it provides us the resources we need to show the coherence of his reading of the modern philosophical tradition, but also because it allows us to demonstrate the profound level of philosophical scrutiny and methodological rigor inherent in his apparent heterodoxy. If the self-development of the former's account of subjectivity is characterized by a recoil from the parallax ontology of transcendental materialism at the core of the cogito's self-positing, the traces of which we can see in the negative contortions of its symbolic space through its entire history, it is only by reconstructing his psychoanalytical interpretation of Schelling as the culmination of German Idealism that we can truly legitimate and assess this claim. 206 Chapter 9 This experience of reading its drafts is properly “traumatic” in the meaning Lacan gives to this term drawing upon its etymological origins in ancient Greek: they bring forth a devastating encounter by which the symbolic support of our personality is “wounded” or “pierced,” during which the coherence of our identity is torn apart as it comes upon its truth.319 The effect is twofold. Firstly, both Schelling's own inability to finish the project, his heap of lost manuscripts, and the subsequent necessity to “invent” a solution to continue his philosophical career and its neglect in the philosophical community as a whole could thus be seen as nothing but a recoil from the Real of our being as finally explicitly brought into the open within his texts. It is thus Schelling who, in an attempt to give a death-dealing materialist response to Hegel's “pan-logicism,” most fully experiences out of all the representatives of the modern tradition the “incomprehensible basis of reality,” that mysterious X that forever haunts transcendentally constituted reality (the Real as a “kink” in the Symbolic), precedes it (the Real as pre-symbolic immediacy lost due to language), and in some modality conditions its very possibility (the Real [R⁄ ] as auto-disruptive substance [N ≠ N] whose self-laceration creates the space for free experience). In an attempt to articulate the irreducibility of freedom, Schelling plunges headfirst into the paradoxical character of subjectivity—not only into the denaturalized state of unruliness we must posit as prior to the emergence of phenomenal reality and culture, the pulsating cauldron of chaotic, heterogeneous forces that first make possible the gesture of infinite withdrawal into the nocturnal irreal self of the world, but the very ambiguity of the latter as an absolute self-positing The Abyss of Unconscious Decision 207 The Abyss of Unconscious Decision 207 Although such a recoil from this traumatic Real may be evident in Descartes’s reification of the subject as a thinking thing, Kant's attempt to ontologize “this I or he or it (the thing) that thinks” or more strongly his inability to delve into obscure foundations of unruliness and diabolic evil, or in Hegel's mature system as a covering up of the madness of the night of the world, it is only by recourse to Schelling that he can retroactively posit such a self-deploying disavowed knowledge that deepens itself through the trajectory of the tradition and that leads to its eventual culmination in psychoanalysis.320 What makes Schelling such an important outlier to this sustained recoil, however, was his ability (for a relatively brief period of time) to completely immerse himself within the frenzy, horror, and pandemonium that are the true origin of subjectivity in such a way that he brings to the fore the entire latent Grundlogik of modern philosophy of the subject preceding him and that is surpassed only by psychoanalysis' thematization of the unconscious and Todestrieb. His own turning away The Abyss of Unconscious Decision 209 The Four Fundamental Concepts of Psychoanalysis appears to be a tribute to Freud, since the four concepts are taken directly from his work. Just as Lacan at that time calls his institute the “Freudian School,” in his seminar he uses the term “Freudian concepts” just to prove that he is not a dissident. But within this “tribute” he tries to go beyond Freud. Not a beyond Freud which leaves Freud behind; it is a beyond Freud which is nevertheless in Freud. Lacan is looking for something in Freud's work of which Freud himself was unaware. Something which we may call “extimate,” as it is so very intimate that Freud himself was not aware of it. So very intimate that this intimacy is extimate. It is an internal beyond.323 But how is Žižek able to accomplish such a feat, seeing that Schelling himself fought against this? 208 Chapter 9 208 Chapter 9 208 Chapter 9 from this insight and the various inconsistencies that lace his texts must be seen as symptomatic of a greater tendency within the movement (and in humans in general) towards the development of defense mechanisms in the face of an inability to integrate the psychoanalytical truth the Real tries to force upon us. If Schelling's own recoil—a return to a pre-modern essentialism—is so severe, it is because he, as the one who experienced the horror of the metaphysical implications of subjectivity most strongly, needed a defense mechanism more radical than the rest. In their “very failure, [the Weltalter] are arguably the acme of German Idealism and, simultaneously, a breakthrough into an unknown domain whose contours became discernible only in the aftermath of German Idealism.”321 Žižek thus sees his philosophy not only as an attempt to reactualize the German Idealist tradition, but also as immanently participating within its own self-deployment. He understands his project as a remodulation of its surface logic by clinically working through what he perceives as its internal tension, so as to construct the self-effacing, transcendental materialism that has been its unconscious truth.322 He is not merely performing an act of hermeneutical retrieval, but (more strongly) taking these ideas into what he considers to be their dialectical “completion” from which they have been hindered by the tradition itself. For Žižek, it is Lacan who gives us the methodological tools we need to “rehabilitate” its fundamental concepts, but which amounts to something more than a mere application of psychoanalytical concepts to various texts in the history of philosophy. The approach is much more provocative. By enacting a kind of therapeutic space within key texts of the middle-late Schelling as the culminating point of German Idealism, the wager of Žižek's philosophy is that, by being open to their own inner movement, he can bring forth that which is in Schelling more than Schelling himself: the extimate, traumatic core lodged deep within his spirit and soul and from which Schelling withdrew into philosophical “paralysis” by integrating (constructing) the originally inassimilable theoretical potential and unsettling metaphysical consequences of the second draft of the Weltalter. The relationship of Žižek to Schelling as the endpoint of German Idealism is therefore structurally identical to that of Lacan to Freud. As Miller puts it: The Abyss of Unconscious Decision 209 9.3 A Mytho-Poetics of Creation and the Seducing Hand of Fantasy The answer lies in the second draft, where Žižek sees a distinctively Hegelia structure that enables him to develop a metapsychological reading of the text, preventing its underlying ontology from succumbing to philosophical commitments that he rejects and identifies as in opposition to the unconscious Grundlogik of German Idealism. In fighting against what he perceives to be the limitations and threat of Hegelian logic, Schelling, in the end, only radicalizes its perverse truth, yet is unable to contain the monster that he unleashes. After developing an astonishing philosophy of freedom, Schelling immediately recoils from its implications by positing a principle of mediation that enables the neutral coexistence of Grund and existence through their mutual grounding in what Schelling in the Freiheitsschrift refers to as the Ungrund, that which in itself is ungrounded and thus simultaneously precedes both and is neither one nor the other. Because Schelling here understands the freedom of unconscious decision (Entscheidung) that separates Grund from existence as a return to the primordial origin of all reality that is the abyss of freedom itself, its conceptual edifice displays a structure of quaternity, which is articulated 210 Chapter 9 210 Chapter 9 Chapter 9 in his thinking largely through a systemization and reconceptionalization of thinkers like Jakob Böhme and Franz Baader, and is thereby able to sidestep the implication of freedom as a cancerous upsurge of pure self- assertion. But insofar as the second draft displays freedom as a kind of self-positing activity that identifies Grund (the will-to-contraction, the No) and existence (the will-to-expansion, the Yes), Žižek jumps at this slight “slip” in Schelling's text, seeing in this discursive inconsistency a possibility of “formalizing” the conceptual movement of the work by “purifying” it of all extraneous theosophic commitments through traces of Hegelian logic he sees operative within it. It is in this sense that Žižek's philosophy is a hybridism of Schellingian ontology and a Hegelian quadruple dialectics of non-reconciliation. For Žižek, Hegel is the superior logician because he has no need to posit a principle of mediation outside of the internal dynamic of Grund and existence. There is no possible return to (or even initial existence of) a state of “originary” health, as typified by the abyss of freedom as independent from the antagonism of the dual principles, for there is nothing that has not always already succumbed to the restlessness of the negative. The Abyss of Unconscious Decision 211 entire logical process: “[t]here is thus no reversal of negativity into positive greatness—the only greatness is 'negativity' itself.”325 In other words, the negativity of the second is entirely inscribed within the first, arising from within its closed immediacy as a kind of tension or contradiction, and is in this regard not separate or distinct from it insofar as there is nothing but the dialectical register of the first. Yet because the second presents us with an internal limit (it itself has no substance of its own) and is thus by definition non-coincident with the first and its operational principles, it is in the same breath minimally distinguished from it, thus engendering a fissure in the logical self-closure of the field in a movement that makes the entire order inconsistent and ill at ease, which in turn opens up a foothold for the possibility of change.326 For Hegel, it is this deadlock of real internal limit—an inassimilable kernel—that ultimately serves as the springboard for all dialectical change by creating the space necessary for the unpredictable self-founding of a new order, but of itself it only becomes explicit in the aftermath of the third as it overthrows the primacy of the first through the paradoxical causality of a retroactive positing of presuppositions (Setzung der Voraussetzungen). There is something like an internal parasitic logic that re-totalizes the entire dialectical framework, so that instead of witnessing a return to the first, an initially subordinate moment degrades its own genetic conditions into its own subordinate moment by means of the unforeseeable and destructive power of negativity, which now reigns supreme.327 With the self-positing of the third (the second positing itself, counting itself, thus making itself the third by a self-doubling), negativity is finally fully “brought to life,” but in such a way that its dark pre-history in the vicissitudes of the previous stage as a purely negative real limit necessarily vanishes from sight, for the contingency of its self-positing has been immanently overcome. But even if the stark remodulation of the (onto)‍logical field within which it occurs, a remodulation that effectuates itself by establishing itself as the supreme category, covers up its steps like an experienced criminal used to getting away with his crime, in the same breath it sets the stage for a new (relatively closed) immediacy and thus the possibility of new unforeseeable dialectical change. 9.3 A Mytho-Poetics of Creation and the Seducing Hand of Fantasy Although textbook Hegelianism presents the third moment of the logic as a synthesis of two previous incompatible conceptual polarities by means of a cancellation of the falsehood and a preservation of the truth contained in each and thereby bringing them into a higher, more comprehensive dialectical standpoint (the banal reading of the equivocal character of the word “Aufhebung”) Žižek thinks this picture misses the true philosophical innovation that we see in the movement from one stage to another.324 The third moment itself is only the second insofar as it hegemonically usurps the position of the first through positing itself as such—which is, in a way, a mere amplification of an already existing interruption in the immanence of the first's logical field, an amplification that destroys its sway from within, but without ever leaving its fold. The dialectical movement from (i) immediacy → (ii) negation → (iii) negation of negation is superior not only because there is no return to the first (something irreducibly different and operatively new emerges, irreversibly reconfiguring the entire [onto]‍logical apparatus through its self-positing) but also because there is no need to posit something outside the self-movement of negativity to explain the y g g g , p dialectical standpoint (the banal reading of the equivocal character of the word “Aufhebung”) Žižek thinks this picture misses the true philosophical innovation that we see in the movement from one stage to another.324 The third moment itself is only the second insofar as it hegemonically usurps the position of the first through positing itself as such—which is, in a way, a mere amplification of an already existing interruption in the immanence of the first's logical field, an amplification that destroys its sway from within, but without ever leaving its fold. The dialectical movement from (i) immediacy → (ii) negation → (iii) negation of negation is superior not only because there is no return to the first (something irreducibly different and operatively new emerges, irreversibly reconfiguring the entire [onto]‍logical apparatus through its self-positing) but also because there is no need to posit something outside the self-movement of negativity to explain the The Abyss of Unconscious Decision 211 The Abyss of Unconscious Decision 211 But we must recognize that this self-positing always comes too late: its upsurge may posit itself at the logical beginning of the movement as necessary, it may make itself the 212 Chapter 9 primary principle, that to which the movement had always tended, but this only becomes visible after the fact and is thus plagued by a devastating belatedness. There is no way notionally to deduce the act of self-positing because, as free, it impossible to predetermine its arrival or even be aware that it could occur at all.328 The reversal characteristic of the third moment befalls being, for “every dialectical passage or reversal is a passage in which the new figure emerges ex nihilo.”329 What we often miss in the necessity of the dialectical movement and what is even obfuscated by it is the fact that every movement of its self-articulation is, in fact, constituted by a series of a contingent acts brought on by negativity, contingencies that only retroactively gain the status of necessity by making themselves necessary.330 The passage from tension to victory is never ontologically guaranteed—and it is precisely this ambiguity intrinsic to the restlessness of negativity that constitutes the Real of the Weltalter. If it was Hegel who gave the former the most profound philosophical articulation in its raw purity in his Science of Logic, it was Schelling who was the first to stumble upon the full range of its metaphysical implications (to which we shall return).331 What is more, this also lets us explain why Hegel could have stopped dead in his tracks in his mature thinking of a triadic relationship between logic, nature, and spirit despite already having at his disposition such a sophisticated account of a metaphysically contingent retroactive restructuring of being from within. Although the inner movement he describes in the Logic gives perfect expression to the quadruple dialectics already hinted at in the night of the world of his Realphilosophie, the problem is that the categories it describes, like Lacan's mathemes, do not supply us with any horizon of meaning. As such, they must be supplemented with the “concrete” symbolic content of the philosophies of nature and spirit, thus creating space in which Hegel could phantastically recoil from his breakthrough insight into the subject as a self-instituting gap in being and its stark consequences for our understanding of the self and the world. The Abyss of Unconscious Decision 211 Hence why the Weltalter is so crucial for grasping the true importance of subjectivity as a contingent, world-changing event: it gives us mytho-poetic content that comes closer to grasping the matter at hand than Hegel's account of the particular sciences does, which rather obfuscates it (just as many empirical accounts continue to do today when faced with the explosion of the Ideal out of the Real). primary principle, that to which the movement had always tended, but this only becomes visible after the fact and is thus plagued by a devastating belatedness. There is no way notionally to deduce the act of self-positing because, as free, it impossible to predetermine its arrival or even be aware that it could occur at all.328 The reversal characteristic of the third moment befalls being, for “every dialectical passage or reversal is a passage in which the new figure emerges ex nihilo.”329 What we often miss in the necessity of the dialectical movement and what is even obfuscated by it is the fact that every movement of its self-articulation is, in fact, constituted by a series of a contingent acts brought on by negativity, contingencies that only retroactively gain the status of necessity by making themselves necessary.330 The passage from tension to victory is never ontologically guaranteed—and it is precisely this ambiguity intrinsic to the restlessness of negativity that constitutes the Real of the Weltalter. If it was Hegel who gave the former the most profound philosophical articulation in its raw purity in his Science of Logic, it was Schelling who was the first to stumble upon the full range of its metaphysical implications (to which we shall return).331 What is more, this also lets us explain why Hegel could have stopped dead in his tracks in his mature thinking of a triadic relationship between logic, nature, and spirit despite already having at his disposition such a sophisticated account of a metaphysically contingent retroactive restructuring of being from within. Although the inner movement he describes in the Logic gives perfect expression to the quadruple dialectics already hinted at in the night of the world of his Realphilosophie, the problem is that the categories it describes, like Lacan's mathemes, do not supply us with any horizon of meaning. The Abyss of Unconscious Decision 213 Hence Schelling can say at the end of the Freiheitsschrift that nature is the first revelation of God.332 But with the highest creature, man, something new emerges in creation as the principles become separable, which leads to the possibility of the free conquering of the dark principle by the light if humans choose to live their lives in imitation of God's spirit as the perfect unity of Grund and existence and, by consequence, to the possibility of a complete revelation of God to Himself insofar as humans then become the image of The immediate problem facing Žižek here is the fact that all of Schelling's middle-late works present themselves as a theogony, an account of the birth of God, based on the anti-dialectical structure of the quaternity, where the last movement is not the expression of something logically irreducible and operatively new immanently emerging from within a system, but a “return” to the first as a kind of direct contact with and raising up to a higher power of a dormant force that is simultaneously the primordial and unfathomable origin of all things. Each text repeats the same movement, although the drafts of the Weltalter—in contradistinction to the Freiheitsschrift—abruptly end before historical time. As the first, the abyss of God's freedom as the impenetrably dark source of divine and created reality rests in the absolute joy of eternity, a blind existence, where it wills nothing because it is the pure virtuality wherein everything is potentially contained, though does not determinately exist. Yet without distinction and duality, the eternal nature as freedom remains unrevealed and thus lacks the fullness of self-knowledge. In order to achieve this, God (who is here not yet a person) must somehow contract finitude and difference, limit His freedom, if He is to have an Other through which He can reveal Himself, thereby establishing the distance necessary to Himself to become a subject capable of owning freedom as a predicate instead of merely being freedom as a pure virtuality. The Abyss of Unconscious Decision 211 As such, they must be supplemented with the “concrete” symbolic content of the philosophies of nature and spirit, thus creating space in which Hegel could phantastically recoil from his breakthrough insight into the subject as a self-instituting gap in being and its stark consequences for our understanding of the self and the world. Hence why the Weltalter is so crucial for grasping the true importance of subjectivity as a contingent, world-changing event: it gives us mytho-poetic content that comes closer to grasping the matter at hand than Hegel's account of the particular sciences does, which rather obfuscates it (just as many empirical accounts continue to do today when faced with the explosion of the Ideal out of the Real). The Abyss of Unconscious Decision 213 The Abyss of Unconscious Decision 213 The Abyss of Unconscious Decision 213 The Abyss of Unconscious Decision 213 The immediate problem facing Žižek here is the fact that all of Schelling's middle-late works present themselves as a theogony, an account of the birth of God, based on the anti-dialectical structure of the quaternity, where the last movement is not the expression of something logically irreducible and operatively new immanently emerging from within a system, but a “return” to the first as a kind of direct contact with and raising up to a higher power of a dormant force that is simultaneously the primordial and unfathomable origin of all things. Each text repeats the same movement, although the drafts of the Weltalter—in contradistinction to the Freiheitsschrift—abruptly end before historical time. As the first, the abyss of God's freedom as the impenetrably dark source of divine and created reality rests in the absolute joy of eternity, a blind existence, where it wills nothing because it is the pure virtuality wherein everything is potentially contained, though does not determinately exist. Yet without distinction and duality, the eternal nature as freedom remains unrevealed and thus lacks the fullness of self-knowledge. In order to achieve this, God (who is here not yet a person) must somehow contract finitude and difference, limit His freedom, if He is to have an Other through which He can reveal Himself, thereby establishing the distance necessary to Himself to become a subject capable of owning freedom as a predicate instead of merely being freedom as a pure virtuality. He breaches this pure virtuality by instituting the conflict of Grund (the No, the darkness of materiality, the contractive energy that holds all together) and existence (the Yes, the light of spirit, the expansive structures that give order to the rulessness of the Grund) within Him, so that He can beget Himself as a self-conscious being and eventually decide to bestow upon His own Grund the status of an independent and productive being, thus becoming God the Creator and allowing this same conflict constitutive of his inner life to be mirrored in all living things. 214 Chapter 9 God (imago dei). With the forward march of history, the goal of creation will be attained when evil is completely vanquished, for this will have meant that we, like God, will have achieved the holy unification of the light and dark principles, insofar as we will have made ourselves subjects capable of owning freedom as a predicate by autonomously choosing the Yes and thus overcoming our separation from God by returning to Him. Material being, which we will have then “divinized” in showing how it is capable of the good in and through us, and personal God will then be reconciled and love—which presents itself as the fourth, the positive counterpart at the end of the system corresponding to the Ungrund as its absolute beginning—will prevail: as nature yearns for the spoken Word, humanity longs for for the destruction of the antagonism of principles in the Future, which proves itself to be a paradoxical “return” to the Past, since once the Present has begun, its beginning is always already lost, so that the only way the Ungrund can reemerge is if the world ends in a Future that is to come. The pure virtuality that contains everything potentially may be irretrievable, but redemption— as love—awaits as a point to which the world tends, for this tendency towards its own annihilation as we know it is part of its metaphysical structure. The inner life of all being thus follows a series of fourfolds that are modelled after the structure intrinsic to the life of God: The Abyss of Unconscious Decision 213 He breaches this pure virtuality by instituting the conflict of Grund (the No, the darkness of materiality, the contractive energy that holds all together) and existence (the Yes, the light of spirit, the expansive structures that give order to the rulessness of the Grund) within Him, so that He can beget Himself as a self-conscious being and eventually decide to bestow upon His own Grund the status of an independent and productive being, thus becoming God the Creator and allowing this same conflict constitutive of his inner life to be mirrored in all living things. Hence Schelling can say at the end of the Freiheitsschrift that nature is the first revelation of God.332 But with the highest creature, man, something new emerges in creation as the principles become separable, which leads to the possibility of the free conquering of the dark principle by the light if humans choose to live their lives in imitation of God's spirit as the perfect unity of Grund and existence and, by consequence, to the possibility of a complete 214 Chapter 9 Ungrun ↓ self-manifest God or complete revelation But how is Schelling able to maintain such a narrative within the second period even in face of a structure that apparently forecloses its very possibility? That which enables Schelling to sustain such a narrative in face of a structure that compromises the dynamic of the quaternity paradoxically coincides with his insight into a parallactic dialectics of restless negativity. The success of Schelling's reaction formation goes hand in hand with the intensity of his plunging into the abyssal origins of subjectivity, since the latter constitutes some kind of primordial trauma whose repression is The Abyss of Unconscious Decision 215 The Abyss of Unconscious Decision 215 simultaneous with the transcendental constitution of reality so that it is only to be expected that such a two-way tension would emerge. Given that the origins of the Symbolic withdraw in their founding gesture, if Schelling is to describe the interior involutions of the Real, and in what sense they could bring forth the conditions of the possibility of an absolutely free subjectivity (either human or divine) Schelling must have recourse to a mytho-poetic medium to talk about that which forever precedes and can never enter the light of consciousness and language. If the subject is “the primordial Big Bang” of experiential reality,333 how are we to grasp what occurred only nanoseconds prior to it? The issue is not the Real-as-excess as that which logically precedes and exceeds symbolization, which spectrally haunts all synthetic constitution, but the very moment in which the Symbolic upsurges; and because such an event is a structurally impossible object of discourse, any discourse that attempts to account for it is by the same token plagued by this very impossibility. Faced with this impasse, we are forced to fall back into mythology to explain that which transcends the bounds of ideality as its core: Does not this step involve “regression” to a version of New Age mythology? The Abyss of Unconscious Decision 215 When, at the key points of their theoretical edifice, Freud and Lacan also resorted to a mythical narrative (Freud's myth of the primordial father in Totem and Taboo, his reference to Plato's myth of androgynous primordial man in Beyond the Pleasure Principle; Lacan's myth of “lamella” in his Four Fundamental Concepts of Psycho-Analysis), they were driven by the same necessity as Schelling: the need for the form of mythical narrative arises when one endeavours to break the circle of the symbolic order and to give an account of its genesis (“origins”) from the Real and its pre-symbolic antagonism.334 Although mythology is rationally justified insofar as we have, as it were, deduced the necessity of the non-deducible and now need a new manner to investigate it, it does not come without its risks: the pre-symbolic act at the very founding gesture of language—the vanishing mediator between nature and culture that cannot be found in either—is that which we can “access” through a kind of self-reflexive speculative fabulation that stages “the birth 216 Chapter 9 216 Chapter 9 from darkness into light,”335 but in such a way that we cannot guarantee a priori that such an endeavour will not be plagued by defence mechanisms, unconscious fantasies and wishes, especially given its essentially traumatic nature. To search for our origins, we must plunge into the abyss, but it can lead us astray: the mytho-poetics of speculative fabulation can just as much help us narrate the pre-history of the Symbolic and the always already lost act that institutes it, the trauma that is the lacerating cut of pure self-positing in its full horror, as it can nourish psychoanalytical defences against it when we approach the ontological catastrophe at our nativity as human subjects. It is exactly here at this juncture of interplay of mytho-poetic fabulation and phantasmic interference within Schelling's second period that Žižek intervenes, using the resources of Lacanian psychoanalysis to traverse its fantasy and thereby restructure its conceptual space so as to bring forth that which is in Schelling so intimately that it is most properly characterized as extimate. But how can Žižek accomplish such a feat? Before we can directly answer this question, it is necessary to summarize the two intertwined levels active in the rational mythology of the Weltalter project in order to locate the unsolved tension from which psychoanalysis offers us a way out. The Abyss of Unconscious Decision 215 Within the stillborn drafts of the Weltalter, Schelling divides the passage from the eternal Past to the Present into three distinct stages. Because Schelling's text simultaneously operates in a theosophic and metapsychological mode, I will quickly summarize Žižek's presentation of each stage. Instead of outlining the various conceptual distinctions and internal differences that occur within the three existent drafts of the Weltalter, I will only be dealing with Žižek's own exegesis, which focuses on the second draft, since as we have seen he largely dismisses the importance of the other two versions. 1. In the absolute beginning prior to God's contraction of material being and the annular rotation of drives, there is a joyous nothingness, a pure potentiality that exists in timeless, inexhaustible rapture. God is not a yet a “He,” but merely an impersonal, anonymous “it”: knowing no conflict, God knows not even itself as radical freedom. For Žižek, in contemporary terms this would be equivalent to the pure void that exists before the vacuum fluctuation declared by quantum cosmology, a The Abyss of Unconscious Decision 217 nothingness that must be declared positively charged because from its (auto)disturbance “something” emerges. What is of utmost importance here is the irreconcilable contrast between this stage and the next: the joyous void of divine non-being is breached by the contraction of finitude and the self-diremption of perfection that it entails. Metapsychologically, this sundering of heavenly symmetry is thus structurally identical to the disruption of the oceanic unity of child and mother that supposedly precedes the Oedipus complex, or the smooth, placid functioning of cyclical nature, which is skewered by the advent of human subjectivity. 2. After the contraction of material being, we have what Žižek calls “Schelling's grandiose 'Wagnerian' vision of God.” Within Schellingian cosmogony, this is so “terrifying” because, instead of the endless joy of divine eternity, we have a God as subject who finds Himself caught within the self-lacerating rage of matter. There is a sense in which God the Almighty is in infinite pain because his freedom has been lost within the torment-ridden movement that is coincident with the moment when the blind rotation of drives falls into an erratic, uncontrollable oscillation. The Abyss of Unconscious Decision 215 As such, God is comparable to a helpless animal stuck in a trap.336 To exemplify this point further, Žižek compares this stage to the unfathomable chaos that occurs after the vacuum fluctuation at the origins of the universe: the contraction of matter into an infinite point of absolute singularity, an incomprehensible upheaval within which the logic of our known physical universe breaks down. In terms of a metapsychology, it can be read as a mytho-poetic description of the ontological short circuit within the instinctual schemata of nature occurring before the eruption of human subjectivity. This moment of ontological standstill coincides with the pre-personal realm of anarchic self-experience seen in the night of the world and expresses the first beginnings of psychotic withdrawal of nature into an irreal self that severs the Innenwelt from Aussenwelt. The Abyss of Unconscious Decision 219 have become completely lost to themselves at a now inaccessible point in the vicissitudes of their dark pre-history. What is so terrifying here is that, instead of the light of consciousness and the universe of meaning, there could have been nothing but the agonizing rotation of drives in their frantic disorder, nothing but the ontological mayhem/madness wherein being would be irrevocably laid to waste in its own breakdown and catastrophism. If being awakens in a moment of primordial trauma, how could one be certain that its defense formation (subjectification through the Symbolic) would work? That it would solve its antagonism? If freedom is to be irreducible, it must posit itself entirely by its own activity from within the clutches of unbreakable determinism, but this comes at a price—and it is exactly around this problematic of the breach of symmetry necessary for a suitable “foundation” of freedom and its alarming metaphysical implications that the existent stillborn drafts circulate and ultimately falter, each offering a different spin irreconcilable with the others. For Žižek, however, the psychoanalytical tension of the Weltalter drafts displays a much more complex structure than that of a mere attempt to cover up the dialectical contingency at the heart of the unconscious decision and its wider implications. What intensifies the problem at hand is that the entire investigation has the fundamental structure of fantasy.337 In the articulation of the absolute beginning, we insert ourselves as a pure gaze into the Real that is prior to our own conception, just as if we were to imagine ourselves as spectators in our funerals watching our friends react to our death—and if we read the mytho-poetic introspective analysis that leads Schelling to such a discussion with this in mind, then we must conclude that it too exhibits the psychoanalytical traits associated with such fantasy constructions, complete with all of the problems that go along with them. 218 Chapter 9 218 Chapter 9 3. Finally, we have God who is able to speak the Word and thus overcome the deadlock that he found Himself lodged within by becoming a full-fledged subjectivity. Ejecting the materiality that He had contracted, pushing it out of him like some kind of foreign body, he bestows upon this now excremental product its own independent existence and thus becomes God the Creator. For Žižek, this corresponds in physics to the primordial Big Bang itself: the beginning of our material universe as the self-expansion of the initially infinitely dense point of matter. Metapsychologically, in the Word we see the unconscious decision (Entscheidung) that separates Grund and existence for the first time. The Symbolic erupts as an attempt to discipline the unruliness of the previous stage, which has immanently disrupted the primacy of organism's autopoietic schemata. The schismatic split that characterizes the essence of the psychoanalytical experience and the actual freedom of the cogito is posited as such for the first time as a response to the complete denaturalization that is the zero-level of the subject. In both the theosophy of creation, the physics of the Big Bang, and the metapsychology of the birth of subjectivity, this stage is something unpredictable that founds something new (God the Creator, material reality, the universe of meaning). The existent drafts of the Weltalter find themselves confronted with the same problem as the Freiheitsschrift, that is, the arbitrariness of the beginning: why does God speak the Word? If the act of unconscious decision cannot be deduced according to notional necessity (were it able to be, Schelling would fall back into the perceived threat of Absolute Idealism that he is fighting against) there is no guarantee that God, after contracting material finitude, will be able to assert His freedom by making it a predicate of Himself: that is, there is no guarantee that the subject will be able to liberate itself from the deadlock of the drives within which it finds itself, in other words, their unruliness. Attempting to articulate a radical philosophy of freedom, Schelling in the second draft has no possible recourse to an explanatory principle capable of saving God or human being from possible infinite diremption: he is unable to foreclose the possibility that both could The Abyss of Unconscious Decision 219 The Abyss of Unconscious Decision 219 For both endeavour to describe the impossible: a linguistically ordered field of experience where there is none, a world of thought where no thinking could possibly exist, the first merely in a “subjective” sense (I cannot witness my own funeral because my own personal ego will never exist after my death in order to be able even to see such events unfolding as I may imagine them), but the second in an “objective” sense (not only is there no one there to witness the emergence of a world of signification, since consciousness presupposes someone has already emerged, but thought itself 220 Chapter 9 220 Chapter 9 as logos has yet even to appear on the scene). When put in this manner, the question that poses itself is how the second, “objective” fabulation can be saved from the various kinds of illusions that may play themselves out in the first, “subjective” mode of fabulation such as those that occur in our daydreaming. In this respect, it is understandable how Schelling could have fallen into a trap: because we can only retroactively posit the material origins of subjectivity from within the Symbolic and the Imaginary and these origins represent the unthinkable basis of thinking itself,338 the descriptions of this natal darkness can serve as a mere screen upon which we project fantasmatic supplements to satisfy unconscious desires. Not only does the very nature of the investigation jeopardize it (since we can structurally never reach the act that brings consciousness into existence, speculation has reached its limit and must pass into dramatic mytho-poetics) but it could easily be abolished through a reduction to the narcissistic orbit of the Imaginary or succumb to various symbolic levels of defence that would prevent the subject from performing its description, even in a self-reflexive mytho-poetic medium. By protecting us from the traumatic Real of our being, something that we of course all want, these supplements can lead us away from the truth for which we are searching. The Abyss of Unconscious Decision 219 But it is precisely here that the strength of psychoanalysis shows itself: as a discourse about discourse, in short, a discourse whose aim is to understand the inherent limitations plaguing all discourse (itself thus included), it is able to bestow upon such theoretical investigation an extra level of self-reflexivity that could help it from falling into such traps: “the way to avoid this utopian reduction of the subject to the impossible gaze witnessing an alternate reality from which it is absent is not to abandon the topos of alternate reality as such, but to reformulate it so as to avoid the mystification of the theosophic mytho- poetic narrative which pretends to render the genesis of the cosmos.”339 Thus, by focusing on the ambiguity of inserting ourselves as a pure gaze into the absolute beginning that we must retroactively posit, that is, the very impossibility of the endeavour itself to succeed in its task, Žižek is able to find a way to entirely cut off the theosophic character of Schelling's text and read it exclusively as a metapsychological account of the emergence Thus, by focusing on the ambiguity of inserting ourselves as a pure gaze into the absolute beginning that we must retroactively posit, that is, the very impossibility of the endeavour itself to succeed in its task, Žižek is able to find a way to entirely cut off the theosophic character of Schelling's text and read it exclusively as a metapsychological account of the emergence of Symbolic from the meaningless Real. Man's inclusion within the divine theo-cosmogonic drama ultimately proves to be an extraneous feature of the The Abyss of Unconscious Decision 221 operative logic of the Weltalter drafts, a code to be deciphered, many features of which can be ignored as a mere phantasmal projection accidentally adjoining itself to an exploration of the abyssal origins of subjectivity. One of Žižek's most provocative claims is that one can see such a phantasmal projection in the attempts to cover up its most groundbreaking insight: the destructive self-movement of negativity in reality that leads to and is radicalized in the self-positing of subjectivity. The Abyss of Unconscious Decision 219 In this way, we can say that paradoxically “it was [Schelling's] very 'regression' from pure philosophical idealism to the pre-modern theosophical problematic which enabled him to overtake modernity itself.”340 But now the question imposes itself upon us: what is revealed when we purify the odyssey that is the Freiheitsschrift and the Weltalter from its mytho-poetic phantasmagoria? operative logic of the Weltalter drafts, a code to be deciphered, many features of which can be ignored as a mere phantasmal projection accidentally adjoining itself to an exploration of the abyssal origins of subjectivity. One of Žižek's most provocative claims is that one can see such a phantasmal projection in the attempts to cover up its most groundbreaking insight: the destructive self-movement of negativity in reality that leads to and is radicalized in the self-positing of subjectivity. In this way, we can say that paradoxically “it was [Schelling's] very 'regression' from pure philosophical idealism to the pre-modern theosophical problematic which enabled him to overtake modernity itself.”340 But now the question imposes itself upon us: what is revealed when we purify the odyssey that is the Freiheitsschrift and the Weltalter from its mytho-poetic phantasmagoria? Notes 294. See Žižek, The Ticklish Subject, p. 55; and The Parallax View, p. 166. 295. Žižek, The Indivisible Remainder, p. 8. 296. Schelling, Freiheitsschrift, p. 276. 296. Schelling, Freiheitsschrift, p. 276. 297. Ibid., p. 278. 297. Ibid., p. 278. 298. Žižek, The Indivisible Remainder, p. 105. 298. Žižek, The Indivisible Remainder, p. 105. 299. Ibid., p. 92. 300. Ibid., p. 103. 300. Ibid., p. 103. 301. Ibid., p. 106. 301. Ibid., p. 106. 302. Žižek, Less Than Nothing, p. 234. 302. Žižek, Less Than Nothing, p. 234. 3. Žižek, The Indivisible Remainder, pp. 35–39. 303. Žižek, The Indivisible Remainder, pp. 35–39. 303. Žižek, The Indivisible Remainder, pp. 35–39. 304. Schelling, Weltalter II, p. 4. 304. Schelling, Weltalter II, p. 4. 305. Žižek, The Indivisible Remainder, p. 38. 305. Žižek, The Indivisible Remainder, p. 38. 306. Ibid., p. 92. 306. Ibid., p. 92. 307. Wirth, “Translator’s Introduction,” in Weltalter III, pp. ix–x. 308. Hegel, The Phenomenology of Spirit, p. 9. 308. Hegel, The Phenomenology of Spirit, p. 9. 309. Žižek, The Indivisible Remainder, pp. 20–21. 310. Schelling, Freiheitsschrift, p. 281. 310. Schelling, Freiheitsschrift, p. 281. 311. Schelling, Weltalter II, p. 121. 311. Schelling, Weltalter II, p. 121. 312. Žižek, The Indivisible Remainder, p. 38. 313. Ibid., p. 92. 313. Ibid., p. 92. 222 Chapter 9 222 Chapter 9 314. Ibid., p. 37. 315. Ibid., p. 39. 316. Schelling, Weltalter II, p. 121. 317. Žižek, The Abyss of Freedom, pp. 3–4. 318. Žižek, The Indivisible Remainder, p. 24. 319. See Laplanche and Pontalis, The Language of Psychoanalysis, trans. D. Nicholson- Smith (London: Karnac Books, 1988), pp. 465–69; and also Lacan, The Seminar of Jacques Lacan, XI: The Four Fundamental Concepts of Psycho-analysis, 1963–1964, ed. Jacques-Alain Miller, trans Alan Sheridan (London: Vintage, 1998), p. 51. 320. Žižek, “Liberation Hurts”; and The Ticklish Subject, p. 48. 321. Žižek, The Abyss of Freedom, pp. 3–4. 321. Žižek, The Abyss of Freedom, pp. 3–4. 322. Žižek, The Indivisible Remainder, p. 92. 323. Miller, “Context and Concepts,” in Reading Seminar XI: Lacan’s Four Fundamental Concepts of Psychoanalysis, ed. Richard Feldstein, Bruce Fink, and Maire Jaanus (Albany: SUNY Press, 1995), pp. 7–8. 324. Žižek, Less Than Nothing, pp. 300ff. 324. Žižek, Less Than Nothing, pp. 300ff. 325. Ibid., p. 198. Compare with pp. 292ff. and 304. 326. Ibid., pp. 293–94. 326. Ibid., pp. 293–94. 326. Ibid., pp. 293–94. 327. Ibid., p. 234. 328. Ibid., p. 285. 329. Ibid., p. 231. 330. Ibid., p. 213. 331. Ibid., p. 274. 332. Schelling, Freiheitsschrift, p. 284. 333. Žižek, The Ticklish Subject, p. 31. 334. Žižek, The Indivisible Remainder, p. 9. 335. Schelling, Freiheitsschrift, p. 239. 336. Žižek, The Indivisible Remainder, p. 23. 337. Ibid., p. 22. 337. Ibid., p. 22. 338. Žižek, Less Than Nothing, p. 645. 339. Ibid., p. 273. 339. Ibid., p. 273. 340. Žižek, The Indivisible Remainder, p. 8. Chapter 10 Radicalizing the Subject Substance Gasping for Breath, the Metaphysics of Quantum Mechanics, and the Žižekian Unconscious Chapter 10 Radicalizing the Subject Substance Gasping for Breath, the Metaphysics of Quantum Mechanics, and the Žižekian Unconscious Although Žižek's transcendental materialism relies upon Schelling to explicate the origins of subjectivity, Hegel is nevertheless omnipresent in Žižek's work. Perceiving a strictly Hegelian structure of self-relating negativity in the second draft of the Weltalter that identifies Grund and existence in a manner inconsistent with its surface structure of quaternity, Žižek offers a psychoanalytical reconstruction of Schelling's aborted masterpiece by showing how Hegelian dialectics is its unconscious truth. Demonstrating this complex and nuanced hybridism of Hegelian logic and Schellingian ontology at the heart of Žižek's philosophy does not merely help us see its originality and singularity; it also enables us to shed light on in what sense his philosophy is a revisionist metaphysics of the subject. The existence of subjectivity not only attests that nature is at best a fragile not-all whose disruptive wounds are more primordial than its positive being. More drastically than this, the absolute, instead of being a fully subsisting reality that exists by means of a self-explanatory surplus, is at its most basic level only minimally indistinguishable from the void of nothingness, whereby the subject becomes one of the names of the eternal disturbance of this void by means of which there is something rather than nothing. This also leads us into a discussion of the paradoxical causality explicit in the self-begetting 224 Chapter 10 Chapter 10 of the Žižekian subject and its repercussions for our understanding of the unconscious. 10.1 From the Psychoanalytical Purification of the Theosophic to Substance Gasping for Breath Žižek does not articulate his own solution to the problematic of how he is able to purify the odyssey that is the Freiheitsschrift and the Weltalter from its mytho-poetic phantasmagoria. The issue at hand is how he can formalize Schelling's philosophy by purifying its theosophic content (the illusion of an originary oceanic bliss and its crucial role in covering up the intrinsically dialectical structure inherent to the Grund by means of a quaternity) by traversing its fantasy. To do this, Žižek relies on the primordiality of the psychoanalytical experience and the irreducibility of negativity in Hegelian logic, the traces of which he sees in the second draft's descriptions of the three stages involved in the movement from the Past to the Present. The resources offered by both allow us to internally reconfigure its conceptual movement by removing this distorting fantasy element through a philosophical reconstruction of the abyssal origins of the subject by tarrying with the Real of Schelling's texts. It is this revamped version of the Weltalter's investigations into the spectral, never-present beginnings of the Symbolic that enables Žižek to draw out the full metaphysical implications of the paradoxical nature of psychoanalytico-Cartesian subjectivity. According to psychoanalytical experience, the zero-level fact in the passage from the Past to the Present has to be the second stage,341 that is, the self-lacerating rage of matter. 10.1 From the Psychoanalytical Purification of the Theosophic to Substance Gasping for Breath Because Žižek identifies the subject with the non-coincidence of substance, its alienation from itself,342 this self-lacerating rage is equivalent to what Lacan refers to as the “organic dehiscence” exhibited in the mirror stage that forms the basis of the ontogenesis of personality.343 But if we are to mytho-poetically explain how such a dismemberment forms the meta-transcendental conditions for the possibility of subjectivity's self-positing, we cannot understand it as a haphazard feature of nature that falls upon it like an alien blow from nowhere—rather, its pure contingency must itself emerge from some kind of immanent, self-effacing possibility always already implicit within it.344 As that which metapsychologically corresponds to the eternal calm of the pure Radicalizing the Subject 225 immanence of substance preceding the deadlock of drives and the struggle to speak the Word, the joyous nothingness of divine eternity is therefore merely a part of the ego's fantasy of desired fullness, a narcissistic and reactive attempt to secure the false status of nature as all in order to protect us from the dark truth implicit in the psychoanalytical experience: that one cannot draw a metaphysical distinction between substance as a nothingness that rejoices in the oceanic bliss of non-experience and the unruly basis of human subjectivity that “disrupts” this unity. For Žižek, the transcendental materialist logic of which we see premonitions in the second draft thus allows us to add precision to the late German Idealist attempts to think substance as subject: the model of subjectivity as a disease within the vital fold of being needs to be slightly modified, for there was never a state of metaphysical harmony and innocence that the going-haywire of human unruliness could have brought into ruin once and for all. 10.1 From the Psychoanalytical Purification of the Theosophic to Substance Gasping for Breath This is exactly why Žižek feels justified in proclaiming the superiority of Hegelian logic and violently remodulating Schelling's middle-late ontology to get rid of its notion of absolute indifference, for in positing an initial state of health, Schelling demonstrates a tendency—a tendency only encouraged by the abyssal void of the Real-as-origin—to construct a rampant fantasy whose imaginary support (certain theosophic details) could protect him from his own insight into the restless movement of negativity in the interior life of being and at the core of the subject's pure act, a fantasy that in itself is filled with holes and inconsistencies as shown by the endless proliferation of stillborn drafts of the Weltalter in an attempt to secure it. That the subject is synonymous with the irrevocable self-sundering of substance does not mean that before the advent of complete denaturalization in our constitutive excess there was no internal obstruction within the former's immanent ebb and flow. If the human being is an irreversible blockage in the vital fold of being, it must represent an amplification of an already existing potentiality in nature. We can see this in various forms—natural disasters, deformed animals, mass extinction, black holes, all of which point to ways in which the originary “harmony” of the world is predicated upon disorder, eruptive disarray, and its inability to sustain itself in perfect symmetry. Knowledge in the Real is never perfect: 226 Chapter 10 The most unsettling aspect of such phenomena is the disturbance caused in what Lacan called “knowledge in the Real”: the “instinctual” knowledge which regulates animal and plant activity. This obscure knowledge can run amok. When winter is too warm, plants and animals misread the temperature as a signal that spring has already begun and so start to behave accordingly, thus not only rendering themselves vulnerable to later onslaughts of cold, but also perturbing the entire rhythm of natural reproduction.345 The most unsettling aspect of such phenomena is the disturbance caused in what Lacan called “knowledge in the Real”: the “instinctual” knowledge which regulates animal and plant activity. This obscure knowledge can run amok. 10.1 From the Psychoanalytical Purification of the Theosophic to Substance Gasping for Breath When winter is too warm, plants and animals misread the temperature as a signal that spring has already begun and so start to behave accordingly, thus not only rendering themselves vulnerable to later onslaughts of cold, but also perturbing the entire rhythm of natural reproduction.345 These examples of destruction in nature must, however, be seen in their raw conceptual materiality. Rather than being justified by Žižek's rhetoric, they point to one of the major achievements of Žižek's dialectics: its ability to think through the philosophical implications of what is normally seen as a mere contingent breakdown of all-present order or a short-lived calamitous outburst of pandemonium in the otherwise smooth flow of things. Žižek refuses such a thesis: for him, contradiction and non-coincidence are to be seen as at the very basis of reality to such an extent that reality's logical closure upon itself is unable to sustain itself and is constitutively torment-ridden with fracture points. Rather than being a totalizing weave that is and thus embraces all things in its soft, calming touch, despite things seeming otherwise in experience (just as turbulent eddies are only possible given the oneness of the ocean that persists through them), substance is dirempt and constantly risks bursting at its seams due to its own internal fragmentation. These wounds in the absolute do not exude the vital blood of substance in such a way that they will, after a brief moment of cosmic fright, heal themselves and once again be subsumed within the economy of a symphonized sexualized dance of lack and plenitude, life and death: substance does not engage in a ritual of bloodletting to gain strength, as if conflict, although internal to the system, only existed so that through its conquering substance could express again and again its infinite, almighty power. On the contrary, the wounds of substance are those places where it touches the void. Torn apart from the inside out, substance in its auto-laceration is in danger of dissipating into nothingness, of no longer being capable of holding itself together. 10.1 From the Psychoanalytical Purification of the Theosophic to Substance Gasping for Breath Here one must think of the fundamental presupposition of Schellingian philosophy: were substance (God) all, were there from the very beginning nothing but Radicalizing the Subject 227 a balanced movement, a pure, all-devouring totality, no subjectivity or experience would be possible.346 In this sense, Žižek is not merely trying to radicalize this insight by reactualizing Schelling's metapsychology so that it does not succumb to its theosophic tendencies, but more primordially trying to make it internally consistent. According to Schelling's own words, if we posit an initial state of health, we cannot explain how subjectivity could emerge. Perfection is only an imaginary, fantasmatic extension of the existential horizon of our broken finitude, that is, a reaction formation, a reaction formation within which we encounter again and again the insupportable negativity that tears apart the world, for after all, the repressed always returns. As the greatest thinker of subjectivity, we begin to grasp why there would be such an immense conflict within Schelling's middle-late period between the irreconcilable extremes of a radical philosophy of freedom and an equally radical philosophy of a theo-cosmogonic drama of divine being seeking self-manifest revelation. They go hand in hand: traumatic insight elicits protective defences, but the latter can never be completely successful. Accordingly, Žižek psychoanalytically modifies Schelling's descriptions of the Past as that elusive X that forever haunts and precedes consciousness so that the real encounter (the horror of substance as subject) can be freed from its imaginary, fantasmatic surface (the theosophic odyssey) and brought into explicit mythologico-symbolization. This has severe consequences. First and foremost, we must remember that at the level of logic, Schelling's mytho-poetic narrative of the Past does not primordially present a chronology of the absolute, even if it is derived from phenomenological experience by the alchemical identification of the highest and lowest and can thus be used to explain the ontogenesis of the individual subject by means of reason by analogy. The “stages” Schelling refers to are purely logical and organized according to priority: the category of linear temporality only emerges with the Present as that which mirrors the structural relations intrinsic to the inner life of God. Consequently, there is no sense in which the joyous nothingness temporally precedes God as subject caught in the throes of the self-lacerating rage of matter. 10.1 From the Psychoanalytical Purification of the Theosophic to Substance Gasping for Breath However, insofar as freedom as a predicate of a subject exists (stage three), the abyss of freedom as pure potentiality—a freedom that is not yet posited as such—must be said to logically precede the rotation of drives prior to full-fledged subjectivity 228 Chapter 10 (stage two). Žižek follows the argument thus far, but then makes a crucial change in an attempt to draw out a truth hidden in Schelling's descriptions of the passage from one moment to the other. Given that there is no temporal separation between the descriptions of the joyous nothingness of non-experience and the infinite, agonizing oscillation of the potencies, or substance and subject, they are, in essence, two sides of the same coin. The “passage” is nothing but a logical conversion: Let us step back for a moment and reformulate the primordial contraction in terms of the passage from a self-contented Will that wants nothing to an actual Will which effectively wants something: the pure potentiality of the primordial Freedom— this blissful tranquillity, this pure enjoyment, of an unassertive, neutral Will which wants nothing—actualizes itself in the guise of a Will which actively, effectively, wants this “nothing”—that is, the annihilation of every positive, determinate content. By means of this purely formal conversion of potentiality into actuality, the blissful peace of primordial Freedom thus changes into pure contraction, into the vortex of “divine madness” which threatens to swallow everything, into the highest affirmation of God's egotism which tolerates nothing outside of itself. 228 Chapter 10 In other words, the blissful peace of primordial Freedom and the all-destructive divine fury which sweeps away every determinate content are one and the same thing, only in a different modality—first in the mode of potentiality, then in the mode of actuality.347 The ultimate paradox of the shift from the bliss of divine eternity, freedom as pure potentiality, to the annular rotation of drives in the Grund that serves as the stepping-stone to freedom as the predicate of a subject is that there is no movement at all—Grund is always already the Ungrund, the “closed” circle of nature is always already the scene of the possible emergence of freedom.348 If we equate freedom with negativity, then we see that the Ungrund is no longer that which neutrally grounds the conflict of the polar principles in order to know itself and reveal itself to itself through them, but rather becomes synonymous with the devastating negativity at the heart of being, the places where it risks touching the void, and that even when it remains non-posited Radicalizing the Subject 229 as such, is always already present within the palpitations of the latter like a cardiac arrest waiting to happen: the ground is always already minimally ungrounded, ridden with tension, bursting at the seams. We see this clearly in the dialectic of the ontogenesis of the subject. Although the movement from (i) the rapture of symmetry that is substance to (ii) the harrowing madness of the drives in the unruliness of the infant appears to be due to the grotesque excess of life that is human being as an unfortunate accident, the matter is more complicated. In the first stage freedom (negativity) is always already there as a logical possibility, but remains for the most part non-posited in the general economy of the dynamics of substance, though it shows its head constantly, so that we just have a relatively closed and blind annular rotation of drives; in the second, however, it finally successfully posits itself in a significant manner (although not as such) as the self-lacerating rage of matter, but in such a way that its rawness risks devouring itself. It needs to be tamed, disciplined, gentrified. 228 Chapter 10 The decisive moment comes with (iii) the act of decision through which freedom as subject (inner limit of substance) is converted into freedom as the predicate of a subject (self- relating negativity), an act that is not only notionally non-deducible because it is, in itself, radically free, but also one that withdraws in its very gesture of self-positing. When this occurs, negativity, instead of being a single part in the totality of material being, turns itself into an independent center that hegemonically dominates the whole to which it once belonged, which in turn forces the negativity always already contained in the first moment finally to become fully explicit. Freedom is not in direct contact with the Ungrund as that which neutrally grounds the conflict of the polar principles as indifference, nor is it a resurgence of the primordial abyss of freedom: it is nothing but this movement in which the second (the negativity of being), relating to itself, counting itself, usurps the position of the first (being) and thus institutes a mere formal reconfiguration of the whole.349 As pure negativity, full-fledged self-standing freedom (self-relating negativity) does not emerge out of nowhere within a harmonious play of forces, because its existence attests that there is freedom (negativity) all the way down. As Žižek says, “the only greatness is 'negativity' itself.”350 What the psychoanalytico-Cartesian subject thus shows us is that the idea of extra/pre-subjective nature as the self-harmonizing Grund of all What the psychoanalytico-Cartesian subject thus shows us is that the idea of extra/pre-subjective nature as the self-harmonizing Grund of all 230 Chapter 10 230 Chapter 10 things, a causally closed play of forces caught within a blind necessity, is a pure fantasy: the beginning is not a solid, inert density, but a seething mass of heterogeneous matter lacking overarching symmetry and balanced movement. Matter is not some kind of impenetrable or irreducible “real stuff” that persists beyond conscious representation and follows eternal laws—at its very core, matter constantly dematerializes itself by opening up its flesh to the void of (virtual) non-being to the point where it is no longer matter but something more: that is, a full-fledged subject. The self-operative logic of nature as Grund consequently demonstrates that the immanence of substance is not a permeating weave of positive being, a never-ending sea whose fullness encompasses all: it is plagued constitutively by the possibility of self-fragmentation, since irrevocable zones of (virtual) non-being shattering the ordered reality of the world and its causal closure always threaten to erupt and turn it into a chaotic pandemonium of disordered, anarchic forces combating for rulership—a state that would, in fact, bring substance itself to its untimely end, for a substance that has globally turned “into a dispersed floating of membra disjecta”351 is no longer a substance at all. The self-lacerating rage of matter thus takes on a new meaning. The libidinal frenzy of the unruliness of human nature does not merely represent a single case of the diseased breakdown of the ontological, but rather the inability of substance to posit itself as all: substance is destined towards auto-destruction; the “ground fails to ground,”352 for it is always on the verge of passing over into hemorrhaging conflict and ravaging antagonism. In this regard, substance is constitutively weak, a precarious all whose fragility is not only always struggling to keep itself together as the contractive energy of all things, but also always capable of the new and the unpredictable at any cosmic moment due to its negativity, for what will be is not necessarily causally contained in what was. 230 Chapter 10 Here we see the extremely Hegelian logic that Žižek extracts from Schelling in the second draft: it is the failure of the first moment (the self-actualization of substance and the indivisibility of its causal self-enclosure, its closed annular rotation) that leads to/is the second (the unruliness of human nature, the unbearable short circuit in the rotation of drives), whose vicissitudes in turn set the stage for the third (the self-positing of the subject through this rupture in the fold of being, which opens up an autonomous logical space that attempts to things, a causally closed play of forces caught within a blind necessity, is a pure fantasy: the beginning is not a solid, inert density, but a seething mass of heterogeneous matter lacking overarching symmetry and balanced movement. Matter is not some kind of impenetrable or irreducible “real stuff” that persists beyond conscious representation and follows eternal laws—at its very core, matter constantly dematerializes itself by opening up its flesh to the void of (virtual) non-being to the point where it is no longer matter but something more: that is, a full-fledged subject. The self-operative logic of nature as Grund consequently demonstrates that the immanence of substance is not a permeating weave of positive being, a never-ending sea whose fullness encompasses all: it is plagued constitutively by the possibility of self-fragmentation, since irrevocable zones of (virtual) non-being shattering the ordered reality of the world and its causal closure always threaten to erupt and turn it into a chaotic pandemonium of disordered, anarchic forces combating for rulership—a state that would, in fact, bring substance itself to its untimely end, for a substance that has globally turned “into a dispersed floating of membra disjecta”351 is no longer a substance at all. The self-lacerating rage of matter thus takes on a new meaning. The libidinal frenzy of the unruliness of human nature does not merely represent a single case of the diseased breakdown of the ontological, but rather the inability of substance to posit itself as all: substance is destined towards auto-destruction; the “ground fails to ground,”352 for it is always on the verge of passing over into hemorrhaging conflict and ravaging antagonism. 230 Chapter 10 In this regard, substance is constitutively weak, a precarious all whose fragility is not only always struggling to keep itself together as the contractive energy of all things, but also always capable of the new and the unpredictable at any cosmic moment due to its negativity, for what will be is not necessarily causally contained in what was. Here we see the extremely Hegelian logic that Žižek extracts from Schelling in the second draft: it is the failure of the first moment (the self-actualization of substance and the indivisibility of its causal self-enclosure, its closed annular rotation) that leads to/is the second (the unruliness of human nature, the unbearable short circuit in the rotation of drives), whose vicissitudes in turn set the stage for the third (the self-positing of the subject through this rupture in the fold of being, which opens up an autonomous logical space that attempts to Radicalizing the Subject 23 suture the hole). The essence of the third moment is the self-negation of the previous one, which gives it a fully developed notional self-reflexivity in such a way that by guaranteeing the identity of Grund and existence Žižek is able to foreclose the possibility of a theosophic quaternity from within the conceptual fabric of Schelling's text. In this sense we can finally comprehend two controversial claims put forth by Žižek: first, that that “the founding gesture [the subject as the vanishing mediator] 'repressed' by the formal envelope of the 'panlogicist' Hegel is the same as the gesture which is 'repressed' by the formal envelope of the 'obscurantist' Schelling,”353 for both reveal in their own way the “unacknowledged” Grundoperation of German Idealism; and, second, that “to articulate clearly the Grundoperation of German Idealism [...] necessitates reference to Lacan; that is to say, our premiss is that the 'royal road' to this Grundoperation involves reading German Idealism through the prism of Lacanian psychoanalytical theory.”354 It is in this respect that Žižek's philosophy is a hybridism of Hegelian logic and Schellingian ontology, for it is a mere attempt at the thematization of its unconscious Grundlogik. The metaphysical basis of freedom is the irremovable possibility of negativity from within any self-totalizing, self-enclosed system. 230 Chapter 10 There are always unavoidable ruptures and breaks within the logical fold of the world, but these are not mere contingent features of the otherwise harmonious symphony of the infinity of being: the activity of the first moment always already “possesses” its own failure as exhibited in the constant proliferation of negativities that can never be fully subsumed within its dynamics, indicating that substance is constitutively minimally non-coincident to itself. Monstrosities and ontological abortions are an inescapable effect of substance's functioning, for function now shows itself to be one with dysfunction. In terms of Žižek's “reactualization” of Schelling, it is here that its most textually violent moment is located. It proclaims that the only way to save the Schellingian legacy is to say that nature as a full, rich creative potency inherent in the dark womb of the world is an illusion. Nature was always already a sickly creature, whose collapse coincides with its own conditions of (im)possibility. It is not only that nature never knew a moment of eternal happiness and joy, but that the dull, inarticulate pressure of its own gasping for breath (spirit, we remember, comes from the Latin 232 Chapter 10 What is to be “deconstructed” is this very notion of nature: the features we refer to in order to emphasize man's unique status—the constitutive imbalance, the “out-of-joint,” on account of which man is an “unnatural” creature, “nature sick unto death”—must somehow be at work in nature itself, Radicalizing the Subject 233 although—as Schelling would have put it—in another, lower power (in the mathematical sense of the term).356 although—as Schelling would have put it—in another, lower power (in the mathematical sense of the term).356 But here we must be careful. Insofar as the subject is an immanent event within the world, that is, insofar as through it being has “gained” the power to look upon itself by internal reflection due to a maximization of this constitutive self-sabotaging tendency within nature and symbolization occurs as a means to deal with this trauma, one cannot conclude that symbolization is a mere defence mechanism constitutive of the human ego. The illusionary world of the Symbolic into which we withdraw to save ourselves from the trauma that is the very essence of our freedom is simultaneous with being's own recoil from itself as it achieves self-disclosure. Awakening into the nightmare that is the psychoanalytical horror of substance as subject in all of its ambiguity, the primordial reaction of the world opening its eyes for the first time is that of hellish panic, a panic whose ultimate fate is the necessity of an ontological passage through madness which we enact as symbolic subjects by (re)constituting reality in an ontologically solipsistic, eternally nocturnal space of signifiers, a universe of meaning. If the self-revelation of being to itself leads to an originary madness, then not only should we understand the proliferation within the latter of self-composing dream-like images, a rhapsody of social and political phantasmagoria that function as the fabric of our own identities, as events in being, but also this very fiction of ontological completion we witness in accounts of “Nature.” Through us, in a moment of fantasy, being sees itself as perfect—or as having been possibly perfect were it not for our intervention—by living out the impossibility of its own illusionary fullness, a fantasy that is a complete perversion of the typical narrative of God reaching full self-consciousness only by means of human activity. 232 Chapter 10 232 Chapter 10 spiritus, “breath,” and is related to spirare, “to breathe”) precedes the very positivity of its being. Substance can only be substance—nature can only be nature—insofar as it is always already internally torn apart by a constitutive moment of auto-laceration that is the site of spirit/subject: “incompleteness [is] already in itself a mode of subjectivity, such that subjectivity is always already part of the Absolute, and reality is not even thinkable without subjectivity.”355 If the ontological dislocation attested to by emergence of the subject is always already a part of the absolute, then the passage from nature to culture is a mere logical conversion—it only requires a certain gesture or incitation to be brought to the fore while nothing changes at the level of “positive” being. Full-fledged subjectivity, and hence the symbolic universe of meaning that emerges as a belated response, may be an unpredictable event whose result institutes a new age of the world, but its ontological basis demonstrates that there is no ultimate opposition between us and the world. The idea of a unified, self-penetrating substance only comes après-coup as part of a fantasy that helps the subject protect itself, for without such a fantasmatic support of fullness we risk losing our very subjective consistency in face of the tragic incompletion of the world. The fiction of “Nature” is, in many ways, unavoidable: we unconsciously create it to save ourselves from recognizing the true basis of subjectivity and its stark implications, namely, the fact that the world, constitutively ravaged by the Ungrund, is metaphysically imbalanced and thus not-all: True “anthropomorphism” resides in the notion of nature tacitly assumed by those who oppose man to nature: nature as a circular “return of the same,” as the determinist kingdom of inexorable “natural laws,” or (more in accordance with “New Age” sensitivity) nature as a harmonious, balanced Whole of cosmic forces derailed by man's hubris, his pathological arrogance. 232 Chapter 10 In this sense, if the theological implications of quantum mechanics, wherein particles can cheat the universe by coming into and out of existence before they are “symbolically” registered, are that we must “posit a God who is omnipotent, but not omniscient,”357 then Žižek's metaphysics compels us to posit a God (nature, the absolute) that is neither omnipotent nor omniscient, yet somehow persists in being But how? 234 Chapter 10 10.2 Eppur si muove: Ontological Dislocation and the Metaphysics of the Void a collapse of the wave function;361 “normal” laws of linear temporality and causality break down as we encounter particles that retroactively “choose” their paths along virtual chains of possibilities and others that can even cheat the universe, coming in and out of existence without the latter even knowing.362 But this not only drastically challenges any sharp distinction between nature and culture,363 but also attests that the micro-universe of quantum particles is strangely “less” than that of the macro-universe that constructs itself from its vicissitudes, in a way that is remarkably similar to how the Kantian subject can only construct a unified world of appearances from the inconsistent fragments of sensation. In an uncanny logical short circuit, it would appear that not only is there no bottom-up causality at the level of experience (transcendental constitution is more real than what Kant calls “a rhapsody of perception,”364 since it founds a coherent field of reality), but even the most fundamental level of the universe is metaphysically more chaotic and constitutively less ontologically complete than the ordered macro-level physical world that science, as according to classical models, describes. Substance is not a given, but an achievement: it is as if all reality is transcendentally constituted, as if nature itself emerges out of this field of quasi-entities. But just as we had to explain the ontogenesis of unruliness, we must also explain the emergence of these ontologically incomplete realities that serve as the basis for the ordered universe, since they cannot be a brute, arbitrary given. The emphasis of the key philosophical question is thus shifted from how the subject can emerge out of the Real qua substance (thought from being) to how the subject can emerge out of the Real qua void (something from nothing), for the more strictly speaking ontological question of ontogenesis thus proves to be irreducibly entangled with the most fundamental of metaphysical questions. The more we analyze reality the more we find a void, because once we reach the level of quanta our conventional conceptions of the ordered cosmos just stop working. 10.2 Eppur si muove: Ontological Dislocation and the Metaphysics of the Void At this conceptual conjuncture, Žižek takes an additional step in his psychoanalytical reconstruction of German Idealism that simultaneously brings into complete culmination its unconscious, disavowed Grundlogik and leads him into a profoundly new variety of metaphysical thinking that is uniquely his own. If in order to explain the emergence of the subject out of substance we have to proclaim that the latter, rather than being an indivisible oneness that weaves all things into an all-encompassing fabric, is predicated upon a site of auto-laceration, then the subject—as a name for this gap in substance that “sets it in motion”—has in turn to be more fully investigated, for we have not yet seen how ontological dislocation could be a creative force. We cannot stay at the level of substance qua nature: the ordered universe that compromises cosmological, geological, and biological time is so ravaged by negativity that the subject in its various vestiges does not merely indicate the places where substance in its trembling and uneasy auto-articulation risks touching the void, that is to say, is in danger of no longer holding itself together, but also the places where the void threatens to (re-)erupt. In this regard, the dynamic processes of the world are not just dependent upon (potential) disarray, but suggest that substance cannot be the last metaphysical word, since it appears on a second glance that the world itself is a response to something more primordial, something that is constantly trying to show its ugly head: ultimately “there is no Substance, only the Real as the absolute gap, non-identity, and particular phenomena (modes) are Ones, so many attempts to stabilize this gap.”358 Risking anachronism,359 Žižek sees a way to reinterpret the German Idealist attempt to think substance as subject through quantum mechanics that sheds light on this problematic. As quantum mechanics teaches us, any metaphysical investigation into the foundation of substance qua nature (the basic material constituents of reality) has a paradoxical result. 10.2 Eppur si muove: Ontological Dislocation and the Metaphysics of the Void Instead of coming upon a dense field of fully constituted realities that form the ultimate building blocks of the universe, “the more we analyze reality, the more we find a void”:360 rather than encountering entities complete in themselves, we see irreducibly indeterminate states lacking being in any traditional sense and from which “hard” reality can only emerge if there is Radicalizing the Subject 23 a collapse of the wave function;361 “normal” laws of linear temporality and causality break down as we encounter particles that retroactively “choose” their paths along virtual chains of possibilities and others that can even cheat the universe, coming in and out of existence without the latter even knowing.362 But this not only drastically challenges any sharp distinction between nature and culture,363 but also attests that the micro-universe of quantum particles is strangely “less” than that of the macro-universe that constructs itself from its vicissitudes, in a way that is remarkably similar to how the Kantian subject can only construct a unified world of appearances from the inconsistent fragments of sensation. In an uncanny logical short circuit, it would appear that not only is there no bottom-up causality at the level of experience (transcendental constitution is more real than what Kant calls “a rhapsody of perception,”364 since it founds a coherent field of reality), but even the most fundamental level of the universe is metaphysically more chaotic and constitutively less ontologically complete than the ordered macro-level physical world that science, as according to classical models, describes. Substance is not a given, but an achievement: it is as if all reality is transcendentally constituted, as if nature itself emerges out of this field of quasi-entities. But just as we had to explain the ontogenesis of unruliness, we must also explain the emergence of these ontologically incomplete realities that serve as the basis for the ordered universe, since they cannot be a brute, arbitrary given. The emphasis of the key philosophical question is thus shifted from how the subject can emerge out of the Real qua substance (thought from being) to how the subject can emerge out of the Real qua void (something from nothing), for the more strictly speaking ontological question of ontogenesis thus proves to be irreducibly entangled with the most fundamental of metaphysical questions. 236 Chapter 10 236 Chapter 10 is no such zero-level: if we go 'all the way down,' we arrive at the Void.”366 This has a surprising consequence, one whose full metaphysical implications quantum mechanics thus summons us to accept: namely, that the closer we get to the origin of all things, the more ontologically incomplete reality is, the less distinguishable its fundamental constituents are from the void, thus forcing us not merely to proclaim that the void is “the only ultimate reality,”367 but more drastically still that “'all there is' is, precisely, not-All, a distorted fragment which is ultimately a 'metonymy of nothing.'”368 In short, what we experience as hard, full reality is at its core a mere vibration of nothingness lacking any true ontological depth, since there exists a certain radical indistinction between being (a structured physical universe) and the void (a structureless zone without any dense ontological determination in any traditional sense).369 But if the building blocks of the world are nothing but variations upon nothingness, then why do they emerge in the first place? And what prevents the universe from imploding upon itself in some kind of triumphant suicidal gesture, leaving us with nothing but the “eternal peace” of the void? Why something rather than nothing? Žižek hints that the answer is to be found in the very tension between the void and this field of quasi-entities that, the further we push them, the more indistinguishable they appear from nothingness itself—using the Higgs field as an example. Physical systems tend towards a state of lowest energy. In another vein, if we take energy away from a system, we should eventually expect to reach a vacuum state where the total energy count would be zero. Yet certain phenomena tell us that “there has to be something (some substance) that we cannot take away from a given system without raising that system’s energy—this 'something' is called the Higgs field: once this field appears in a vessel that has been pumped empty, and whose temperature has been lowered as much as possible, its energy will be further lowered.”370 Incredibly, once a physical system's energy has been lowered to the point where it it is on the brink of zero, this “something” appears, a “something” that requires less energy than nothing. 10.2 Eppur si muove: Ontological Dislocation and the Metaphysics of the Void Not only do we here recognize that “there simply is no basic level,” that “divisions go on indefinitely,” whereby “the quantum level marks the beginning of the 'blurring' of 'basic' full reality,”365 but also that “[o]ne should thus reject the ‘positive’ ontology that presupposes some zero-level of reality where things 'really happen' and dismisses the higher levels as mere abbreviations, illusory self-perception, and so forth. There 236 Chapter 10 Consequently, “'nothingness (the void, being deprived of all substance) and the lowest level of energy paradoxically no longer coincide, that is, it is 'cheaper' (it costs the system less energy) to persist in 'something' than to dwell in 'nothing,' at the lowest level of tension, or in the void, the dissolution of Radicalizing the Subject 237 all being.”371 But here we should radicalize this paradox by extending it to the ultimate metaphysical level of reality. It is not merely that we cannot bring any physical system to a zero-level of energy without the Higgs field positing itself: what the latter suggests is that nothingness is minimally “inconsistent” with itself and that it is this very “inconsistency” that is responsible for the emergence of something out of nothing. The rapture of void, the bliss of an eternity freed from all tension, does not merely come at too high a price for us as creatures living in this world, but is, strictly speaking, impossible. Metaphysics is thus starkly remodulated: What if we posit that “Things-in-themselves” emerge against the background of the Void of Nothingness, the way this Void is conceived in quantum physics, as not just a negative void, but the portent of all possible reality? This is the only true consistent “transcendental materialism” which is possible after the Kantian transcendental idealism. For a true dialectician, the ultimate mystery is not “Why is there something rather than nothing?” but “Why is there nothing rather than something?”372 Although Žižek makes use of the Higgs field mainly as an image, in Less Than Nothing his expansive engagement with quantum mechanics sheds new light on its precise conceptual role in his own thinking. Not only does this engagement enable him to draw out underlying ontological implications from quantum mechanics' fundamental insights, but it more strongly gives us sufficient resources from which we can extract a more strictly speaking metaphysical argument from his discussion of the Higgs field, which attests to how Žižek potentially supplies quantum mechanics with a wider, non- naturalistic foundation while simultaneously founding a new alternative in metaphysics argumentatively independent from its framework. As that which (theoretically) controls whether forces and particles behave differently, the Higgs field has two modes: it is either “switched off” (inoperative) or “switched on” (operative). 236 Chapter 10 While in the first, given that the system is in a state of pure vacuum, forces and particles cannot be distinguished, in the second symmetry between particles and forces are broken so that differentiations among them can occur. However, the paradox lies precisely in the following: what is so unique about the Higgs field is that it is favorable 238 Chapter 10 for it to be “switched on” (operative), for if a system is in a state of pure vacuum “the Higgs field still has to spend some energy—nothing comes for free; it is not the zero-point at which the universe is just 'resting in itself' in total release—the nothing has to be sustained by an investment of energy.”373 Because the pure vacuum requires the expenditure of energy, in order to solve this paradox we are forced to substitute it with another by “introduc[ing] a distinction between two vacuums”: first, there is the “false” vacuum in which the Higgs field is switched off, i.e., there is pure symmetry with no differentiated particles or forces; this vacuum is “false” because it can only be sustained by a certain amount of energy expenditure. Then, there is the “true” vacuum in which, although the Higgs field is switched on and the symmetry is broken, i.e. there is a certain differentiation of particles and forces, the amount of energy spent is zero. In other words, energetically, the Higgs field is in a state of inactivity, of absolute repose. At the beginning, there is the false vacuum; this vacuum is disturbed and the symmetry is broken because, as with every energetic system, the Higgs field tends towards the minimization of its energy expenditure. This is why “there is something and not nothing”: because, energetically, something is cheaper than nothing.374 What is crucial to note with the Higgs field is that the two vacuums whose existence it posits are not by any means equal: rather than encountering a mere polarity, a two-sided principle that brings together a delicate dance of opposites like light and day, life and death, fullness and lack, into equilibrium, we see a constitutive imbalance. 238 Chapter 10 Once we apply this principle cosmologically as a metaphysical principle, instead of having an eternal repetition of creation (breaking of the symmetries) and its destruction (return to the void), reality and its disappearance into the abyss, we come across a “displaced One, a One which is, as it were, retarded with regard to itself, always already 'fallen,' its symmetry always already broken.”375 As such, there is nothing but creation or reality because the “pure” vacuum wherein one would expect absolute repose is “false,” that is, stricto sensu impossible—it structurally must have always already passed over into and Radicalizing the Subject 239 became the “true” vacuum; and although this would appear to make the “false” vacuum theoretically superfluous (it never had, or could, exist) “this tension between the two vacuums [is to] be maintained: the 'false vacuum' cannot simply be dismissed as a mere illusion, leaving only the 'true' vacuum, so that the only true peace is that of incessant activity, of balanced circular motion—the 'true' vacuum itself remains forever a traumatic disturbance.”376 But why? For the precise reason that without this primordial antagonism we could not explain the minimal distinction between the void and its vibrations, between the nothing and the ontologically incomplete realities barely distinguishable from it—in short, how the symmetries between particles and forces could have been broken in the first place. Were we only to have the “true” vacuum, then finitude, materiality, and ultimately experience would be a mere illusion, for there would be no difference between the symmetry of the void and its disturbance, leaving nothing but a nirvana- like principle of nothingness to which all things are reducible. But once we witness the irreducibility of the antagonism within the void itself between its two modes, reality becomes less a seeming that we have to break through than an intrinsic part of the void that necessarily arises as a response to the primordial metaphysical trauma that is the perturbation of the “true” vacuum and whose essence is thus preserved. Even if nothing and the metonymy of nothing is all there is, this presupposes that the latter does not logically collapse into the former although the exact boundary between them is blurred. 240 Chapter 10 Drawing upon insights gained by contemporary science, however, he says that in order to explain the mere existence of things, we must posit that nothingness necessarily fails “to be,” for any attempt to have a purely vacuous void paradoxically costs more energy than things existing against the background of this void. Here we encounter “the primacy of the inner split,”378 whereby irresolvable conflict is at the origin of all things: the absolute is nothing but this fragile de-substantialized process that “arises” out of the self-splitting of a positively charged void, a split that befalls it from within through its own failure “to be” what it is without that very split. There is no primordial fullness, no positive hard reality that self-unfolds according to a creative principle of actualization;379 there is nothing but a pure ontological dislocation of which we can only say eppur si muove (“and yet it moves”). But insofar as this gap cannot be mediated with the absolute, it presents itself as “the non-dialectical ground of negativity,” so that “[t]he old metaphysical problem of how to name the nameless abyss pops up here in the context of how to name the primordial gap: contradiction, antagonism, symbolic castration, parallax, diffraction, complementarity, up to difference.”380 But the name that is perhaps best suited to this is the subject. 240 Chapter 10 Chapter 10 ontology into a dynamic, self-articulating whole. For him, the primordial fact from which metaphysics must begin is the fact that nothingness is. Drawing upon insights gained by contemporary science, however, he says that in order to explain the mere existence of things, we must posit that nothingness necessarily fails “to be,” for any attempt to have a purely vacuous void paradoxically costs more energy than things existing against the background of this void. Here we encounter “the primacy of the inner split,”378 whereby irresolvable conflict is at the origin of all things: the absolute is nothing but this fragile de-substantialized process that “arises” out of the self-splitting of a positively charged void, a split that befalls it from within through its own failure “to be” what it is without that very split. There is no primordial fullness, no positive hard reality that self-unfolds according to a creative principle of actualization;379 there is nothing but a pure ontological dislocation of which we can only say eppur si muove (“and yet it moves”). But insofar as this gap cannot be mediated with the absolute, it presents itself as “the non-dialectical ground of negativity,” so that “[t]he old metaphysical problem of how to name the nameless abyss pops up here in the context of how to name the primordial gap: contradiction, antagonism, symbolic castration, parallax, diffraction, complementarity, up to difference.”380 But the name that is perhaps best suited to this is the subject. This would inscribe this metaphysics of the void into the legacy of the German Idealist attempt to think substance as subject, while bringing us simultaneously beyond it and into a new sphere, a new materialism.381 If German Idealism has taught us that epistemic ambiguities in idealism (how can the subject overcome its own synthetic mediation of the world and reach the latter an sich?) occur because our division from being is identical with being's own division to itself, quantum mechanics calls us to radicalize this notion of the subject even further and claim that it exhibits the same structure as the split responsible for all things: This, perhaps, is how one can imagine the zero-level of ontology into a dynamic, self-articulating whole. For him, the primordial fact from which metaphysics must begin is the fact that nothingness is. 238 Chapter 10 Hence, the reason Žižek can say that if “[t]he answer to 'Why is there Something rather than Nothing?' is thus that there is only Nothing, and all processes take place 'from Nothing through Nothing to Nothing,'” then “this nothing is not the Oriental or mystical Void of peace, but the nothingness of a pure gap (antagonism, tension, 'contradiction'), the pure form of dislocation ontologically preceding any dislocated content,”377 thus radically changing our very notion of nothingness itself. The Higgs field thus not only offers us an interesting naturalistic explanation of the world when used in cosmology but more strongly hints The Higgs field thus not only offers us an interesting naturalistic explanation of the world when used in cosmology, but more strongly hints at a new variety of metaphysics. Fleshing out its broad consequences, Žižek is not only able to offer a new account of the emergence of things that is inspired by though independent from quantum mechanics, but can also radicalize his ontology of the subject, thereby weaving metaphysics and 240 Chapter 10 240 Chapter 10 This would inscribe this metaphysics of the void into the legacy of the German Idealist attempt to think substance as subject, while bringing us simultaneously beyond it and into a new sphere, a new materialism.381 If German Idealism has taught us that epistemic ambiguities in idealism (how can the subject overcome its own synthetic mediation of the world and reach the latter an sich?) occur because our division from being is identical with being's own division to itself, quantum mechanics calls us to radicalize this notion of the subject even further and claim that it exhibits the same structure as the split responsible for all things: If German Idealism has taught us that epistemic ambiguities in idealism (how can the subject overcome its own synthetic mediation of the world and reach the latter an sich?) occur because our division from being is identical with being's own division to itself, quantum mechanics calls us to radicalize this notion of the subject even further and claim that it exhibits the same structure as the split responsible for all things: This, perhaps, is how one can imagine the zero-level of creation: a red diving line cuts through the thick darkness of the void, and on this line, a fuzzy something appears, the object-cause of desire—perhaps, for some, a woman's naked body (as on the cover of this book). Does this image not supply the minimal coordinates of the subject-object axis, the Radicalizing the Subject 241 truly primordial axis of evil: the red line which cuts through the darkness is the subject, and the body its object?382 truly primordial axis of evil: the red line which cuts through the darkness is the subject, and the body its object?382 If in Žižek's strictly metaphysical thinking the emphasis shifts from how the subject can emerge out of the Real qua substance (thought from being) to how the subject can emerge out of the Real qua void (something from nothing), it is because there is no radical difference between the creation of the world and the creation of the world of meaning. Both exhibit the same catastrophic cutting, so that the primary question becomes that of the originary catastrophe itself rather than just one of its specific “instantiations” in human freedom. 240 Chapter 10 The fact that the answer for both (the Real is always already subject) is identical proclaims that there is only the Real of the gap, that the splitting precedes what is split. Žižek's paradoxical conclusion is that if the subject is “the primordial Big Bang,”383 it cannot merely be that of the universe of meaning, but must also be that of all that there is. The great lesson to be drawn from the metaphysical archaeology of the subject is that “[w]hat, ultimately, 'there is' is only the absolute Difference, the self-repelling Gap”:384 although substance qua nature proves itself to be not-all (traces of which we see in German Idealist accounts of negativity and most radically in contemporary science), so that we must posit the logical precedence of the void to which its indivisibility is always already contrasted and thus impossiblized, nevertheless just like substance qua nature this originary void in itself also proves to be split—a split that is not merely a mere catastrophic cut, but a rupturing antagonism imbued with an “energetic” or “energizing” force, a force that is somehow less than nothing. This has two drastic consequences. First, the complex order of the cosmos may at some point in time be reduced to unimaginable chaos (being nothing but a heterogenous play of powers) but never to a nirvana-like nothingness, for before the moment at which an absolute zero would be reached the void would, as it were, break its own symmetry in advance and prevent its own repose. We cannot escape the fragile and macabre dance of the not-all: non-coincidence is the pulsating heart of all reality, a heart whose diastoles and systoles may whimper under their own weight and threaten to collapse upon themselves at any moment in one great and final apocalyptic turmoil, yet cannot, because this turmoil costs the universe less energy than the pure virtuality of the void. Second, if thinking substance as subject reveals that If in Žižek's strictly metaphysical thinking the emphasis shifts from how the subject can emerge out of the Real qua substance (thought from being) to how the subject can emerge out of the Real qua void (something from nothing), it is because there is no radical difference between the creation of the world and the creation of the world of meaning. 240 Chapter 10 Both exhibit the same catastrophic cutting, so that the primary question becomes that of the originary catastrophe itself rather than just one of its specific “instantiations” in human freedom. The fact that the answer for both (the Real is always already subject) is identical proclaims that there is only the Real of the gap, that the splitting precedes what is split. Žižek's paradoxical conclusion is that if the subject is “the primordial Big Bang,”383 it cannot merely be that of the universe of meaning, but must also be that of all that there is. The great lesson to be drawn from the metaphysical archaeology of the subject is that “[w]hat, ultimately, 'there is' is only the absolute Difference, the self-repelling Gap”:384 although substance qua nature proves itself to be not-all (traces of which we see in German Idealist accounts of negativity and most radically in contemporary science), so that we must posit the logical precedence of the void to which its indivisibility is always already contrasted and thus impossiblized, nevertheless just like substance qua nature this originary void in itself also proves to be split—a split that is not merely a mere catastrophic cut, but a rupturing antagonism imbued with an “energetic” or “energizing” force, a force that is somehow less than nothing. This has two drastic consequences. First, the complex order of the cosmos may at some point in time be reduced to unimaginable chaos (being nothing but a heterogenous play of powers) but never to a nirvana-like nothingness, for before the moment at which an absolute zero would be reached the void would, as it were, break its own symmetry in advance and prevent its own repose. We cannot escape the fragile and macabre dance of the not-all: non-coincidence is the pulsating heart of all reality, a heart whose diastoles and systoles may whimper under their own weight and threaten to collapse upon themselves at any moment in one great and final apocalyptic turmoil, yet cannot, because this turmoil costs the universe less energy than the pure virtuality of the void. 240 Chapter 10 Second, if thinking substance as subject reveals that 242 Chapter 10 242 Chapter 10 Chapter 10 242 nature itself is anything but a powerful, creative source, a pure affirmation displaying ontological closure à la Spinoza, then the void in being imposed upon us by the metaphysical archaeology of the subject demonstrates something much more radical than the fact that the human subject is not a mere accidental breakdown of the natural order, but actually depends upon an inborn negativity that impossiblizes its attempt at a self-articulating totality from the outset (“we should accept that nature is 'unnatural,' a freak show of contingent disturbances with no inner rhyme or reason”385). It further demonstrates that it is not the human subject that is the ultimate ontological catastrophe, but reality as such: There is nothing, basically. I mean it quite literally. But then how do things emerge? Here I feel a kind of spontaneous affinity with quantum physics, where, you know, the idea there is that [the] universe is a void, but a kind of a positively charged void—and then particular things appear when the balance of the void is disturbed. And I like this idea spontaneously very much, that the fact that it's not just nothing—things are out there—it means that something went terribly wrong, that what we call creation is a kind of a cosmic imbalance, cosmic catastrophe, that things exist by mistake.386 However, if what we call creation, the primordial Big Bang, is some kind of cosmic mishap, then this mishap must in some sense have been unavoidable. The ontological catastrophe that is creation is a necessity because nothingness itself fails “to be” nothingness and through its failure never ceases to create “something”; and since this “something” is paradoxically less than nothing, the infinite proliferation of ontologically incomplete quasi-entities is only minimally distinguishable from the purely vacuous void itself. The world does not find its origin in a willed creation, an impersonal emanation from a sphere of consummate being overflowing in itself, or the uncontainable productivity of substance qua nature as absolute power—no, the world comes to be in an originary disaster, a primordial metaphysical cataclysm that is always already occurring, it being impossible to pinpoint a logical moment within which nothingness could have succeeded at “being” itself. Yet the split that is the subject in both its modes should not be considered a loss, but rather a liberation. Radicalizing the Subject 243 is paradoxically “a deprivation, a gesture of taking away which is in itself a giving, productive, generative, opening up and sustaining the space in which something(s) can appear,”387 even if these something(s) only exist because ontological catastrophe is uncannily less than nothing. is paradoxically “a deprivation, a gesture of taking away which is in itself a giving, productive, generative, opening up and sustaining the space in which something(s) can appear,”387 even if these something(s) only exist because ontological catastrophe is uncannily less than nothing. The veritable horror of substance as subject—ontological catastrophe— revealed through the metaphysical archaeology of the psychoanalytico- Cartesian subject is therefore twofold. At the first level, it indicates that nature was always already a sickly creature, its rhythms always already disordered, unsteady, broken. Within the “passage” from drives to desire, substance to subject, no positive content is added, nothing changes at the level of the Real qua Real. What the subject imposes upon us is the realization of the constitutive contingency that lies at the centre of creation, the fragility of the seemingly ordered cosmos that has arisen before us, so that the immanent causality of nature is seen to be predicated upon its potential internal breakdown. On the second level, the metaphysical archaeology of the subject tells us that if we remain here, we have not gone far enough. If we are truly to understand how the not-all of substance could sustain itself in its own precarious being and, more fundamentally, how it could have emerged in the first place, we must radicalize the subject understood as an underlying dysfunctioning of substance's dynamics. If, as Žižek maintains, experience is revelatory of the fact that reality must be not-all, then it also contains a still deeper truth: reminding us that substance fails to ground itself, that it is riddled with holes, that it opens up the logical space of the substance and its contrast, the void, forcing us to explore the latter. We have thus come full circle: an ontological account of the emergence of the subject (the arising of representation out of being) has morphed into a discussion concerning the ultimate metaphysical structure of the world (the upsurge of “something[s]” out of nothing), because the two questions are seen to be intimately linked, the former automatically leading to the latter. 240 Chapter 10 Ontological catastrophe Radicalizing the Subject 243 Radicalizing the Subject 243 And falling upon this intuition, Žižek asks what if the same structure is at play at both levels, so that the answer to the latter will prove to be similar to that of the former, namely, that the movement from the Real of void to creation is not as initially problematic as it may appear because the Real of void shows itself to be always already tainted by the Real of gap. Ontological catastrophe is the zero-level of reality because Chapter 10 244 244 there is a necessity of the primordial void of which mystics speak to be disturbed, a necessity that is not due to the notional necessity of right- wing Hegelian theology (in order for the Idea to actualize itself as infinite freedom, it must sacrifice itself in nature so that it can fully return to itself in spirit after arising from its own ashes) or the conditional necessity of the late Schellingian quaternity (if the Ungrund were to become self-manifest to itself, then it would have to arbitrarily limit its primordial freedom in the kabbalistic act of contracting finitude) but a necessity that derives from the impotence of the void “to be” a purely vacuous void, a necessity that is synonymous with the absolute non-coincidence of nothingness to nothingness that is motor of all things. The language of catastrophe is in this sense completely justified, for creation does not present itself as intrinsically beautiful and creative or purposeful, but rather as a monstrous seat of ontological abortions and terrors devoid of sense though from time to time capable of miracles. Rather than the world being given to us by the self- overflowing exuberance of the Good or the personal hand of God, it moves from nothing through nothing to nothing due to the self-repelling gap of the void, the internal split of a positively charged nothingness, which denies all positivity to that which it sets in motion: “[p]erhaps this gap separating the two vacuums is then the ultimate word (or one of them, at least) that we can pronounce on the universe: a kind of primordial ontological dislocation or différance on account of which, no matter how peaceful things may appear sub specie aeternitatis, the universe is out of joint and eppur si muove.”388 10.3 The Act of Uneasy Self-Begetting: Entscheidung and the Paradoxical Self-Positing of Freedom By plunging into the abyssal origins of subjectivity Žižek is not just led to a new, disquieting form of transcendental materialism that offers an original account of the relationship between the Real and the Ideal, with stark implications for our understanding of substance, the emergence of order, and even the beginnings of the cosmos. He is also led to develop a theory of the unconscious that challenges both the traditional Freudian and Lacanian accounts, and even the one developed by Schelling that he draws upon. This theory deserves to be highlighted in some detail because it is revelatory of one of the most difficult and provocative aspects of Žižek's philosophy: Radicalizing the Subject 245 that is, the pure act at the origin of the subject that is simultaneous with the ontological passage through madness that forces upon us the rational necessity of speculative fabulation to embark upon a metaphysical archeology of the subject. Schelling's account of the birth of subjectivity is more than a theory of the meta-transcendental state of affairs that must be in place if subjectivity is to arise out of nature with a triumphant cry, for central to Schelling's ontogenetic narrative is that the former, though providing necessary conditions for the birth of the subject in/out of nature, does not supply its sufficient conditions. To explicate the moment of ontological judgement that institutes the pure difference between Grund and existence and with it the entire universe of human meaning (the Present), Schelling must concern himself with the very moment of unconscious decision (Entscheidung) by which subjectivity paradoxically liberates itself out of the immanent field of being to which it once “belonged” in a moment that is a self-caused immaculate conception insofar as this self-positing, which cannot be deduced according to notional necessity, is an arbitrary act of pure freedom. What is of paramount importance is the root of the term, which in English displays a similar play on words: Entscheidung as Ent-Scheidung, decision as de-scission. Since the German suffix -ung refers to a process, Entscheidung designates a “de-scissioning” at the basis of self-consciousness and language as that which, by creating the Present, banishes the Past into the abyssal dregs of forgotten and inaccessible time. 10.3 The Act of Uneasy Self-Begetting: Entscheidung and the Paradoxical Self-Positing of Freedom What Žižek focuses on is precisely the formal structure of the act itself, its activity of severing the Real into the parallax of the unconscious drives of nature and phenomenal reality, whereby the act itself is primordially repressed as necessary for the dawning of full-fledged subjectivity and becomes an impossible object for any discourse. Recognizing that the Entscheidung itself is that which originarily constitutes the conscious/unconscious distinction, Žižek argues that one of the fundamental breakthroughs of the Weltalter is its demonstration that drives themselves are, strictly speaking, nonconscious: if the very conscious/ unconscious distinction only occurs with the utterance of the Word (there cannot be a ground without a grounded: prior to the grounded, the ground cannot be posited as ground as such, for it is merely a self-subsisting, semi-closed system of materiality, rather than the ground of existence) then it semi-closed system of materiality, rather than the ground of existence) then it 246 Chapter 10 6 Chapter 10 246 Chapter 10 His original addition to psychoanalysis is the formation of a conception of “the subject [which] at its most elementary is indeed 'beyond the unconscious': an empty form deprived even of unconscious formation encapsulating a variety of libidinal investments.”389 Radicalizing the Subject 247 Here again we see just how much Žižek's account of the subject is highly reliant upon his “modification” of the Weltalter in a way that challenges his so-called Hegelianism. Here again we see just how much Žižek's account of the subject is highly reliant upon his “modification” of the Weltalter in a way that challenges his so-called Hegelianism. Žižek's controversial wager is that there is something more primordial within Schelling's descriptions of the birth of the subject out of the utter twilight of pre-personal being than an account of the self-transformation of the “unconscious” spirit of nature as it rises towards the openness of self-revelation. In a move similar to psychoanalysis' claim that the material processes of our organs cannot be, strictly speaking, unconscious (physiological stimuli and reflexes are of a radically different nature),390 Žižek levels out the richness of the Schellingian account of nature to a mere material autopoiesis that is unable to explain the true seat of personality. This is also why Žižek is so adamant that the unconscious is not to be equated with the set of irrational drives that structurally oppose and yet affect the self-transparency of rationality as we see in various forms of Lebensphilosophie.391 What Schelling's account of decision as the metapsychological event par excellence proclaims is that there is no point of positive juncture between nature as Grund and the subject of the unconscious, insofar as decision takes over/usurps the logically primary position of the Grund through its own self-positing freedom, but in a manner that its self-positing is more fundamental than, and even erases, its dark pre-history: the libidinal-material chaos within nature does not come close to establishing the unconscious proper because the latter is never completely subsumable within the dynamic movement of natural history or laws, for it institutes itself into the fabric of being by means of a self-caused immaculate conception. 246 Chapter 10 would be philosophically fallacious to call this energetic rotation of energy the unconscious proper or the true foundation of subjectivity, even if it incites (or to put it differently negatively carves up the room for) the latter's founding gesture. The result is that the Žižekian-Schellingian subject of the unconscious is radically non-coincident with both the id-forces of the body in its primary mode (the Real of Triebe can only be unconscious as a secondary effect of the self-positing of the act of de-scission[ing] as such) and the more-than-conscious matrix of the Symbolic (the self-generating play of language and culture only emerges after the founding gesture that marks the beginning of transcendental self-reflexivity). That is, the unconscious is the very gap, the irreconcilable parallax, between both registers: it is synonymous with the subject itself as the impossible in-between that binds together materialism (being) and idealism (thought) in their non- relationality by protruding out of yet being simultaneously spectrally present in both. As an irreducible, self-positing negativity that institutes the realm of culture and eternally separates it from nature, the subject of the unconscious is the true site of freedom. Here lies the challenge to Freud and Lacan, who broadly could be said to have respectively located the unconscious in the biological movement of instinctual energy and the alienating effects of language. For Žižek one must presuppose a more primordial level of unconscious activity than that of the biologically closed libidinal energetics within the corpo-Real of the body or that which emerges through the split between the subject of enunciation and the enunciating subject caused by the unpredictable reverberations of meaning within the infinite web of language: namely, an act that exploits the libidinal frenzy of the Real of the human body, the unruliness represented by the Todestrieb, to ground the self-generation and self-proliferation of the automatic machinery of language as a reaction formation. In this sense, Žižek's reactualization of the Schellingian unconscious is an attempt to sublate both the traditional Freudian and Lacanian accounts within a higher dialectical unity by showing their dependence on another, more fundamental conceptual level. 246 Chapter 10 If freedom is to be truly free, then we must not be able to deduce it according to notional necessity; it is as if, when substance risks touching the void in the painful oscillation of drives constitutive of the unruliness of the human organism, the subject creates itself out of nothing, with all the paradox that entails: In the psychoanalytical perspective, of course, this primordial act of free self-positing cannot but appear as the Real of a fantasy-construction: the status of the primordial act is analogous to that of the Freudian parricide—although it never effectively took place within temporal reality, one has 248 Chapter 10 248 Chapter 10 This recollective interiorization (Er-inner-ung) is the direct effect of the subject's uneasy self-begetting, the aftermath of its self-caused immaculate conception in/out of matter that exploits the immanent negativity of the latter for the institution of its “miracle”:394 this recollection is not a mere remembering of something in cosmic memory, an always already existing but non-actualized self awaiting the dawning of consciousness in the world—the very gesture of interiorization is that which creates what is interiorized, that is to say, the self in which the world withdraws out of its bloody night, in such a way that the recollected self or interiority retroactively posits itself as that which was always at the starting point.395 Even a self-caused immaculate conception in being demonstrates a threatening belatedness, for it is stuck in a dialectical contradiction that cannot be resolved: “'[r]econciliation' between subject and substance means acceptance of this radical lack of any firm foundational point: the subject is not at its own origin, is secondary, dependent upon its substantial presuppositions; but these presuppositions do not have a substantial consistency of their own and are always retroactively posited.”396 The only successful ontogenesis is one that underlines the very impossibility, the upper limit, of ontogenesis as such: what is essential is not a transition (whether it be understood in terms of teleological development or the potential productivity of breakdown, as we see in the night of the world) but the pure difference that is the full-fledged subject (which is responsible for “the Radicalizing the Subject 249 madness of the passage to the Symbolic itself”397) which institutes a “gap which makes impossible any account of the rise of the New in terms of a continuous narrative.”398 But we must be very attentive to the conceptual movement if we are not to lose its radicality. Insofar as the chaotic oscillation of drives we witness in unruliness only becomes the Grund of existence after the act of decision, drives only become unconscious in the aftermath of the act itself. Due to the latter's irreducible self-positing, consciousness and the Real of drives qua unconscious both emerge in one magical brushstroke that retroactively constitutes its own evolutionary past—covers up its contingency—by subsuming the ontogenetic pre-history of the subject as part of its own self-effectuation through the paradoxical causality of the Freudian Nachträglichkeit/Lacanian après-coup/Hegelian positing of presuppositions. 248 Chapter 10 to presuppose it hypothetically in order to account for the consistency of the temporal process. The paradox of the primordial act is the same as that of Baron Münchhausen pulling himself out of the swamp by lifting himself by the hair—in both cases, the subject is somehow already here prior to existence and then, by way of free act, creates-posits himself, his own being.392 The subject's freely posited withdrawal into self is intrinsically paradoxical, for the very act of self-positing creates the very self that is at the origin of said positing. This is why we cannot escape the Baron Münchhausen dilemma: the subject is miraculously present at its own birth.393 But how? To follow Žižek and draw upon a Hegelian logical category, when full-fledged subjectivity emerges in being it can be said to come into existence by a process of recollecting or interiorizing itself. 250 Chapter 10 Real, we are not warranted to make certain claims we have been forced to make: when the subject asserts itself at the beginning of its own pre-logical genesis (posits its own presupposition), it is not actually giving birth to itself (which implies a poietic production or genesis) in some kind of temporal loop, but directly creates itself at the instant of its own upsurge. The example of Baron Münchhausen's pulling himself up out of the swamp by his own hair expresses the intrinsic difficulty posed to any transcendental materialist account of the subject, for the subject does not exist before the very act of self-positing that it nonetheless paradoxically enacts—hence why it all happens in one magic brushstroke. The subject has no history except at the level of speculative fabulation (rationally justified mythology), for it is only after the subject's self-positing that we can raise the question of its origins: “what escapes our grasp is not the way things were before the arrival of the New, but the very birth of the New, the New as it was 'in itself,' from the perspective of the Old, before it managed to 'posit its presuppositions,'”399 which is why Žižek can say that the true arche-fossil for correlationism is not occurrences in cosmological or geological time before the transcendental, but the very transcendental itself.400 In this sense, the difficulty of the task of speculative fabulation finds itself reflected upon itself: that is to say, doubled. Not only do there exist psychoanalytical defence mechanisms to cover up the abyss of unconscious decision at the “heart” of subjectivity by means of fantasy constructions, but because the only way we have access to the latter is through a mytho-poetic ontogenesis of the subject's dark pre-history in the ontological vicissitudes of being, there is always the tendency to swap the real trauma (the pure contingency of the act as absolute spontaneity) for a fake (the painful oscillation of drives in the human infant that shows nature's sickness unto death in humanity). Even Schelling has a tendency to make us forget that the Grund, “this monstrous apparition with hundreds of hands, this vortex that threatens to swallow everything, is a lure, a defence against the abyss of the pure act,”401 which is why psychoanalysis is so important. 248 Chapter 10 Here, however, we notice the difficulty of the Real-as-origin: since the abyssal beginning of the subject—the act of decision—effaces itself in the simultaneous gesture of converting the Real of drives into the unconscious as a libidinal system and founding consciousness, the birthplace of the subject becomes a mere posit that is in and of itself never accessible in language; and, as that which only shows itself in its very withdrawal (“sous rature”), it is denied the right of even “indirectly” shining through the cracks of the Ideal. It can only be narrated. Although we must posit the ontogenetic condition of desire (a pre-logical antagonism in the Real-as- excess) as that which precedes and in a certain sense renders possible the self-positing of the decision, the latter proves itself to be not only superior by saying “No!” to substance, thereby setting up a new age of the world within which the Past is always already “lost” through symbolization (an act that institutes the hegemony of the Real-as-lack), but also to be ontologically primary, despite being ontogenetically secondary. In a paradoxical movement where the cause-and-effect chain loses its grip, the self-unfolding causality of nature as substance is “torn apart” in the upsurge of freedom as self- relating negativity, which demands primordiality at all costs, even making being infinitely non-coincident to itself. It must be remembered, however, that this movement from Grund to subjectivity, from the breakdown of nature into a writhing mass of heterogeneous forces to the abyss of unconscious decision, must be described at the level of mytho-poetics. At the level of the 250 Chapter 10 Although this secondary trauma must still be considered as having explanatory merit—it is, after all, a necessary condition for the primary trauma—the point is to pierce through one trauma (ontological catastrophe as the immanent breakdown of substance) to arrive at a more primordial one (ontological Radicalizing the Subject 251 catastrophe as the pure difference that severs being in two). The “repressed spectral 'virtual history' is not the 'truth' [...] but the fantasy that fills in the void of the act [...] the secret narrative that tells its story is purely fantasmatic,”402 which is why: Schelling's fundamental move is thus not simply to ground the ontologically structured universe of logos in the horrible vortex of the Real; if we read him carefully, there is a premonition in his work that this terrifying vortex of the pre-ontological Real itself is (accessible to us only in the guise of) a fantasmatic narrative, a lure destined to distract us from the true traumatic cut, that of the abyssal act of Ent-Scheidung.403 This is why Schelling's inability to complete his middle-late project of an ontology of freedom is of such paramount importance to Žižek, for “[t]he repeated failure of his Weltalter drafts signals precisely Schelling's honesty as a thinker—[not only] the fact that he was radical enough to acknowledge the impossibility of grounding the act or decision in a proto-cosmic myth,” but also that he was “compelled to posit an uncanny act of Ent-Scheidung (decision or separation), an act in a way more primordial than the Real of the 'eternal Past' itself.”404 Focusing on the necessary posit of the convulsing labyrinth that is the pre-symbolic Real-as-excess in human unruliness, and plunging ourselves into it, we risk forgetting the abyss of the Real-as-origin that stares us in the face. The painful oscillations of the Grund in unruliness merely defer us from the true terror that is the pure act instituting the Symbolic's infinitely self-reflexive play of signifiers in which we live and which guarantees that it possesses no direct connection to the extra-notional world. Because this act absolutizes the short circuit between the Innenwelt and Aussenwelt, we can only encounter its abyssal origins in their very gesture of withdrawal. 250 Chapter 10 It is not nature, even in its mode of self-erasure (N ≠ N), that is the first revelation,405 but rather, the void within which the Entscheidung spins—and we can only catch glimpses of the latter as the impossible in-between negatively tying together nature and culture, the Real and the Symbolic, and thereby hope to develop a comprehensive theory of subjectivity inclusive of a new, paradoxical form of metaphysics from within the mytho-poetics of the subject's slow, unsteady, and painful “emergence” out of the vicissitudes 252 Chapter 10 252 Chapter 10 Chapter 10 252 C 252 of being. But all of this has an interesting implication. If the Symbolic is a self-organizing system that freely constitutes the fabric of experience without any contact with objective reality, then this suggests that, from within its ontologically solipsistic dance of cybernetic ciphering, we can break through the impenetrable dusk of psychosis as we (in a reflexive albeit fabulative gesture) see it as the madness it is in a careful reconstruction of its trauma. Here, however, we encounter one of the most fundamental and perhaps paradoxical conclusions of Žižek's parallax ontology. It must be recalled that the trauma at the birthplace of the human subject is not only coincident with its freedom:406 that is, its withdrawal from one's natural environment into an irreal, virtual self,407 but its exploration necessarily demonstrates the structure of fantasy as we insert ourselves as a pure gaze in the moment of our own birth.408 Not only can materialism only justify itself in “the shadow cast by [self-grounding] idealism's insurmountable incompleteness,”409 but more radically, if we follow the true horror of substance as subject at the core of the latter, then we are forced to conclude that a metaphysics is only possible as a form of “successful” psychotic thinking,410 a thinking that, from within the clutches of idealism as an insurmountable ontological psychosis and using the very energy and internal limitations of this psychosis, manages to succeed in achieving the impossible coincidence of subjective and objective reality: that is to say, in developing a comprehensive metaphysics of the Real. A cure to our correlationist imprisonment being excluded from the outset, philosophy has no hope of offering a therapy. 250 Chapter 10 What is more, if such a form of thinking is to be truly successful, not only must we find a way to overcome the realism-idealism debate within idealism, but we must also do so from the side of realism by writing the great epic of being as the eternal Past to come to terms with the ontological passage through madness at the latter's foundation, a task that requires a self-conscious mythologizing, fictionalization, or retrospective narration and is only achievable by entering the abyss of the spectral Real-as-origin and fending off the thrust of unconscious desires that try to protect us from it in order to draw out its stark metaphysical consequences. Only a scientific, psychoanalytically guided fabulation allows one to catch sight of the vanishing mediator that enacts the withdrawal into the night of the world. But how is a radical Radicalizing the Subject 253 Radicalizing the Subject 253 subjective idealism capable of such a feat? And as what brand of metaphysics should we baptize it? Notes 341. For a discussion of the passage, see chapter 9. 342. Žižek, The Ticklish Subject, pp. 88–89. 343. Lacan, “The Mirror Stage as Formative of the I Function,” Écrits, p. 96/77. 343. Lacan, “The Mirror Stage as Formative of the I Function,” Écrits, p. 96/77. 344. See chapter 4. 345. Žižek, Living in the End Times, pp. 350–51. I capitalize “the Real” for consistency. 346. Schelling, Weltalter III, p. 219. 347. Žižek, The Indivisible Remainder, p. 23. 348. Johnson, Žižek’s Ontology, p. 92. 349. Žižek, Less Than Nothing, p. 197. 350. Ibid., p. 198. Compare with pp. 292ff. and p. 304. 350. Ibid., p. 198. Compare with pp. 292ff. and p. 304. 351. Žižek, The Ticklish Subject, p. 33. 351. Žižek, The Ticklish Subject, p. 33. 352. Johnson, Žižek’s Ontology, p. 92. 353. Žižek, The Indivisible Remainder, p. 92. 353. Žižek, The Indivisible Remainder, p. 92. Ibid. 354. Ibid. 355. Žižek, Less Than Nothing, p. 905. 355. Žižek, Less Than Nothing, p. 905. 356. Žižek, The Indivisible Remainder, p. 220. See also p. 223. 357. Žižek, Less Than Nothing, p. 923. 358. Ibid., p. 377. 358. Ibid., p. 377. 359. Ibid., p. 947. 359. Ibid., p. 947. 360. Ibid., p. 925. 360. Ibid., p. 925. 361. Ibid., p. 724. 361. Ibid., p. 724. 362. See, ibid., pp. 918–19; and The Indivisible Remainder, pp. 220–31. 363. See chapter 5. 363. See chapter 5. 364. Kant, The Critique of Pure Reason, A 156/B195. 365. Žižek, Less Than Nothing, p. 726. 365. Žižek, Less Than Nothing, p. 726. 366. Ibid., p. 730. 366. Ibid., p. 730. 367. Ibid., p. 726. 367. Ibid., p. 726. 368. Ibid., p. 641. 369. Ibid., p. 60. 254 Chapter 10 370. Žižek, The Puppet and the Dwarf: The Perverse Core of Christianity (Cambridge: MIT Press, 2003), p. 93. 371. Ibid. 371. Ibid. 372. Žižek, Less Than Nothing, p. 925. 373. Ibid., p. 944. 373. Ibid., p. 944. 374. Ibid., pp. 944–45. 375. Ibid., p. 949. 375. Ibid., p. 949. 376. Ibid., pp. 949–50. 376. Ibid., pp. 949–50. 377. Ibid., p. 38. 378. Ibid., p. 386. 379. Žižek, Living in the End Times, p. 232. 380. Žižek, Less Than Nothing, p. 950. 381. Ibid., p. 60. 381. Ibid., p. 60. 382. Ibid. 382. Ibid. 383. Žižek, The Ticklish Subject, p. 31. 384. Žižek, Less Than Nothing, p. 378. 385. Ibid., p. 298. 385. Ibid., p. 298. 385. Ibid., p. Notes 298. 386. Žižek!, dir. Atra Taylor (Zeitgeist Video, 2007). 386. Žižek!, dir. Atra Taylor (Zeitgeist Video, 2007). 387. Žižek, The Puppet and the Dwarf, p. 93. 388. Žižek, Less Than Nothing, p. 377. 388. Žižek, Less Than Nothing, p. 377. 389. Žižek, Living in the End Times, p. 311. 390. Freud, “Instincts and Their Vicissitudes” (1915), SE, XIV, p. 118. 391. See Žižek, The Indivisible Remainder, pp. 28 and 174. 392. Ibid., p. 19. 393. Ibid. 393. Ibid. 394. Žižek and Daly, Conversations with Žižek, p. 59. 394. Žižek and Daly, Conversations with Žižek, p. 59. 95. Žižek, Less Than Nothing, p. 235. 395. Žižek, Less Than Nothing, p. 235. 395. Žižek, Less Than Nothing, p. 235. 396. Žižek, Living in the End Times, p. 229. 397. Žižek, Less Than Nothing, p. 331. 398. Ibid., p. 273. 99. Ibid. 399. Ibid. 399. Ibid. 400. Ibid., p. 644. 400. Ibid., p. 644. 401. Žižek, The Fragile Absolute, p. 70. 401. Žižek, The Fragile Absolute, p. 70. 402. Žižek, Less Than Nothing, p. 273. 402. Žižek, Less Than Nothing, p. 273. 403. Ibid., p. 275 (taken initially from The Fragile Absolute, p. 73). 404. Ibid., p. 274. Radicalizing the Subject 255 Radicalizing the Subject 255 405. Schelling, Freiheitsschrift, p. 284. 406. Žižek, Less Than Nothing, p. 265. 407. Žižek, The Ticklish Subject, p. 35. 408. Žižek, The Indivisible Remainder, p. 22. 409. Johnston, Žižek’s Ontology, p. 19. 410. Here I am playing with Henri Maldiney’s reading of Hegel’s Phenomenology as a “successful depressive thinking” and taking up certain gestures already hinted at in Jean-Christophe Goddard’s interpretation of this passage. Compare Maldiney, Penser l’homme et la folie, p. 27; and Goddard, Mysticisme et folie. Essai sur la sim- plicité (Paris: Desclée de Brouwer, 2002), pp. 83–84. I return to these points in chapter 12. III Overcoming Idealism III Overcoming Idealism III Chapter 11 From Radical Idealism to Critical Metaphysics How Idealists Write Being's Poem Chapter 11 From Radical Idealism to Critical Metaphysics How Idealists Write Being's Poem Žižek's transcendental materialism understands itself as an uncanny variation on the late German Idealist theme of providing an ontogenesis of the subject consistent with transcendental idealism. But being more than a rethinking of its central problems, it presents itself as a highly rigorous psychoanalytical reconstruction of its unconscious Grundlogik, which reveals itself as a disavowed insight into the identification of subjectivity with an immanent rupture in being so devastating that not only does being lose all direct access to itself, but we must actually posit ontological dislocation as the primary metaphysical fact. In so doing, however, it would appear that there is a potentially fatal inconsistency in Žižek as he oscillates between an inconsistent idealism (the Hegelian notional or symbolic Real-as-lack) and a self-sabotaging materialism (the Schellingian pre/extra-symbolic Real- as-excess). What is more, pointing to the immense difficulty of describing the emergent parallax of being and thinking—the ontological passage of madness—from within the ontological solipsism of thinking itself, Žižek argues for the necessity of a form of thinking that would enable being and thinking to be fully reconciled from the side of being itself through the mytho-poetic narrativization of the moment of parallax. If radical idealism can succeed at overcoming itself and writing being's poem, it will prove itself capable of being maximally idealistic and realistic in a single gesture that leaves behind the speculative throes of correlationism by rendering correlation itself Chapter 11 260 immanent to the absolute, thus becoming a critical metaphysics that has much to offer today to the so-called speculative turn. From Radical Idealism to Critical Metaphysics 261 idealism: it is not only that concepts do not need to adequate themselves with objects subsisting in a world that exists despite me; more radically, the free ciphering of the Real by the Symbolic means that signification has nothing to do with objective reality in itself, for signifiers only participate in an endless chain of self-relation that precludes access to the “outside” world. There is no room possible for a realist epistemology within Lacan because the very link between signifier and extra-linguistic object is cut. And although Žižek remains unsatisfied with taking such a theoretical edifice as a given and seeks to understand the ontological/meta-transcendental event that it must imply (thus recognizing the ultimate need for giving an account of the Symbolic if psychoanalysis is to find a proper scientific footing) the constraints that Lacan himself bestows upon discourse make Žižek's project appear intrinsically problematic. Doesn't the Real in itself prior to or outside of language remain essentially unknowable (because it is always already constituted by a subject, therefore produced by it, and never without its taint)? Isn't the pre- or extra-symbolic Real an impossible concept, for only that which can be spoken in language exists? Nevertheless, Žižek offers more than another sophisticated form of structuralist linguistic idealism. If Lacanian psychoanalysis proclaims that the Real can only appear as a lack within the ciphering activity of language, Žižek attempts to break out of the correlationist circle of language by showing that the very inconsistencies of thinking offer a solution to the problem of how we are able to reach the Real even if we are trapped within the play of appearance appearing to itself. Because the Real we encounter in thinking the world is never “simply subjective, [whereby] it would present a case of the hollow playing of the subject with itself, and we would never reach the level of objective reality,”413 thinking's inability to close in on itself shows that the pre/extra-Symbolic must insist within it. However, such a formulation does not directly address the minimal transcendental conditions of the possibility of attaining an access to the Real “it itself” from within the Symbolic. Adrian Johnston's formulation of the paradoxical materialism-idealism relation is highly revealing of the deadlock Žižek faces: “materialism [...] formulates itself vis-à-vis the deadlocks internal to radical transcendental idealism. 11.1 Lacan and the Prison-House of the Symbolic As an account of the subject's own immaculate self-begetting from within the material flux of being as that which institutes the genesis of the Symbolic, Žižek's transcendental materialism is an attempt to explicate the ontological origins of a self-grounding idealism wherein ontology has been rendered seemingly impossible. Insofar as the Symbolic is able to generate meaning without external reference, it refuses all forms of realism: access to a Real outside of the correlation being/language is proclaimed a naïve position to be rejected due to the immense linguistic power that constructs our reality. Yet even if our entrapment within the self-referential, masturbatory play of signifiers is absolute, the ontological solipsism that it entails is precisely that: namely, an ontological solipsism whose very existence suggests that this very inability is somehow internal to the interior play of forces constitutive of being. The issue at hand is that “the transcendental standpoint is in a sense irreducible, for one cannot look 'objectively' at oneself and locate oneself in reality; and the task is to think this impossibility itself as an ontological fact, not only as an epistemological limitation.”411 In this minimalist sense, images and words cannot be seen as mere parasites that contingently latch themselves onto being and disturb its otherwise smooth flow, but must be negatively indicative of some self-sabotaging tendency always already at work in its heart of hearts: “[t]he symbolic order is not a cause which intervenes from the outside, violently derailing the human animal and thus setting in motion its becoming-human.”412 In other words, to account for the very “consistency” of the Symbolic we are called upon to write the great metaphysical epic of that forgotten eternal Past preceding our coming into the world as speaking subjects. Such a conceptual move, however, poses an epistemological problem. Within the Lacanian registers, the Real necessarily appears as a lack. As soon as the Symbolic emerges as a self-replicating, self-evolving differential system that transcendentally constitutes the condition of the possibility of phenomenal reality, any direct contact with the Real qua ontological field is lost. The Lacanian thesis is a variation of structuralist linguistic From Radical Idealism to Critical Metaphysics 261 From Radical Idealism to Critical Metaphysics 261 shadow cast by idealism's insurmountable incompleteness.”414 The problem is the following: even if the immanent “breakdowns” of solipsistic self- enclosure—the internal obstructions of language's psychotic dance— represent the inability of the subject to posit itself as an autocratic all hallucinating its world, they only point to a negative experience of the Real and do not suffice to offer a positive articulation of the ontological qua ontological. Of course ciphering sometimes runs into knots, but this very blockage can only be understood in the Symbolic by the Symbolic and in such a way that we never leave its prison. How could a kink in the Symbolic be revelatory of the pre- or extra-symbolic Real that somehow unpredictably upsurges within its sphere? Even given the paradoxical mode of the double-feature of inclusion/exclusion, internal/external, and presence/absence that defines the Real, it is still, formally speaking, a lack, nothing more than a notional antagonism in its ciphering code. It is unclear how this logical torsion, an internal hindrance in ideality, could serve as a foundation for a new materialist metaphysics, for there is no such thing as an outside or Other to symbolic mediation to which we can have access: “the Lacanian Real—the Thing—is not so much the inert presence that 'curves' the symbolic space (introducing gaps and inconsistencies in it), but, rather, the effect of these gaps and inconsistencies.”415 If it is understood as the alien presence of some extra-ideal presence within synthesis, it can only be understood as such insofar as it is posited as such—or, as Fichte says, insofar as there is a transference of the productive activity of the subject to the not-I, but such a transference, although enabling us to give a consistent account of why representations often fail due to some “alien presence” that obstructs them (the necessity of a realist moment in a critical idealism), never leaves the constraints of a subjective idealism. We appear stuck in the most rampant form of correlationism. Even if the Real can negatively show itself through an internal tension pressurizing the Ideal from within, we can never reach the ontological as such. There is always the correlation language/being wherein being itself is reduced to that which is constructed in the symbolic field of discourse, regardless of there being elements that appear within the latter that it cannot control. From Radical Idealism to Critical Metaphysics 261 On this account, materialism is philosophically tenable only as the spectral inverse of idealism, accompanying it as the 262 Chapter 11 262 Chapter 11 Chapter 11 262 shadow cast by idealism's insurmountable incompleteness.”414 The problem is the following: even if the immanent “breakdowns” of solipsistic self- enclosure—the internal obstructions of language's psychotic dance— represent the inability of the subject to posit itself as an autocratic all hallucinating its world, they only point to a negative experience of the Real and do not suffice to offer a positive articulation of the ontological qua ontological. Of course ciphering sometimes runs into knots, but this very blockage can only be understood in the Symbolic by the Symbolic and in such a way that we never leave its prison. How could a kink in the Symbolic be revelatory of the pre- or extra-symbolic Real that somehow unpredictably upsurges within its sphere? Even given the paradoxical mode of the double-feature of inclusion/exclusion, internal/external, and presence/absence that defines the Real, it is still, formally speaking, a lack, nothing more than a notional antagonism in its ciphering code. It is unclear how this logical torsion, an internal hindrance in ideality, could serve as a foundation for a new materialist metaphysics, for there is no such thing as an outside or Other to symbolic mediation to which we can have access: “the Lacanian Real—the Thing—is not so much the inert presence that 'curves' the symbolic space (introducing gaps and inconsistencies in it), but, rather, the effect of these gaps and inconsistencies.”415 If it is understood as the alien presence of some extra-ideal presence within synthesis, it can only be understood as such insofar as it is posited as such—or, as Fichte says, insofar as there is a transference of the productive activity of the subject to the not-I, but such a transference, although enabling us to give a consistent account of why representations often fail due to some “alien presence” that obstructs them (the necessity of a realist moment in a critical idealism), never leaves the constraints of a subjective idealism. We appear stuck in the most rampant form of correlationism. From Radical Idealism to Critical Metaphysics 263 From Radical Idealism to Critical Metaphysics 263 this never leads to any knowledge of the Real in itself. From within the originary psychosis of a self-grounding idealism, there can be no overlapping of subjective and objective reality, for the very emergence of the subject as an ontological lacuna has as its founding gesture an irreconcilable rupture between the Innenwelt and Aussenwelt. Despite this, however, Žižek embarks upon an exploration of how a structuralist self-enclosed system preventing any direct engagement with the Real could have arisen by using the very fact of the Symbolic as a manner of proceeding. If the Symbolic is an ontological solipsism, then the lack of access to being we have must be understood as an event in being. In this sense, the Real-as-lack must ultimately open onto a metaphysics of the Real as that which precedes and exceeds the Symbolic and the Imaginary. But Žižek's Lacanian commitments would seem to render such a move unjustifiable. It is uncertain how he can balance his own radical idealism with his other materialist tendencies—indeed, his attempt to inscribe the former within the latter, to make the epistemic limitations of linguistic idealism synonymous with being's non-coincidence to self, appears to intensify the problem. How can a “negative materialism” based on the infinite conflict between mind and body found a new metaphysics? If we take as our starting point the (non-)relation between system (materialism) and freedom (idealism) as revealed by the psychoanalytical experience, then doesn't the logical category of the disjunctive “and” explicit in their contradiction prohibit us from delving into the retroactively posited past of the Real-as-origin to see how the latter explodes out of being as an event? How can we overcome the withdrawal into the nocturnal self of the world at the very birth of subjectivity so as even to see this withdrawal for what it is? From Radical Idealism to Critical Metaphysics 261 We may forever approach it as the negative magnitude contorting our notional apparatus as produced by the latter's very inconsistency, but 11.2 The Hegelian Real-as-Lack: The Painful March of Ontological Solipsism The problem at hand is how to understand the separate theses that (i) the inconsistency of thinking methodologically justifies an overcoming of the realism-idealism debate from within idealism and (ii) given the fact that the Symbolic is always already minimally outside itself we can embark upon an explication of its origins without ever leaving it. Endeavoring to give Lacanian psychoanalysis the support it needs—a comprehensive metaphysics—it is perhaps to be expected that Žižek falls into such a Chapter 11 264 theoretically difficult position. Lacan argues that we are stuck in language and, accordingly, that access to the ontological is impossible. Yet such a claim, if it cannot account for the ontological genesis of that which in the first place impossiblizes our access to the ontological, risks being merely a dogmatically asserted statement. This tension between the two major theoretical roles that the Real must assume—a notional lack and a symbolically excessive activity that is independent to all forms of correlation or access conditions—explains the constant oscillation in Žižek's position as he wavers between his strictly Lacanian and larger metaphysical and ontological commitments, even if the latter are always mediated by the former. Although most of his comments on Schelling, for instance, bespeak the possibility and necessity of delving into the Real-as-excess,416 understood as the pre-symbolic antagonism that gives rise to the transcendental matrices of the subject (the “orgasm of forces”) he often refers to the purely fantasmatic character of such inquiry due to the impossibility of reaching the absolute beginning of language417 and its necessary mytho- poetic component given its resistance to rational speculation,418 often without acknowledging the problem that this could appear to pose to the endeavor of establishing the ontological grounding of the subject. Given that he identifies “the key question” of philosophy as that of “how thought is possible in a universe of matter,” so that we should focus our efforts on “the very rise of representation or appearing out of the flat stupidity of being” if we are to avoid “a regression to a 'naive' ontology of spheres or levels,”419 the conceptual contours of this problematic deserve to be investigated in full. This is further necessitated by the fact that many fundamental Žižekian concepts directly exhibit this tension-ridden simultaneity of two opposed directions between the Real-as-lack and the Real-as-excess. From Radical Idealism to Critical Metaphysics 265 utilization of the concept—which he presents as a reworking of Kant's theory of unruliness—is then used to displace the mature Hegelian dialectical triad and argue for its inability to explain the passage from nature to culture,422 thereby further intensifying its speculative reach. How can he move from one register to the other? Despite this uncertain wavering between the purely logical and metaphysical or ontological value of Žižek's reading of the night of the world, other categories that Žižek extracts from Hegel show why he, as a rule, would generally have a preference for Hegel over Schelling, even in face of the obvious debt to the latter in his own transcendental materialism. The Hegelian concept “tarrying with the negative,” for instance, intrinsically displays the structure of the Real-as-lack and is thus, strictly speaking, completely compatible with Lacanian epistemology, whereas the categories he finds in Schelling pose a more immediate problem. Emerging out of Hegel's critique and extension of the Kantian thing-in-itself, tarrying with the negative is an attempt to show that the latter, as a theoretical posit, is superfluous. Objects give themselves to consciousness, but it is an illegitimate move to say that, beyond their appearing, there is an inner core of the thing that is hiding, ever out of reach of the transcendental ego's limited synthesizing powers, because even this infinite elsewhere of subject- independent interiority is itself only possible from within the manifold field of phenomenal experience: that is, it is itself an appearance. This becomes most evident in the experience of non-coincidence between our concepts and that which they “represent.” Within the inconsistency of the immanent structure of knowledge exhibited by these types of encounters, the object “in itself” shows itself through the form of a negative determination that burdens experience. We could say that what we come across here is the raw positivity of the object that obstructs our idealization and forces us to adapt to it, but only with the qualification that this positivity is revealed within the shadows cast by idealization's failure in such a way that it can only be brought forth or posited retroactively by means of a modification of the matrix of concepts that constitute our mediation of the world so that the original paradox or blockage disappears. 11.2 The Hegelian Real-as-Lack: The Painful March of Ontological Solipsism His discussions of the night of the world in The Ticklish Subject, for instance, have a tendency to treat the Real as the other side of transcendental imagination, and hence as a logical rather than an ontological concept (Žižek warns us that “it is crucial to 'close the circle': we never exit the circle of imagination”420), although he also bestows upon the notion a clear metaphysical reach by locating the destructive force of understanding preceding its synthesizing From Radical Idealism to Critical Metaphysics 265 From Radical Idealism to Critical Metaphysics 265 In the aftermath of this epistemic remodulation, we see that what we initially experienced as an internal or structural deformation of ideal space was the negative 266 Chapter 11 Chapter 11 266 refraction of the object's “true” nature such that the Real, less than a solid thing that obstructs ideality from the outside, is a pure effect. This has two implications: firstly, we do not need to overcome the split between phenomenal appearance and the thing-in-itself because this very split arises from within appearance itself, so appearance is “always more than appearance,” as it were. Secondly, the noumenon as a “transcendence” that gives itself of itself to experience is reduced to a mere defence against the potential horror of the immanent, uncontrollable flux of pure appearance, for if a transcendent object causes our representations, then our representations are guaranteed to constitute a minimally smooth fabric. As an illusion of a place wherein all contradiction is always already resolved in a reality complete unto itself, the noumenon helps bestow a sense of order and unity to the structure of experience even where it does not of itself display any, thereby taking the edge off of ideal fragmentation. From Radical Idealism to Critical Metaphysics 267 through which it (only haphazardly) grasps (fragments of) the latter. The issue is not that thinking cannot comprehend being, but that, since it is inscribed within the latter, thinking can obtain no transcendent gaze upon it by which it can totalize it into a complete system. Thinking is necessarily incomplete—there is an irremovable lacuna in every truth-claim—because it can never get rid of the subjective gaze, a gaze that, while making knowledge finite, simultaneously renders it possible in the first place. Following this train of thought to its logical conclusion, we must assert that not only can we never know the system of the world, but if thinking itself is a part of the world, and there is only one world, there is no god’s-eye point of view from which the world can never turn back upon itself and close itself. The gaze of the subject is a non-suturable gap in being—this is what it means to say that “phenomenalization, appearance, 'illusion,' split, finitude, Understanding, and so on, [...] are inherent to the life of the Absolute itself.”426 Through its own subjective movement, thought comes to realize that there is an outside to thinking posited from within as that which insists and persists as the extimate core of all discourse. That is to say, thought is a constant struggle insofar as its struggle with itself is never a mere masturbatory play, but is simultaneously a struggle with the outside world. From Radical Idealism to Critical Metaphysics 265 The infinite (the perfect ontology of the noumenal extra-subjective being) only emerges as a response to the radical finitude of phenomenal reality (ideal obstruction in our dealings with reality), whereby “every positive figure of the In-itself is a 'positivization' of negativity, a fantasmatic formation we construct to order to fill in the gap of negativity”423: there is only the restlessness of the negative, the incessant internal fracturing of experience due to idealism's intrinsic limitations and inability to posit itself as a complete all, which always leads to symbolic dismemberment, so that noumena, now understood as the negativity of phenomena, their internal inconsistency, “designate the In-itself as it appears to us, embedded in phenomenal reality [...] there is no mysterious gap separating us from the unknown, the unknown is simply unknown, indifferent to being-known.”424 In other words, what is on the other side of the screen of consciousness is not another reality, but “the same reality we find in front of the screen”: Žižek tells us to think of the illusion of a theatre stage, where what is responsible for the illusion is not the machinery backstage to which we have no access while the play is going on, but the very theatre stage itself, for even if the backstage and its mechanics are visible not only do we realize that the secretive reality normally “beyond our grasp” is exactly the same as the one being staged but the deceptive From Radical Idealism to Critical Metaphysics 267 From Radical Idealism to Critical Metaphysics 267 Its operationally and epistemologically closed ciphering of the world is capable of knowledge because the very inside-outside distinction becomes intra-discursive, that is, immanent to thinking itself because it is sustained by the activity of thinking, so that there is a paradoxical coinciding of the purely subjective and the purely objective: “[e]very tension between Notion and reality, every relationship of the Notion to what appears as its irreducible Other encountered in the sensible, extra- notional experience, already is an intra-notional tension i.e., already implies a minimal notional determination of this 'otherness.'”427 Although we are trapped in idealization and the Real-as-lack is our doomed fate, there is no need to mourn the loss of some kind of immediate being in itself, some Oedipal womb of nature from which we have fallen: what Hegel shows is that, from within the level of the logical self-articulation of the reflective notional constructs of thinking themselves and the symbolic space of self-generating meaning they engender, we can “reach” objective reality because there is no significant gap between thinking and being at the level of thinking. The Symbolic is that which opens up access to being: we provide a freely 268 Chapter 11 Chapter 11 developed notional construct that creates the norm for an intelligible field, a field that then may fall into inconsistency when said notional construct fails to render its corresponding phenomenal reality consistent, but inasmuch as the failure of said notional construct enables us to modify it we thus can be said to slowly and patiently track truth over time. Once we have seen that “[t]he opposition between idealistic and realistic philosophy is therefore without meaning,”428 we can develop a metaphysics critically rather than dogmatically. This dissolves the worry regarding how we can have access to the world from within the clutches of subjective thinking. “out there” because of the inconsistencies and gaps in the symbolic order.430 With this insight gained Žižek, like Hegel, discovers an epistemological foothold—the productive space of ideal inconsistency (in the quote above Žižek is talking about our access to the Real from within the Symbolic rather than its ontological constitution)—from within which he can find the resources required to develop a new metaphysics. It is less idealism that poses a problem for the latter than the traps that it (unconsciously) creates for us as we attempt to catch a glimpse what “lies beyond” phenomenal reality: the inevitable and necessary symbolic dismemberment of a self- grounding idealism that fails to fully become itself not only creates the possibility of error, but since the first showing of the Real is always an internal deformation of ideal structure (a pure negative form lacking content), it itself risks being riddled with a thick layer of fantasmatic projections and unconscious desires. That which allows idealism to overcome itself also can hinder the speculative process. Yet, despite this, the Real-as- lack and the Real-as-excess do not stand in opposition to one another. Although the former is always epistemologically superior or primary, being the transcendental condition of the possibility of access to the latter, we are nevertheless capable of attaining that which is pre- or extra-Symbolic not only despite but more primordially by dint of the Symbolic's clutches. This is why, on Žižek's reading, Hegel's Science of Logic, while never leaving the matrix of self-thinking thought, can coincide with metaphysics and describe pre-subjective reality in its raw categorial purity and dialectical movement. Ontological solipsism is only apparent, for materialism justifies itself in the cracks of a radical idealism: the very condition of possibility of discourse means that discourse is always more than itself, even if that means that its very possibility coincides with its impossibility. Fichte refers to such a theoretical position—a critical idealism brought to fruition—as a real-idealism or an ideal-realism.431 Although his own position may fail to execute this adequately, it can serve nevertheless as the most consequent description of a true, successful idealism. “out there” because of the inconsistencies and gaps in the symbolic order.430 As Žižek correctly points out, “[t]he irony of the history of philosophy is that the line of philosophers who struggle against the sophistic tradition ends with Hegel, the 'last philosopher,' who, in a way, is also the ultimate sophist, embracing the self- With this insight gained Žižek, like Hegel, discovers an epistemological foothold—the productive space of ideal inconsistency (in the quote above Žižek is talking about our access to the Real from within the Symbolic rather than its ontological constitution)—from within which he can find the resources required to develop a new metaphysics. It is less idealism that poses a problem for the latter than the traps that it (unconsciously) creates for us as we attempt to catch a glimpse what “lies beyond” phenomenal reality: the inevitable and necessary symbolic dismemberment of a self- grounding idealism that fails to fully become itself not only creates the possibility of error, but since the first showing of the Real is always an internal deformation of ideal structure (a pure negative form lacking content), it itself risks being riddled with a thick layer of fantasmatic projections and unconscious desires. That which allows idealism to overcome itself also can hinder the speculative process. Yet, despite this, the Real-as- lack and the Real-as-excess do not stand in opposition to one another. Although the former is always epistemologically superior or primary, being the transcendental condition of the possibility of access to the latter, we are nevertheless capable of attaining that which is pre- or extra-Symbolic not only despite but more primordially by dint of the Symbolic's clutches. This is why, on Žižek's reading, Hegel's Science of Logic, while never leaving the matrix of self-thinking thought, can coincide with metaphysics and describe pre-subjective reality in its raw categorial purity and dialectical movement. Ontological solipsism is only apparent, for materialism justifies itself in the cracks of a radical idealism: the very condition of possibility of discourse means that discourse is always more than itself, even if that means that its very possibility coincides with its impossibility. Fichte refers to such a theoretical position—a critical idealism brought to fruition—as a real-idealism or an ideal-realism.431 Although his own position may fail to execute this adequately, it can serve nevertheless as the most consequent description of a true, successful idealism. From Radical Idealism to Critical Metaphysics 267 To say that the Real is a product of thought is not to lapse into a Berkeleyan form of idealism wherein reality is simply created by the subject: “the Real is not some kind of primordial Being which is lost,” but rather “what we cannot get rid of, what always sticks on as the remainder of the symbolic operation.”429 As an aftereffect of this symbolic operation, it ensures that we have an indirect but methodologically secure entry point into the world by means of the inconsistencies that our notional apparatus generates in the freely determined self-generation of the universe of meaning, inconsistencies that unexpectedly let us develop an objective discourse. But the limits of idealism entail that idealism is always already more than itself—thought itself is utterly incapable of positing itself as a self- enclosed positivity that simply creates its own universe of meaning; it becomes contaminated, as it were, by a constitutive “outside” as soon as it tries to posit itself in its own self-determining freedom, so that it must constantly struggle with this outside. This constitutive failure on behalf of thinking successfully to posit itself as all guarantees that there is a subject- independent reality that we experience and can speculatively describe: There is a Real not because the Symbolic cannot grasp its external Real, but because the Symbolic cannot fully become itself. There is being (reality) because the symbolic system is inconsistent, flawed, for the Real is an impasse of formalization. This thesis must be given its full “idealist” weight: it is not only that reality is too rich, so that every formalization fails to grasp it, stumbles over it; the Real is nothing but an impasse of formalization—there is dense reality From Radical Idealism to Critical Metaphysics 269 From Radical Idealism to Critical Metaphysics 269 “out there” because of the inconsistencies and gaps in the symbolic order.430 “out there” because of the inconsistencies and gaps in the symbolic order.430 As Žižek correctly points out, “[t]he irony of the history of philosophy is that the line of philosophers who struggle against the sophistic tradition ends with Hegel, the 'last philosopher,' who, in a way, is also the ultimate sophist, embracing the self- y p p y y y This is why, on Žižek's reading, Hegel's Science of Logic, while never leaving the matrix of self-thinking thought, can coincide with metaphysics and describe pre-subjective reality in its raw categorial purity and dialectical movement. Ontological solipsism is only apparent, for materialism justifies itself in the cracks of a radical idealism: the very condition of possibility of discourse means that discourse is always more than itself, even if that means that its very possibility coincides with its impossibility. Fichte refers to such a theoretical position—a critical idealism brought to fruition—as a real-idealism or an ideal-realism.431 Although his own position may fail to execute this adequately, it can serve nevertheless as the most consequent description of a true, successful idealism. As Žižek correctly points out, “[t]he irony of the history of philosophy is that the line of philosophers who struggle against the sophistic tradition ends with Hegel, the 'last philosopher,' who, in a way, is also the ultimate sophist, embracing the self- 270 Chapter 11 70 Chapter 11 270 C 270 referential play of the Symbolic with no external support of its truth.”432 Hegel can accomplish this prima facie paradoxical feat because he is able to demonstrate that accepting the impossibility of leaving the “correlationist circle” does not fall into a naïve idealism whereby objective reality is reduced to nothing, but rather shows how even the self-referential nature of thinking itself always already depends upon and is entangled with the world, thereby attesting that the split between knowledge in itself and for us exists not because we are separated from the world, but because we are a part of it: “the very limitation of our knowing—its inevitably distorted, inconsistent character—bears witness to our inclusion in reality.”433 In this respect, idealism (reflection, notional constructs, language as such) creates the space of reasons in virtue of which things can present themselves to us as they are in reality in itself. This presenting, however, requires a stage upon which their theatrical appearance can be performed, a stage that produces the illusion of a backstage to which we do not have access. From Radical Idealism to Critical Metaphysics 271 the effects of the Symbolic's self-enclosure is that, in point of fact, it allows things to appear intelligibly through the reflective mediation of language. All we have to do is take the appropriate attitude toward the inner limitations of phenomenal reality as such. Paradoxically, a realist epistemology does not make a realist. This is why the choice between idealism and realism is false for the idealist. “out there” because of the inconsistencies and gaps in the symbolic order.430 Instead of merely separating us from the world, the reflexivity of the Ideal thereby allows objects to have meaning for us as something more than objects to be used by specialized biological or natural needs. We symbolize them, grant them a place in discourse, a discourse whose failures make it seem as if a world out there directly attacks our concepts and theoretical models when, in fact, we never exit discourse at all, for only its self-sustaining matrix can sustain phenomenal reality as a universe of meaning. This signifies, moreover, that to pose the question of being qua being, there must be a difference between us and being, for being qua being can only show itself to a finite thinker for whom there exists a distance within which being can be phenomenalized or phenomenalize itself by means of notional constructs. It is important to note, however, that in order to get being right, we must also be able to get it wrong, the minimal condition of which is satisfied by the ontological madness that is the basic structure of ideality as that which prevents us from having a direct “immersion” in the world and as such opens up the possibility of replicating being within itself; the self-stipulating norms of discourse internally guide the constitution of phenomena while their inconsistency demonstrates that we are capable of objectively describing From Radical Idealism to Critical Metaphysics 271 272 Chapter 11 of Žižek is his nuanced vision of the world that is able to use idealism's resources not only to overcome its own apparent limitations from within, but also to demonstrate how, if this self-overcoming is successfully executed, our understanding of being is simultaneously remodulated. Idealism and realism, transcendentalism and metaphysics, are not mutually opposed if you think them through in relation to one another, for the former forces us to come to grasp what it means for thinking to exist in an irreducible manner, a fact that has stark consequences for our understanding of the world at large. After a long period in contemporary philosophy where there was a general disdain for speculation, what distinguishes Žižek so radically from others who have also raised the question of the possibility of a new metaphysics (perhaps most notably Deleuze and Badiou) and those who are now active in establishing this possibility is his call for a critical metaphysics and its superiority over a mere return to dogmatic philosophizing as a means of overcoming the heritage of what has recently come to be known since Meillassoux's After Finitude as correlationism.438 For Žižek, the urgent call for a philosophy that can combat the apparent speculative throes of the irreducibility of the correlation in our knowledge of the world (if the intelligibility of any specific empirical truth-claim depends upon the subject for whom such a truth-claim has meaning in the first place, how can we even speak about that which occurred before the existence of such a subject without falling into ontic nonsense?) does not have its origin in paradoxes concerning “ancestral” statements concerning what the universe must have been like prior to humanity, as Meillassoux would like us to believe,439 this having been proven to be an intrinsic possibility opened up by the logical space of the correlation as such, but rather what the universe must be like if something like humanity and its transcendental constituting powers are to arise at all. The issue is how the Real could have come to appear to itself—and although this may appear to risk an anthropomorphization of nature (“[w]e should apply here something like a weak anthropic principle: how should the Real be structured so that it allows for the emergence of subjectivity [...]?”440), one should proceed cautiously. 11.3 A Call for a Critical Metaphysics At this juncture we must underline one important feature of this argument for a self-grounding idealism that intrinsically contains an irreducibly real moment. If the Symbolic freely constitutes phenomenal reality “with no external support of its truth,”434 then this suggests that from within its ontologically solipsistic dance of cybernetic ciphering, we can break through the impenetrable dusk of psychosis as we find, in an innovative theoretical gesture, a secure foothold from which to found a new science of being. But this means that we should not merely overcome radical idealism from within idealism (an epistemological sublation of the correlation): we must also overcome it from the side of being by showing how the ambiguities of idealism are in fact a part of the world's fundamental structure through an account of how being comes to appearance/thinking/phenomenalization (an ontological inscription of the correlation). If we can succeed, then realism and idealism will have become intimately dialectically linked. Not only would this entail a strong theory of thinking insofar as the latter would be inscribed within the fold of being as an irreducible event rather than as a mere illusory feature, but also a profoundly rich metaphysics inclusive of both realism and idealism given that both would now reciprocally ground one another (in realistic and idealistic terms) in a completely self-sufficient and self-reflexive whole. That is to say, we would have a new variation on the late German Idealist theme of the unification of system and freedom: or, in other words, another take on the Kantian heritage and how it radically changes the field of philosophizing. In this respect, whereas much of contemporary philosophy's understanding of idealism fails to take it seriously, often equating it with a form of Berkeleyanism (a tradition that runs from Kant's first critics to Lenin435 and taken up once again by Moore436 and most recently by Meillassoux437), one of the greatest strengths 272 Chapter 11 Žižek's metaphysical archaeology of the psychoanalytical subject is an attempt to think the intersection of the Real and the Symbolic, the coldness of being and the From Radical Idealism to Critical Metaphysics 273 fervor of humanity, because if images and words, and by implication thinking, exist, they must exist in the world. The universe is inclusive, not exclusive, of humanity: a true speculative philosophy should comprehend both the Real in its pure non-correlationality (the nonhuman) and how correlation comes to pass in being (the human). Perhaps unexpectedly, the price we pay for this theoretical gain of re-inscribing humanity into nature, that is, the latter's minimal anthropomorphization, is a simultaneous denaturalization of nature and a dehumanization of humanity. Not only is nature now reduced to “a freak show of contingent disturbances with no inner rhyme or reason,”441 but even if humanity still retains a certain qualitatively distinct status in contradistinction to other things in virtue of its autonomy, what we normally take as the great and sublime achievements of thinking are, in fact, grounded in a mere virtual re-compensation for our traumatic disruption from the Real. What thus makes Žižek's speculative real-idealism/ideal-realism (to borrow that German Idealist leitmotif) so penetrating and deserving of attention today is its ability to combine a profoundly idealist epistemology with a dynamic realist metaphysics in one single gesture, which shows us one path that contemporary metaphysics could take: namely, a critical one. In this regard, if Meillassoux's critique of correlationism is a call to station ourselves after (Kantian) finitude, it must be recalled that this is precisely what Schelling and Hegel did in their own critique of correlationism avant la lettre. Žižek is able to balance the real and ideal poles in such a nuanced way because of his precise and original take on the breakthrough inaugurated by transcendental philosophy, a breakthrough that for him presents itself as the unthought (Lacanian) cause at the heart of German Idealism. 272 Chapter 11 One of his central claims is that if we read Kant closely, we see hints that what truly fascinates him is not how the subject brings forth its own universe of meaning as a new kind of metaphysical agent capable of guaranteeing the universality and necessity of experience/empirical truth, the two being identical for Kant, but “something quite different,” something that sets the stage for the “few decades [of German Idealism that] represent a breathtaking concentration of the intensity of thinking” within which “more happened than in centuries or even millennia of the 'normal' development of human thought.”442 Commenting on Kant's description of the Copernican Chapter 11 From Radical Idealism to Critical Metaphysics 275 relationality and whereby one leads to the other: “[w]e can also see in what way two lacks overlap in this impossible object: the constitutive lack of the subject (what the subject has to lose in order to emerge as the subject of the signifier) and the lack in the Other itself (what has to be excluded from reality so that reality can appear).”447 Although the transcendental (our subjective position) objectively exists in the world, it nevertheless appears to itself in its first mode as merely subjective. To overcome itself and pass over into a metaphysics, it must do so from within; it does so by drawing attention to the fact that, beneath the correlation of (the conscious) subject and reality, “there is the more primordial correlation of the subject (of the unconscious) and its Real/ impossible objectal counterpoint, S-a.”448 Although this may seem to be just another correlation (as Heidegger only institutes the correlation being/Dasein as more originary than that of consciousness/world, doesn't Žižek do something similar?) this is a false appearance. Rather than being a strict correlation in Meillassouxian terms this binary points towards the emergence of correlation within being: “this impossible/Real object is the very mode of inscription of the subject into trans-subjective reality; as such, it is not transcendental but (what Derrida would have called) arche- transcendental, an attempt to circumscribe the 'subject in becoming,' the trans-subjective process of the emergence of the subject.”449 Žižek's wager, one that he shares with the entirety of post-Kantian idealism as a critical metaphysics fighting against any dogmatic breed thereof, is that if we are to truly break free from correlationism, no return to a “naïve” realism is possible. Not only is the latter always open to critique insofar as it could remain correlational in a hidden way (isn't its specific image of reality related to a subject?450), so that the specific nature of the correlation, namely the ideal conditions of the possibility of intelligibility of any theory, should always be thematized before embarking on speculation, but more primordially it fails to give us of a grasp of how thought is situated/ comes to pass in being and, therefore, the very ontological conditions of the possibility of its own status as a theory. Chapter 11 274 revolution (the experiment of seeing “if he might not have greater success if he made the observer revolve and left the stars at rest”443), which Kant identifies with his own transcendentalist position, Žižek notes: The precise German terms (“die Zuschauer sich drehen”—not so much turn around another centre as turn or rotate around themselves) make it clear what interests Kant: the subject loses its substantial stability or identity and is reduced to the pure substanceless void of the self-rotating abyssal vortex called “transcendental apperception.”444 What initially appears as a rampant subjectivism wherein the ego reigns above the world proves on closer inspection to be something infinitely more complex, for the self-grounding field of phenomenal reality only exists because it itself revolves around the positively charged void of I. The breakthrough of transcendental idealism is precisely the discovery of this zone of negativity within the subject, that X that can never be fully appropriated into transcendental constitution and yet somehow engenders its very possibility, whereby it shows itself to be not so much a displacing of the substantial unity of reality from the external world to the conceptual forms of cognitive construction (a subjective Ptolemaic counterrevolution against Galileo’s de-centering of the medieval world of teleology445) as the opening up of a new understanding of being, that is, of the radical ontological incompletion of reality, the breakdown of substance, provoked by the meta-transcendental conditions of thought as such. As the late German Idealist reappropriation of the subject demonstrates, what is truly unique in transcendental idealism is that the real and ideal poles are unsettled in one brush stroke: linked to the Todestrieb that destroys the homeostasis of nature and das Unbehagen in der Kultur that prevents our second nature from becoming a new substantialist order, the “objectal status” of the subject is that which is “no longer” ontological and “not yet” symbolic because it cannot be contained in either register in its pure form;446 it is what Žižek sometimes refers to as the absent centre that, by protruding out of all ontological and symbolic structures, negatively ties them together in its very undecidability. Here, and only here, the real and the ideal poles intersect, so that both are given their equal due because of an unapproachable X, a je ne sais quoi, that relates them in their very non- From Radical Idealism to Critical Metaphysics 275 276 Chapter 11 Real, and thus breaks the circle of transcendental correlationism.”451 In this manner, we should search for how the transcendental hints towards its dark beginnings (the pure act of the Real-as-origin and the unruliness of drives in the Real-as-excess that precedes it), the emphasis being on the ontogenetic process of becoming more than on the specificity of the transcendental constitution of reality for us, so that the fundamental issue is no longer so much how can we attain knowledge of the absolute (this already being accomplished by the first methodological step, as for instance in Hegel) but how does our subjective viewpoint fit into it as something that objectively exists: “[t]he true question is therefore how I (as the site where reality appears to itself) emerge in 'objective' reality (or, more pointedly, how can a universe of meaning arise in the meaningless Real).”452 With this, we have come full circle and hit upon the great merit of Žižek's philosophy. Having already overcome idealism from within, and having opened up the space for a speculative philosophy, he can develop a theory that is capable of being maximally realist and idealist and therefore best suited as a self- explanatory theory of the metaphysical “totality” of the world, insofar as it can simultaneously supply the ontological conditions of the possibility of its own status as a theory and the ideal conditions of possibility of its own intelligibility in one sweeping move, thus making the theory itself extremely self-referential in its structure. In short, the theory displays complete systematic self-enclosure: it explains itself as a theory in both the real and ideal registers in such a manner that both depend upon and mutually ground one another in a self-articulating whole; it has succeeded at developing “a concept of the world or the Real which is capable of accounting for the replication of reality within itself.”453 What Žižek teaches us, a lesson already brought to the fore in late German Idealism but since forgotten, is that radical idealism is not a closure to the absolute. It is rather a new approach towards it, a new way of relating to it—and to see it as such merely requires a parallax shift in perception. 11.4 Being's Poem: Speculative Philosophy and the From Radical Idealism to Critical Metaphysics 275 Concerning the latter, merely explaining subjectivity as a purely contingent emergence amongst others à l Meillassoux bodes no better: “one should locate traces of this contingency in a kind of umbilical cord which links the subject to its pre-subjective 11.4 Being's Poem: Speculative Philosophy and the Mytho-Poetic Parallax Shift If Žižek is right to say that the founding gesture of idealism is an ontological passage through madness, then it would not go far enough if we were to admit that the opposition between realism and idealism has been already From Radical Idealism to Critical Metaphysics 277 resolved at the level of idealism. Something is missing, for the real event that immediately precedes the autarchy of the Ideal, and whose exploration would enable this opposition to be also resolved at the level of realism (thus radicalizing and guaranteeing what Fichte referred to as a real-idealism or ideal-realism), is an impossible object of discourse. Conceding that notional antagonism can indeed be spectrally expressive of objective reality, and thus enable us to speculate about reality within ideality, to fully explain how we can have contact with the world where there is properly speaking no contact at all we must nevertheless acknowledge that we can never even indirectly reach the exact moment at which being begins to exist in the modality of the Ideal. The difficulty is much more severe than that the Real is always already minimally symbolized, since the Real-as-origin expresses two fundamental theoretical problems. First, insofar as its abyss of unconscious decision represents a pure self-instituting difference that unpredictably splits the world into two new logically irreconcilable registers, it cannot be deduced from the auto-movement of the Real; there is no “transition,” but only a self-caused “leap” that forever evades complete conceptual or natural dialectic mediation. Second, because this act withdraws in the very gesture of giving birth to the Symbolic, it lies stricto sensu “beyond” the grasp of the latter and can never appear within it, not even negatively. It is in this precise manner that the subject is neither Real nor Symbolic and is only expressible through a series of paradoxical avatars unable to bestow content upon it (the disjunctive “and,” the “in-between,” the “abyss of freedom,” the “vanishing mediator,” and so on). A philosophical discourse about the subject is thus intrinsically paradoxical because the latter is “a non-provable presupposition, something whose existence cannot be demonstrated but only inferred through the failure of its direct demonstration.”454 In short, it can only be investigated at the level of mytho-poetics. 11.4 Being's Poem: Speculative Philosophy and the Mytho-Poetic Parallax Shift But what intensifies the problematic of such a metaphysical archeology of the subject is that without such a mytho-poetic narrativization of its impossible Past we would be unable fully to explicate how the Symbolic can in fact relate to reality in itself, for without it we cannot perform the parallax shift of “transposing the ll it can only be investigated at the level of mytho-poetics. But what intensifies the problematic of such a metaphysical archeology of the subject is that without such a mytho-poetic narrativization of its impossible Past we would be unable fully to explicate how the Symbolic can in fact relate to reality in itself, for without it we cannot perform the parallax shift of “transposing the tragic gap that separates the reflecting subject from pre-reflexive Being into this Being itself,” whereby “the problem becomes its own solution: it is our very division from absolute Being which unites us with it, since this division 8 Chapter 11 From Radical Idealism to Critical Metaphysics 279 The system of being and thinking has closed upon itself in one final self-referential gesture. Ž Because the objectal status of the subject escapes any straightforward causal explanation and lies outside of all correlation insofar as it is responsible for its very upsurge, the only way to reach it is by means of a mytho-poetics of speculative fabulation. And given that this realistically non-deducible and idealistically inaccessible zone coincides with that very place in which the subject is inscribed within being as one creature amongst others (and is thus that which would enable us to pass without any immanent obstacle from the real pole to the ideal pole and vice versa) the parallax shift from the negative limitation of knowledge to the positive structure of the absolute itself requires more than mere rational ideal discourse. Only then can we “relate the In-itself to the split in the subject,”457 for “what Lacan calls the objet a, the subject's impossible-Real objectal counterpart, is precisely such an 'imagined' (fantasmatic, virtual) object which never positively existed in reality—it emerges through its loss, it is directly created as a fossil.”458 In this sense, Žižek's philosophy is paradoxical precisely because it attempts to think the unthinkable, that is, the cogito ergo sum as “I am that impossible piece of the Real where I cannot think”459 that uncannily corresponds to that space within which the meaningless Real contingently awakens and opens its eyes for the first time. Chapter 11 278 is immanent to Being.”455 In other words, the real and the ideal sides of overcoming idealism are intimately connected and cannot be discussed in isolation from one another: in order for idealism to completely surmount its own apparent limitations, it must be able to come full circle and show how, from within realism, its own ambiguities are not merely epistemological but also ontological; it must show what it means for thinking to be a part of the world, a manner in which the world relates to itself, no matter how paradoxically. In creating itself by an act of immaculate conception, the Real-as-origin of the pure act at the birth of full-fledged subjectivity retroactively takes over what we come to know as the Real-as-excess of the drives. In a contradictory moment in which cause-and-effect relations are torn apart, the effect becomes greater than and autonomous from its cause, even going so far as to write a virtual possibility into the eternal dregs of the Past that never existed prior to its having been written there through an act of positing of its own presuppositions. The subject creates its own past in the same instance in which it begets itself out of nothing, so that the true “arche-fossils” are not ancestral statements concerning what occurred billions of years ago before the emergence of the thinking subject or life itself, but the objectal status of the subject: what Lacan asserts is precisely the irreducible (constitutive) discord, or non-correlation, between subject and reality: in order for the subject to emerge, the impossible object-that- is-subject must be excluded from reality, since it is this very exclusion which opens up the space for the subject. [...] The true problem of correlationism is not whether we can reach the In-itself the way it is outside of any correlation (or the way the Old is outside its perception from the standpoint of the New); but the true problem is to think the New itself “in becoming.” The fossil is not the Old the way it was in itself, the true fossil is the subject itself in its impossible objectal status—the fossil is myself, the way the terrified cat sees me when it looks at me. This is what truly escapes correlation, not the In-itself of the object, but the subject as object.456 From Radical Idealism to Critical Metaphysics 279 From Radical Idealism to Critical Metaphysics 279 Because the objectal status of the subject escapes any straightforward causal explanation and lies outside of all correlation insofar as it is responsible for its very upsurge, the only way to reach it is by means of a mytho-poetics of speculative fabulation. And given that this realistically non-deducible and idealistically inaccessible zone coincides with that very place in which the subject is inscribed within being as one creature amongst others (and is thus that which would enable us to pass without any immanent obstacle from the real pole to the ideal pole and vice versa) the parallax shift from the negative limitation of knowledge to the positive structure of the absolute itself requires more than mere rational ideal discourse. Only then can we “relate the In-itself to the split in the subject,”457 for “what Lacan calls the objet a, the subject's impossible-Real objectal counterpart, is precisely such an 'imagined' (fantasmatic, virtual) object which never positively existed in reality—it emerges through its loss, it is directly created as a fossil.”458 In this sense, Žižek's philosophy is paradoxical precisely because it attempts to think the unthinkable, that is, the cogito ergo sum as “I am that impossible piece of the Real where I cannot think”459 that uncannily corresponds to that space within which the meaningless Real contingently awakens and opens its eyes for the first time. In so doing, the metaphysical archaeology of the subject it offers endeavors to demonstrate that what the subject “loses” in order to become a subject coincides with what is excluded from reality so that reality can appear to itself, in such a way that the Real is thereby transformed from being a primordial being to which we have lost access due to its symbolization into something of which we cannot shake ourselves, no matter how hard we try, because through a mere formal reversal the gaze of the subject is seen to be the gaze of the world upon itself, the ambiguities and difficulties of the former being always already those of the latter: what we see is that “the narrative [we are telling] is not merely the subject coping with its division from Being, it is simultaneously the story Being is telling itself about itself,” so that realism and idealism are no longer in opposition, but stand in a self-sufficient totality inclusive of both as immanent to the life of substance. From Radical Idealism to Critical Metaphysics 279 In so doing, the metaphysical archaeology of the subject it offers endeavors to demonstrate that what the subject “loses” in order to become a subject coincides with what is excluded from reality so that reality can appear to itself, in such a way that the Real is thereby transformed from being a primordial being to which we have lost access due to its symbolization into something of which we cannot shake ourselves, no matter how hard we try, because through a mere formal reversal the gaze of the subject is seen to be the gaze of the world upon itself, the ambiguities and difficulties of the former being always already those of the latter: what we see is that “the narrative [we are telling] is not merely the subject coping with its division from Being, it is simultaneously the story Being is telling itself about itself,” so that realism and idealism are no longer in opposition, but stand in a self-sufficient totality inclusive of both as immanent to the life of substance. The system of being and thinking has closed upon itself in one final self-referential gesture. In Žižek, we must narrativize the movement from being to thinking if we In Žižek, we must narrativize the movement from being to thinking if we wish to reconcile the two and completely escape the speculative throes of 280 Chapter 11 Chapter 11 280 correlationism, since at the level of content there is always a minimal “non- dialectizable” difference of one to the other (a division) that prevents such a move in purely rational discourse. But we must highlight the precise logical structure of this solution if we are to come to terms with the originality, daringness, and potential problems of Žižek's position. Given that the exact real event that instigates being's coming into appearance/thinking/ phenomenalization is the primordial ontological trauma that is the subject as object, we must in a mytho-poetic register (the subject in its objectal status being forever elusive) show that this trauma is not a mere accidental, haphazard occurrence in the personal history of an individual subject, but rather reveals itself as a constitutive, yet disrupting part of a greater trauma within being itself. From Radical Idealism to Critical Metaphysics 281 our apparently purely epistemological limitations are intimately linked to the ontological grounding of our notional apparatus. But is such a mytho- poetic narrativization a sufficient basis for a new speculative philosophy? As we will see in the next chapter, delving into this question leads us to three potentially fatal issues with Žižek's critical metaphysics, all emerging from its fundamental concept of ontological catastrophe as the vanishing meditator between the Real and the Ideal. Does Žižek have the resources necessary to combat them? And if so, at what price? From Radical Idealism to Critical Metaphysics 279 But this (Žižekian) dialectical reconciliation is not a complete sublation of the opposition between realism and idealism, a complete break with the paradoxes of correlation, for the problematic nature of the latter is something that must be accounted for rather than explained away. As with all dialectical movement, “reconciliation is a reconciliation with the irreducibility of the antinomy, and it is in this way that the antinomy loses its antagonistic character,”460 so that in this case, reconciliation only truly occurs when we realize that there is no reconciliation (a complete solution) possible because what we take as our finitude should be inscribed into the thing itself (being) as its non-coincidence to self; and although this reconciliation can only thus come to pass at the level of mythological form, and never at that of content, instead of focusing on the impossibility of idealist representation to capture that which is being described, namely the objectal status of the subject, the solution paradoxically “shifts the focus to how (as Lacan put it) the signifier itself falls into the Real, that is, how the signifying intervention (narrativization) intervenes into the Real, how it brings about the resolution of a real antagonism,”461 thus working against its own impossibility: “the narrative path directly renders the life of Being itself.”462 Though we can never “exit the circle of imagination”463 to capture the abyss of unconscious decision at its real origin, by writing being's poem we can see, due to a mytho- poetic parallax shift that the poem we are writing coincides with the one From Radical Idealism to Critical Metaphysics 281 Notes 411. Žižek, Less Than Nothing, p. 239. 412. Ibid., p. 562. 412. Ibid., p. 562. 412. Ibid., p. 562. 413. Žižek, The Abyss of Freedom, p. 45. 414. Johnston, Žižek’s Ontology, p. 19. 414. Johnston, Žižek’s Ontology, p. 19. 415. Žižek, The Puppet and the Dwarf, p. 66. 416. See Žižek, The Ticklish Subject, p. 55; and The Parallax View, p. 166. 417. For example, Žižek, The Indivisible Remainder, pp. 22–23. 418. See, for instance, ibid., p. 9. 418. See, for instance, ibid., p. 9. 419. Žižek, Less Than Nothing, p. 905. 419. Žižek, Less Than Nothing, p. 905. 420. Žižek, The Ticklish Subject, p. 33. 420. Žižek, The Ticklish Subject, p. 33. 421. Ibid., p. 31. 421. Ibid., p. 31. 422. Ibid., pp. 79ff. 423. Žižek, Less Than Nothing, p. 282. 424. Ibid., p. 283. 424. Ibid., p. 283. 425. Ibid., pp. 374–75. 426. Žižek, The Ticklish Subject, pp. 88–89. 427. Žižek, Tarrying with the Negative, p. 20. 428. Hegel, The Science of Logic, p. 124. 429. Žižek, Less Than Nothing, pp. 645–46. 430. Ibid. 431. Fichte, Science of Knowledge, p. 247. 432. Žižek, Less Than Nothing, pp. 76–77. 433. Ibid., p. 390. 434. Ibid., p. 77. 282 Chapter 11 282 Chapter 11 435. References to such are scattered throughout Lenin’s Materialism and Empirio- Criticism (Peking: Foreign Languages Press, 1972). 436. See Moore, “Refutation of Idealism,” Mind 12 (1903). 437. This is at the heart of Meillassoux’s critique of correlationism via the arche-fossil in After Finitude: An Essay on the Necessity of Contingency, trans. Ray Brassier (New York: Continuum, 2008). 438. For a similar in spirit account of the distinction between critical and dogmatic metaphysics, see Gabriel, Das Absolute und die Welt in Schellings, p. 8. For the basic definition of “correlationism,” see Meillassoux, After Finitude, pp. 4–6. 439. Meillassoux, After Finitude, pp. 9–13. 440. Žižek, Less Than Nothing, p. 905. 440. Žižek, Less Than Nothing, p. 905. 441. Ibid., p. 298. 441. Ibid., p. 298. 442. Ibid., p. 8. 442. Ibid., p. 8. 443. Kant, Critique of Pure Reason, B xvi. 443. Kant, Critique of Pure Reason, B xvi. 444. Žižek, Less Than Nothing, p. 631. 444. Žižek, Less Than Nothing, p. 631. 445. Meillassoux, After Finitude, pp. 118–19. 446. Žižek, The Ticklish Subject, p. 36. 447. Žižek, Less Than Nothing, p. 645. 448. Ibid., p. 642. 448. Ibid., p. 642. 449. Ibid. 449. Ibid. 450. Ibid. 450. Ibid. 451. Ibid., pp. 642–43. 451. Ibid., pp. 642–43. 452. Ibid., p. 924. 452. Ibid., p. 924. 453. Gabriel and Žižek, “Introduction: A Plea for a Return to Post-Kantian Idealism,” in Mythology, Madness and Laughter, p. 13. 454. Žižek, Less Than Nothing, p. 730. 454. Žižek, Less Than Nothing, p. 730. 455. Ibid., p. 15. 455. Ibid., p. 15. 456. Ibid., p. 644. 456. Ibid., p. 644. 457. Ibid. 457. Ibid. 458. Ibid., p. 645. 459. Ibid. 460. Ibid., p. 950. 460. Ibid., p. 950. 461. Ibid., p. 16. 461. Ibid., p. 16. 462. Ibid. 463. Žižek, The Ticklish Subject, p. 33. Chapter 12 The Deadlocks of Ontological Catastrophe The Cases of Naturphilosophie, Anton-Babinski Syndrome, and Tarte à la crème 12.1 The Schellingian Real-as-Excess: Iain Hamilton Grant, Naturphilosophie, and the Interior Involutions of Being But is mytho-poetics the only option here? Although Žižek follows Hegel's defence of radical idealism as capable of a speculative account of extra- notional reality, a potentially fatal problem arises internally within Žižek's own position as soon as we take a closer look at Žižek's other major interlocutor: Schelling. Ultimately unsatisfied with Hegel's mature account of the passage from nature to culture, Žižek turns to Schelling to explicate the genesis of the Symbolic. What Hegel missed was the paradoxical essence of this very movement, a dialectical movement that causes dialectics to collapse upon itself. With the birth of subjectivity, we see a snag in substance that prevents the absolute from self-actualizing itself so that, instead of being the Idea completely returning to itself out of its otherness in nature, which would present us with a reconciliation of nature and spirit in the Idea, nature's complete self-sublation in spirit, culture is actually a mere secondary (“virtual, artificial, symbolic, not substantially natural”464) response to the primordial ontological trauma that lies at the core of the human being. For Žižek, this means that culture is the always failed attempt at reconciliation: the Idea is nothing other than this very act of its own returning to itself, this movement being constitutive of that to which is returned;465 for it to reach its end would herald its death. Representing a recoil into a psychotic, irreal space, not only do the subjective and objective/ mind and world thereby fall into infinite conflict with one another, but it becomes impossible to explain why the Ideal emerges. According to Žižek it is Schelling who, fighting against the perceived threat of Hegelian Absolute Idealism, gives the most detailed account of this immanent self-sundering of being into its real and ideal poles in his account of the Grund as the ever elusive, eternal Past of consciousness, and the pure act of unconscious decision underlying the birth of the universe of human meaning. Schelling's position is paradoxical: from within a solipsistic space (transcendental idealism) he tries to develop a philosophical language capable not only of piercing the primacy of the Real-as-lack and explaining its monstrous pre-history within pre-symbolic antagonism (materialism), a logically prior but directly unreachable modality of the Real that precedes and exceeds the Symbolic, but also of describing the inexplicable self-positing of subjectivity But is mytho-poetics the only option here? Chapter 12 The Deadlocks of Ontological Catastrophe The Cases of Naturphilosophie, Anton-Babinski Syndrome, and Tarte à la crème Žižek's theoretical philosophy aims to be a critical metaphysics capable of simultaneously overcoming radical idealism from within idealism (an epistemological sublation of the correlation) and from within realism (an ontological inscription of the correlation). However, at this juncture three potential problems emerge from various directions. The first direction is that of Schelling's Naturphilosophie. Not only does Schelling proclaim that, insofar as the subject is anything but an ontological catastrophe, thought is inscribed within being in such a way that we have no need to overcome radical idealism from within itself, it also challenges Žižek's own psychoanalytical reactualization of Schelling. The second direction is that of the skeptic, who can invent a thought experiment to demonstrate that it is perhaps impossible to develop any positive metaphysics from within a differential system of signifiers without any external reference. The third direction is that of the very basis of ideality itself understood as a psychotic withdrawal into the night of the world, the overcoming of which demands the seemingly impossible task of developing a paradoxical form of “successful” psychotic thinking capable of penetrating the impenetrable dusk of its own psychosis. How does Žižek stand up to such critiques? Is his radical idealism truly capable of providing us with a comprehensive metaphysics? 284 Chapter 12 The Deadlocks of Ontological Catastrophe 285 The Deadlocks of Ontological Catastrophe 285 (idealism). After all, even if we can be said to have access to both the ancestral past of the Real prior to subjectivity and the Real that surrounds us “outside” of language through the very inconsistency of our notional apparatus, the methodology of which Hegel offers us through an analysis of the dialectic of phenomenal appearance and the very structure of symbolic thought, nevertheless the precise moment in which the subject institutes itself into being poses a problem to such a self-overcoming of radical idealism, because it is a leaping point in the Real into a new age of the world that is always “beyond” the Symbolic as its irretrievable origin. Because Schelling realizes the impossibility of a purely speculative account of the subject's emergence, and thus the necessity of a mytho-poetics, it is he and not Hegel who most adequately realizes the quadruple logic of dialectics at the core of German Idealism. But even with his highly methodological psychoanalytical construction of the unconscious Grundlogik of German Idealism, for anyone who is familiar with Schelling's vast corpus it is unclear how Žižek can appropriate Schelling for his own project without potentially destabilizing his most fundamental theoretical category: that of the ontological catastrophe. Schelling for his own project without potentially destabilizing his most fundamental theoretical category: that of the ontological catastrophe. Although the two major concepts Žižek utilizes—denaturalized unruliness (the dark pre-history of subjectivity in the vicissitudes of being) and the unconscious decision (the separating Ent-Scheidung whose effect is the institution of a metaphysically disjunctive “and”)—are meant to explain how we get entrapped in the Symbolic and the matrix of idealization, Žižek's own writings on Schelling do not seem fully to take into account the possibility that the latter might not be able to be so easily translated into the framework that he sees as basic to the entire tradition. His division of Schelling's thinking into three distinct stages—the Schelling1 of a quasi-Spinozistic philosophy of absolute indifference, Schelling2 of a radical materialist ontology of freedom, and Schelling3 of the philosophy of mythology and revelation—already hints at an irremovable tension. It is uncertain that the materialism-idealism relationship we see in the Schelling of the Freiheitsschrift and the Weltalter can be read as a mytho-poetics of the birth of a radical transcendental idealism in the manner Žižek suggests. 12.1 The Schellingian Real-as-Excess: Iain Hamilton Grant, Naturphilosophie, and the Interior Involutions of Being Although Žižek follows Hegel's defence of radical idealism as capable of a speculative account of extra- notional reality, a potentially fatal problem arises internally within Žižek's own position as soon as we take a closer look at Žižek's other major interlocutor: Schelling. Ultimately unsatisfied with Hegel's mature account of the passage from nature to culture, Žižek turns to Schelling to explicate the genesis of the Symbolic. What Hegel missed was the paradoxical essence of this very movement, a dialectical movement that causes dialectics to collapse upon itself. With the birth of subjectivity, we see a snag in substance that prevents the absolute from self-actualizing itself so that, instead of being the Idea completely returning to itself out of its otherness in nature, which would present us with a reconciliation of nature and spirit in the Idea, nature's complete self-sublation in spirit, culture is actually a mere secondary (“virtual, artificial, symbolic, not substantially natural”464) response to the primordial ontological trauma that lies at the core of the human being. For Žižek, this means that culture is the always failed attempt at reconciliation: the Idea is nothing other than this very act of its own returning to itself, this movement being constitutive of that to which is returned;465 for it to reach its end would herald its death. Representing a recoil into a psychotic, irreal space, not only do the subjective and objective/ mind and world thereby fall into infinite conflict with one another, but it becomes impossible to explain why the Ideal emerges. According to Žižek it is Schelling who, fighting against the perceived threat of Hegelian Absolute Idealism, gives the most detailed account of this immanent self-sundering of being into its real and ideal poles in his account of the Grund as the ever elusive, eternal Past of consciousness, and the pure act of unconscious decision underlying the birth of the universe of human meaning. Schelling's position is paradoxical: from within a solipsistic space (transcendental idealism) he tries to develop a philosophical language capable not only of piercing the primacy of the Real-as-lack and explaining its monstrous pre-history within pre-symbolic antagonism (materialism), a logically prior but directly unreachable modality of the Real that precedes and exceeds the Symbolic, but also of describing the inexplicable self-positing of subjectivity The Deadlocks of Ontological Catastrophe 285 When we look at Schelling's thought, even as Žižek himself presents it, we are tempted to say that, if Hegel is able to show that the absolute opposition between 286 Chapter 12 Chapter 12 286 286 idealist and realist philosophy is without meaning from within idealism, Schelling could be said to do the same from within realism and without needing to pass through the former. This is further supported by the fact that Schelling never gives us a dialectical analysis of phenomenal appearance or the structure of symbolic thought in the way Hegel does and describes his own idealism in the Darstellung as “real” or “objective,” for its principal idea is not to use thought's inconsistencies to find a new starting point for speculative philosophy, but rather to directly investigate the origins of thought itself from its dark nonconscious ground,466 a point repeated by the “higher realism” of the Freiheitsschrift467 and the emergence of ideality in the Stuttgarter Privatvorlesungen out of minerals.468 The argument that Schelling2 is an ephemeral rupture risks succumbing to arbitrariness, since now we find a manner to unify the central problematic of the Naturphilosophie and the “theosophic” philosophy of freedom. Given that one of the key tools for the development of Žižek's own metaphysics is the extracting of an unconscious Grundlogik underlying German Idealism, if Schelling's philosophy turns out to be more continuous than Žižek's analysis lets on, then his psychoanalytical construction of the German Idealist tradition could be jeopardized. For Schelling, we do not just “tarry with the negative.” The X that eludes consciousness, one of the centrepieces of Schelling's thought, is never just a mere formal limit: it is an attempt to express the subject-independent interiority of nature to which we have access despite the mediating activity of consciousness precisely because the subject and its ideational capacities are a part of nature, one way through which nature relates to itself as ground. Schelling refuses to separate the ontological in-itself of precognitive or extra- symbolic reality from the epistemological sphere of idealist representations, arguing that the two must be intimately connected if philosophy is to find a secure basis. If there is an identity between the Real and the Ideal, the problem of their relation to one another is relegated to a metaphysical or naturephilosophical level rather than a strictly epistemic or idealist one. The Deadlocks of Ontological Catastrophe 285 Whereas in the middle-late period this idea of identity is expressed by the notion of the Mitwissenschaft (“co-science”) of creation, it is more clearly for our purposes articulated in the earlier Ideas for a Philosophy of Nature, where Schelling argues for the necessity of a dialectically positive For Schelling, we do not just “tarry with the negative.” The X that eludes consciousness, one of the centrepieces of Schelling's thought, is never just a mere formal limit: it is an attempt to express the subject-independent interiority of nature to which we have access despite the mediating activity of consciousness precisely because the subject and its ideational capacities are a part of nature, one way through which nature relates to itself as ground. The Deadlocks of Ontological Catastrophe 287 288 Chapter 12 288 Chapter 12 from the Latin speculari (to watch over), it is a realism that attempts to, through a scrutinizing surveillance, account for the immanent pulsations of the universe as it transforms bodies of matter into the complex field of living being and eventually thought as ideal self-mediation, so that the Real is not only always excessive to the Ideal, that which can never be brought into it due to nature's raw productivity, but the latter also loses its theoretical primacy insofar as nature in the stirrings of its nocturnal ground itself becomes the true a priori. It is interesting to bring up this aspect of Schelling's philosophy, not just because it could be a weak point in Žižek's psychoanalytical reading, but also because it is a specific manner in which Žižek's metaphysical problematic enters into direct debate with the current speculative turn. In this regard, Iain Hamilton Grant's own transcendental materialism is in complete opposition to that of Žižek, so that contrasting the two allows us simultaneously to bring to the fore both the daring character of the latter's position and its potential internal limitations. Taking up Schelling's diagnosis that modern philosophy exhibits an agonizing deficiency—that nature does not exist for it470—Grant puts forward the argument that this diagnosis is just as sound today as ever: “[i]nsofar therefore as the antithetical couple ‘Plato- Kant’ that lay at the heart of the immediate postkantian context continues to organize metaphysics, contemporary philosophy is importantly and immediately postkantian.”471 Through a careful reconstruction of Schelling's philosophical career with an eye towards its explicit naturephilosophical content and the natural scientific context that surrounded and inspired it, Grant's thesis is that by following its spirit, we may finally find a way to leave behind us the Kantian heritage insofar as Schelling offers us, instead of a two-world metaphysics that results in an “eliminative practicism” (the irreducibility of culture to nature), a “one-world physics capable of the Idea.”472 We cannot cut the world in two, for there is no great divide: even to understand culture as culture, we have to understand its genesis from its ground within nature, for culture itself is originally natural. The Deadlocks of Ontological Catastrophe 287 interactivity between mind and matter if transcendental idealism is to have a proper founding, which goes in the face of both the Hegelian proof of the insignificance of an opposition between idealistic and realistic philosophy and a Žižekian metaphysics of the disjunctive “and”: For what we want is not that Nature should coincide with the laws of our mind by chance (as if through some third intermediary), but that she herself, necessarily and originally, should not only express, but even realize, the laws of our mind, and that she is, and is called, Nature only insofar as she does so. Nature should be Mind made visible, Mind the invisible Nature. Here then in the absolute identity of Mind in us and Nature outside us, the problem of the possibility of a Nature external to us must be resolved. The final goal of our further research is, therefore, this idea of Nature.469 It is exactly this problematic that reverberates throughout the entirety of Schelling's thinking, even when he attempts to outline the tension- ridden oscillations of the Yes and the No, the light and the dark principles, constitutive of the self-operative logic of the Grund. Rather than offering some kind of paradoxical eruptive logic balancing materialism and idealism through their infinite conflict, Schelling's philosophy can very easily be read as a passionate attempt to show that the forces underlying human spiritual-transcendental activity are nothing more than the already existent potencies of nature arisen to a higher “power” through nature's auto- development. If the starting point of Žižek's transcendental materialism is a self-grounding idealism (which shows his distinctive Lacanian-Hegelian presuppositions), Schelling's own transcendental materialism is a self- articulating realism (which shows how Schelling has been influenced by the evolutionary dynamism of natural scientists such as Kielmeyer), the stark point separating them being that whereas in the former materialism is always a spectral materialism developed in the cracks of idealism, in the latter idealism becomes, as it were, a mere conditioned phenomenon. In this strict sense, if Schelling's philosophy is an account of the self-unfolding of the powers of nature according to their inner movement, it is because it is a speculative realism understood in its original etymological meaning: deriving The Deadlocks of Ontological Catastrophe 289 organizations according to its own cryptic inner process. According to Grant, Schelling's revolutionary thesis is that the only way we can explicate ideality is to see it as just one specific expression of nature's productivity, one among many possible products of nature as a priori. There is just a difference of degree and not of type between, say, atoms, chemicals, and free ethical subjects, insofar as there is a natural history of mind to which we must have recourse to explain its apparent autonomy. Far from being an idealism that grounds itself from within the infinite self-reflexivity of thinking “with no external support of its truth,”473 Schelling's idealism is a naturephilosophical investigation into the interior involutions of being, the latter being equally inclusive of thought's transcendental activity as the somatic constitution of physical bodies. Nature is a dark dynamicity that brings philosophy far away from the practico-concrete sphere of an anthropocentric universe into the enigmatic palpating powers that gave birth to it in their antagonism and the forgotten aeons of the abyssal dregs of cosmic time that have preceded us. In this manner, Grant is taking Schelling literally at his word: organizations according to its own cryptic inner process. According to Grant, Schelling's revolutionary thesis is that the only way we can explicate ideality is to see it as just one specific expression of nature's productivity, one among many possible products of nature as a priori. There is just a difference of degree and not of type between, say, atoms, chemicals, and free ethical subjects, insofar as there is a natural history of mind to which we must have recourse to explain its apparent autonomy. Far from being an idealism that grounds itself from within the infinite self-reflexivity of thinking “with no external support of its truth,”473 Schelling's idealism is a naturephilosophical investigation into the interior involutions of being, the latter being equally inclusive of thought's transcendental activity as the somatic constitution of physical bodies. Nature is a dark dynamicity that brings philosophy far away from the practico-concrete sphere of an anthropocentric universe into the enigmatic palpating powers that gave birth to it in their antagonism and the forgotten aeons of the abyssal dregs of cosmic time that have preceded us. 288 Chapter 12 There is no absolute split between the Real and the Ideal, mind and matter, the dead movement of inanimate objects and the life of organic being—there are only the unconditioned, groundless powers of nature as a pulsating all that creates specific bodies and their various, innumerable, and unpredictable The Deadlocks of Ontological Catastrophe 289 The Deadlocks of Ontological Catastrophe 289 In this manner, Grant is taking Schelling literally at his word: A great work of the ancient world stands before us as an incomprehensible whole until we find traces of its manner of growth and gradual development. How much more must this be the case with such a multifariously assembled individual as the earth! What entirely different intricacies and folds must take place here! Even the smallest grain of sand must contain determinations within itself that we cannot exhaust until we have laid out the entire course of creative nature leading up to it. Everything is only the work of time, and it is only through time that each thing receives its particular character and meaning.474 Although Grant and Žižek are reading Schelling in different, largely incommensurable ways—the former by a remarkable reconstruction of Schelling's naturephilosophical and scientific context, the other by a violent psychoanalytical overhauling of the entire German Idealist tradition—the fact that they both fall upon the same figure to elaborate their own materialist position is extraordinarily thought provoking. First and foremost, it suggests that the role of Schelling in Žižek's thinking poses Chapter 12 290 Chapter 12 290 a potential problem for the latter's own transcendental materialist ontology of the subject, and not merely for external reasons: it challenges the latter's very notion of a metaphysics of the disjunctive “and.” The great contribution of Schelling's Naturphilosophie was to enact a complete displacement of the human subject, for it imposes upon us the counterintuitive task of a geology of morals that fights against any complete separation of the human (the realm of free acting) from the natural (the realm of necessity). The stark implications of this, as Grant himself notes, were clearly perceived by Eschenmayer, a Fichtean natural scientist, who after reading the Freiheitsschrift fell into a paroxysm of horror due to its primary soul- wrenching implication: “your essay on human freedom seems to me a complete transformation of ethics into physics, a consumption of the free by the necessary, of feeling by understanding, of the moral by the natural, and above all a complete depotentiation of the higher into the lower order of things.”475 Commenting on this passage, Grant writes: We can imagine Eschenmayer’s shock: why does this work on the subject of freedom contain so much geology? Why is the turba gentium [...], the world-disorder or species- riots, presented as the ground of freedom? [... The Deadlocks of Ontological Catastrophe 291 contemporary philosophies of finitude—he is in many ways completely justified, but in so doing he risks opening himself to the criticism that he misses how Schelling proceeds in this endeavour as well as its larger philosophical consequences, which presents two immediate major problems in his own usage of Schelling. First, if Grant is correct, Schelling rejects from the outset the very idea of a self-enclosed transcendental framework and its concomitant mind-body dualism, the ontological emergence of which is, according to Žižek, the fundamental philosophical obsession of the middle-late Schelling. If the unconscious Grundlogik of German Idealism is constituted by the dynamic of a self-grounding idealism and a spectral materialism grounded in the former, then Schelling's precise place within it would seem to be uncertain. Second, if Žižek's transcendental materialism assumes the birth of the I out of the not-I as an impossible event with no true precursor in the ancestral past of the pulsating fires of the heavens, the sluggishly slow evolution of geological formations, or even the forces of evolution in biological life-forms (none of its apparent brethren come close to its pure difference), in such a way that the self-positing of freedom literally cuts the absolute in two in an inexplicable manner, then we must conclude that Schelling's own metaphysics does not automatically result in a metaphysics wherein substance is split at its core, and thus ontological dislocation is the primordial fact. Contra Fichte, Schelling explicitly claims that there is a continuity between the I and the not-I, that the passage is one of a transition, not a leap. If psychoanalysis is to work in a therapeutic setting, an encounter with the Real can only truly be brought forth if all the intended meanings of the subject have been understood for what they are; otherwise a psychoanalytical interpretation does not work, because it does not hit the cause (which is why Lacanian analysts rarely offer interpretations). Moreover, even if we agree that Žižek is right to contend that the theosophical content of the middle-late Schelling is a mythological component that we can formalize in order to render its content more explicit (which Grant's work also implicitly does, given that he does not explicitly deal with the theological aspect of the Freiheitsschrift or the Weltalter), it is uncertain that he can so easily get rid of the naturephilosophical implications of these texts. The Deadlocks of Ontological Catastrophe 289 B]ecause the consequences of the dependence of transcendental physics on dynamic naturalism impose upon Schelling’s reconditioned transcendentalism the demand that the All be grounded in the “subject of nature itself,” i.e., in the forces. Accordingly, the more disorderly the phenomenon, the darker and more abyssal the ground. This is why the inquiry into human freedom must (a) specify the attachment of this power of infinite evolution to a finite phenomenon (human), and (b) consider the ground of such a freedom as derivative of the “self-operation of the ground” or the “will of the deep” in the geological series: the potentiating series through which such a freedom must (repeatedly) evolve must therefore present the expression of geological potencies in practical intelligence.476 In this respect, when Žižek claims that it is Schelling who gives the most detailed description of the ontogenesis of subjectivity in the philosophical tradition—which makes him the father of dialectical materialism and The Deadlocks of Ontological Catastrophe 291 The Deadlocks of Ontological Catastrophe 291 But the stakes are much higher than those of the philological-textual fidelity of an interpretation in this case: if Schelling's Chapter 12 292 Chapter 12 The Deadlocks of Ontological Catastrophe 293 at a conception of 'nature as subject,'” this investigation is never a mere reconstruction within the ideal series of the Real, but is rather a movement of the Real itself, for “such a philosophical system does not therefore seek a fixed point from which to gain leverage on an external world, nor to rise above it, but is itself a 'genetic' [...] movement in and on this world, unconditionally.”479 Schelling expresses this identity most daringly when he says that “to philosophize about nature means to create nature.”480 “Mental” activity is always already a part of nature and therefore a part of its own auto- development by being one of its emergent attributes, but nature, being the truly and absolutely a priori, is unconditional and thereby guarantees that thought is never limited to the mere ideational or physiological constraints of conditioned particulars insofar as it is an expression of nature's productivity rather than embodied in the fixity of one of its products. In this way, for Schelling, we are primordially “connected” with nature as the pre-Symbolic, insofar as the Real and the Ideal remain identified at an essential level, the consequence being that the subject is not a dialectically non-sublatable in-between that exists as the psychotic withdrawal of the world into self. Accordingly, Žižek's reliance upon Schellingian ontology risks problematizing his own position, for if Žižek draws upon Schelling as a partner for the elaboration of the impossible genesis of the transcendental out of an orgasm of forces within the pre-symbolic Real, this immediately draws our attention to other possibilities of understanding the subject that do not present the latter as an irrevocable moment of ontological catastrophe in the flux of material being. This would force us to rethink the very nature of the psychoanalytical experience and the essence of the disjunctive “and” that is central to Žižek's own parallax ontology—and in this manner, not only does its theoretical first principle begin to tremble, but the very primacy of the Lacanian psychoanalysis that serves as its starting point is put into question. Chapter 12 292 Cha 292 philosophy presents a framework incompatible with the one Žižek sees in it and his own psychoanalytical reconstruction extensively relies upon this for its own argumentative vitality, Žižek's own development of a metaphysics of the Real by an engagement with Schelling is not only potentially misguided, but could also lose sight of various other resources explicit in Schelling that could be used to rethink the materialism-idealism relationship. Elaborating on Grant's argument, we see that Schelling completely bypasses the problem of the materialism-idealism relationship because idealism is never a purely self-referential play. The whole concept of a “spectral” materialism just has no place here; speculative philosophy has a stricto sensu non-idealistic foundation. As Grant succinctly puts it, what is at stake is the “impersonal coincidence of the transcendentally generated universal and self-generating nature [... and] Schelling’s hypothesis is, in other words, that there is a naturalistic or physicalist ground of philosophy,”477 a ground that does not implicate an eliminative materialism wherein all is reduced to empirically observable bodies—somatism—but in such a way that genuine philosophy “consists in the dynamic elaboration of the identity of nature and Ideas.”478 In this manner, if one reads Žižek's own transcendental materialism alongside that which Grant develops from his own reactualization of Schelling, one is presented with an alternative to Žižek's own metaphysics of the not-all, one not centred in the Ideal as being's irreconcilable self-division, but rather one based upon the fragile productivity of nature as it contingently and continually takes on new forms (and destroys others), a productivity that in no way has man as its summit, but will create new creatures (and monsters) without end because there is no stasis, but only a restless movement of the depths, a beautiful and macabre dance of great delicacy and improvisation whose actors simultaneously whimper and laugh under its weight. Lacanian psychoanalysis prohibits this Schellingian move because it would require that the chain of signification constitutive of human language be not based on an operationally closed system with no natural grounding, but could actually open up onto the world as it is in its own interior involutions because it would be, as it were, one with it. Although we philosophize about nature by “following a procedure of successive unconditioning performed by thought-operations about nature,” a process that allows us to “arrive The Deadlocks of Ontological Catastrophe 293 It is highly revelatory that to make use of Schelling for his project in the first place Žižek can only focus on two works (the Freiheitsschrift and the second draft of the Weltalter) because of their apparently disavowed Hegelian structure as that which would enable one to extract from them a self-operative logic establishing the primacy of the Real- as lack through the abyss of unconscious decision 294 Chapter 12 We can begin to see why Žižek's proclamation that his project is Hegelian, but never Schellingian (despite the fact that the Freiheitsschrift and the Weltalter contain the most vivid description of the emergence of the Symbolic), is multilayered. First and foremost, Žižek takes radical idealism as the only true beginning for philosophy insofar as we can only interact with the world through the medium of thought, making correlationism basic to our experience. If the cracks within ideality epistemologically enable us to develop a spectral materialism, then the irreducibility of the Real-as-lack paradoxically does not prohibit us from having access to being as the Real-as-excess because this very concept thereby becomes internal to our notional apparatus. In this manner, Žižek is quite justified in saying that his project is Hegelian given that—on his own reading of Hegel at least—it strictly speaking shares this identical starting point and draws from it the exact same consequences, a move that in turn allows him to avoid the problem of expressing too strong a reliance on and debt to Schelling, which could potentially bring his own thinking uncomfortably close to everything he denies: the non-Freudian unconscious (in its Bergsonian, Jungian, Deleuzian, etc., forms), “pre-modern” cosmology, or Romantic theories of nature. Yet, this does not by any means solve the ambiguity of the Hegel-Schelling relationship in his thinking, for as we have seen, what is crucial to Žižek's own reading of Schelling is that Schelling's own theoretico-epistemological framework, at least in the second period of his thinking, is unknown to itself the same as that of Hegel. Although Žižek has to do great violence to Schelling to extract a Hegelian dialectical structure of negativity in his texts, nevertheless the interpretation he presents is extremely internally consistent and methodologically sound. If the thoroughness of a position like Grant's is a challenge to Žižek, then Žižek's own reading of Schelling is equally a challenge to Grant's and other canonical and non-canonical interpretations that exist. The game goes both ways—and because the conceptual terrain within which both operate is vastly different, it is not evident how we are to decide upon the favourability of one interpretation over another However one has to underline that this If the thoroughness of a position like Grant's is a challenge to Žižek, then Žižek's own reading of Schelling is equally a challenge to Grant's and other canonical and non-canonical interpretations that exist. The Deadlocks of Ontological Catastrophe 295 tradition, but an unconscious truth that can be seen to deploy itself through them. Accordingly, not only is there absolutely no contradiction in saying that Žižek's philosophy is a hybridism of Hegelian logic and Schellingian ontology insofar as it is precisely this hybridism that can retroactively be seen to be the traumatic core at the formative heart of the tradition itself, but reflecting upon the intrinsic ambiguity of the Hegel-Schelling relationship helps us reveal the originality and daringness of Žižek's critical metaphysics. 294 Chapter 12 The game goes both ways—and because the conceptual terrain within which both operate is vastly different, it is not evident how we are to decide upon the favourability of one interpretation over another. However, one has to underline that this oscillation between Schelling and Hegel is not so much an inconsistency or sleight-of-hand gesture on Žižek's part, the reason being that what interests him is less the specific differences between the historical thinkers of the The Deadlocks of Ontological Catastrophe 295 96 Chapter 12 However, the very inconsistency of our notional apparatus allows us to develop knowledge of reality in itself, for as soon as we “apply” a category to the world and it shows itself as inadequate, we see that the field of appearing is always more than appearance, whereby the noumenal now appears as the self-limitation of the phenomenal. Our experience of the world is not a full-blown hallucination: we can use these experiences of breakdown to our advantage in order to explore a world that only seems to be infinitely “beyond” our reach. The Real-as-excess becomes an intra-discursive category, so that epistemic limitations of knowledge negatively demonstrate our inclusion into and thus capacity of understanding the world at large, rather than our imprisonment in a socially constructed universe of discourse. It is precisely because of this that Žižek says that “the true problem is not how to reach the Real when we are confined to the interplay of the (inconsistent) multitude of appearances, but, more radically, the properly Hegelian one: how does appearance itself emerge from the interplay of the Real?”481 Second, given that we are in some sense a part of the absolute, our failure to reach it has to coincide with a failure of the absolute itself. Žižek's wager is that if we do fail in reaching the absolute in thought, this cannot be due merely to the finitude of our notional apparatus: “[i]f we can think our knowledge of reality (i.e., the way reality appears to us) as radically failed, as radically different from the Absolute, then this gap (between the for-us and the in-itself) must be part of the Absolute itself, so that the very feature that seemed forever to keep us away from the Absolute is the only feature which directly unites us with the Absolute.”482 The point is not to “'overcome' the gap [...] but to take note of how this gap is internal to [the Absolute]”:483 arguing that our inability to overcome our entrapment in the Symbolic and find our place within being is already the very lost object we are looking for, Žižek turns epistemological limit into positive ontological condition by inscribing the limitation of knowledge into the world as an event immanent in the latter: that is, by making it an (auto-)‍limitation of the absolute itself. 12.2 Anton-Babinski Syndrome: Slavoj Žižek's Paradoxical Overcoming of Idealism Although Žižek's appropriation of Schelling could be perceived as highly problematic, insofar as the latter may not so easily fit into the proto- structuralist framework Žižek sees as operative in German Idealism, another problem immediately arises that potentially hits the core of his overcoming of radical idealism. Even if we accept the legitimacy of his reading of Schelling, it is unlikely that Žižek's own Hegelian attempt to show the insignificance of any absolute opposition between idealist and realist philosophies from within idealism would satisfy a realist. If we never leave the clutches of idealism, then any knowledge that we possess would never be able to reach the absolute in its pure non-correlationality to the subject. We are always entrapped in the masturbatory play of signifiers in their incessant sliding. Moreover, the Real-as-excess as what precedes and exceeds consciousness is an explicitly impossible concept. Even Žižek's “materialist” response to idealism has as its fundamental task to bring to the fore this intrinsic impossibility: remodulating the Schellingian act of unconscious decision, its seeks to show how the shift from the Real-as-excess (pre- symbolic antagonism) to the Real-as-lack (symbolic imprisonment) is the ultimate ontological parallax by a speculative fabulation of the always lost and inaccessible moment of the auto-disruption of the noumenal realm. At its best, realist metaphysics appears to be reduced to mythology. Žižek's overcoming of idealism articulates itself in two distinct moments. First, since the Real-as-lack is the logical zero-level of any philosophy due to the insurmountability of the Symbolic, to arrive at some kind of knowledge of an “extra-”notional zone of experience (the Real-as-excess) Chapter 12 96 Chapter 12 296 would appear a priori foreclosed. However, the very inconsistency of our notional apparatus allows us to develop knowledge of reality in itself, for as soon as we “apply” a category to the world and it shows itself as inadequate, we see that the field of appearing is always more than appearance, whereby the noumenal now appears as the self-limitation of the phenomenal. Our experience of the world is not a full-blown hallucination: we can use these experiences of breakdown to our advantage in order to explore a world that only seems to be infinitely “beyond” our reach. The Real-as-excess becomes an intra-discursive category, so that epistemic limitations of knowledge negatively demonstrate our inclusion into and thus capacity of understanding the world at large, rather than our imprisonment in a socially constructed universe of discourse. It is precisely because of this that Žižek says that “the true problem is not how to reach the Real when we are confined to the interplay of the (inconsistent) multitude of appearances, but, more radically, the properly Hegelian one: how does appearance itself emerge from the interplay of the Real?”481 Second, given that we are in some sense a part of the absolute, our failure to reach it has to coincide with a failure of the absolute itself. Žižek's wager is that if we do fail in reaching the absolute in thought, this cannot be due merely to the finitude of our notional apparatus: “[i]f we can think our knowledge of reality (i.e., the way reality appears to us) as radically failed, as radically different from the Absolute, then this gap (between the for-us and the in-itself) must be part of the Absolute itself, so that the very feature that seemed forever to keep us away from the Absolute is the only feature which directly unites us with the Absolute.”482 The point is not to “'overcome' the gap [...] but to take note of how this gap is internal to [the Absolute]”:483 arguing that our inability to overcome our entrapment in the Symbolic and find our place within being is already the very lost object we are looking for, Žižek turns epistemological limit into positive ontological condition by inscribing the limitation of knowledge i h ld i i h l h i b ki i would appear a priori foreclosed. The Deadlocks of Ontological Catastrophe 297 reality; moving from one to the other just requires a certain switch of perspectives, a parallax shift, whose very possibility we can only explain by making the very irreconcilable split between idealism and materialism the imperceptible truth of both. Žižek's name for that which can strangely mediate between them because it is neither idealistic nor materialistic yet is included in both as excluded (“include me out!”) is the subject as an insurmountable ontological and symbolic lacuna. Consequently, the Symbolic is always already more than itself because it points to its dark origins in being's passage through madness—and even if the precise moment in which the world withdraws into its nocturnal, irreal self is forever lost in the universe of meaning it brings forth, we are nevertheless justified in mytho-poetically fabulating the act of decision that induces our collective psychosis, because the Symbolic can never do away with its origins. Although the undecidable ambiguity of the Real is a problem Žižek inherits from Lacan, a problem similar to that faced by many forms of idealism, Žižek throughout his writings remains true to his great master's attempt to desubstantialize the Real, but with an important twist, a twist that accentuates the theoretical challenge of his critical metaphysics. What most clearly distinguishes Žižek's project from that of Lacan is his refusal to take our lack of access to the Real as a brute fact and his subsequent endeavour to inscribe it within being. The question arises, however, of whether Žižek's account of this emergent ontological parallax is even philosophically possible, given his epistemological commitments. Since phenomenal reality emerges only after being has sundered itself, whereby our access to the Real must be mediated by transcendental constitution, Žižek's double claim that the internal inconsistency of idealism is that which allows us to overcome it from within and that our inability to reach the thing itself is already that which we are looking for poses two important difficulties to his project that, for many, may not be adequately resolved or seem outright problematic. On the one hand, to switch epistemological limit into positive ontological condition by a mere parallax shift of perspective appears in many ways to be a mere sleight-of-hand argument. 96 Chapter 12 If idealism is some form of ontological solipsism, then it must be revelatory of a zone wherein the absolute is irrevocably non-coincident to itself. In another vein, this means that any radically self-grounding idealism is always already a materialism, the two being nothing more than supplementary views on the same underlying The Deadlocks of Ontological Catastrophe 297 The Deadlocks of Ontological Catastrophe 297 Given that this precise moment where our division from the absolute coincides with the self-division of the absolute (so that the story we are telling about being is simultaneously the story that being is telling to itself) can only be 298 Chapter 12 298 narrativized at the level of mytho-poetics, which reconciles substance and subject at the level of form and never at the level of content, it is unclear that the claim that the problem its own solution does the argumentative work it purports to do. To put it bluntly, since this precise moment defies any proper speculative explanation, it merely covers up the underlying issue that we are facing: that is, how we could have access to being in the first place. On the other, it is unclear that we can really collapse the distinction between realism and idealism by making the very distinction itself intra-conceptual or intra-discursive insofar as this move fails to sufficiently provide the conditions under which we could develop a truly speculative account of reality in itself that is not always already entrapped within the ambiguities of symbolization. In both cases, a realist would be quick to argue that we have done everything but leave the correlationalist circle, that we are stuck in a constituted world for us. For the former, it could not be said that we have some kind of “access” to reality through the immanent obstructions of the Symbolic as that which indicates the spectral presence of an extra-notional reality posited from within it, for the Real is a mere effect of the Symbolic. Does this go far enough in establishing the groundwork for a new metaphysics? If we call the Real an internal limit or limitation of the Symbolic, we must be careful, because the Real is not so much a limit in the sense of a border that separates two distinct yet commensurable terrains, or a limitation in the sense of a restricting condition coming from an exterior force that one ought to overcome. Rather, the adjective “internal” is of utmost importance here because it stresses that the Real is completely immanent to the Symbolic's very idealizing activity in such a way that there is no outside except an outside that is paradoxically posited as inside. The Deadlocks of Ontological Catastrophe 297 If the Symbolic functions within a psychotic withdrawal from the world, not only does it freely (re)‍constitute reality according to an autonomous, self-referential play or ciphering, but any obstruction that occurs within it would only be due to its freedom. In its first guise, the Real is nothing other than “a purely formal parallax gap or impossibility,” “the rupture or gap which makes the order of discourses always and constitutively inconsistent and non-totalizable,”484 so that even if we are permitted to call this negative encounter with the Real a “positive running up against” the exterior world that operates as if it “touches the Real,”485 in the same breath we must qualify this statement 300 Chapter 12 Lost in visual madness, they can never indirectly see the world shine through the inconsistencies of their hallucination. Here the case of Anton-Babinski syndrome should be evoked as a possible skeptical argument against Žižek's overcoming of radical idealism. A rare medical phenomenon, the syndrome is a symptom of brain damage (usually from a stroke) in the occipital lobe. What is so peculiar is that people who suffer from it, although cortically blind, claim that they can see. In their speech and general behaviour there is often, at first, no sign of blindness—family members and the medical team typically only begin to notice something is amiss when the patient begins to stumble into various physical objects in their path, whether it be tripping over a coffee table in front of him, walking into a wall, or describing things that are not really there. Not only do patients continue to refuse to admit their blindness despite all the inexorable obstructions in the all-out hallucination of their own visible field of experience, but, more primordially, it is clear that no amount of tarrying with the negative offered by the latter's internal short- circuiting would ever enable them to develop a “spectral” vision of the world in itself of which they have been deprived through organic devastation. To deal with the incomprehensible agony caused by such constant disturbances in their psychotically self-sufficient and imagined perception of subjective reality, those who suffer from Anton-Babinski syndrome actually find ways of giving support to its free generation by falsifying their memories, a process that in the medical community is called confabulation. The Deadlocks of Ontological Catastrophe 299 insofar as this can only be brought forth in the aftermath of symbolic distortion, that is, après-coup. This recognition of an indirect confrontation with a constitutive outside internal to the Symbolic is in itself just another symbolization: any “materialism” that could be developed by means of it will always already be entrapped within its ambiguities, so that this “materialism” is nothing but a mere retroactive adjustment of ideality to accommodate for its internal inconsistencies, for it is only from within the Symbolic that we see the Real as the residue of a failed attempt to synthesize an “extra”-notional reality. As Adrian Johnston puts it: It's not that there is no Real that isn't immanent to the Symbolic. Instead, the non-immanent Real is accessible exclusively through the deadlocks and inconsistencies immanent to the Symbolic [...]. The Real-as-presupposed [the Real-as-excess as posited in/by the Symbolic] actually exists “for us” only insofar as it indirectly shines through the cracks in the façade of Imaginary-Symbolic reality, insofar as it is asymptotically approached by the parlêtre along the fault lines of this reality's inner conflicts.486 But “...indirectly shines through...” is a misleading metaphor: nothing breaks through the prison of language. As Žižek says, “we do not touch the Real by way of breaking out of the prison of language and gaining access to the external transcendent referent [...]. We touch the Real-in-itself in our very failure to touch it.”487 This is what Žižek emphasizes when he posits an ontological passage through madness at the beginning of the Symbolic, for once it has occurred there is no contact with the world that is possible. If we take Lacan and Žižek at their word, we can never truly liberate ourselves from the psychotically self-sustaining construction of reality that is the Symbolic's autonomous idealization. Even if the latter does not equate to an omnipotent, non-limited hallucination of our world of experience—a Godlike primary process—nevertheless there is no escape from our collective hallucination of reality: impenetrable in its density, omnipresent in its extension, nothing is left untouched by this tenebrous realm of transcendental phantasmagoria within which we live and breathe as speaking subjects. The light of being is unable to radiate through the holes of the all- encompassing web of the Symbolic. 300 Chapter 12 Here the case of Anton-Babinski syndrome should be evoked as a possible skeptical argument against Žižek's overcoming of radical idealism. A rare medical phenomenon, the syndrome is a symptom of brain damage (usually from a stroke) in the occipital lobe. What is so peculiar is that people who suffer from it, although cortically blind, claim that they can see. In their speech and general behaviour there is often, at first, no sign of blindness—family members and the medical team typically only begin to notice something is amiss when the patient begins to stumble into various physical objects in their path, whether it be tripping over a coffee table in front of him, walking into a wall, or describing things that are not really there. Not only do patients continue to refuse to admit their blindness despite all the inexorable obstructions in the all-out hallucination of their own visible field of experience, but, more primordially, it is clear that no amount of tarrying with the negative offered by the latter's internal short- circuiting would ever enable them to develop a “spectral” vision of the world in itself of which they have been deprived through organic devastation. To deal with the incomprehensible agony caused by such constant disturbances in their psychotically self-sufficient and imagined perception of subjective reality, those who suffer from Anton-Babinski syndrome actually find ways of giving support to its free generation by falsifying their memories, a process that in the medical community is called confabulation. In other words, even if a patient, realizing their condition, were to think that they are actually in the process of developing a sound mental map of the physical universe that is around them through the aid of their mishaps as a means of retroactively readjusting their imaginary field, and this not only with the hope of learning to navigate within it, but also to overcome their blindness by making the absolute opposition between a hallucinated world produced in the void of blindness and a vision of objective reality caused by retina input without meaning within their hallucination, it must be concluded that they could never assure themselves that this “spectral” seeing captures the world nor whether it is not just another hallucination that has been produced to save themselves from the psychological trauma of their own blindness. The Deadlocks of Ontological Catastrophe 301 What complicates this philosophical issue is the fact that in all mytho-poetic narratives where the very “origins” of the Symbolic in the Real are at stake, the event in question that institutes the movement from one to the other “never effectively took place within temporal reality, [although] one has to presuppose it hypothetically in order to account for the consistency of the temporal process.”488 The result is that the event of the decision that violently separates Grund from existence potentially never occurred: it could be nothing but a fantasmatic, retroactive posit But isn't this precisely the same situation we find ourselves in with respect to Žižek's attempt to break the correlationalist circle? The only possibility for an ontological grounding of the psychoanalytico-Cartesian subject being a phantom-like vision of the world building itself within the internal obstructions of the Symbolic's ciphering of the world, it would seem that just as it is impossible for those suffering from Anton-Babinski syndrome to spectrally construct a vision of the world that has been lost to them due to their lack of sight, so it is impossible that one could achieve some kind of paradoxical coincidence of the subjective and the objective capable of positive truth from within the nocturnal night of the world. If the self-overcoming of radical idealism proves insufficient to ground a new speculative philosophy, then the only alternative left is a pure mytho-poetic fabulation of the obscure origins of the Symbolic, the latter's ontological solipsism always already pointing beyond itself to an inaccessible material event that haunts it but that remains forever inaccessible. Unable to sublate the opposition of realism and idealism from within idealism, we could still, by writing being's poem, provide a mythological account of how our division from being is the same as being's division to itself, thereby hinting towards how this opposition is always already reconciled from within realism, making the problem itself moot: we must tell a story that inscribes our failure to reach the absolute in the absolute itself, so that which appears to keep us from the absolute is in actuality the only thing that ties us to it. Our madness is being's own. 300 Chapter 12 In other words, even if a patient, realizing their condition, were to think that they are actually in the process of developing a sound mental map of the physical universe that is around them through the aid of their mishaps as a means of retroactively readjusting their imaginary field, and this not only with the hope of learning to navigate within it, but also to overcome their blindness by making the absolute opposition between a hallucinated world produced in the void of blindness and a vision of objective reality caused by retina input without meaning within their hallucination, it must be concluded that they could never assure themselves that this “spectral” seeing captures the world nor whether it is not just another hallucination that has been produced to save themselves from the psychological trauma of their own blindness. Lost in visual madness, they can never indirectly see the world shine through the inconsistencies of their hallucination. The Deadlocks of Ontological Catastrophe 301 The Deadlocks of Ontological Catastrophe 301 But isn't this precisely the same situation we find ourselves in with respect to Žižek's attempt to break the correlationalist circle? The only possibility for an ontological grounding of the psychoanalytico-Cartesian subject being a phantom-like vision of the world building itself within the internal obstructions of the Symbolic's ciphering of the world, it would seem that just as it is impossible for those suffering from Anton-Babinski syndrome to spectrally construct a vision of the world that has been lost to them due to their lack of sight, so it is impossible that one could achieve some kind of paradoxical coincidence of the subjective and the objective capable of positive truth from within the nocturnal night of the world. If the self-overcoming of radical idealism proves insufficient to ground a new speculative philosophy, then the only alternative left is a pure mytho-poetic fabulation of the obscure origins of the Symbolic, the latter's ontological solipsism always already pointing beyond itself to an inaccessible material event that haunts it but that remains forever inaccessible. Unable to sublate the opposition of realism and idealism from within idealism, we could still, by writing being's poem, provide a mythological account of how our division from being is the same as being's division to itself, thereby hinting towards how this opposition is always already reconciled from within realism, making the problem itself moot: we must tell a story that inscribes our failure to reach the absolute in the absolute itself, so that which appears to keep us from the absolute is in actuality the only thing that ties us to it. Our madness is being's own. But if such a medium of expression presents itself as the rational necessity of a non-rational discourse to explain discourse as such, then just as those suffering from Anton-Babinski syndrome create false memories to guarantee the consistency of their self-sufficient hallucination, so too does all speculative fabulation risk always being nothing more than a confabulation. The Deadlocks of Ontological Catastrophe 301 But if such a medium of expression presents itself as the rational necessity of a non-rational discourse to explain discourse as such, then just as those suffering from Anton-Babinski syndrome create false memories to guarantee the consistency of their self-sufficient hallucination, so too does all speculative fabulation risk always being nothing more than a confabulation. What complicates this philosophical issue is the fact that in all mytho-poetic narratives where the very “origins” of the Symbolic in the Real are at stake, the event in question that institutes the movement from one to the other “never effectively took place within temporal reality, [although] one has to presuppose it hypothetically in order to account for the consistency of the temporal process.”488 The result is that the event of the decision that violently separates Grund from existence potentially Chapter 12 302 necessary for the internal consistency of our universe of meaning, so that the distinction between philosophy and fantasy/defence mechanism risks being blurred. As a consequence, Žižek's Schellingian “obscurantist idealist” manner of “deducing” this act from the pre-Symbolic could only be true insofar as it gestures towards the fundamental horror underlying subjectivity, just as the empirically false memories unearthed by those with false memory syndrome (being seduced, child sexual abuse) often merely reveal an underlying deadlock haunting a patient (that there is no sexual relationship). In this regard, not only is it unclear how we could truly test one mytho- poetic fabulation against another so as to guarantee their scientificity, but whether they have any metaphysical or ontological merit as such. Given that our freedom means that we are forever stuck within a Given that our freedom means that we are forever stuck within a constitutive psychosis, the withdrawal of the world into the eternal darkness of its irreal self, the very category of truth here has been so starkly modified that Žižek's own philosophy risks undercutting the very ground it seeks. The subject is reduced to a mere spinning in the void of freedom, a void whose very emergence appears to render itself inexplicable and problematize any knowledge of the “outside” world. The Deadlocks of Ontological Catastrophe 301 A realist will not only always find the reduction of the thought/being opposition to an intra-conceptual distinction an insufficient basis for a positive knowledge of the ontological and its vicissitudes, but will also reject myth as a speculative science insofar as correlationism has been preserved rather than overcome, for without the prior self-overcoming of idealism, the best mytho-poetics can do in the framework of a radical subjective idealism is to reconcile substance and subject at the level of mere mythological form rather than that of content. If idealism is co-incident with an ontological passage through madness, how could we develop a form of linguistic thinking able to overcome the psychotic withdrawal from objective reality that appears to be its very meta- transcendental condition of possibility to describe its event in being? The Deadlocks of Ontological Catastrophe a transcendental materialist account of its emergence out of the not-I? Or is it not Fichte who, by refusing to fall upon the speculative potential opened up by the Anstoß and sticking to the internal dynamics of subjectification, ultimately has the last laugh in the history of post-Kantianism as a paradoxical attempt to develop a new metaphysics in the wake of idealism? Could he have uncannily predicted this dilemma? Perhaps it is in this precise sense that we should read Fichte's incomprehension of his critics, an incomprehension designed not so much to show his disgust at the childish laughter of established scholars at the apparent absurdity of his position (“Fichte, do you really think that air and light are a priori transcendental conditions of human freedom?”) as to directly express by public ridicule his own laughter at the absurdity of their position (“Established scholars, you really think you can break free of correlationism and develop a speculative philosophy?”): I tell them that I have given here an a priori deduction of air and light. They answer me: “Air and light a priori, just think of it! Ha ha ha! Ha ha ha! Ha ha ha! Come on, laugh along with us! Ha ha ha! Ha ha ha! Ha ha ha! Air and light a priori: tarte à la crème, ha ha ha! Air and light a priori! Tarte à la crème, ha ha ha! Air and light a priori! Tarte à la crème, ha ha ha!” et cetera ad infinitum. Stunned, I look around me. Where did I lose my way? I thought that I had entered the republic of scholars. Have I fallen into a madhouse instead?489 For Fichte, true madness is not the psychotic withdrawal at the founding gesture of subjectivity, but rather rejecting its implications—in short, acting as if it never happened. 12.3 Fichte's Laughter, Henri Maldiney, and the Necessity of a “Successful” Psychotic Thinking Spinning in the void of freedom—isn't this the Fichtean position? Does Žižek truly succeed in overcoming the theoretical impossibility forced upon us by the pure I and develop what Fichte thought was contrary to reason: namely, The Deadlocks of Ontological Catastrophe The Deadlocks of Ontological Catastrophe If the ontological solipsism of the Ideal reduces all reality to a mere image, so that all “is transformed into a fabulous dream, without there being any life the dream is about, without there being a mind which dreams; a dream which hangs together in a dream of itself,”490 rather than bemoaning the loss of being, we should realize the implications of this inexplicable leap into freedom, that is, that the phenomenal world “absolutely creates itself [...] in a genesis out of nothing.”491 Just like the Fichtean transcendental Wissenschaftslehrer is able to come to the realization that life as we know it is nothing but a void doubled in Chapter 12 304 on itself, a dream of a dream, so too the Žižekian transcendental materialist is able from within the throes of originary psychosis to see this psychosis for what it is. But he does not stop there. While the Wissenschaftslehrer proclaims that the only thing left for us to do is to actively create, through the infinity of imagination, the groundless images necessary to fully actualize our freedom in concrete striving, the transcendental materialist pauses for a moment at this insight: if we can see that our life is a dream of a dream, if we can understand psychosis as psychotic, then there must be a minimal level of distance possible, as it were, between us and the transcendental (re)constitution of reality as a collective hallucination—and it is precisely this distance that enables us to thematize the entire process for what it is both in terms of the internal dynamics of subjectification and its wider inscription within being. In short, the theoretical gesture at the heart of Žižek's project is the following: if we can recognize our symbolic entrapment as entrapment, then our idealist psychosis is not only non-coincident with itself, but must in certain instances be “unable” to fully lock us within its cage. The very reason why we even know that there is a free transcendental constructionism fabricating our world of experience in the first place is that this constructionism fails and is unable to absolutely create itself: radical idealism fails to be radical idealism because it is haunted by seemingly non-ideal constraints, so that in this immanent failure it opens up the space for a new form of materialism insofar as it demonstrates that the Symbolic is always already minimally outside itself. The Deadlocks of Ontological Catastrophe In this sense, the Real-as-lack, as that which was apparently at the very root of the realist objection to being able to overcome correlationism from within idealism, is of irreducible importance since it enables us to enact a metaphysical archaeology of the subject, and thus mytho-poetically fabulate a picture of its emergence from a pre-symbolic antagonism that sets the stage for the free idealization of the world. For otherwise, we cannot explicate how we can see psychosis as psychotic in the first place. It is the only possible Archimedean point from which we could be saved from confabulation by a constant tarrying with its traumatic piercing. Yet a speculative fabulation is merely that—a fabulation: recognizing the limits of rational inquiry for describing the exact moment of withdrawal into self at the commencement of the universe of The Deadlocks of Ontological Catastrophe 305 The Deadlocks of Ontological Catastrophe 305 For their very incompatibility here is due to an extremely close proximity,” for given that the phenomenological aims to be the (self-) thematization of the phenomenal, their very distinction risks dissipating into nothing as a certain undecidability emerges.494 The task of the phenomenological science of psychosis is to let psychosis live in its fullness, to show its true meaning. schizophrenia as the object of a science). But this presupposition is, in fact, a Hegelian presupposition: “let oneself be carried by the very nature of things” is a literal repetition of the definition of science in the Preface of The Phenomenology of Spirit, where it is said that, in the dialectical method, we must let ourselves sink into the content at hand, thereby “letting it move spontaneously of its own nature.”493 We must always presuppose an interior life of the object whose essence will then be freely and spiritually internalized/mediated/idealized by the concept. Even if this presupposition appears harmless, it has a strange consequence in this context because of the specific object under investigation by the phenomenologist: “[i]t is not enough to bear witness to the incompatibility between science and psychosis. For their very incompatibility here is due to an extremely close proximity,” for given that the phenomenological aims to be the (self-) thematization of the phenomenal, their very distinction risks dissipating into nothing as a certain undecidability emerges.494 The task of the phenomenological science of psychosis is to let psychosis live in its fullness, to show its true meaning. It is this methodological ambiguity that in turn enables Maldiney to reap a wealth of resources from the experience of psychosis. For Maldiney, the similarity that Hegel's dialectical method in The Phenomenology of Spirit bears to Binswanger's phenomenological approach to schizophrenia and thus, by implication, to his own to psychosis is not just limited to how they define their way of proceeding. Rather, it can also be seen at the very level of the investigation of their objects, so that there exists an extremely close proximity between the two levels of investigation that is constantly in danger of conflating them, but with an important twist. The Deadlocks of Ontological Catastrophe 305 In this sense, there is a distinction to be made between (the) phenomenal experience (of schizophrenia) and (the) phenomenological experience (of Chapter 12 306 schizophrenia as the object of a science). But this presupposition is, in fact, a Hegelian presupposition: “let oneself be carried by the very nature of things” is a literal repetition of the definition of science in the Preface of The Phenomenology of Spirit, where it is said that, in the dialectical method, we must let ourselves sink into the content at hand, thereby “letting it move spontaneously of its own nature.”493 We must always presuppose an interior life of the object whose essence will then be freely and spiritually internalized/mediated/idealized by the concept. Even if this presupposition appears harmless, it has a strange consequence in this context because of the specific object under investigation by the phenomenologist: “[i]t is not enough to bear witness to the incompatibility between science and psychosis. For their very incompatibility here is due to an extremely close proximity,” for given that the phenomenological aims to be the (self-) thematization of the phenomenal, their very distinction risks dissipating into nothing as a certain undecidability emerges.494 The task of the phenomenological science of psychosis is to let psychosis live in its fullness, to show its true meaning. schizophrenia as the object of a science). But this presupposition is, in fact, a Hegelian presupposition: “let oneself be carried by the very nature of things” is a literal repetition of the definition of science in the Preface of The Phenomenology of Spirit, where it is said that, in the dialectical method, we must let ourselves sink into the content at hand, thereby “letting it move spontaneously of its own nature.”493 We must always presuppose an interior life of the object whose essence will then be freely and spiritually internalized/mediated/idealized by the concept. Even if this presupposition appears harmless, it has a strange consequence in this context because of the specific object under investigation by the phenomenologist: “[i]t is not enough to bear witness to the incompatibility between science and psychosis. The Deadlocks of Ontological Catastrophe 305 intrinsic inaccessibility of its object. Even if such a medium is justified by means of the Symbolic's own failure, which shows itself in a parallax shift of perspective as a disruptive ontological occurrence, we must concede that the actual narrative cannot truly articulate the miraculous advent of subjectivity in the Real. By delving into the impossible, the best it gives us is a sideways glance into the always absent origin. The scientific legitimacy of mytho-poetics relies, for its theoretical force, largely on Žižek's solution to the realism-idealism debate from within idealism. To embark upon a mythologico-metaphysical archeology of the subject is to try to come to terms with the unfathomable zone in between the pure Real and the Symbolic that lies paradoxically in both and neither. But to describe the passage from one to the other is stricto sensu impossible because such a passage that can be nothing other than an unpredictable event that arises ex nihilo within the Real itself and which simultaneously is always already withdrawn from the very logical space that could rationally investigate it. But to see this as an impossibility in the Real (“the leaping point”) and not just of the Symbolic (its “origin” in unconscious decision) presupposes that the question has changed from how we can gain access to the Real through the Symbolic to the ambiguous genesis of the latter out of the former. But has Žižek given us an adequate foothold from within which we can escape correlationism and answer this? Henri Maldiney, a little known French phenomenologist who rethinks human transcendence through the experience of psychosis, can give us some useful if controversial resources to draw out the intrinsically paradoxical nature of this inquiry. Discussing the introduction to Ludwig Binswanger's case studies on schizophrenia,492 Maldiney outlines a peculiarity in the former's phenomenological method. Rejecting the possibility of understanding schizophrenia directly, either through positivistic methodology or an immediate experience (what the person says being inadequate to express their illness), one has, as Biswanger notes, to “let oneself be carried by the very nature of things,” that is, presuppose an inner, self-articulating structuration of the phenomenon that will reveal itself through a careful description as that which lets its phenomenological essence mediate itself to us. The Deadlocks of Ontological Catastrophe 305 In the case of a phenomenology of psychosis, by letting psychosis speak the fullness of its essence by letting oneself be carried away by the in-dwelling logic underlying its phenomenality, what is at stake is not merely to understand how a psychotic crisis is a singular event responsible for the existential demise of an individual, but more primordially how this demise is revelatory of a failed transcendence and thus continues to participate in the very ambiguity and enigma at the heart of transcendence itself, even if in an out-of- joint manner, for “[i]ts dramatic testifies (pathei mathos) to that which is irreducible in man.”495 If this is the case, then we can question psychosis The Deadlocks of Ontological Catastrophe 307 The Deadlocks of Ontological Catastrophe 307 But each object giving way to another object, those who suffer 308 Chapter 12 Chapter 12 from depression are forced to persevere in this indefinite path—a circle without beginning and end, a circle in which their thinking is ensnared, has become their only horizon.497 For Maldiney, the primordial lost object that the Phenomenology searches for is “in reality existence” itself:498 that is, human transcendence as a capacity of welcoming the completely unexpected and utterly new as revealed to us in the very flesh of sensation wherein subject and object are constituted after the fact by means of a single movement in the flux of a pure appearing that knows no bounds and no a priori. Maldiney relates this not only to the originary impression (ursprüngliche Empfindung) that Hölderlin identifies at the origin of his poetry, a primordial experience that demands a complete transformation—an asubjective becoming-other—wherein the world emerges at each moment as something never seen before,499 but also to the pure present that is the true place within which subjectification and temporalization take place.500 As a result, the key to understanding transcendence is not ideational-conceptual mediation (the gnosologic: the encyclopedia, the systematic), but rather sensibility (the pathic: the eruption of the unpredictable, being held out into the Open), which leads Maldiney to claim that the present is ecstatic—not in the sense analyzed by Hegel, but as an event that is “an outpouring, a gushing, of the new. All of this is just to name the Originary. It is the originarity of the present which founds at each moment the reality of time; and it is its novelty that renders time irreversible.”501 Because Hegel's critique of sense certainty excludes any access to this primordial self-giving outside of conceptual mediation, it is as if the very source of all unity and stability has been obstructed. The paradox is that, although consciousness has lost its primordial object, which should lead to psychopathology, “the Hegelian Aufhebung reproduces—in its own register—the transcendence of existence; but it can only reproduce it (in terms of a substitute of an Ersatz)—and this is the decisive characteristic— because existence has already been lost.”502 It is in this manner that the Phenomenology exhibits a contradictory existential form of “successful” depressive thinking. The Deadlocks of Ontological Catastrophe 307 in order to bring to the fore the various existentialia operative in the very process of subjectification and temporalization central to human existence: “[w]hether his illness [maladie] is organic or vesanic, for man it is first of all a human trail; and it is only possible to understand the latter if one first of all knows what it means 'to be man.'”496 In this sense, the experience of psychosis as a failed transcendence is beneficial for coming to terms with what a successful transcendence would be, by opening up the room for its (self-)thematization. In other words, if there is a certain ambiguity between the phenomenal (psychosis) and the phenomenological (its scientific essence) in this case, it is because the latter shows us that the former is always a possibility for us, an intimate potential of being human, so that the advent of the Real (in the sense of Maldiney) at the core of the subject is indifferent to the success or failure of the latter. The Phenomenology, however, does not present us with a failed transcendence whose very failure highlights irreducible features of the drama of existence, as in Biswanger's case studies or Maldiney's own work on psychosis. But neither does it present us with a straightforwardly successful transcendence (if such a thing even exists) for the movement of consciousness it depicts is identical to the fundamental structure of depression, so that the question itself emerges as to how the Phenomenology can even arrive at dynamic unity and stability if the consciousness it describes is intrinsically depressive in nature: The principle of Aufhebung, “to abolish and to preserve,” is consistent with the general scheme of depressive existence. Its double meaning agrees with, amongst other things, the double dimension of the depressive dramatic as explained by psychoanalytic theory, according to which the “relation to the object” serves to signify being-in-the-world with the same unilaterality that we see in Hegel. From the psychoanalytical perspective, depression is constituted by an uncertain relation to the primordial object, to which the subject remains attached even though it has been detached from it. Compelled, after the loss of the primordial object, to search for another, in the quest for a new object it is always in search of the lost object. The Deadlocks of Ontological Catastrophe 309 To say that the Phenomenology is a form of successful depressive thinking is thus to say that it has apparently immanently overcome depression without ever leaving its clutches on existence by encountering the the Phenomenology this ambiguity is expanded from the distinction between the phenomenal and the phenomenological to the distinction between the phenomenal and phenomenological and the investigator within which this distinction is enacted, so that the in-dwelling phenomenological essence of the phenomenality they investigate can be brought forth. Since the phenomenal here is conscious experience in its depressive existence, the investigator, by assuming the phenomenological attitude, is performing an ideal the Phenomenology this ambiguity is expanded from the distinction between the phenomenal and the phenomenological to the distinction between the phenomenal and phenomenological and the investigator within which this distinction is enacted, so that the in-dwelling phenomenological essence of the phenomenality they investigate can be brought forth. Since the phenomenal here is conscious experience in its depressive existence, the investigator, by assuming the phenomenological attitude, is performing an ideal reconstruction of his or her own consciousness with said structure (which is simultaneously a self-construction of a depressive consciousness within the concept) into a scientific knowledge that is the system of experience. In this sense, the phenomenological level cannot be isolated from the phenomenal any more than it can in the phenomenology of psychosis, but now with an important precision to be made: the phenomenal experience is here the direct self-experience of the consciousness that goes through the odyssey that is the Phenomenology, but in such a way that it is as if phenomenal experience becomes phenomenological to itself, thematizes itself, in an act of gaining distance from itself, for the investigator who is exploring what it means to be a depressive consciousness is implicated in the very process. In short, there is ultimately no distinction between us and the object of investigation, because we are participating in the very thing that we are investigating. The Deadlocks of Ontological Catastrophe 309 the Phenomenology this ambiguity is expanded from the distinction between the phenomenal and the phenomenological to the distinction between the phenomenal and phenomenological and the investigator within which this distinction is enacted, so that the in-dwelling phenomenological essence of the phenomenality they investigate can be brought forth. Since the phenomenal here is conscious experience in its depressive existence, the investigator, by assuming the phenomenological attitude, is performing an ideal reconstruction of his or her own consciousness with said structure (which is simultaneously a self-construction of a depressive consciousness within the concept) into a scientific knowledge that is the system of experience. In this sense, the phenomenological level cannot be isolated from the phenomenal any more than it can in the phenomenology of psychosis, but now with an important precision to be made: the phenomenal experience is here the direct self-experience of the consciousness that goes through the odyssey that is the Phenomenology, but in such a way that it is as if phenomenal experience becomes phenomenological to itself, thematizes itself, in an act of gaining distance from itself, for the investigator who is exploring what it means to be a depressive consciousness is implicated in the very process. In short, there is ultimately no distinction between us and the object of investigation, because we are participating in the very thing that we are investigating. Accordingly, if the Phenomenology reveals that the fundamental structure of consciousness is a depressive structure, for those who embark upon it it is simultaneously a transformation of the mode of existing in transcendence that is depression into a unique style of living, wherein the same structure we see in the psychopathological state of depression is made into that which bestows upon consciousness a profound and never-ending source of energy while resting all the while depressive. If the phenomenology of psychosis brings to the fore various existentialia operative in the very process of subjectification and temporalization so that we see a successful transcendence in its contours, the Phenomenology offers a therapeutic realization of a similar kind of vitality that the latter provides, but within its very failure. The Deadlocks of Ontological Catastrophe 307 The problem is only intensified when one takes into account the The problem is only intensified when one takes into account the methodological ambiguity Maldiney emphasizes in any phenomenology of psychosis between the phenomenal and the phenomenological, insofar as in The Deadlocks of Ontological Catastrophe 309 310 Chapter 12 Consequently, we see three structural levels of depression in Hegel's Phenomenology: the basal depressive structure of consciousness—the loss of the primordial object—which is the zero-level of its transcendence as such (originary depression or depression0, failed transcendence); the psychopathological response that brings a particular person to an existential standstill, which we see in psychiatry (clinical depression or depression1); and lastly the dialectical system of experience that, although depressive, is somehow successfully so, since even if its trial insists in experience it becomes a positive basis for a newfound energy and dynamism rather than a catalyst for decay (a form of successful depressive thinking, which is not a successful depression and is different in nature from depression1). Following a suggestion taken up by Jean-Christophe Goddard, we can expand the idea as follows: it is not merely that the Phenomenology is a paradoxical form of successful depressive thinking, but more radically, it sketches a form of successful psychotic thinking in general.503 Insofar as the event to which we are exposed by being held out into the Open in the pathos of sensation is the utterly new and unpredictable of which we can say nothing in advance, to give oneself over to it in its pure self-outpouring must itself be a form of originary psychosis, so that we see two possible responses: either from within an originary psychosis inflicting human transcendence as such we learn to be successfully psychotic or we opt for a path that would end in a pathological response to this primordial experience at the core of our being by levelling out its trauma instead of using it as the ground for the very impetus of one's subjective life in the world (a capacity Maldiney refers to as transpassibility). Just as Hegel's Phenomenology could be described as an attempt to develop a form of successful depressive thinking, so too it could be ventured that Žižek's transcendental materialism may be an attempt to develop a form of successful psychotic thinking, but psychosis taken here not in Maldiney's unique understanding of it, but in the technical definition that Žižek instills it with as the world's withdrawal into its nocturnal self. The Deadlocks of Ontological Catastrophe 309 Accordingly, if the Phenomenology reveals that the fundamental structure of consciousness is a depressive structure, for those who embark upon it it is simultaneously a transformation of the mode of existing in transcendence that is depression into a unique style of living, wherein the same structure we see in the psychopathological state of depression is made into that which bestows upon consciousness a profound and never-ending source of energy while resting all the while depressive. If the phenomenology of psychosis brings to the fore various existentialia operative in the very process of subjectification and temporalization so that we see a successful transcendence in its contours, the Phenomenology offers a therapeutic realization of a similar kind of vitality that the latter provides, but within its very failure. To say that the Phenomenology is a form of successful depressive thinking is thus to say that it has apparently immanently overcome depression without ever leaving its clutches on existence by encountering the crisis head-on and coming out strong. The Deadlocks of Ontological Catastrophe 311 spontaneously of its own nature, that is, by reconstructing it ideally and scientifically in the concept through a mytho-poetic medium. But if the entirety of the Symbolic is some kind of virtual recompensation for this loss of objective reality, then this originary psychosis, essential to what it is to be a subject, must be primordially repressed if it is to be successful. We must forget that the very fabric of culture is nothing but the deluded ravings of the asylum, since otherwise we are confronted with the very monstrosity— ontological catastrophe, the passage of being through madness—that it was meant to cover up. This is precisely why the psychoanalytical experience is of irreducible importance for Žižek, for we could only hope to get beyond the various defence mechanisms underlying the constitution of our fantasmatic reality and catch a glimpse of their abyssal origin if this reality were wrought with piercing holes: if in our everyday lives we are completely lost in the transcendentally hallucinated world fabricated by the Symbolic, then only the upsurge of the Real as indicative of the infinite disharmony between mind and body can enable us to gain the necessary distance towards our self-loss in psychosis and thereby render possible its free (albeit mythological) internalization within the concept. But the Real not only opens up the space necessary for speculative fabulation as a faculty for explicating how we got “trapped” in ontological solipsism; it also lets us find a way, from within our constitutive psychosis, to minimally overcome this very entrapment. As that which is irreducible to the autonomous construction of the world of experience, the Real creates a realist moment within idealism itself. With these two elements achieved, we could be said to have developed a form of successful psychotic thinking: that is to say, a thinking that, from within its own psychosis, would not be limited by the latter in the same way that a form of successful depressive thinking would not be limited by its depression and would have gained a vitality similar to successful transcendence, despite its remaining depressive in structure. A f l h i hi ki h i i i i i ll A successful psychotic thinking—such an expression is intrinsically ambiguous and reveals the insurmountable difficulty any radical idealism has to overcome its own limitations. 310 Chapter 12 Taking as its object the thematization of the inner structure of psychosis, which, since “madness signals the unconstrained explosion at the very core of human being,”504 can only be done in and through psychosis, it seeks to use psychosis to pierce through its own impenetrable dusk by letting it move The Deadlocks of Ontological Catastrophe 311 The Deadlocks of Ontological Catastrophe 311 If our starting point is a self-grounding subjective idealism, we can never truly get “behind” it from within the universe of ideality, even if this idealism is wrought with fracture lines, inner tension, and agonizing cracks, for its symbolic (re)constitution of 312 Chapter 12 reality functions with no need of an “external support of its truth.”505 Instead of representing that by which the light of being sneaks in despite the correlationalist prison, these can only indicate a torsion, an immanent implosion, from within ideal self-enclosure. By thematizing these places of non-coincidence we can perhaps come to terms with our entrapment and see it for what it is (namely, an entrapment). Yet it is not clear we can hope for much more if these skeptical reflections hold true: a successful psychotic thinking does not result in any strict overcoming of the epistemological constraints psychosis imposes upon us; it is only from within the Symbolic that we see the Real as the residue of a failed attempt of synthesis, thereby rendering it always already minimally symbolized, idealized, always already necessarily lost, there being nothing but the Symbolic and its self-referential play. The paradoxical overlapping of the purely subjective and the purely objective is precisely that: paradoxical. It does not allow for any encounter of the pure Real within the Symbolic, but is rather cognizant of the fact that, since the Real is an impossible concept, any attempt to describe its extra-notional character must be done from within its clutches. Knowledge of reality in itself becomes reduced to a retroactive readjustment of the Symbolic. We see this most clearly in the very expression itself. If a successful psychotic thinking is not a successful psychosis (a cure) but still exhibits the fundamental structure of psychosis, just as a successful depressive thinking in no way means that depression has been left behind and is always searching for the lost object, what we have attained would be at best the upper limit of the dialectic of appearance as such, which has finally collapsed upon itself in one great final cry giving voice to a contradictory combination of absolute power and utmost impotence. The Deadlocks of Ontological Catastrophe 311 In recognizing the very limit of ideal synthesis, even if we are in a certain sense minimally beyond it (the “realist” moment) we can only admit the limit as an impasse with no beyond or content (even the “real” moment is completely determined by the throes of idealist entrapment). But is this sufficient to establish a new metaphysics? To speak metaphorically, it is as if, on Žižek's account, in radical idealism we see the four walls that surround us as a barrier not due to some window to the outside by which we know this site as the prison that it is (the ontological solipsism of the constitutive reality functions with no need of an “external support of its truth.”505 Instead of representing that by which the light of being sneaks in despite the correlationalist prison, these can only indicate a torsion, an immanent implosion, from within ideal self-enclosure. By thematizing these places of non-coincidence we can perhaps come to terms with our entrapment and see it for what it is (namely, an entrapment). Yet it is not clear we can hope for much more if these skeptical reflections hold true: a successful psychotic thinking does not result in any strict overcoming of the epistemological constraints psychosis imposes upon us; it is only from within the Symbolic that we see the Real as the residue of a failed attempt of synthesis, thereby rendering it always already minimally symbolized, idealized, always already necessarily lost, there being nothing but the Symbolic and its self-referential play. The paradoxical overlapping of the purely subjective and the purely objective is precisely that: paradoxical. It does not allow for any encounter of the pure Real within the Symbolic, but is rather cognizant of the fact that, since the Real is an impossible concept, any attempt to describe its extra-notional character must be done from within its clutches. Knowledge of reality in itself becomes reduced to a retroactive readjustment of the Symbolic. We see this most clearly in the very expression itself. The Deadlocks of Ontological Catastrophe 313 due to their very shaking we feel that we are incarcerated in an infinitely claustrophobic space from which we will never emancipate ourselves. From the cracks in the wall there may seep through faint, trembling voices, but in the painstaking process of reconstructing their mumbled words they risk becoming identical with those voices that we hallucinate in our solitude. And even if we succeed at spectrally envisioning this outside by a careful translation of their garbled noise into structured speech, could we be said finally to have liberated ourselves by making the very distinction between liberty and imprisonment void? If a successful form of psychotic thinking is an intrinsically paradoxical concept, it is perhaps because radical idealism is itself intrinsically paradoxical. To say that we can never reach reality, that we are forever stuck within the human universe of meaning, is not merely problematic to ordinary natural and scientific consciousness because it goes against our basic intuitions, but in a more primordially discomforting way: for it proclaims an originary withdrawal from objective reality at the very foundation of what it is to be human, thus potentially reducing the world of experience to the rampant free play of phantasmagoria. For someone who adheres to such a radical idealism, it would appear that even if the inconsistency of our notional apparatus may always be insufficient for a realist to overcome the ambiguity of a successful psychotic thinking, and thus to find resources to establish a strong realism from within idealism itself, we can still embark upon a speculative fabulation of ontological catastrophe as the necessary condition of the possibility of the subject. If a successful form of psychotic thinking is an intrinsically paradoxical concept, it is perhaps because radical idealism is itself intrinsically paradoxical. To say that we can never reach reality, that we are forever stuck within the human universe of meaning, is not merely problematic to ordinary natural and scientific consciousness because it goes against our basic intuitions, but in a more primordially discomforting way: for it proclaims an originary withdrawal from objective reality at the very foundation of what it is to be human, thus potentially reducing the world of experience to the rampant free play of phantasmagoria. The Deadlocks of Ontological Catastrophe 311 If a successful psychotic thinking is not a successful psychosis (a cure) but still exhibits the fundamental structure of psychosis, just as a successful depressive thinking in no way means that depression has been left behind and is always searching for the lost object, what we have attained would be at best the upper limit of the dialectic of appearance as such, which has finally collapsed upon itself in one great final cry giving voice to a contradictory combination of absolute power and utmost impotence. In recognizing the very limit of ideal synthesis, even if we are in a certain sense minimally beyond it (the “realist” moment) we can only admit the limit as an impasse with no beyond or content (even the “real” moment is completely determined by the throes of idealist entrapment). But is this sufficient to establish a new metaphysics? To speak metaphorically, it is as if, on Žižek's account, in radical idealism we see the four walls that surround us as a barrier not due to some window to the outside by which we know this site as the prison that it is (the ontological solipsism of the constitutive psychosis of the Symbolic) and could envisage an escape, but rather The Deadlocks of Ontological Catastrophe 313 Chapter 12 314 of its object, for we have fallen into a new age of mythology that has been rationally justified. Yet language is always more language (this is the major consequence of the Real), and if ontology and metaphysics have been rendered impossible, insofar as we can see this fact for what it is, we must have the right to investigate it in a non-rational discourse, no matter what the status of idealism's self-overcoming. Although a transcendental materialism developed après-coup in the enclosed terrain of a radical idealism may always leave the realist unsatisfied as to its very possibility, turning epistemological limit into positive ontological condition via a mytho-poetic narrativization of being's self- division does allow us to make minimal ontological claims that go beyond mythological imagery. If the Symbolic does present itself as quasi-full- blown constructionism of reality that cuts all ties with an extra-notional, extra-linguistic outside, then the very founding gesture of the Symbolic's self-containment must be structurally homologous with that of psychosis understood as a withdrawal from social reality into an irreal self, and thus must attest to an originary madness at the very ontogenetic basis of human subjectivity. In this respect, were one to refuse the move to mythology as a means of overcoming radical idealism as a mere confabulation that assures the consistency of its order by the production of false memories, the latter would nevertheless reveal a deadlock haunting subjectivity: the deadlock of ontological catastrophe. If the Symbolic exists, then the world is, at best, a fragmented totality whimpering under its own weight—a totality that, unable to posit itself as all, in the case of at least one creature (man) is forced to withdraw into a nocturnal irreal self due to a primordial moment of metaphysical trauma in its heart of hearts, thereby establishing the primary role of dislocation in ontology. The Deadlocks of Ontological Catastrophe 313 For someone who adheres to such a radical idealism, it would appear that even if the inconsistency of our notional apparatus may always be insufficient for a realist to overcome the ambiguity of a successful psychotic thinking, and thus to find resources to establish a strong realism from within idealism itself, we can still embark upon a speculative fabulation of ontological catastrophe as the necessary condition of the possibility of the subject. For we must nevertheless be able to explicate the fact that we can see our life as a dream of a dream, our ontological psychosis as psychotic, for the Symbolic fails to posit itself as all, and must therefore be always already minimally outside itself. Just as Schelling declares that many things take place before the beginning proper, so Žižek contends that a lot happens in the indemonstrable Real-as-origin haunting the Symbolic. The abyss of unconscious decision vibrates with an immense energy, and when we catch a glimpse of this impossibility in a mytho-poetics of the Symbolic's genesis, we simultaneously see a glimmer in the corner of our eye of why idealism For we must nevertheless be able to explicate the fact that we can see our life as a dream of a dream, our ontological psychosis as psychotic, for the Symbolic fails to posit itself as all, and must therefore be always already minimally outside itself. Just as Schelling declares that many things take place before the beginning proper, so Žižek contends that a lot happens in the indemonstrable Real-as-origin haunting the Symbolic. The abyss of unconscious decision vibrates with an immense energy, and when we catch a glimpse of this impossibility in a mytho-poetics of the Symbolic's genesis, we simultaneously see a glimmer in the corner of our eye of why idealism is such an explosive event in being, despite the ontological solipsism of idealism itself. But such an epiphany is limited by the very impossibility Chapter 12 The Deadlocks of Ontological Catastrophe 315 declares that something must have gone horribly wrong in the life of the absolute: for no God, no divine nature, could have wanted this to be our fate, since for both, our fate is tied up with theirs. Chapter 12 Even if we might not be able to say more without writing a great fabulative epic, many details of which may succumb to confabulation and fantasmatic figures, the mere existence of a radically self-grounding idealism would not only demonstrate that reality split itself into two irreconcilable logical zones insofar as thought must exist, but more primordially that ontological catastrophe, whereby being is infinitely divided from itself and doomed to wander in a world of images that are images of nothing, is irreducible in the transcendental explanation of the conditions of the possibility of experience and the Symbolic. Idealism The Deadlocks of Ontological Catastrophe 315 Notes 464. Žižek, Less Than Nothing, p. 314. 465. Ibid., pp. 234–35. 465. Ibid., pp. 234–35. 465. Ibid., pp. 234–35. 466. Schelling, Darstellung, I, 4, p. 106. 466. Schelling, Darstellung, I, 4, p. 106. 467. Schelling, Freiheitsschrift, p. 232. 467. Schelling, Freiheitsschrift, p. 232. 468. Schelling, Stuttgart Seminars, pp. 216ff. 469. Schelling, Ideas for a Philosophy of Nature, trans. Errol E. Harris and Peter Heath (Cambridge: Cambridge University Press, 1995), pp. 41–42. 470. Schelling, Freiheitsschrift, p. 236. 470. Schelling, Freiheitsschrift, p. 236. 471. Grant, Philosophies of Nature after Schelling, pp. viii–ix. 472. Ibid., p. ix. 472. Ibid., p. ix. 473. Žižek, Less Than Nothing, p. 77. 474. Schelling, Weltalter II, pp. 121–22. 475. Schelling, “Briefwechsel mit Eschenmayer bezüglich der Abhandlung ‘Philosophische Untersuchungen über das Wesen er menschlichen Freiheit’,” Schellings sämmtliche Werke, I, 8, p. 150; translation taken from Iain Hamilton Grant, Philosophies of Nature after Schelling, p. 202. 476. Grant, Philosophies of Nature after Schelling, pp. 202–3. 477. Ibid., p. 2. 477. Ibid., p. 2. 478. Ibid., p. 62. 478. Ibid., p. 62. 479. Ibid., pp. 1–2. 479. Ibid., pp. 1–2. 480. Schelling, First Outline of a System of the Philosophy of Nature, trans. Keith R. Peterson (Albany: State University of New York Press, 2004), p. 14. 481. Žižek, The Parallax View, p. 106. 481. Žižek, The Parallax View, p. 106. 482. Žižek and Woodard, “Interview,” The Speculative Turn: Continental Materialism and Realism, pp. 412–13. 483. Ibid., p. 413. 483. Ibid., p. 413. 484. Ibid., p. 409. 484. Ibid., p. 409. 485. Cf. Johnson, Žižek’s Ontology, p. 148. 485. Cf. Johnson, Žižek’s Ontology, p. 148. 486. Ibid., p. 152. 486. Ibid., p. 152. 487. Žižek, Less Than Nothing, p. 959. 487. Žižek, Less Than Nothing, p. 959. 488. Žižek, The Indivisible Remainder, p. 19. 316 Chapter 12 316 Chapter 12 489. Fichte, Fichte: Early Philosophical Writings, pp. 347–48. Also quoted by Žižek, “Fichte’s Laughter,” in Madness, Mythology and Laughter, p. 160, but with a differ- ent interpretation than what I offer here. 490. Fichte, The Vocation of Man, p. 64. For my discussion, see chapter 6. 491. Fichte, Wissenschaftslehre 1805, pp. 127–28. 491. Fichte, Wissenschaftslehre 1805, pp. 127–28. 492. Maldiney, Penser l’homme et la folie, p. 9. 492. Maldiney, Penser l’homme et la folie, p. 9. 493. Hegel, Phenomenology of Spirit, p. 36; cited by Maldiney, Penser l’homme et la folie, p. 263. 494. Goddard, Mysticisme et folie, p. 83. 494. Goddard, Mysticisme et folie, p. 83. 495. Maldiney, Penser l’homme et la folie, p. 7. 496. Ibid., p. 263. 497. Ibid., p. 27. Ibid., p. 27. Ibid., p. 29. 498. Ibid., p. 29. 498. Ibid., p. 29. 499. 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Levi Bryant, Nick Srnicek, and Graham Materialism and Realism. Ed. Levi Bryant, Nick Srnicek, and Graham Harman. Melbourne: re.press, 2011. 406–15. Harman. Melbourne: re.press, 2011. 406–15. Žižek! Dir. Atra Taylor. DVD. Zeitgeist Video: 2007. Žižek! Dir. Atra Taylor. DVD. Zeitgeist Video: 2007. This book is an original investigation into Slavoj Žižek’s return to German Idealism in the wake of Lacanian psychoanalysis. As is well known, Žižek creates productive friction between these traditions by isolating their mutually compatible notions of the death drive, paving the way for Žižek’s highly original model of the subject. Joseph Carew systematizes the stark metaphysical conse- quences of Žižek’s account. If the emergence of the Symbolic out of the Real marks the advent of a completely self-enclosed struc- tural system, then we must posit the absolute This book is an original investigation into Slavoj Žižek’s return to German Idealism in the wake of Lacanian psychoanalysis. As is well known, Žižek creates productive friction between these traditions by isolating their mutually compatible notions of the death drive, paving the way for Žižek’s highly original model of the subject. Joseph Carew systematizes the stark metaphysical conse- quences of Žižek’s account. If the emergence of the Symbolic out of the Real marks the advent of a completely self-enclosed struc- tural system, then we must posit the absolute as a fragile not-all wrought by negativity and antagonism. OPEN HUMANITIES PRESS Cover design by Katherine Gillieson · Illustration by Tammy Lu OPEN HUMANITIES PRESS Cover design by Katherine Gillieson · Illustration by Tammy Lu Cover design by Katherine Gillieson · Illustration by Tammy Lu
https://openalex.org/W2291961953	https://arts.units.it/bitstream/11368/2871483/2/Excitation%20of%20vibrational%20modes%20in%20the%20ionization%20of%20water%20molecule%20by%20XUV-X-ray%20radiation.pdf	English	null	Excitation of vibrational modes in the ionization of water molecule by XUV/X-ray radiation	Journal of physics. Conference series	2,015	cc-by	1,160	Home Search Collections Journals About Contact us My IOPscience Excitation of vibrational modes in the ionization of water molecule by XUV/X-ray radiation Excitation of vibrational modes in the ionization of water molecule by XUV/X-ray radiation This content has been downloaded from IOPscience. Please scroll down to see the full text. 2015 J. Phys.: Conf. Ser. 635 112098 (http://iopscience.iop.org/1742-6596/635/11/112098) View the table of contents for this issue, or go to the journal homepage for more Download details: Selma Engin† 1, Jes´us Gonz´alez-V´azquez†, In´es Corral†, Alicia Palacios†, David Ayuso†, Piero Decleva∗⋆, and Fernando Mart´ın†‡ Selma Engin† 1, Jes´us Gonz´alez-V´azquez†, In´es Corral†, Alicia Palacios†, David Ayuso†, Piero Decleva∗⋆, and Fernando Mart´ın†‡ Selma Engin† 1, Jes´us Gonz´alez-V´azquez†, In´es Corral†, Alicia Palacios†, Da Piero Decleva∗⋆, and Fernando Mart´ın†‡ † Departamento de Qu´ımica, M´odulo 13, Universidad Aut´onoma de Madrid, Cantoblanco 28049 Madrid, Spain ‡ Instituto Madrile˜no de Estudios Avanzados en Nanociencia, Cantoblanco 28049 Madrid, Spain ∗Dipartimento di Scienze Chimiche e Faramaceutiche, Universit`a di Trieste, 34127 Trieste, Italy ⋆CNR-IOM, Trieste, Italy Synopsis We present a theoretical study of the vibrationally resolved core photoionization of the water molecule up to high photon energies. In order to understand the role of the coupled electron-nuclear motion in polyatomic molecules, we thus have implemented a new methodology to describe all vibrational modes of a polyatomic molecule. We show our preliminary results on the O(1s) photoionization, with special focus on the vibrationally resolved cross sections in a large range of photon energies, reaching up to 1500 eV. ing the static-exchange DFT method developed by P. Decleva and collaborators [3]. The reli- ability of our approach is tested by comparing our calculated O(1s) vibrationally resolved pho- toelectron spectrum with available experimental data for a photon energy of 590 eV [4]. We ﬁnd a good agreement with the vibrational progression at 590 eV, where the dominant contribution is that of the bending mode of the core-ionized wa- ter molecule. We further analyze the v-ratios as a function of photon energy, i.e. the ratios between the photoionization cross sections for individual vibrational states. For all normal modes, we observe oscillations around the Franck-Condon factor value, similar to those found in previous works for other diatomic and polyatomic targets (CO, CH4, BF3, etc) [1, 2], whose origin comes from diﬀraction of the ejected electron by the surrounding H atoms. Most theoretical studies of coupled electron and nuclear dynamics in photoionization prob- lems have been restricted to diatomic molecules [1] or to a single vibrational mode of polyatomic molecules [2]. In some cases, a reasonable ap- proximation is to reduce the nuclear degrees of freedom to one principal normal mode. This was shown in photoionization problems for highly symmetric targets such that CH4 or BF3 [2], whose symmetric stretching mode was the main active mode upon the photon absorption. How- ever, such simpliﬁcation is not valid in most prob- lems. Content from this work may be used under the terms of the Creative Commons Attribution 3.0 licence. Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI. Published under licence by IOP Publishing Ltd 1 IP Address: 87.30.54.86 This content was downloaded on 02/11/2016 at 15:02 Please note that terms and conditions apply. You may also be interested in: Model potentials in liquid water ionization by fast electron impact M L de Sanctis, M-F Politis, R Vuilleumier et al. Double ionization of water molecule induced by swift H+ A C Tavares, H Luna, W Wolff et al. Raman Spectroscopy Investigation of the Polar Vibrational Modes in CuB2O4 V Tomov, P M Rafailov and L Yankova Double differential cross sections for ionization of water molecules by ion impact C A Tachino, J M Monti, O A Fojón et al. New state of the water molecule Ad ti f th t l l l h f h ff t it t You may also be interested in: Model potentials in liquid water ionization by fast electron impact M L de Sanctis, M-F Politis, R Vuilleumier et al. Model potentials in liquid water ionization by fast electron impact M L de Sanctis, M-F Politis, R Vuilleumier et al. Raman Spectroscopy Investigation of the Polar Vibrational Modes in CuB2O4 V Tomov, P M Rafailov and L Yankova Double differential cross sections for ionization of water molecules by ion impact C A Tachino, J M Monti, O A Fojón et al. New state of the water molecule Adsorption of the water molecule on monolayer graphene surface has effect on its optical properties Y F Peng, J Wang, Z S Lu et al. Erratum: On variational arguments for vibrational modes near jamming Le Yan, Eric DeGiuli and Matthieu Wyart XXIX International Conference on Photonic, Electronic, and Atomic Collisions (ICPEAC2015) IOP Publishing Journal of Physics: Conference Series 635 (2015) 112098 doi:10.1088/1742-6596/635/11/112098 Selma Engin† 1, Jes´us Gonz´alez-V´azquez†, In´es Corral†, Alicia Palacios†, David Ayuso†, Piero Decleva∗⋆, and Fernando Mart´ın†‡ The aim of this study is to extend the methodology previously developed in those molecular targets, which used a one-dimensional vibrational wave function, to include an arbitrary number of nuclear degrees of freedom. We thus seek to achieve a complete description of vibra- tional motion, although, for simplicity our ﬁrst implementation uses Jacobi coordinates, which are particularly suitable for non-linear triatomic molecules. In the present study, we report re- sults for the O(1s) photoionization of the wa- ter molecule induced by synchroton radiation, where all normal modes are simultaneously ac- counted for : bending, symmetric stretching and asymmetric stretching. Accurate vibrationally resolved cross sections require the computation of dipole matrix elements for the transition from the ground state of the neutral to the core-ionized water molecule. These dipole elements are ob- tained for the full grid of nuclear coordinates us- 1E-mail: selma.engin@uam.es [4] R. Sankari et al., Chem. Phys. Lett. 380 647-653 (2003) [3] M. Stener et al., J. Chem. Phys. 112 10871 (2000) M. Stener et al., Chem. Phys. Lett. 351 469 (2002) D. Toﬀoli et al., Chem. Phys. 276 2543 (2002) [2] E. Pl´esiat et al., Phys. Rev. A 85 023409 (2012) K. Ueda et al., J. Chem. Phys. 139 124306 (2013) References [1] E. Kukk et al., Phys. Rev. A 88 033412 (2013) D. Ayuso et al., J. Elec. Spec. Rel. Phen. 195 320-326 (2014) [2] E. Pl´esiat et al., Phys. Rev. A 85 023409 (2012) K. Ueda et al., J. Chem. Phys. 139 124306 (2013) [2] E. Pl´esiat et al., Phys. Rev. A 85 023409 (2012) K. Ueda et al., J. Chem. Phys. 139 124306 (2013) [2] E. Pl´esiat et al., Phys. Rev. A 85 023409 (2012) K. Ueda et al., J. Chem. Phys. 139 124306 (2013) [3] M. Stener et al., J. Chem. Phys. 112 10871 (2000) M. Stener et al., Chem. Phys. Lett. 351 469 (2002) D. Toﬀoli et al., Chem. Phys. 276 2543 (2002) [4] R. Sankari et al., Chem. Phys. Lett. 380 647-653 (2003) [4] R. Sankari et al., Chem. Phys. Lett. 380 647-653 (2003) 1E-mail: selma.engin@uam.es
https://openalex.org/W4235784950	https://peerj.com/articles/cs-189v0.3/submission	English	null	Peer Review #2 of "Evaluating named entity recognition tools for extracting social networks from novels (v0.2)"	null	2,019	cc-by	21,200	Computer Science Computer Science Computer Science Computer Science Manuscript to be reviewed 1We follow (Sainte-Beuve, 1910) here in deﬁning a classic novel not as one written by the ancient Greeks or Romans (‘the classics’) but to canonical works. Evaluating named entity recognition tools 1 for extracting social networks from novels 2 Niels Dekker1, Tobias Kuhn1, and Marieke van Erp2 3 1Department of Computer Science, Vrije Universiteit Amsterdam, Amsterdam, The 4 Netherlands 5 2DHLab, KNAW Humanities Cluster, Amsterdam, The Netherlands 6 Corresponding author: 7 Marieke van Erp2 8 Email address: marieke.van.erp@dh.huc.knaw.nl 9 Email address: marieke.van.erp@dh.huc.knaw.nl 9 Email address: marieke.van.erp@dh.huc.knaw.nl 9 ABSTRACT 10 The analysis of literary works has experienced a surge in computer-assisted processing. To obtain insights into the community structures and social interactions portrayed in novels, the creation of social networks from novels has gained popularity. Many methods rely on identifying named entities and relations for the construction of these networks, but many of these tools are not speciﬁcally created for the literary domain. Furthermore, many of the studies on information extraction from literature typically focus on 19th and early 20th century source material. Because of this, it is unclear if these techniques are as suitable to modern-day literature as they are to those older novels. We present a study in which we evaluate natural language processing tools for the automatic extraction of social networks from novels as well as their network structure. We ﬁnd that there are no signiﬁcant differences between old and modern novels but that both are subject to a large amount of variance. Furthermore, we identify several issues that complicate named entity recognition in our set of novels and we present methods to remedy these. We see this work as a step in creating more culturally-aware AI systems. Computer Science Manuscript to be reviewed Evaluating named entity recognition tools for extracting social networks from novels 1 Department of Computer Science, Vrije Universiteit Amsterdam, Amsterdam, The Ne 2 DHLab, KNAW Humanities Cluster, Amsterdam, The Netherlands Corresponding Author: Marieke van Erp Email address: marieke.van.erp@dh.huc.knaw.nl The analysis of literary works has experienced a surge in computer-assisted processing. To obtain insights into the community structures and social interactions portrayed in novels, the creation of social networks from novels has gained popularity. Many methods rely on identifying named entities and relations for the construction of these networks, but many of these tools are not speciﬁcally created for the literary domain. Furthermore, many of the studies on information extraction from literature typically focus on 19th and early 20th century source material. Because of this, it is unclear if these techniques are as suitable to modern-day literature as they are to those older novels. We present a study in which we evaluate natural language processing tools for the automatic extraction of social networks from novels as well as their network structure. We ﬁnd that there are no signiﬁcant diﬀerences between old and modern novels but that both are subject to a large amount of variance. Furthermore, we identify several issues that complicate named entity recognition in our set of novels and we present methods to remedy these. We see this work as a step in creating more culturally-aware AI systems. PeerJ Comput. Sci. reviewing PDF \| (CS-2018:08:30671:2:0:NEW 7 Mar 2019) Computer Science Computer Science Manuscript to be reviewed are obtained from Project Gutenberg,2 where such public domain books are available for free. 41 While beneﬁcial for the accessibility and reproducibility of the studies in question, more recent 42 novels may not imitate these classic novels with respect to structure or style. It is therefore 43 possible that classic novels have social networks that have a structure that is very different from 44 more recent literature. They might differ, for example, in their overall number of characters, in 45 the typical number of social ties any given character has, in the presence or absence of densely 46 connected clusters, or in how closely connected any two characters are on average. Moreover, 47 changes along dimensions such as writing style, vocabulary, and sentence length could prove to 48 be either beneﬁcial or detrimental to the performance of natural language processing techniques. 49 This may lead to different results even if the actual network structures remained the same. Vala 50 et al. (2015) did compare 18th and 19th century novels on the number of characters that appear 51 in the story, but found no signiﬁcant difference between the two. Furthermore, an exploration 52 of extracted networks can also be used to assess the quality of the extracted information and 53 investigate the structure of the expression of social ties in a novel. 54 Thus far, we have not found any studies that explore how named entity recognition tools 55 perform on a diverse corpus of ﬁction literature. In this study, we evaluate four different tools on 56 a set of classic novels which have been used for network extraction and analyses in prior work, 57 as well as more recent ﬁction literature (henceforth referred to as modern novels). We need such 58 an evaluation to assess the robustness of these tools to variation in language over time (Biber and 59 Finegan, 1989) and across literary genres. Comparing social networks extracted from corpora 60 consisting of classic and modern novels may give us some insights into what characteristics of 61 literary text may aid or hinder automatic social network extraction and provide indications of 62 cultural change. 63 As previous work (e.g. Ardanuy and Sporleder (2014)) has included works from different 64 genres, in this work we decided to focus on the fantasy/science ﬁction domain to smooth 65 potential genre differences in our modern books. 1 INTRODUCTION 25 The characters and their relations can be seen as the backbone of any story, and explicitly creating 26 and analysing a network from these relationships can provide insights into the community 27 structures and social interactions portrayed in novels (Moretti, 2013). Quantitative approaches 28 to social network analysis to examine the overall structure of these social ties, are borrowed 29 from modern sociology and have found their way into many other research ﬁelds such as 30 computer science, history and literary studies (Scott, 2012). Elson et al. (2010), Lee and Yeung 31 (2012), Agarwal et al. (2013), and Ardanuy and Sporleder (2014) have all proposed methods for 32 automatic social network extraction from literary sources. The most commonly used approach 33 for extracting such networks, is to ﬁrst identify characters in the novel through Named Entity 34 Recognition (NER) and then identifying relationships between the characters through for example 35 measuring how often two or more characters are mentioned in the same sentence or paragraph. 36 Many studies use off-the-shelf named entity recognisers, which are not necessarily optimised 37 for the literary domain and do not take into account the surrounding cultural context. Furthermore, 38 to the best of our knowledge, such studies focus on social network extraction from 19th and 39 early 20th century novels (which we refer to as classic novels).1 Typically, these classic novels 40 1 PeerJ Comput. Sci. reviewing PDF \| (CS-2018:08:30671:2:0:NEW 7 Mar 2019) 2http://gutenberg.org/ 3https://github.com/dbamman/book-nlp – commit: 81d7a31 4A gazetteer is a list of names 2http://gutenberg.org/ 3 Manuscript to be reviewed unlabelled data via clustering and gazetteers that outperformed other state-of-the-art named 85 entity recognition (NER) tools on their within and out-domain evaluations. 86 unlabelled data via clustering and gazetteers that outperformed other state-of-the-art named 85 entity recognition (NER) tools on their within and out-domain evaluations. 86 unlabelled data via clustering and gazetteers that outperformed other state-of-the-art named 85 entity recognition (NER) tools on their within and out-domain evaluations. 86 To answer the second research question, we use the recognised named entities to create a 87 co-occurrence network for each novel. Network analysis measures are then employed to compare 88 the extracted networks from the classic and modern novels to investigate whether the networks 89 from the different sets of novels exhibit major differences. 90 The contributions of this paper are: (1) a comparison and an analysis of four named entity 91 recognition on 20 classic and 20 modern novels; (2) a comparison and an analysis of social 92 network analysis measures on networks automatically extracted from 20 classic and 20 modern 93 novels; (3) experiments and recommendations for boosting performance on recognising entities 94 in novels; and (4) an annotated gold standard dataset with entities and coreferences of 20 classic 95 and 20 modern novels. 96 The remainder of this paper is organised as follows. We ﬁrst discuss related work Section 2. 97 Next, we describe our approach and methods in Section 3. We present our evaluation of four 98 different named entity recognition systems on 20 classic and 20 modern novels in Section 4, 99 followed by the creation and analysis of social networks in Section 5. We discuss issues that we 100 encountered in the identiﬁcation of ﬁctional characters and showcase some methods to boost 101 performance in Section 6. We conclude by suggesting directions for future work in Section 7. 102 The code for all experiments as well as annotated data can be found at https://github. 103 com/Niels-Dekker/Out-with-the-Old-and-in-with-the-Novel. 104 To answer the second research question, we use the recognised named entities to create a 87 co-occurrence network for each novel. Network analysis measures are then employed to compare 88 the extracted networks from the classic and modern novels to investigate whether the networks 89 from the different sets of novels exhibit major differences. Named Entity Recognition 112 Named Entity Recognition 112 The ﬁrst and foremost challenge in creating a social network of literary characters is identifying 113 the characters. Named Entity Recognition is often used to identify passages in text that identify 114 things by a name. Furthermore, identiﬁed passages are often also classiﬁed into various categories 115 such as person, location, and organisation. Typically, this approach is also used to identify 116 miscellaneous numerical mentions such as dates, times, monetary values and percentages. 117 Elson et al. (2010), Ardanuy and Sporleder (2014), Bamman et al. (2014) and Vala et al. 118 (2015) all use the Stanford NER tagger (Finkel et al., 2005) to identify characters in literary ﬁction. 119 On a collection of Sherlock Holmes novels, these studies perform Named Entity Recognition 120 tasks with F1-scores between: .45 and .54. Vala et al. (2015) propose that the main difﬁculty 121 with this collection is the multitude of minor characters, a problem which we expect to be also 122 present in our collections of classic and modern novels. 123 A big difference between the news domain (for which most language technology tools have 124 been created) and the literary domain, is that names do not have to follow the same ‘rules’ as 125 names in the real world. This topic is explored in the Namescape project by De Does et al. 126 (2017).5 In this project, 1 million tokens taken from 550 Dutch novels were manually annotated. 127 A distinction between ﬁrst and last names was made in order to test whether different name parts 128 are used with different effects. A named entity recogniser was trained speciﬁcally for this corpus 129 The ﬁrst and foremost challenge in creating a social network of literary characters is identifying 113 the characters. Named Entity Recognition is often used to identify passages in text that identify 114 things by a name. Furthermore, identiﬁed passages are often also classiﬁed into various categories 115 such as person, location, and organisation. Typically, this approach is also used to identify 116 miscellaneous numerical mentions such as dates, times, monetary values and percentages. 117 Elson et al. (2010), Ardanuy and Sporleder (2014), Bamman et al. (2014) and Vala et al. 118 (2015) all use the Stanford NER tagger (Finkel et al., 2005) to identify characters in literary ﬁction. 2 RELATED WORK 105 As mentioned in Section 1, we have not found any other studies that compared the performances 106 of social network extraction on classic and modern novels; or compared the structures of these 107 networks. This section therefore focuses on the techniques used on classic literature. In ﬁrst part 108 of this section, we will describe how other studies extract and cluster characters. In the second 109 part, we outline what different choices can be made for the creation of a network, and motivate 110 our choices for this study. 111 Manuscript to be reviewed In our evaluation, we devote extra attention to 66 the comparison between classic and modern fantasy/science ﬁction in our corpus. 67 We deﬁne the following research questions: 68 • To what extent are off-the-shelf named entity recognition tools suitable for identifying 69 ﬁctional characters in novels? 70 • To what extent are off-the-shelf named entity recognition tools suitable for identifying ﬁctional characters in novels? • Which differences or similarities can be discovered between social networks extracted for different novels? To answer our ﬁrst research question, we ﬁrst evaluate four named entity recognisers on 20 73 classic and 20 modern fantasy/science ﬁction novels. In each of these novels, the ﬁrst chapter is 74 manually annotated with named entities and coreference relations. The named entity recognisers 75 we evaluate are: 1) BookNLP (Bamman et al., 2014)3 which is speciﬁcally tailored to identify 76 and cluster literary characters, and has been used to extract entities from a corpus of 15,099 77 English novels. At the time of writing, this tool was cited 80 times. 2) Stanford NER version 78 3.8.0 (Finkel et al., 2005), one of the most popular named entity recognisers in the NLP research 79 community, cited 2,648 times at the time of writing. 3) Illinois Named Entity Tagger version 80 3.0.23 (Ratinov and Roth, 2009), a computationally efﬁcient tagger that uses a combination 81 of machine learning, gazetteers,4 and additional features extracted from unlabelled data. At 82 the time of writing, the system was downloaded over 10,000 times. Our last system (4) is 83 IXA-Pipe-NERC version 1.1.1 (Agerri and Rigau, 2016), a competitive classiﬁer that employs 84 2/26 PeerJ Comput. Sci. reviewing PDF \| (CS-2018:08:30671:2:0:NEW 7 Mar 2019) Manuscript to be reviewed 90 The contributions of this paper are: (1) a comparison and an analysis of four named entity 91 recognition on 20 classic and 20 modern novels; (2) a comparison and an analysis of social 92 network analysis measures on networks automatically extracted from 20 classic and 20 modern 93 novels; (3) experiments and recommendations for boosting performance on recognising entities 94 in novels; and (4) an annotated gold standard dataset with entities and coreferences of 20 classic 95 and 20 modern novels. 96 The remainder of this paper is organised as follows. We ﬁrst discuss related work Section 2. 97 Next, we describe our approach and methods in Section 3. We present our evaluation of four 98 different named entity recognition systems on 20 classic and 20 modern novels in Section 4, 99 followed by the creation and analysis of social networks in Section 5. We discuss issues that we 100 encountered in the identiﬁcation of ﬁctional characters and showcase some methods to boost 101 performance in Section 6. We conclude by suggesting directions for future work in Section 7. 102 The code for all experiments as well as annotated data can be found at https://github. 103 com/Niels-Dekker/Out-with-the-Old-and-in-with-the-Novel. 104 5http://blog.namescape.nl/ Named Entity Recognition 112 119 On a collection of Sherlock Holmes novels, these studies perform Named Entity Recognition 120 tasks with F1-scores between: .45 and .54. Vala et al. (2015) propose that the main difﬁculty 121 with this collection is the multitude of minor characters, a problem which we expect to be also 122 present in our collections of classic and modern novels. 123 A big difference between the news domain (for which most language technology tools have 124 been created) and the literary domain, is that names do not have to follow the same ‘rules’ as 125 names in the real world. This topic is explored in the Namescape project by De Does et al. 126 (2017).5 In this project, 1 million tokens taken from 550 Dutch novels were manually annotated. 127 A distinction between ﬁrst and last names was made in order to test whether different name parts 128 are used with different effects. A named entity recogniser was trained speciﬁcally for this corpus 129 3/26 PeerJ Comput. Sci. reviewing PDF \| (CS-2018:08:30671:2:0:NEW 7 Mar 2019) Coreference resolution 135 One difﬁculty of character detection is the variety of aliases one character might go by, or; 136 coreference resolution. For example, George Martin’s Tyrion Lannister, might alternatively be 137 mentioned as Ser Tyrion Lannister, Lord Tyrion, Tyrion, The Imp or The Halfman. In the vast 138 majority of cases, it is desirable to collapse those character references into one character entity. 139 However, in some cases, retaining some distinction between character references can be useful: 140 we provide an example of this in Subsection 5.4. 141 Two distinct approaches attempt to address this difﬁculty, (1) omit parts of a multi-word 142 name, or (2) compile a list of aliases. The former approach leaves out honoriﬁcs such as the Ser 143 and Lord in the above example in order to cluster the names of one character. To automate this 144 clustering step, some work has been done by Bamman et al. (2014) and Ardanuy and Sporleder 145 (2014). While useful, the former approach alone provides no solace for the matching of the last 146 two example aliases; where no part of the character’s name is present. The latter approach thus 147 suggests to manually compile a list of aliases for each character with the aid of external resources 148 or annotators. This method is utilised by Elson et al. (2010) and Lee and Yeung (2012). In Van 149 Dalen-Oskam et al. (2014), wikiﬁcation (i.e. attempting to match recognised names to Wikipedia 150 resources) is used. Obviously this is most useful for characters that are famous enough to have a 151 Wikipedia page. The authors state in their error analysis Van Dalen-Oskam et al. (2014, Section 152 3.2) that titles that are most likely from the fantasy domain are most difﬁcult to resolve, which 153 already hints at some differences between names in different genres. 154 Manuscript to be reviewed by Van Dalen-Oskam et al. (2014), obtaining an F1 score of 0.936 for persons. The corpus 130 contains fragments of novels written between the 17th and 20th century, but as the corpus and 131 tools are not available, we cannot investigate its depth or compare it directly to our work. Other 132 approaches attempt to use the identiﬁcation of locations and physical proximity to improve the 133 creation of a social network (Lee and Yeung, 2012). 134 Anaphora resolution 155 To identify as many character references as possible, it is important to take into account that not 156 all references to a character actually mention the character’s name. In fact, Bamman et al. (2014) 157 show that 74% of character references come in the form of a pronouns such as he, him, his, she, 158 her and hers in a collection of 15,099 English novels. To capture these references, the anaphoric 159 pronoun is typically matched to its antecedent by using the linear word distance between the 160 two, and by matching the gender of anaphora to that of the antecedent. The linear word distance 161 can be, for example, the number of words between the pronoun and the nearest characters. For 162 unusual names, as often found in science ﬁction and fantasy, identiﬁcation of the gender may be 163 problematic. 164 Network Analysis 182 Social network analysis draws upon network theory for its network analysis measures (Scott, 183 2012). The application of these measures to networks extracted from literature has been demon- 184 strated insightful in assessing the relationships of characters in for example ‘Alice in Wonder- 185 land’ (Agarwal et al., 2012) and ‘Beowulf’, the ‘Iliad’ and ‘T´ain B´o Cuailnge’ (‘The Cattle Raid 186 of Cooley’, an Irish epic) (Mac Carron and Kenna, 2012). Network analysis can also play a 187 role in authorship attribution e.g.(Amancio, 2015; Akimushkin et al., 2017) and characterising a 188 novel (Elson et al., 2010). 189 Social network analysis draws upon network theory for its network analysis measures (Scott, 183 2012). The application of these measures to networks extracted from literature has been demon- 184 strated insightful in assessing the relationships of characters in for example ‘Alice in Wonder- 185 land’ (Agarwal et al., 2012) and ‘Beowulf’, the ‘Iliad’ and ‘T´ain B´o Cuailnge’ (‘The Cattle Raid 186 of Cooley’, an Irish epic) (Mac Carron and Kenna, 2012). Network analysis can also play a 187 role in authorship attribution e.g.(Amancio, 2015; Akimushkin et al., 2017) and characterising a 188 novel (Elson et al., 2010). 189 Network Creation 165 For a social network of literary characters, characters are represented by the nodes, whereas 166 the edges indicate to some interaction or relationship. While the deﬁnition of a character is 167 uniformly accepted in the literature, the deﬁnition of an interaction varies per approach. In 168 previous research, two main approaches can be identiﬁed to deﬁne such an edge. On the one 169 hand, conversational networks are used in approaches by Chambers and Jurafsky (2008), Elson 170 and McKeown (2010) and He et al. (2013). This approach focuses on the identiﬁcation of 171 speakers and listeners, and connecting each speaker and listener to the quoted piece of dialogue 172 they utter or receive. On the other hand, co-occurrence networks (as used by Ardanuy and 173 Sporleder (2014) and Fernandez et al. (2015)) are created by connecting characters if they occur 174 in the same body of text. While conversational networks can provide a good view of who 175 speaks directly to whom, Ardanuy and Sporleder (2014) argue that “...much of the interaction 176 4/26 PeerJ Comput. Sci. reviewing PDF \| (CS-2018:08:30671:2:0:NEW 7 Mar 2019) 6The Guardian: https://www.theguardian.com/books/2003/oct/12/features.fiction Last retrieved: 30 October 2017 7https://www.gutenberg.org/ 8bestfantasybooks.com/top25-fantasy-books.php Last retrieved: 30 October 2017 Manuscript to be reviewed in novels is done off-dialogue through the description of the narrator or indirect interactions” 177 (p. 34). What value to assign to the edges depends on the end-goal of the study. For example, 178 Fernandez et al. (2015) assign a negative or positive sentiment score to the edges between each 179 character-pair in order to ultimately predict the protagonist and antagonist of the text. Ardanuy 180 and Sporleder (2014) used weighted edges to indicate how often two characters interact. 181 in novels is done off-dialogue through the description of the narrator or indirect interactions” 177 (p. 34). What value to assign to the edges depends on the end-goal of the study. For example, 178 Fernandez et al. (2015) assign a negative or positive sentiment score to the edges between each 179 character-pair in order to ultimately predict the protagonist and antagonist of the text. Ardanuy 180 and Sporleder (2014) used weighted edges to indicate how often two characters interact. 181 in novels is done off-dialogue through the description of the narrator or indirect interactions” 177 (p. 34). What value to assign to the edges depends on the end-goal of the study. For example, 178 Fernandez et al. (2015) assign a negative or positive sentiment score to the edges between each 179 character-pair in order to ultimately predict the protagonist and antagonist of the text. Ardanuy 180 and Sporleder (2014) used weighted edges to indicate how often two characters interact. 181 3 MATERIALS AND DATA PREPARATION 190 For the study presented here, we are interested in the recognition and identiﬁcation of persons 191 mentioned in classic and modern novels for the construction of the social network of these 192 ﬁctitious characters. We use off-the-shelf state-of-the-art entity recognition tools in an automatic 193 pipeline without manually created alias lists or similar techniques. For the network construction, 194 we follow Ardanuy and Sporleder (2014) and apply their co-occurrence approach for the genera- 195 tion of the social network links with weighted edges that indicate how often two characters are 196 mentioned together. We leave the consideration of negative weights and sentiments for future 197 work. Before we will explain the details of the used entity recognition tools, how they compare 198 for the given task, and how their results can be used to build and analyse the respective social 199 networks, we explain ﬁrst the details of our selected corpus, how we preprocessed the data, and 200 how we collected the annotations for the evaluation. 201 3.1 Corpus Selection 202 3.1 Corpus Selection 202 Our dataset consists of 40 novels – 20 classic and 20 modern novels – the speciﬁcs of which are 203 presented in Table A2 in the Appendix. Any selection of sources is bound to be unrepresentative 204 in terms of some characteristics but we have attempted to balance breadth and depth in our 205 dataset. Furthermore, we have based ourselves on selections made by other researchers for the 206 classics and compilations by others for the modern books. 207 For the classic set, the selection was based on Guardian’s Top 100 all-time classic novels.6 208 Wherever possible, we selected books that were (1) analysed in related work (as mentioned in 209 Subsection 2) and (2) available through Project Gutenberg.7 210 For the modern set, the books were selected by reference to a list compiled by BestFantasy- 211 BooksCom.8 For our ﬁnal selection of these novels, we deliberately made some adjustments 212 to get a wider selection. That is, some of the books in this list are part of a series. If we were 213 to include all the books of the upvoted series, our list would consist of only 4 different series. 214 We therefore chose to include only the ﬁrst book of each of such series. As the newer books 215 are unavailable on Gutenberg, these were purchased online. These digital texts are generally 216 provided in .epub or .mobi format. In order to reliably convert these ﬁles into plain text format, 217 6The Guardian: https://www.theguardian.com/books/2003/oct/12/features.fiction Last retrieved: 30 October 2017 7https://www.gutenberg.org/ 8bestfantasybooks.com/top25-fantasy-books.php Last retrieved: 30 October 2017 Our dataset consists of 40 novels – 20 classic and 20 modern novels – the speciﬁcs of which are 203 presented in Table A2 in the Appendix. Any selection of sources is bound to be unrepresentative 204 in terms of some characteristics but we have attempted to balance breadth and depth in our 205 dataset. Furthermore, we have based ourselves on selections made by other researchers for the 206 classics and compilations by others for the modern books. 8bestfantasybooks.com/top25-fantasy-books.php Last retrieved: 30 October 2017 Annotation Data 241 To evaluate the performance for each novel, a gold standard was created manually. Two annotators 242 (not the authors of this article) were asked to evaluate 10 books from each category. For each 243 document, approximately one chapter was annotated with entity co-occurrences. Because the 244 length of the ﬁrst chapter ﬂuctuated between 84 and 1,442 sentences, we selected an average 245 of 300 sentences for each book that was close to a chapter-boundary. For example, for Alice 246 in Wonderland, the third chapter ended on the 315th sentence, so the ﬁrst three chapters were 247 extracted for annotation. While not perfect, we attempted to strike a balance between comparable 248 annotation lengths for each book, without cutting off mid-chapter. 249 id Preceding context Focus sentence Subsequent context # Person 1 Person 2 541 Bran reached out hesitantly. “Go on,” Robb told him. “You can touch him.” 2 Robb Stark Bran Stark Table 1. Annotation Example. Table 1. Annotation Example. 3.2 Data Preprocessing 223 To ensure that all the harvested text ﬁles were ready for processing, we ﬁrstly ensured that the 224 encoding for all the documents was the same, in order to avoid issues down the line. In addition, 225 all information that is not directly relevant to the story of the novel was stripped. Even while 226 peripheral information in some books – such as appendices or glossaries – can provide useful 227 information about character relationships, we decided to focus on the story content and thus 228 discard this information. Where applicable, the following peripheral information was manually 229 removed: (1) reviews by fellow writers, (2) dedications or acknowledgements, (3) publishing 230 information, (4) table of contents, (5) chapter headings and page numbers, and (6) appendices 231 and/or glossaries. 232 During this clean-up phase, we encountered some encoding issues that came with the 233 conversion to plain text ﬁles. Especially in the modern novels, some novels used inconsistent or 234 odd quotation marks. This issue was addressed by replacing the inconsistent quotation marks 235 with neutral quotations that are identical in form, regardless of whether if it is used as opening or 236 closing quotation mark. 237 Manuscript to be reviewed we used Calibre9– a free and open-source e-book conversion tool. This conversion was mostly 218 without any hurdles, but some issues were encountered in terms of encoding, as is discussed in 219 the next section. Due to copyright restrictions, we cannot share this full dataset but our gold 220 standard annotations of the ﬁrst chapter of each are provided on this project’s Github page. 221 The ISBN numbers of the editions used in our study can be found in Table A2 the Appendix. 222 3.3 Annotation 238 Because of limitations in time and scope, we only annotated approximately 1 chapter of each 239 novel. In this subsection, we describe the annotation process. 240 Because of limitations in time and scope, we only annotated approximately 1 chapter of each 239 novel. In this subsection, we describe the annotation process. 240 Because of limitations in time and scope, we only annotated approximately 1 chapter of each 239 novel. In this subsection, we describe the annotation process. 240 3.1 Corpus Selection 202 207 For the classic set, the selection was based on Guardian’s Top 100 all-time classic novels.6 208 Wherever possible, we selected books that were (1) analysed in related work (as mentioned in 209 Subsection 2) and (2) available through Project Gutenberg.7 210 For the classic set, the selection was based on Guardian’s Top 100 all-time classic novels.6 208 Wherever possible, we selected books that were (1) analysed in related work (as mentioned in 209 Subsection 2) and (2) available through Project Gutenberg 7 210 ) ( ) g j g For the modern set, the books were selected by reference to a list compiled by BestFantasy- 211 BooksCom.8 For our ﬁnal selection of these novels, we deliberately made some adjustments 212 to get a wider selection. That is, some of the books in this list are part of a series. If we were 213 to include all the books of the upvoted series, our list would consist of only 4 different series. 214 We therefore chose to include only the ﬁrst book of each of such series. As the newer books 215 are unavailable on Gutenberg, these were purchased online. These digital texts are generally 216 provided in .epub or .mobi format. In order to reliably convert these ﬁles into plain text format, 217 For the modern set, the books were selected by reference to a list compiled by BestFantasy- 211 BooksCom.8 For our ﬁnal selection of these novels, we deliberately made some adjustments 212 to get a wider selection. That is, some of the books in this list are part of a series. If we were 213 to include all the books of the upvoted series, our list would consist of only 4 different series. 214 We therefore chose to include only the ﬁrst book of each of such series. As the newer books 215 are unavailable on Gutenberg, these were purchased online. These digital texts are generally 216 provided in .epub or .mobi format. In order to reliably convert these ﬁles into plain text format, 217 8bestfantasybooks.com/top25-fantasy-books.php Last retrieved: 30 October 2017 5/26 PeerJ Comput. Sci. reviewing PDF \| (CS-2018:08:30671:2:0:NEW 7 Mar 2019) Annotation Instructions 250 For each document, the annotators were asked to annotate each sentence for the occurrence of 251 characters. That is, for each sentence, identify all the characters in it. To describe this process, an 252 example containing a single sentence from A Game of Thrones is included in Table 1. The id of 253 9https://calibre-ebook.com/ – version 2.78 6/26 PeerJ Comput. Sci. reviewing PDF \| (CS-2018:08:30671:2:0:NEW 7 Mar 2019) Manuscript to be reviewed 278 Guideline Example Ignore generic pronouns “Everyone knows; you don’t mess with me!” Ignore exclamations “For Christ’s sake!” Ignore generic “Bilbo didn’t know what noun phrases to tell the wizard.” Include non-human “His name is Buckbeak, named characters he’s a hippogriph.” Table 2. Annotation Instructions Guideline Example Ignore generic pronouns “Everyone knows; you don’t mess with me!” Ignore exclamations “For Christ’s sake!” Ignore generic “Bilbo didn’t know what noun phrases to tell the wizard.” Include non-human “His name is Buckbeak, named characters he’s a hippogriph.” Table 2. Annotation Instructions Table 2. Annotation Instructions Table 2. Annotation Instructions 4 NAMED ENTITY RECOGNITION EXPERIMENTS AND RESULTS 279 We evaluate the performance of four different named entity recognition systems on the anno- 280 tated novels: BookNLP (Bamman et al., 2014), Stanford NER(Finkel et al., 2005), Illinois 281 Tagger (Ratinov and Roth, 2009) and IXA-Pipe-NERC (Agerri and Rigau, 2016). The BookNLP 282 pipeline uses the 2014-01-04 release of Stanford NER tagger (Finkel et al., 2005) internally with 283 the 7-class ontonotes model. As there have been several releases, and we focus on entities of 284 type Person, we also evaluate the 2017-06-09 Stanford NER 4-class CoNLL model. 285 The results of the different Named Entity Recognition systems are presented in Table 3 286 Manuscript to be reviewed the sentence is later used to match the annotated sentence to its system-generated counterpart for 254 performance evaluation. The focus sentence is the sentence that corresponds to this id, and is the 255 sentence for which the annotator is supposed to identify all characters. As context, the annotators 256 are provided with the preceding and subsequent sentences. In this example, the contextual 257 sentences could be used to resolve the ‘him’ in the focus sentence to ‘Bran’. To indicate how 258 many persons are present, the annotators were asked to ﬁll in the corresponding number (#) of 259 people – with a maximum of 10 characters per sentence. Depending on this number of people 260 identiﬁed, subsequent ﬁelds became available to the annotator to ﬁll in the character names. 261 To speed up the annotation, an initial list of characters was created by applying the BookNLP 262 pipeline to each novel. The annotators were instructed to map the characters in the text to the 263 provided list to the best of their ability. If the annotator assessed that a person appears in a 264 sentence, but is unsure of this character’s identity, the annotators would mark this character as 265 default. In addition, the annotators were encouraged to add characters, should they be certain 266 that this character does not appear in the pre-compiled list, but occurs in the text nonetheless. 267 Such characters were given a speciﬁc tag to ensure that we could retrieve them later for analysis. 268 Lastly, if the annotator is under the impression that two characters in the list refer to the same 269 person, the annotators were instructed to pick one and stick to that. Lastly, the annotators were 270 provided with the peripheral annotation instructions found in Table 2. 271 While this identiﬁcation process did include anaphora resolution of singular pronouns – such 272 as resolving ‘him’ to ‘Bran’ – the annotators were instructed to ignore plural pronoun references. 273 Plural pronoun resolution remains a difﬁcult topic in the creation of social networks, as family 274 members may sometimes be mentioned individually, and sometimes their family as a whole. 275 Identifying group membership, and modelling that in the social network structure is not covered 276 by any of the tools we include in our analysis or the related work referenced in Section 2 and 277 therefore left to future work. he results of the different Named Entity Recognition systems are presented in Table 3 Manuscript to be reviewed 323 As the BookNLP pipeline in the majority of the cases outperforms the other systems and 317 includes coreference resolution and character clustering, we further utilise this system to create 318 our networks. The results of the BookNLP pipeline including the coreference and clustering are 319 presented in Table A4. One of the main differences in that table is that if popular entities are not 320 recognised by the system they are penalised heavier because the coreferent mentions are also not 321 recognised and linked to the correct entities. This results in scores that are generally somewhat 322 lower, but the task that is measured is also more complex. 323 As the BookNLP pipeline in the majority of the cases outperforms the other systems and 317 includes coreference resolution and character clustering, we further utilise this system to create 318 our networks. The results of the BookNLP pipeline including the coreference and clustering are 319 presented in Table A4. One of the main differences in that table is that if popular entities are not 320 recognised by the system they are penalised heavier because the coreferent mentions are also not 321 recognised and linked to the correct entities. This results in scores that are generally somewhat 322 lower, but the task that is measured is also more complex. 323 4 NAMED ENTITY RECOGNITION EXPERIMENTS AND RESULTS 279 We evaluate the performance of four different named entity recognition systems on the anno- 280 tated novels: BookNLP (Bamman et al., 2014), Stanford NER(Finkel et al., 2005), Illinois 281 Tagger (Ratinov and Roth, 2009) and IXA-Pipe-NERC (Agerri and Rigau, 2016). The BookNLP 282 pipeline uses the 2014-01-04 release of Stanford NER tagger (Finkel et al., 2005) internally with 283 the 7-class ontonotes model. As there have been several releases, and we focus on entities of 284 type Person, we also evaluate the 2017-06-09 Stanford NER 4-class CoNLL model. 285 The results of the different Named Entity Recognition systems are presented in Table 3 286 We evaluate the performance of four different named entity recognition systems on the anno- 280 tated novels: BookNLP (Bamman et al., 2014), Stanford NER(Finkel et al., 2005), Illinois 281 Tagger (Ratinov and Roth, 2009) and IXA-Pipe-NERC (Agerri and Rigau, 2016). The BookNLP 282 pipeline uses the 2014-01-04 release of Stanford NER tagger (Finkel et al., 2005) internally with 283 the 7-class ontonotes model. As there have been several releases, and we focus on entities of 284 type Person, we also evaluate the 2017-06-09 Stanford NER 4-class CoNLL model. 285 yp The results of the different Named Entity Recognition systems are presented in Table 3 286 yp The results of the different Named Entity Recognition systems are presented in Table 3 286 7/26 PeerJ Comput. Sci. reviewing PDF \| (CS-2018:08:30671:2:0:NEW 7 Mar 2019) 10https://www.clips.uantwerpen.be/conll2002/ner/bin/conlleval.txt Last retrieved: 30 October 2017 Manuscript to be reviewed Upon inspection, we ﬁnd 303 that the annotated chapter of this book contains only 5 different characters among which “The 304 When comparing the F1 scores of the 1st person novels to the 3rd person novels in Tables 312 3 and 4, we ﬁnd that the 1st person novels perform signiﬁcantly worse than their 3rd person 313 counterparts, at p < .01. These ﬁndings are in line with the ﬁndings of Elson et al. (2010). 314 When comparing the F1 scores of the 1st person novels to the 3rd person novels in Tables 312 3 and 4, we ﬁnd that the 1st person novels perform signiﬁcantly worse than their 3rd person 313 counterparts, at p < .01. These ﬁndings are in line with the ﬁndings of Elson et al. (2010). 314 When comparing the F1 scores of the 1st person novels to the 3rd person novels in Tables 312 3 d 4 ﬁd th t th 1st l f i iﬁ tl th th i 3rd 3 and 4, we ﬁnd that the 1 person novels perform signiﬁcantly worse than their 3 person 313 counterparts, at p < .01. These ﬁndings are in line with the ﬁndings of Elson et al. (2010). 314 counterparts, at p < .01. These ﬁndings are in line with the ﬁndings of Elson et al. (2010). 314 In Section 6, we delve further into particular difﬁculties that ﬁction presents named 5 recognition with and showcase solutions that do not require retraining the entity models. 316 recognition with and showcase solutions that do not require retraining the entity models 6 As the BookNLP pipeline in the majority of the cases outperforms the other systems and 317 includes coreference resolution and character clustering, we further utilise this system to create 318 our networks. The results of the BookNLP pipeline including the coreference and clustering are 319 presented in Table A4. One of the main differences in that table is that if popular entities are not 320 recognised by the system they are penalised heavier because the coreferent mentions are also not 321 recognised and linked to the correct entities. This results in scores that are generally somewhat 322 lower, but the task that is measured is also more complex. Manuscript to be reviewed for the classic novels, and Table 4 for the modern novels. All results are computed using the 287 evaluation script used in the CoNLL 2002 and 2003 NER campaigns using the phrase-based 288 evaluation setup.10 The systems are evaluated according to micro-averaged precision, recall and 289 F1 measure. Precision is the percentage of named entities found by the system that were correct. 290 Recall is the percentage of named entities present in the text that are retrieved by the system. The 291 F1 measure is the harmonic mean of the precision and recall scores. In a phrase-based evaluation 292 setup, the system only scores a point if the complete entity is correctly identiﬁed, thus if in a 293 named entity consisting of multiple tokens only two out of three tokens are correctly identiﬁed, 294 the system does not obtain any points. 295 The BookNLP and IXA-Pipe-NERC systems require that part of speech tagging is performed 296 prior to named entity recognition, we use the modules included in the respective systems for this. 297 For Stanford NER and Illinois NE Tagger plain text is offered to the NER systems. 298 The BookNLP and IXA-Pipe-NERC systems require that part of speech tagging is performed 296 prior to named entity recognition, we use the modules included in the respective systems for this. 297 For Stanford NER and Illinois NE Tagger plain text is offered to the NER system 298 For Stanford NER and Illinois NE Tagger plain text is offered to the NER systems. 298 As the standard deviations on the bottom rows of Tables 3 and 4 indicate, the results on the 299 different books vary greatly. However, the different NER systems generally do perform similarly 300 on the same novels, indicating that difﬁculties in recognising named entities in particular books 301 is a characteristic of the novels rather than the systems. An exception is Brave New World on 302 which BookNLP performs quite well, but the others underperform. Upon inspection, we ﬁnd 303 that the annotated chapter of this book contains only 5 different characters among which “The 304 Director” which occurs 19 times. This entity is consistently missed by the systems resulting in a 305 high penalty. Furthermore, the ‘Mr.’ in ‘Mr. Foster’ (occurring 31 times) is often not recognised 306 as in some NE models titles are excluded. Manuscript to be reviewed An exception is Brave New World on 302 which BookNLP performs quite well, but the others underperform. Upon inspection, we ﬁnd 303 that the annotated chapter of this book contains only 5 different characters among which “The 304 Director” which occurs 19 times. This entity is consistently missed by the systems resulting in a 305 high penalty. Furthermore, the ‘Mr.’ in ‘Mr. Foster’ (occurring 31 times) is often not recognised 306 As the standard deviations on the bottom rows of Tables 3 and 4 indicate, the results on the 299 different books vary greatly. However, the different NER systems generally do perform similarly 300 on the same novels, indicating that difﬁculties in recognising named entities in particular books 301 , different books vary greatly. However, the different NER systems generally do perform similarly 300 on the same novels, indicating that difﬁculties in recognising named entities in particular books 301 is a characteristic of the novels rather than the systems. An exception is Brave New World on 302 which BookNLP performs quite well but the others underperform Upon inspection we ﬁnd 303 different books vary greatly. However, the different NER systems generally do perform similarly 300 on the same novels, indicating that difﬁculties in recognising named entities in particular books 301 is a characteristic of the novels rather than the systems. An exception is Brave New World on 302 which BookNLP performs quite well, but the others underperform. Upon inspection, we ﬁnd 303 that the annotated chapter of this book contains only 5 different characters among which “The 304 Director” which occurs 19 times. This entity is consistently missed by the systems resulting in a 305 high penalty. Furthermore, the ‘Mr.’ in ‘Mr. Foster’ (occurring 31 times) is often not recognised 306 as in some NE models titles are excluded. A token-based evaluation of Illinois NE Tagger on 307 , g g g p is a characteristic of the novels rather than the systems. An exception is Brave New World on 302 v y p v N w W which BookNLP performs quite well, but the others underperform. Manuscript to be reviewed A token-based evaluation of Illinois NE Tagger on 307 this novel for example yields a F1 score of 51.91. The same issue is at hand with Dr. Jekyll and 308 Mr. Hyde and Dracula. Although the main NER module in BookNLP is driven by Stanford 309 NER, we suspect that additional domain adaptations in this package account for this performance 310 difference. 311 d As the standard deviations on the bottom rows of Tables 3 and 4 indicate, the results on the 299 different books vary greatly. However, the different NER systems generally do perform similarly 300 on the same novels, indicating that difﬁculties in recognising named entities in particular books 301 is a characteristic of the novels rather than the systems. An exception is Brave New World on 302 which BookNLP performs quite well, but the others underperform. Upon inspection, we ﬁnd 303 that the annotated chapter of this book contains only 5 different characters among which “The 304 Director” which occurs 19 times. This entity is consistently missed by the systems resulting in a 305 high penalty. Furthermore, the ‘Mr.’ in ‘Mr. Foster’ (occurring 31 times) is often not recognised 306 as in some NE models titles are excluded. A token-based evaluation of Illinois NE Tagger on 307 this novel for example yields a F1 score of 51.91. The same issue is at hand with Dr. Jekyll and 308 Mr. Hyde and Dracula. Although the main NER module in BookNLP is driven by Stanford 309 NER, we suspect that additional domain adaptations in this package account for this performance 310 difference. 311 As the standard deviations on the bottom rows of Tables 3 and 4 indicate, the results on the 299 different books vary greatly. However, the different NER systems generally do perform similarly 300 on the same novels, indicating that difﬁculties in recognising named entities in particular books 301 is a characteristic of the novels rather than the systems An exception is Brave New World on 302 As the standard deviations on the bottom rows of Tables 3 and 4 indicate, the results on the 299 different books vary greatly. However, the different NER systems generally do perform similarly 300 on the same novels, indicating that difﬁculties in recognising named entities in particular books 301 is a characteristic of the novels rather than the systems. 5 NETWORK ANALYSIS 324 In this section, we explain how the networks were created using the recognised named entities 325 (Subsection 5.1), followed by an explanation of network analysis measures that we applied to 326 compare the networks (Subsection 5.2). We discuss the results of the analysis (Subsection 5.3), 327 as well as present an exploration of the network of one novel in particular to illustrate how 328 a visualisation of a network can highlight particular characteristics of the interactions in the 329 selected novel (Subsection 5.4). 330 8/26 PeerJ Comput. Sci. reviewing PDF \| (CS-2018:08:30671:2:0:NEW 7 Mar 2019) Computer Science Computer Science Manuscript to be reviewed BookNLP Stanford NER Illinois NER IXA-NERC Title P R F1 P R F1 P R F1 P R F1 1984 92.31 70.59 80.00 89.29 73.53 80.65 93.55 85.29 89.23 93.55 85.29 89.23 A Study in Scarlet⊙ 25.00 30.77 27.59 22.22 30.77 25.81 14.29 15.38 14.81 20.00 23.08 21.43 Alice in Wonderland 89.13 55.78 68.62 83.33 57.82 68.27 87.07 87.07 87.07 84.30 69.39 76.12 Brave New World 82.93 60.71 70.00 7.50 5.36 6.25 7.69 5.36 6.32 2.63 1.79 2.13 David Copperﬁeld⊙ 29.41 35.71 32.26 54.02 67.14 59.87 58.82 71.43 64.52 14.47 15.71 15.07 Dracula⊙ 5.00 20.00 8.00 4.00 20.00 6.67 12.50 60.00 20.69 10.53 40.00 16.67 Emma 86.96 93.02 89.89 25.90 27.91 26.87 26.81 28.68 27.72 30.22 32.56 31.34 Frankenstein⊙ 52.00 76.47 61.90 37.93 64.71 47.83 30.77 47.06 37.21 34.62 52.94 41.86 Huckleberry Finn 86.84 98.51 92.31 81.08 89.55 85.11 77.92 89.55 83.33 79.71 82.09 80.88 Dr. Jekyll and Mr. Hyde 86.36 82.61 84.44 18.18 17.39 17.78 21.74 21.74 21.74 13.64 13.04 13.33 Moby Dick⊙ 67.65 74.19 70.77 63.89 74.19 68.66 68.42 83.87 75.36 37.84 45.16 41.18 Oliver Twist 85.61 94.44 89.81 36.30 42.06 38.97 44.32 33.62 38.24 34.69 40.48 37.36 Pride and Prejudice 79.26 94.69 86.29 32.33 38.05 34.96 29.37 32.74 30.96 33.87 37.17 35.44 The Call of the Wild 80.65 30.49 44.25 86.36 46.34 60.32 89.47 82.93 86.08 88.14 63.41 73.76 The Count of Monte Cristo 78.22 89.77 83.60 67.95 60.23 63.86 79.80 89.77 84.49 72.31 53.41 61.44 The Fellowship of the Ring 73.39 72.15 72.77 66.12 68.35 67.22 56.52 38.40 45.73 63.33 56.12 59.51 The Three Musketeers 65.71 29.49 40.71 63.64 35.90 45.90 45.45 25.64 32.12 73.68 35.90 48.28 The Way We Live Now 73.33 92.77 81.91 49.52 62.65 55.32 28.18 37.35 32.12 43.30 50.60 46.67 Ulysses 76.74 94.29 84.62 70.10 97.14 81.44 71.28 95.71 81.71 72.29 85.71 78.43 Vanity Fair 67.30 65.44 66.36 32.46 34.10 33.26 32.61 34.56 33.56 53.12 47.00 49.88 Mean µ 70.16 68.95 67.72 52.03 53.00 51.13 51.37 55.98 52.26 49.26 48.29 47.61 Standard Deviation σ 24.03 26.27 24.25 27.27 25.24 24.93 28.68 30.16 29.17 29.70 24.71 26.50 Table 3. Precision (P), Recall (R) and F1 scores of different NER systems on classic novels. The highest scores in each column are highlighted in boldface, and the lowest scores in italics. Novels written in 1st person are marked with ⊙. PeerJ Comput. Sci. Computer Science Computer Science Manuscript to be reviewed Manuscript to be reviewed BookNLP Stanford NER Illinois NER IXA-NERC Title P R F1 P R F1 P R F1 P R F1 A Game of Thrones 97.98 62.99 76.68 92.73 66.23 77.27 93.51 93.51 93.51 92.08 60.39 72.94 Assassin’s Apprentice⊙ 63.33 38.38 47.80 61.19 41.41 49.90 61.45 40.40 48.78 53.12 34.34 41.72 Elantris 82.00 89.78 85.71 76.97 92.70 84.11 83.12 97.08 89.56 76.52 64.23 69.84 Gardens of the Moon 35.29 34.29 34.78 39.02 45.71 42.11 40.43 54.29 46.34 44.44 45.71 45.07 Harry Potter 83.80 90.36 86.96 61.24 65.66 63.37 58.43 58.43 58.43 54.94 53.61 54.27 Magician 72.92 42.17 53.44 65.57 48.19 55.56 77.67 96.39 86.02 63.10 63.86 63.47 Mistborn 96.46 81.95 88.62 93.22 82.71 87.65 90.07 95.49 92.70 94.05 59.40 72.81 Prince of Thorns 69.23 62.07 65.45 64.29 62.07 63.16 60.00 51.72 55.56 72.73 55.17 62.75 Storm Front⊙ 65.00 65.00 65.00 68.42 65.00 66.67 64.71 55.00 59.46 63.16 60.00 61.54 The Black Company⊙ 77.27 96.23 85.71 29.41 9.43 14.29 67.39 58.49 62.63 60.87 26.42 36.84 The Black Prism 90.29 90.29 90.29 88.35 88.35 88.35 88.68 91.26 89.95 87.21 72.82 79.37 The Blade Itself 62.50 71.43 66.67 71.43 71.43 71.43 52.63 71.43 60.61 55.56 35.71 43.48 The Colour of Magic 83.33 37.50 51.72 84.00 52.50 64.62 71.43 25.00 37.04 77.78 35.00 48.28 The Gunslinger 64.71 100.00 78.57 64.71 100.00 78.57 61.76 95.45 75.00 59.38 86.36 70.37 The Lies of Locke Lamora 86.16 74.05 79.65 87.58 76.22 81.50 86.79 74.59 80.23 88.19 68.65 77.20 The Name of the wind 85.88 74.49 79.78 87.36 77.55 82.16 78.82 68.37 73.22 85.92 62.24 72.19 The Painted Man 87.02 71.70 78.62 86.47 72.33 78.77 80.81 87.42 83.99 83.09 71.07 76.61 The Way of Kings 80.72 87.01 83.75 75.82 89.61 82.14 70.10 88.31 78.16 66.67 49.35 56.72 The Wheel of Time 66.67 45.86 54.34 70.93 77.71 74.16 58.05 87.26 69.72 66.67 57.32 61.64 Way of Shadows 53.85 77.78 63.64 48.72 70.37 57.58 45.45 92.59 60.98 42.86 44.44 43.64 Mean µ 75.22 69.67 70.86 70.87 67.76 68.17 69.57 74.12 70.09 69.42 55.30 60.54 Standard Deviation σ 15.34 20.73 15.86 17.53 20.95 18.08 15.12 21.57 16.67 15.63 15.02 13.50 Table 4. Precision (P), Recall (R) and F1 scores of different NER systems on modern novels. The highest scores in each column are highlighted in boldface, and the lowest scores in italics. Novels written in 1st person are marked with ⊙. PeerJ Comput. Sci. Computer Science reviewing PDF \| (CS-2018:08:30671:2:0:NEW 7 Mar 2019) 9/26 PeerJ Comput. Sci. reviewing PDF \| (CS-2018:08:30671:2:0:NEW 7 Mar 2019) Manuscript to be reviewed 5.1 Network Construction 331 As explained in Section 2, we opt for the co-occurrence rather than the conversational method 332 for ﬁnding the edges of our networks. The body of text that is used to deﬁne a co-occurrence 333 differs per approach. Whereas Fernandez et al. (2015) deﬁne such a relation if characters are 334 mentioned in the same sentence, Ardanuy and Sporleder (2014) use a paragraph for the same 335 deﬁnition. We consider the delineation of what constitutes a paragraph to be too vague for the 336 purpose of this study. While paragraphs are arguably better at conveying who interacts with 337 whom, simply because of their increased length, it also brings forth an extra complexity in terms 338 of their deﬁnition. Traditionally, paragraphs would be separated from another by means of a 339 newline followed by an indented ﬁrst line of the next paragraph. While this format holds for 340 a part of our collection, it is not uniform. Other paragraph formats simply add vertical white 341 space, or depend solely on the content (Bringhurst, 2004). Especially because the text ﬁles in 342 our approach originate from different online sources – each with their own accepted format – 343 we decided that the added ambiguity should be avoided. For this study, we therefore deﬁne 344 that a co-occurrence relationship between two characters exists if they are mentioned in the 345 same sentence. For a co-occurrence of more than two characters, we follow Elson et al. (2010). 346 That is, a multi-way co-occurrence between four characters is broken down into six bilateral 347 co-occurrences. 348 For the construction of each social network, the co-occurrences are translated to nodes for 349 characters and edges for relationships between the characters. We thus create a static, undirected 350 and weighted graph. For the weight of each edge, we follow Ardanuy and Sporleder (2014). 351 That is, each edge is assigned a weight depending on the number of interactions between two 352 characters. For the construction of the network, we used NetworkX11 and Gephi12 to visualise 353 the networks. 354 To ground the network analysis to be presented below, we gathered some overall statistics of 355 the network creation process shown in Table A3 on page 24. Manuscript to be reviewed reviewing PDF \| (CS-2018:08:30671:2:0:NEW 7 Mar 2019) 10/26 PeerJ Comput. Sci. reviewing PDF \| (CS-2018:08:30671:2:0:NEW 7 Mar 2019) Manuscript to be reviewed As mentioned in Subsection 3.3, if 356 the annotator decided that a character was deﬁnitely present, but unable to assert which character, 357 the occurrence was marked as default. The fraction of defaults represents what portion of all 358 identiﬁed characters was marked with default. The fraction of unidentiﬁed characters represents 359 the percentage of characters that were not retrieved by the system, but had to be added by the 360 annotators. Next, we present some overall statistics such as sentence length, the average number 361 of persons in a sentence, and the average fraction of sentences that mention a person. Lastly, 362 we kept track of the total number of annotated sentences, the total number of unique characters 363 and character mentions. The only difference that could be identiﬁed between classes is the 364 average sentence length, which was signiﬁcant at p < .01. The sentences in classic books are 365 signiﬁcantly longer than in modern novels, suggesting that there is indeed some difference in 366 writing style. However, other than that, none of the other measures differ signiﬁcantly. This is 367 useful information, as it helps support that the novels used in either class are comparable, despite 368 their age-gap. 369 12https://gephi.org/ – v0.9.1 11https://networkx.github.io/ – v1.11 5.2 Network Features 370 We analyse the following eight network features: 371 We analyse the following eight network features: 371 1. Average degree is the mean degree of all the nodes in the network. The degree of a node 372 is deﬁned as the number of other nodes the node is connected to. If the degree of a node 373 is 0, the node is connected to no other nodes. The degree of a node in a social network 374 is thus is measure of its social ‘activity’ (Wasserman and Faust, 1994). A high value – 375 1. Average degree is the mean degree of all the nodes in the network. The degree of a node 372 is deﬁned as the number of other nodes the node is connected to. If the degree of a node 373 is 0, the node is connected to no other nodes. The degree of a node in a social network 374 is thus is measure of its social ‘activity’ (Wasserman and Faust, 1994). A high value – 375 1. Average degree is the mean degree of all the nodes in the network. The degree of a node 372 is deﬁned as the number of other nodes the node is connected to. If the degree of a node 373 is 0, the node is connected to no other nodes. The degree of a node in a social network 374 is thus is measure of its social ‘activity’ (Wasserman and Faust, 1994). A high value – 375 11/26 PeerJ Comput. Sci. reviewing PDF \| (CS-2018:08:30671:2:0:NEW 7 Mar 2019) Manuscript to be reviewed Graph density is the fraction of edges compared to the total number of possible edges. 402 It thus indicates how complete the network is, where completeness would constitute all 403 nodes being directly connected by an edge. This is often used in social network analysis 404 to represent how closely the participants of the network are connected (Scott, 2012). 405 6. Modularity is used to represent community structure. The modularity of a network is 406 “...the number of edges falling within groups minus the expected number in an equivalent 407 network with edges placed at random” (Newman, 2006). Newman shows modularity can 408 be used as an optimisation metric to approximate the number of community structures 409 found in the network. To identify the community structures, we used the Louvain algorithm 410 (Blondel et al., 2008). The identiﬁcation of community structures in graph is useful, 411 because the nodes in the same community are more likely to have other properties in 412 common (Danon et al., 2005). It would therefore be interesting to see if differences can be 413 observed between the prevalence of communities between the classic and modern novels. 414 7. Connected components are the number of distinct graph compartments. That is, a graph 415 component is a subgraph in which any two vertices are connected to each other by paths, 416 and which is connected to no additional vertices in the supergraph. In other words, it 417 is not possible to traverse from one component to another. In most social communities, 418 one ‘giant component’ can typically be identiﬁed, which contains the majority of all 419 vertices (Kumar et al., 2010). A higher number of connected components would indicate 420 a higher number of isolated communities. This is different from modularity in the sense 421 that components are more strict. If only a single edge goes out from a subgraph to the 422 12/26 put. Sci. reviewing PDF \| (CS-2018:08:30671:2:0:NEW 7 Mar 2019) 2. Average Weighted Degree is fairly similar to the average degree, but especially in the sense of social networks, a distinction must be made. It differs in the sense that the weighted degree takes into account the weight of each of the connecting edges. Manuscript to be reviewed It 399 is in essence the longest, shortest path that can be found between any two nodes in the 400 network, and is indicative of the linear size of the network (Wasserman and Faust, 1994). 401 4. Network Diameter is the longest possible distance between two nodes in the network. It 399 is in essence the longest, shortest path that can be found between any two nodes in the 400 network, and is indicative of the linear size of the network (Wasserman and Faust, 1994). 401 4. Network Diameter is the longest possible distance between two nodes in the network. It 399 is in essence the longest, shortest path that can be found between any two nodes in the 400 network, and is indicative of the linear size of the network (Wasserman and Faust, 1994). 401 5. Graph density is the fraction of edges compared to the total number of possible edges. 402 It thus indicates how complete the network is, where completeness would constitute all 403 nodes being directly connected by an edge. This is often used in social network analysis 404 to represent how closely the participants of the network are connected (Scott, 2012). 405 5. Graph density is the fraction of edges compared to the total number of possible edges. 402 It thus indicates how complete the network is, where completeness would constitute all 403 nodes being directly connected by an edge. This is often used in social network analysis 404 to represent how closely the participants of the network are connected (Scott, 2012). 405 6. Modularity is used to represent community structure. The modularity of a network is 406 “...the number of edges falling within groups minus the expected number in an equivalent 407 network with edges placed at random” (Newman, 2006). Newman shows modularity can 408 be used as an optimisation metric to approximate the number of community structures 409 found in the network. To identify the community structures, we used the Louvain algorithm 410 (Blondel et al., 2008). The identiﬁcation of community structures in graph is useful, 411 because the nodes in the same community are more likely to have other properties in 412 common (Danon et al., 2005). It would therefore be interesting to see if differences can be 413 observed between the prevalence of communities between the classic and modern novels. 414 7. Connected components are the number of distinct graph compartments. Manuscript to be reviewed If only a single edge goes out from a subgraph to the 422 12/26 put. Sci. reviewing PDF \| (CS-2018:08:30671:2:0:NEW 7 Mar 2019) Contrarily, a low value – e.g. in 1984 – indicates that the characters only interact with a 377 small number of other characters. 378 2. Average Weighted Degree is fairly similar to the average degree, but especially in the 379 sense of social networks, a distinction must be made. It differs in the sense that the 380 weighted degree takes into account the weight of each of the connecting edges. Whereas a 381 character in our social network could have a high degree – indicating a high level of social 382 activity – if the weights of all those connected edges are relatively small, this suggests 383 only superﬁcial contact. Conversely, while the degree of a character could be low – e.g. 384 the character is only connected to two other characters – the two edges could have very 385 large weights, indicating a deep social connection between the characters. Newman (2006) 386 underlines the importance of this distinction in his work on scientiﬁc collaborations. To 387 continue the examples of Ulysses and 1984; while their average degrees are vastly different 388 (with Ulysses being the highest of its class and 1984 the lowest), their average weighted 389 degrees are comparable. 390 3. Average Path Length is the mean of all the possible shortest paths between each node 391 in the network; also known as the geodesic distance. If there is no path connecting two 392 nodes, this distance is inﬁnite and the two nodes are part of different graph components 393 (see item 7, Connected Components on the next page). The shortest path between two 394 nodes can be found by using Dijkstra’s algorithm (Dijkstra, 1959). The path length is 395 typically an indication of how efﬁciently information is relayed through the network. A 396 network with a low path length would indicate that the people in the network can reach 397 each other through a relatively small number of steps. 398 4. Network Diameter is the longest possible distance between two nodes in the network. It 399 is in essence the longest, shortest path that can be found between any two nodes in the 400 network, and is indicative of the linear size of the network (Wasserman and Faust, 1994). 401 5. Manuscript to be reviewed e.g. in Ulysses – indicates that the characters interact with many different other characters. Contrarily, a low value – e.g. in 1984 – indicates that the characters only interact with a small number of other characters. e.g. in Ulysses – indicates that the characters interact with many different other characters. Contrarily, a low value – e.g. in 1984 – indicates that the characters only interact with a small number of other characters. e.g. in Ulysses – indicates that the characters interact with many different other characters. Contrarily, a low value – e.g. in 1984 – indicates that the characters only interact with a small number of other characters. e.g. in Ulysses – indicates that the characters interact with many different other characters. 376 Contrarily, a low value – e.g. in 1984 – indicates that the characters only interact with a 377 small number of other characters. 378 2. Average Weighted Degree is fairly similar to the average degree, but especially in the 379 sense of social networks, a distinction must be made. It differs in the sense that the 380 weighted degree takes into account the weight of each of the connecting edges. Whereas a 381 character in our social network could have a high degree – indicating a high level of social 382 activity – if the weights of all those connected edges are relatively small, this suggests 383 only superﬁcial contact. Conversely, while the degree of a character could be low – e.g. 384 the character is only connected to two other characters – the two edges could have very 385 large weights, indicating a deep social connection between the characters. Newman (2006) 386 underlines the importance of this distinction in his work on scientiﬁc collaborations. To 387 continue the examples of Ulysses and 1984; while their average degrees are vastly different 388 (with Ulysses being the highest of its class and 1984 the lowest), their average weighted 389 degrees are comparable. 390 3. Average Path Length is the mean of all the possible shortest paths between each node 391 in the network; also known as the geodesic distance. If there is no path connecting two 392 nodes, this distance is inﬁnite and the two nodes are part of different graph components 393 (see item 7, Connected Components on the next page). The shortest path between two 394 nodes can be found by using Dijkstra’s algorithm (Dijkstra, 1959). Manuscript to be reviewed The path length is 395 typically an indication of how efﬁciently information is relayed through the network. A 396 network with a low path length would indicate that the people in the network can reach 397 each other through a relatively small number of steps. 398 4. Network Diameter is the longest possible distance between two nodes in the network. It 399 is in essence the longest, shortest path that can be found between any two nodes in the 400 network, and is indicative of the linear size of the network (Wasserman and Faust, 1994). 401 5. Graph density is the fraction of edges compared to the total number of possible edges. 402 It thus indicates how complete the network is, where completeness would constitute all 403 nodes being directly connected by an edge. This is often used in social network analysis 404 to represent how closely the participants of the network are connected (Scott, 2012). 405 6. Modularity is used to represent community structure. The modularity of a network is 406 “...the number of edges falling within groups minus the expected number in an equivalent 407 network with edges placed at random” (Newman, 2006). Newman shows modularity can 408 be used as an optimisation metric to approximate the number of community structures 409 found in the network. To identify the community structures, we used the Louvain algorithm 410 (Blondel et al., 2008). The identiﬁcation of community structures in graph is useful, 411 because the nodes in the same community are more likely to have other properties in 412 common (Danon et al., 2005). It would therefore be interesting to see if differences can be 413 observed between the prevalence of communities between the classic and modern novels. 414 7. Connected components are the number of distinct graph compartments. That is, a graph 415 component is a subgraph in which any two vertices are connected to each other by paths, 416 and which is connected to no additional vertices in the supergraph. In other words, it 417 is not possible to traverse from one component to another. In most social communities, 418 one ‘giant component’ can typically be identiﬁed, which contains the majority of all 419 vertices (Kumar et al., 2010). A higher number of connected components would indicate 420 a higher number of isolated communities. This is different from modularity in the sense 421 that components are more strict. Manuscript to be reviewed Whereas a character in our social network could have a high degree – indicating a high level of social activity – if the weights of all those connected edges are relatively small, this suggests only superﬁcial contact. Conversely, while the degree of a character could be low – e.g. the character is only connected to two other characters – the two edges could have very large weights, indicating a deep social connection between the characters. Newman (2006) underlines the importance of this distinction in his work on scientiﬁc collaborations. To continue the examples of Ulysses and 1984; while their average degrees are vastly different (with Ulysses being the highest of its class and 1984 the lowest), their average weighted degrees are comparable. 3. Average Path Length is the mean of all the possible shortest paths between each node 391 in the network; also known as the geodesic distance. If there is no path connecting two 392 nodes, this distance is inﬁnite and the two nodes are part of different graph components 393 (see item 7, Connected Components on the next page). The shortest path between two 394 nodes can be found by using Dijkstra’s algorithm (Dijkstra, 1959). The path length is 395 typically an indication of how efﬁciently information is relayed through the network. A 396 network with a low path length would indicate that the people in the network can reach 397 each other through a relatively small number of steps. 398 3. Average Path Length is the mean of all the possible shortest paths between each node 391 in the network; also known as the geodesic distance. If there is no path connecting two 392 nodes, this distance is inﬁnite and the two nodes are part of different graph components 393 (see item 7, Connected Components on the next page). The shortest path between two 394 nodes can be found by using Dijkstra’s algorithm (Dijkstra, 1959). The path length is 395 typically an indication of how efﬁciently information is relayed through the network. A 396 network with a low path length would indicate that the people in the network can reach 397 each other through a relatively small number of steps. 398 4. Network Diameter is the longest possible distance between two nodes in the network. Manuscript to be reviewed That is, a graph 415 component is a subgraph in which any two vertices are connected to each other by paths, 416 and which is connected to no additional vertices in the supergraph. In other words, it 417 is not possible to traverse from one component to another. In most social communities, 418 one ‘giant component’ can typically be identiﬁed, which contains the majority of all 419 vertices (Kumar et al., 2010). A higher number of connected components would indicate 420 a higher number of isolated communities. This is different from modularity in the sense 421 that components are more strict. If only a single edge goes out from a subgraph to the 422 12/26 PeerJ Comput. Sci. reviewing PDF \| (CS-2018:08:30671:2:0:NEW 7 Mar 2019) 5.3 Results of Network Analysis 431 While we can observe large variation overall, there is no clear difference between the two 439 classes, i.e. between classic and modern novels. None of the evaluated network features differ 440 signiﬁcantly between these classes. Graph density is the feature that comes closest to being 441 signiﬁcant (p = 0.09), with our classic novels on average exhibiting denser networks than the 442 modern ones. 443 In order to better interpret these values, and in order to ﬁnd out whether this variance in 444 network features is by itself a characteristic property of social networks exposed in novels, or 445 whether this is true for social networks in general, we need a point for comparison. For that 446 purpose, we compare our network results to metrics that have been reported for other social 447 network in the literature. Table 5 shows ten such networks for comparison, including three small 448 networks on karate club members, football players, and email users (Telesford et al., 2011), three 449 medium-sized networks of mathematicians, a larger group of email users, and actors (Boccaletti 450 et al., 2006), and four large networks of online platforms (Mislove et al., 2007). 451 We can see that social networks reported elsewhere exhibit a wide variation as well, showing 452 (unsurprisingly) an even much wider range for the network size, with the reported online social 453 networks reaching millions of nodes. Our networks from novels are on the lower end of the size 454 range, with the smallest ones being smaller than the smallest network of our comparison set 455 (Karate). This directly explains why the path lengths are also on the lower end of the range, but 456 with a considerable overlap. With respect to the average degree, our novel networks are covered 457 by the range given by these comparison networks, with even the outliers of our dataset being 458 less extreme than the most extreme cases of the comparison networks. The same holds for the 459 clustering coefﬁcient, except for the outlier for a very small network with a clustering coefﬁcient 460 of 0 (Alice in Wonderland). In summary, we can say that social networks from novels appear 461 to be no different than social networks in general in showing a high variation in basically all 462 network features across different networks. 5.3 Results of Network Analysis 431 While networks differ much individually, there is no 463 signiﬁcant fundamental difference between classic and modern novels. 464 Manuscript to be reviewed supergraph, it is no longer considered a separate component. Modularity attempts to identify those communities that are basically ‘almost’ separate components. supergraph, it is no longer considered a separate component. Modularity attempts to 423 identify those communities that are basically ‘almost’ separate components. 424 8. Average clustering coefﬁcient is the mean of all clustering coefﬁcients. The clustering 425 coefﬁcient of a node can perhaps best be described as ‘all-my-neighbours-know-each- 426 other’. Social networks with a high clustering coefﬁcient (and low average path length) 427 may exhibit small world13 properties (Watts and Strogatz, 1998). The small world 428 phenomenon was originally described by Stanley Milgram in his perennial work on social 429 networks (Travers and Milgram, 1967). 430 Manuscript to be reviewed The character Dany does not truly interact 479 with the characters of this main cluster, but is rather name-dropped in conversations between 480 characters in that cluster. Her character ‘co-occurs’ with the characters that drop her name and 481 edges are created to represent that. 482 the largest network of the modern novels, which is A Game of Thrones. A visualisation of that 468 network is shown in Figure 1. We see that it is a quite dense network with many connections 469 (it has the highest average degree of all modern novels; see Table A5) and a complex structure. 470 Despite this complexity, the relationship between the main characters of this novel can easily 471 be identiﬁed from this visualisation, and one can clearly identify social clusters. Such informal 472 visual explorations should then of course be substantiated with formal analyses, i.e. by ranking 473 the edges of the network by their weights and by applying a clustering algorithm in the case of 474 the two given examples. As the readers of this novel might have alraedy spotted, Dany resides in 475 a completely different part of the world in this novel, which explains her distance from rest of 476 the network. Moreover, in A Game of Thrones, this character does not at any point physically 477 interact with any of the characters in the larger cluster. This highlights a caveat of the use of 478 co-occurrence networks over conversational networks. The character Dany does not truly interact 479 with the characters of this main cluster, but is rather name-dropped in conversations between 480 characters in that cluster. Her character ‘co-occurs’ with the characters that drop her name and 481 edges are created to represent that. 482 To stick with the example of Dany, we can also identify two seemingly separate characters, 483 Dany and Daenerys Targaryen in Figure 1. These names actually refer to the same entity. 484 As mentioned in Section 2, this issue may be addressed by creating a list of aliases for each 485 character. Some online sources exist that can help expedite this process, but we would argue these 486 sources are not applicable to our modern novels. Whereas 19th century novels typically have 487 characters with more traditional names such as Elizabeth Bennet, modern fantasy novels have 488 unconventional names such as Daenerys Targaryan. 14MetaCPAN is a search engine for Perl code and documentation: https://metacpan.org/source/ BRIANL/Lingua-EN-Nickname-1.14/nicknames.txt Last Retrieved: 30 October 2017 Manuscript to be reviewed average clustering average network via nodes degree coefﬁcient path length Karate Telesford et al. (2011) 35† 4.46 0.55 2.41† Football Telesford et al. (2011) 115 10.66 0.40 2.51 E-mail Telesford et al. (2011) 1 133 9.62 0.22 3.60 Math1999 Boccaletti et al. (2006) 58 516 5.00 0.15 8.46⋄ e-mail Boccaletti et al. (2006) 59 812 2.88 0.03† 4.95 Actors Boccaletti et al. (2006) 225 226 61.00⋄ 0.79⋄ 3.65 YouTube Mislove et al. (2007) 1 157 827 1.81 0.14 5.10 Flickr Mislove et al. (2007) 1 846 198 1.76 0.31 5.67 Orkut Mislove et al. (2007) 3 072 441 1.50† 0.17 4.25 LiveJournal Mislove et al. (2007) 5 284 457⋄ 1.62 0.33 5.88 maximum 522 15.77 0.81 3.33 classic novels mean 106 6.14 0.60 2.49 minimum 10 1.66 0.00 1.53 maximum 314 10.50 0.75 4.06 modern novels mean 99 5.50 0.56 2.68 minimum 27 3.00 0.42 2.22 Table 5. Comparison to other social networks. The highest scores in each column are highlighted with a ⋄and the lowest scores with a † for the comparison networks. Table 5. Comparison to other social networks. The highest scores in each column are highlighted with a ⋄and the lowest scores with a † for the comparison networks. the largest network of the modern novels, which is A Game of Thrones. A visualisation of that 468 network is shown in Figure 1. We see that it is a quite dense network with many connections 469 (it has the highest average degree of all modern novels; see Table A5) and a complex structure. 470 Despite this complexity, the relationship between the main characters of this novel can easily 471 be identiﬁed from this visualisation, and one can clearly identify social clusters. Such informal 472 visual explorations should then of course be substantiated with formal analyses, i.e. by ranking 473 the edges of the network by their weights and by applying a clustering algorithm in the case of 474 the two given examples. As the readers of this novel might have alraedy spotted, Dany resides in 475 a completely different part of the world in this novel, which explains her distance from rest of 476 the network. Moreover, in A Game of Thrones, this character does not at any point physically 477 interact with any of the characters in the larger cluster. This highlights a caveat of the use of 478 co-occurrence networks over conversational networks. 5.4 Network Exploration 465 5.4 Network Exploration 465 In addition to the formal analysis above, we show here a more informal exploration of one of the 466 networks in order to give a more intuitive explanation of our results. For that purpose, we selected 467 13https://en.wikipedia.org/wiki/Small-world_experiment 13/26 PeerJ Comput. Sci. reviewing PDF \| (CS-2018:08:30671:2:0:NEW 7 Mar 2019) Manuscript to be reviewed External sources such as on metaCPAN14 489 can help to connect Elizabeth to nicknames such as Lizzy, but there are no sources that can do 490 this for Daenerys and Dany. Even if there was such a source, the question remains whether if it 491 14/26 PeerJ Comput. Sci. Manuscript to be reviewed reviewing PDF \| (CS-2018:08:30671:2:0:NEW 7 Mar 2019) Computer Science Manuscript to be reviewed Joseth Joseth Harys Ser Harys Ser Brackens Brackens Lord Robb Lord Robb Cohollo Cohollo Piper Ser Marq Piper Ser Marq Hullen Hullen Tommen Prince Tommen Prince Trant Meryn Ser Trant Meryn Ser Hightower Ser Gerold Hightower Ser Gerold Lord Vance Lord Vance Dareon Dareon Arya Horseface Arya Horseface Lord Hornwood Lord Hornwood Robert Baratheon Robert Baratheon Cotter Pyke Cotter Pyke Caron Lord Bryce Caron Lord Bryce Elia Elia Stark Sansa Stark Sansa Mott Master Mott Master Aggo Aggo Rodrik Cassel Ser Rodrik Cassel Ser Thoros Thoros Lyanna Lyanna Ser Donnel Ser Donnel Nymeria Nymeria Sherrer Sherrer Tarly Sam Tarly Sam Jhiqui Jhiqui Alyssa Arryn Alyssa Arryn Jyck Jyck Yoren Yoren Frey Lady Frey Lady Rayder Mance Rayder Mance Pyp Pyp Manderly Ser Wylis Manderly Ser Wylis Chella Chella Jhogo Jhogo Chiggen Chiggen Dontos Ser Dontos Ser Bronze Yohn Royce Bronze Yohn Royce Chett Chett Visenya Visenya Cassel Jory Cassel Jory Grenn Grenn Lord Slynt Lord Slynt Hal Mollen Hal Mollen Ned Stark Ned Stark Stark Brandon Stark Brandon Mikken Mikken Greyjoy Balon Greyjoy Balon Morrec Morrec Tomard Tomard Danwell Danwell Mya Stone Mya Stone Heartsbane Heartsbane Jaremy Ser Rykker Jaremy Ser Rykker Egen Ser Vardis Egen Ser Vardis Godwyn Godwyn Castle Black Castle Black Lord Dondarrion Beric Lord Dondarrion Beric Brynden Blackfish Brynden Blackfish Maester Luwin Maester Luwin Maester Aemon Maester Aemon Craven Craven Mord Mord Matt Matt Clegane Sandor Clegane Sandor Shae Shae Harrenhal Harrenhal Lord Nestor Royce Lord Nestor Royce Pentoshi Pentoshi Toad Toad Porther Porther Lord lord Tyrion Lord lord Tyrion Mago Mago Vargo Hoat Vargo Hoat Rickon Rickon Eroeh Eroeh Lord Arryn Lord Arryn Quaro Quaro Lord Piper Lord Piper Lysa Lady Arryn Lysa Lady Arryn Braavosi Braavosi Matthar Matthar Bracken Jonos Lord Bracken Jonos Lord Lord Steward Lord Steward Manderly Ser Wendel Manderly Ser Wendel Tregar Tregar Timett Timett Santagar Ser Aron Santagar Ser Aron Barristan Selmy Ser Barristan Selmy Ser Payne Ser Ilyn Payne Ser Ilyn Boy Moon Boy Moon Perwyn Ser Perwyn Ser Lord Mallister Jason Lord Mallister Jason Samwell Tarly Samwell Tarly Poole Vayon Poole Vayon Jofftey Jofftey Beth Beth Gared Gared Moreo Moreo Whent Oswell Ser Whent Oswell Ser Forel Syrio Forel Syrio Dany Dany Kurleket Kurleket Greatjon Greatjon Lannister Tyrion Lannister Tyrion Ser Moore Mandon Ser Moore Mandon Lord Wyman Lord Wyman Hardin Hardin Dorne Dorne Lord Jon Lord Jon Stannis Baratheon Lord Stannis Baratheon Lord Jeren Jeren Ulf Ulf Fat Tom Fat Tom Jaime Ser Lannister Jaime Ser Lannister Ogo Khal Ogo Khal Moat Cailin Moat Cailin Cassel Martyn Cassel Martyn Alliser Ser Thorne Alliser Ser Thorne Farlen Farlen Lord Robert Lord Robert Lys Lys Lord Rowan Lord Rowan Jeyne Poole Jeyne Poole Tyroshi Tyroshi Conn Conn Maegor Maegor Haggo Haggo Vale Vale Edmure Ser Tully Edmure Ser Tully Highgarden Highgarden Gage Gage Hill Horn Hill Horn Coratt Coratt Heddle Masha Heddle Masha Maege Mormont Maege Mormont Lady Catelyn Stark Lady Catelyn Stark Cayn Cayn Ben Stark Ben Stark Marillion Marillion Lady Mormont Lady Mormont King King Robert Arryn Robert Arryn Gendry Gendry Xho Jalabhar Xho Jalabhar Khaleesi Khaleesi Lord Baratheon Renly Lord Baratheon Renly Alyn Alyn Lord Baelish Petyr Lord Baelish Petyr Lady Sansa Lady Sansa Mirri Maz Duur Mirri Maz Duur Lord Frey Walder Lord Frey Walder Father Father Ser Addam Marbrand Ser Addam Marbrand Hugh Ser Hugh Ser Old Nan Old Nan Lharys Lharys Jacks Jacks Rhaegar Targaryen Rhaegar Targaryen Joffrey Prince Joffrey Prince Boros Ser Blount Boros Ser Blount Vance Karyl Vance Karyl Joff Joff Arthur Dayne Ser Arthur Dayne Ser Mordane Septa Mordane Septa Ser Tallhart Helman Ser Tallhart Helman Lord Tytos Blackwood Lord Tytos Blackwood Tywin Lord Lannister Tywin Lord Lannister Yi Ti Yi Ti Jen Ben Jen Ben Halder Halder Shagga Shagga Arryn Jon Arryn Jon Dolf Dolf Baelor Baelor Gunthor Gunthor Tyrell Ser Loras Tyrell Ser Loras Lannister Ser Kevan Lannister Ser Kevan Stevron Frey Ser Stevron Frey Ser Tanda Lady Tanda Lady Raymun Darry Ser Raymun Darry Ser Shaggydog Shaggydog Lord Tully Hoster Lord Tully Hoster Arys Ser Arys Ser Flowers Jafer Flowers Jafer Willis Ser Wode Willis Ser Wode Dawn Dawn Heward Heward Willem Darry Willem Darry Fogo Fogo Malleon Malleon Will Will Rhaggat Khal Rhaggat Khal Mycah Mycah Jaggot Jaggot Flement Brax Ser Flement Brax Ser Umar Umar Robar Ser Robar Ser Naerys Naerys Cheyk Cheyk Tobho Mott Tobho Mott Benjen Stark Benjen Stark Mohor Mohor Littlefinger Littlefinger Lord Tyrell Lord Tyrell Brynden Ser Tully Brynden Ser Tully Hali Hali Myrcella Myrcella Stiv Stiv Othell Yarwyck Othell Yarwyck Greyjoy Theon Greyjoy Theon Irri Irri Maester Pycelle Maester Pycelle Grey Wind Grey Wind Quorin Halfhand Quorin Halfhand Jaehaerys Jaehaerys Lord Cerwyn Lord Cerwyn Clydas Clydas Rakharo Rakharo Dywen Dywen Magister Illyrio Magister Illyrio Torrhen Torrhen Aegon Targaryen Aegon Targaryen Bowen Marsh Bowen Marsh Daryn Hornwood Daryn Hornwood Riverrun Riverrun Clegane Gregor Ser Clegane Gregor Ser Snow Jon Snow Jon Rast Rast Aerys Targaryen Aerys Targaryen Drogo Khal Drogo Khal Viserys Targaryen Viserys Targaryen Qotho Qotho Whent Lady Whent Lady Hobb Three-Finger Hobb Three-Finger Dothraki Dothraki Royce Ser Andar Royce Ser Andar Karyl Ser Karyl Ser Hake Hake Lance Lance Hosteen Hosteen Mace Tyrell Mace Tyrell Lord Hunter Lord Hunter Hallis Mollen Hallis Mollen Dothrak Vaes Dothrak Vaes Daeren Targaryen Daeren Targaryen Lord Lefford Lord Lefford Volantis Volantis Glover Galbart Glover Galbart Rhaego Rhaego Bolton Roose Bolton Roose Catelyn Tully Catelyn Tully Lannister Cersei Lannister Cersei Joss Joss Waymar Ser Royce Waymar Ser Royce Lothor Brune Lothor Brune Lord Tarly Randyll Lord Tarly Randyll Derik Lord Derik Lord Jared Frey Ser Jared Frey Ser Tyrosh Tyrosh Ser Swann Balon Ser Swann Balon Lord Varys Lord Varys Bran Bran Harrion Karstark Harrion Karstark Jhaqo Jhaqo Doreah Doreah Haider Haider bush bush Janos Slynt Janos Slynt Brothers Moon Brothers Moon Arya Stark Arya Stark Daenerys Targaryen Daenerys Targaryen Corbray Lyn Ser Corbray Lyn Ser Hodor Hodor Robett Glover Robett Glover Harwin Harwin Arbor Arbor Lord Karstark Rickard Lord Karstark Rickard Bronn Bronn Hobber Ser Hobber Ser Khal Jommo Khal Jommo Horas Ser Horas Ser Lord Mormont Lord Mormont Desmond Desmond Starks Starks Robb Stark Robb Stark Lord Lord Albett Albett Noye Donal Noye Donal Jorah Ser Mormont Jorah Ser Mormont Figure 1. Manuscript to be reviewed Social network of G.R.R. Martin’s A Game of Thrones Manuscript to be review omputer Science Figure 1. Social network of G.R.R. Martin’s A Game of Thrones is desirable to collapse those characters. Especially in A Game of Thrones, the mentions of Dany 492 and Daenerys Targaryen occur in entirely different contexts. Whereas references to Dany occur 493 in an environment that is largely friendly towards her; her formal name of Daenerys Targaryen 494 is mostly used by her enemies (in her absence). Rather than simply collapsing the two characters 495 as one, it might be useful to be able to retain that distinction. This is a design choice that will 496 depend on the type of research question one wants to answer by analysing the social networks. 497 Manuscript to be reviewed Original Adjusted Blue Richard Croaker Thomas Curly Daniel Dancing Edward Mercy Charles One-Eye Timothy Silent James Walleye William Table 6. Unidentiﬁed names in The Black Company replaced by generic English names. Table 6. Unidentiﬁed names in The Black Company replaced by generic English names shows all names affected for The Black Company. 510 Secondly, we noticed that persons with special characters in their names can prove difﬁcult 511 to retrieve. For example, names such as d’Artagnan in The Three Musketeers or Shai’Tan in 512 The Wheel of Time were hard to recognise for the systems. To test this, we replaced all names 513 in our corpus such as d’Artagnan or Shai’Tan with Dartagnan and Shaitan. By applying these 514 transformations to our corpus, we found that the performances could be improved, uncovering 515 some of the issues that plague named entity recognition. As can be observed in Figure 2, not all 516 of the novels were affected by these transformations. Out of the 40 novels used in this study, 517 we were able to improve the performance for 14. While the issue of the apostrophed afﬁx was 518 not as recurrent in our corpus as the real-word names, its impact on performance is troublesome 519 nonetheless. Clearly, two novels are more affected by these transformations than the others, 520 namely: The Black Company and the The Three Musketeers. To further sketch these issues, we 521 delve a bit deeper into these two speciﬁc novels. 522 These name transformations show that the real-word names and names with special characters 523 were indeed problematic and put forth a problem for future studies to tackle. As illustrated 524 by Figure 2, the aforementioned issues are also present in the classic novels typically used by 525 related works (such as The Three Musketeers). This begs the question of the scope of these 526 problems. To the best of our knowledge, similar works have not identiﬁed this issue to affect their 527 performances, but we have shown that with a relatively simple workaround, the performance can 528 be drastically improved. It would thus be interesting to evaluate how much these studies suffer 529 from the same issue. Lastly, as manually replacing names is clearly far from ideal, we would 530 like to encourage future work to ﬁnd a more robust approach to resolve this issue. 531 The Black Company 532 This fantasy novel describes the dealings of an elite mercenary unit – The Black Company – and 533 its members, all of which go by code names such as the ones in Table 6. With a preliminary F1 534 score of 06.85 (see Table A4), The Black Company did not do very well. We found this book 535 had the highest percentage of unidentiﬁed characters of our collection. Out of the 14 characters 536 found by our annotators, only 5 were identiﬁed by the pipeline. Interestingly enough, 8 out of the 537 9 unidentiﬁed characters in this novel have names that correspond to regular nouns. By applying 538 our name transformation alone, the F1 score rose from 06.85 to the highest in our collection to 539 90. 540 6 DISCUSSION AND PERFORMANCE BOOSTING OPTIONS 498 In analysing the output of the different NER systems, we found that some types of characters 499 were particularly difﬁcult to recognise. Firstly, we found a number of unidentiﬁed names that 500 are so called word names (i.e. terms that also occur in dictionaries, for example to denote nouns 501 such as Grace or Rebel). We suspected that this might hinder the named entity recognition, 502 which is why we collected all such names in our corpus in Table A1 on page 22, and highlighted 503 such word names with a †. This table shows that approximately 50% of all unidentiﬁed names 504 in our entire corpus consist at least partially of a word name, which underpins that this issue is 505 potentially widely spread. In order to verify this, we replaced all potentially problematic names 506 in the source material by generic English names. We made sure not to add names that were 507 already assigned to other characters in the novel, and we ensured that these names were not also 508 regular nouns. An example of these changed character names can be found in Table 6, which 509 15/26 PeerJ Comput. Sci. reviewing PDF \| (CS-2018:08:30671:2:0:NEW 7 Mar 2019) Computer Science Computer Science Manuscript to be reviewed 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Assassin's Apprentice Gardens of the Moon The Black Company The Blade Itself The Colour of Magic The Gunslinger The Lies of Locke Lamora The Name of the Wind The Wheel of Time Alice in Wonderland Brave New World The Call of the Wild The Three Musketeers Vanity Fair Performance in F1-score Default Apostrophe Real Names Modern Classic Figure 2. Effect of transformations on all affected classic and modern novels in F1score in using the BookNLP pipeline (includes co-reference resolution) Assassin's Apprentice Gardens of the Moon Figure 2. Effect of transformations on all affected classic and modern novels in F1score in using the BookNLP pipeline (includes co-reference resolution) Three Musketeers performs the second worst of our corpus, and the worst in its class. By 544 simply replacing names such as d’Artagnan with Dartagnan the F1 score rose from 13.91 to 545 53, suggesting that the apostrophed name was indeed the main issue. To visualise this, we have 546 included ﬁgures of both The Three Musketeer networks – before and after the ﬁx – in Figures 3 547 and 4. As can be observed in Figure 3, the main character of the novel is hardly represented in 548 this network, which is not indicative of the actual story. The importance of resolving the issue of 549 apostrophed named is made clear in Figure 4, where the main character is properly represented. 550 The Three Musketeers 541 This classic piece recounts the adventures of a young man named d’Artagnan, after he leaves 542 home to join the Musketeers of the Guard. With an F1 score of 13.91 (see Table A4), The 543 home to join the Musketeers of the Guard. With an F1 score of 13.91 (see Table A4), The 543 16/26 PeerJ Comput. Sci. reviewing PDF \| (CS-2018:08:30671:2:0:NEW 7 Mar 2019) 7 CONCLUSION & FUTURE WORK 551 Tremouille... de M. Tremouille... Norman Norman de M. Bassompier... de M. Bassompier... IV Henry IV Henry Villiers George Villiers George Bearnais Bearnais I Charles I Charles Pierre Pierre monsieur Aramis ... monsieur Aramis ... Jussac Jussac Denis Denis Gascons Gascons Coquenard Madame Coquenard Madame Crevecoeur Crevecoeur Picard Picard pope Pope pope Pope de M. Treville de M. Treville de Marie Medicis de Marie Medicis Lorraine Lorraine #N/A #N/A Cardinal Monsieur Cardinal Monsieur Fourreau Fourreau Bicarat Bicarat Marie Michon MAR... Marie Michon MAR... Lord de Winter Lord de Winter Milady de De Win... Milady de De Win... M. dâArtagnan M. dâArtagnan Duke Duke Messieurs Porthos Messieurs Porthos Kitty Kitty Figure 3. Social network of The Three Musketeers without adjustment for apostrophed names. Manuscript to be reviewed mputer Science gure 3. Social network of The Three Musketeers without adjustment for apostrophed names To answer our primary research question, we evaluated four state-of-the-art named entity 560 recognition systems on 20 classic and 20 modern science ﬁction/fantasy novels. In our study, 561 we found no signiﬁcant difference in performance of the named entity recognisers on classic 562 novels and modern novels. We did ﬁnd that novels written in 3rd person perspective perform 563 signiﬁcantly better than those written in 1st person, which is in line with ﬁndings in related 564 studies. In addition, we observed a large amount of variance within each class, even despite 565 our limitation for the modern novels to the fantasy/science ﬁction genre. We also identiﬁed 566 some recurring problems that hindered named entity recognition. We delved deeper into two 567 such problematic novels, and ﬁnd two main issues that overarch both classes. Firstly, we found 568 that word names such as Mercy are more difﬁcult to identify to the systems. We showed that 569 replacing problematic word names by generic placeholders can increase performance on affected 570 novels. Secondly, we found that apostrophed names such as d’Artagnan also prove difﬁcult 571 to automatically identify. With fairly simple methods that capture some cultural background 572 knowledge, we circumvented the above two issues to drastically increase the performance of the 573 used pipeline. To the best of our knowledge, none of the related studies discussed in Section 2 574 acknowledge the presence of these issues. 7 CONCLUSION & FUTURE WORK 551 In this study, we set out to close a gap in the literature when it comes to the evaluation of named 552 entity recognition for the creation of social networks from ﬁction literature. In our exploration 553 of related work, we found no other studies that attempt to compare networks from classic and 554 modern ﬁction. To ﬁll this gap, we attempted to answer the following two research questions: 555 • To what extent are off-the-shelf named entity recognition tools suitable for identifying 556 ﬁctional characters in novels? 557 • To what extent are off-the-shelf named entity recognition tools suitable for identifying 556 ﬁctional characters in novels? 557 • Which differences or similarities can be discovered between social networks extracted for 558 different novels? 559 17/26 PeerJ Comput. Sci. reviewing PDF \| (CS-2018:08:30671:2:0:NEW 7 Mar 2019) Computer Science Manuscript to be reviewed Chalais Chalais M. Bonacieux M. Bonacieux de M. Busigny de M. Busigny Houdiniere La Houdiniere La John Felton John Felton Bois-Tracy de Ma... Bois-Tracy de Ma... de M. Schomberg de M. Schomberg Lubin Lubin Porthos Monsieur Porthos Monsieur la Harpe de Rue la Harpe de Rue Rochellais Rochellais Richelieu de Richelieu de de Busigny Monsi... de Busigny Monsi... Milady Clarik Milady Clarik Rochefort Rochefort Grimaud Monsieur Grimaud Monsieur M. Coquenard M. Coquenard de Treville Mons... de Treville Mons... Mr. Felton Mr. Felton Montague Montague dâArtagnan Mon... dâArtagnan Mon... Buckingham de Mo... Buckingham de Mo... de Monsieur Voit... de Monsieur Voit... Monsieur Bernajo... Monsieur Bernajo... III Henry III Henry Monsieur Dessess... Monsieur Dessess... de Chevreuse Mad... de Chevreuse Mad... Donna Estafania Donna Estafania Lord Duke Lord Duke Quixote Don Quixote Don Lorme de Marion Lorme de Marion de Cahusac Monsi... de Cahusac Monsi... Bazin Bazin Chevalier Monsie... Chevalier Monsie... Musketeer Musketeer Constance Bonaci... Constance Bonaci... M. Dessessart M. Dessessart Germain Germain de M. Cavois de M. Cavois Judith Judith Gascon Gascon Mousqueton Mousqueton Monsieur Athos Monsieur Athos Duke Monsieur Duke Monsieur Charlotte Backson Charlotte Backson Bethune Bethune Planchet Monsieur Planchet Monsieur Louis XIII Louis XIII Bonacieux Madame Bonacieux Madame de Benserade Mon... de Benserade Mon... Gervais Gervais Meung Meung Chesnaye La Chesnaye La Bonacieux Monsie... Bonacieux Monsie... Chrysostom Chrysostom Wardes de De M. Wardes de De M. Coquenard Monsie... Coquenard Monsie... Patrick Patrick Berry Berry Mande Mande Laporte M. Laporte M. de M. Laffemas de M. Laffemas Laporte Monsieur Laporte Monsieur Louis XIV Louis XIV Anne Anne de M. 7 CONCLUSION & FUTURE WORK 551 We would thus like to encourage future work to 575 evaluate the impact of these two issues on existing studies, and call to develop a more robust 576 approach to tackle them in future studies. 577 To answer our secondary research question, we created social networks for each of the 578 novels in our collection and calculated several networks features with which we compared the 579 two classes. As with the named entity recognition experiments, no major differences were 580 found between the classic and modern novels. Again, we found that the distribution of network 581 measures within a class was subject to high variance, which holds for our collection of both 582 classic and modern novels. We therefore recommend that future work focuses on determining 583 particular characteristics that can inﬂuence these analyses ﬁrst and then perform a comparative 584 analysis between subsets to see if this similarity between classes holds when the variance is 585 reduced. Future studies could therefore attempt to compare classic and modern novels in the 586 same genre or narration type (e.g. ﬁrst-person vs third-person perspective). Lastly, different 587 types of networks that for example collapse characters that occur under different names (cf. Dany 588 and Daenerys) as well as dealing with plural pronouns and group membership (e.g. characters 589 18/26 PeerJ Comput. Sci. reviewing PDF \| (CS-2018:08:30671:2:0:NEW 7 Mar 2019) Computer Science Manuscript to be reviewed Chalais Chalais M. Bonacieux M. Bonacieux de M. Busigny de M. Busigny Houdiniere La Houdiniere La John Felton John Felton Bois-Tracy de Ma... Bois-Tracy de Ma... de M. Schomberg de M. Schomberg Lubin Lubin Porthos Monsieur Porthos Monsieur la Harpe de Rue la Harpe de Rue Rochellais Rochellais de Marie Medicis de Marie Medicis de Busigny Monsi... de Busigny Monsi... Milady Clarik Milady Clarik Rochefort Rochefort Grimaud Monsieur Grimaud Monsieur M. Coquenard M. Coquenard de Treville Mons... de Treville Mons... Commissary Monsi... Commissary Monsi... Mr. Felton Mr. Felton Montague Montague Buckingham de Mo... Buckingham de Mo... de Monsieur Voit... de Monsieur Voit... M. Dartagnan M. Dartagnan Monsieur Bernajo... Monsieur Bernajo... III Henry III Henry Monsieur Dessess... Monsieur Dessess... de Chevreuse Mad... de Chevreuse Mad... Donna Estafania Donna Estafania Lord Duke Lord Duke Quixote Don Quixote Don Lorme de Marion Lorme de Marion de Cahusac Monsi... de Cahusac Monsi... Bazin Bazin Chevalier Monsie... Chevalier Monsie... Musketeer Musketeer M. Dessessart M. Dessessart Germain Germain de M. Cavois de M. 7 CONCLUSION & FUTURE WORK 551 Cavois Judith Judith Monsieur Dartagn... Monsieur Dartagn... Gascon Gascon Mousqueton Mousqueton Monsieur Athos Monsieur Athos Duke Monsieur Duke Monsieur Charlotte Backson Charlotte Backson Bethune Bethune Planchet Monsieur Planchet Monsieur Louis XIII Louis XIII Milady de Winter Milady de Winter Bonacieux Madame Bonacieux Madame de Benserade Mon... de Benserade Mon... Gervais Gervais Meung Meung Chesnaye La Chesnaye La Bonacieux Monsie... Bonacieux Monsie... Chrysostom Chrysostom Wardes de De M. Wardes de De M. Coquenard Monsie... Coquenard Monsie... Patrick Patrick Lord de De Winter Lord de De Winter Berry Berry Mande Mande Laporte M. Laporte M. Richelieu de Richelieu de Godeau Godeau Laporte Monsieur Laporte Monsieur Louis XIV Louis XIV Anne Anne de M. Tremouille... de M. Tremouille... Norman Norman de M. Bassompier... de M. Bassompier... IV Henry IV Henry Villiers George Villiers George de M. Laffemas de M. Laffemas Bearnais Bearnais Pierre Pierre monsieur Aramis ... monsieur Aramis ... Jussac Jussac Denis Denis Gascons Gascons Crevecoeur Crevecoeur Picard Picard pope Pope pope Pope de M. Treville de M. Treville de Monsieur Cavo... de Monsieur Cavo... Lorraine Lorraine Dangouleme Duc Dangouleme Duc #N/A #N/A Cardinal Monsieur Cardinal Monsieur Fourreau Fourreau Bicarat Bicarat Marie Michon MAR... Marie Michon MAR... I Charles I Charles Duke Duke Villeroy Villeroy Messieurs Porthos Messieurs Porthos Kitty Kitty Bonacieux Consta... Bonacieux Consta... Figure 4. Social network of The Three Musketeers with adjustment for apostrophed names. Manuscript to be reviewed mputer Science Figure 4. Social network of The Three Musketeers with adjustment for apostrophed names. sometimes mentioned individually and sometimes as part of a group) are currently unsolved 590 problems for language technology and knowledge representation. 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In Odijk, J. and van Hessen, A., editors, CLARIN in 626 the Low Countries, page 361?370. Ubiquity Press. License: CC-BY 4.0. 627 Dijkstra, E. W. (1959). A note on two problems in connexion with graphs. Numerische 628 mathematik, 1(1):269–271. 629 Elson, D. K., Dames, N., and McKeown, K. R. (2010). Extracting social networks from literary 630 ﬁction. In Proceedings of the 48th annual meeting of the association for computational 631 linguistics, pages 138–147. Association for Computational Linguistics. 632 Elson, D. K. and McKeown, K. (2010). Automatic attribution of quoted speech in literary 633 narrative. In The Twenty-Fourth AAAI Conference on Artiﬁcial Intelligence (AAAI-2010). 634 Association for the Advancement of Artiﬁcial Intelligence. 635 Fernandez, M., Peterson, M., and Ulmer, B. (2015). Extracting social network from literature to 636 predict antagonist and protagonist. Technical report, Stanford University. 637 Finkel, J. R., Grenager, T., and Manning, C. (2005). 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Social network analysis of 594 alice in wonderland. In Proceedings of the NAACL-HLT 2012 Workshop on computational 595 linguistics for literature, pages 88–96. 596 Agarwal, A., Kotalwar, A., and Rambow, O. (2013). Automatic extraction of social networks 597 from literary text: A case study on alice in wonderland. In Sixth International Joint Conference 598 on Natural Language Processing (IJNLP 2013), pages 1202–1208. 599 Agerri, R. and Rigau, G. (2016). Robust multilingual named entity recognition with shallow 600 semi-supervised features. Artiﬁcial Intelligence, 238:63–82. 601 Akimushkin, C., Amancio, D. R., and Oliveira Jr, O. N. (2017). Text authorship identiﬁed using 602 the dynamics of word co-occurrence networks. PloS one, 12(1):e0170527. 603 Amancio, D. R. (2015). Probing the topological properties of complex networks modeling short 604 written texts. PloS one, 10(2):e0118394. 605 Ardanuy, M. C. and Sporleder, C. (2014). Structure-based clustering of novels. In Proceedings 606 of the EACL Workshop on Computational Linguistics for Literature, pages 31–39. 607 Bamman, D., Underwood, T., and Smith, N. A. (2014). A bayesian mixed effects model 608 of literary character. In Proceedings of The 52nd Annual Meeting of the Association for 609 Computational Linguistics (ACL 2014), pages 370–379. 610 p g p g Biber, D. and Finegan, E. (1989). Drift and the evolution of english style: A history of three 611 genres. Language, 65(3):487 – 517. 612 Blondel, V. D., Guillaume, J.-L., Lambiotte, R., and Lefebvre, E. (2008). Fast unfolding of 613 communities in large networks. Journal of statistical mechanics: theory and experiment, 614 2008(10):P10008. 615 19/26 PeerJ Comput. Sci. reviewing PDF \| (CS-2018:08:30671:2:0:NEW 7 Mar 2019) Manuscript to be reviewed Universal properties of mythological networks. EPL 650 (Europhysics Letters), 99(2):28002. 651 Mislove, A., Marcon, M., Gummadi, K. P., Druschel, P., and Bhattacharjee, B. (2007). Mea- 652 surement and analysis of online social networks. In Proceedings of the 7th ACM SIGCOMM 653 conference on Internet measurement, pages 29–42. ACM. 654 Moretti, F. (2013). Distant reading. Verso Books. 5 Moretti, F. (2013). Distant reading. Verso Books. 655 Newman, M. E. (2006). Modularity and community structure in networks. Proceedings of the 656 national academy of sciences, 103(23):8577–8582. 657 Ratinov, L. and Roth, D. (2009). Design challenges and misconceptions in named entity 658 recognition. In Proceedings of the Thirteenth Conference on Computational Natural Language 659 Learning (CoNLL-2009), pages 147–155, Boulder, Colorado. Association for Computational 660 Linguistics. 661 Sainte-Beuve, C. A. (1910). What is a classic? In Eliot, C. W., editor, Literary and Philosophical 662 E F h G d It li ol me 32 of Th H d l i P F Collier & Son Sainte-Beuve, C. A. (1910). What is a classic? In Eliot, C. W., editor, Literary and Philosophical 662 Essays: French, German and Italian, volume 32 of The Harvard classics. P. F. Collier & Son 663 Sainte-Beuve, C. A. (1910). What is a classic? In Eliot, C. W., editor, Literary and Philosophical 662 Essays: French, German and Italian, volume 32 of The Harvard classics. P. F. Collier & Son 663 Corporation. 664 Corporation. 664 20/26 PeerJ Comput. Sci. reviewing PDF \| (CS-2018:08:30671:2:0:NEW 7 Mar 2019) PeerJ Comput. Sci. reviewing PDF \| (CS-2018:08:30671:2:0:NEW 7 Mar 2019) Table A1. Characters that were not identiﬁed by the system, supplied by the annotators. Characters whose names (partly) consist of a real word – such as ‘Curly’ or ‘Mercy’ – are marked with a †. Checked against http://dictionary.com. Manuscript to be reviewed Scott, J. (2012). Social network analysis. Sage. 665 Telesford, Q. K., Joyce, K. E., Hayasaka, S., Burdette, J. H., and Laurienti, P. J. (2011). The 666 ubiquity of small-world networks. Brain connectivity, 1(5):367–375. 667 Travers, J. and Milgram, S. (1967). The small world problem. Phychology Today, 1:61– 68 Vala, H., Jurgens, D., Piper, A., and Ruths, D. (2015). Mr. bennet, his coachman, and the 669 archbishop walk into a bar but only one of them gets recognized: On the difﬁculty of detecting 670 characters in literary texts. In EMNLP, pages 769–774. 671 van Dalen-Oskam, K., de Does, J., Marx, M., Sijaranamual, I., Depuydt, K., Verheij, B., and Geir- 672 naert, V. (2014). Named entity recognition and resolution for literary studies. Computational 673 Linguistics in the Netherlands Journal, 4:121–136. 674 Linguistics in the Netherlands Journal, 4:121–136. 674 Wasserman, S. and Faust, K. (1994). Social network analysis: Methods and applications, 675 volume 8. Cambridge university press. 676 Watts, D. J. and Strogatz, S. H. (1998). Collective dynamics of small-world-networks. nature, 677 393(6684):440–442. 678 21/26 PeerJ Comput. Sci. reviewing PDF \| (CS-2018:08:30671:2:0:NEW 7 Mar 2019) Table A2. Classic and modern novels included in this study. The short E-book numbers are the catalog entry of novels obtained from Gutenberg. Novels obtained through online purchase are denoted by the longer ISBNs. Manuscript to be reviewed APPENDIX: ADDITIONAL STATISTICS 679 APPENDIX: ADDITIONAL STATISTICS 679 Classic Modern Ada Howard Mrs. Billington Archmage of Ymitury† Manie Algy Joanna Mrs. Birch† August† Meena Alice Johnny Mrs. Crisp† Bil Baker† Mercy† Anna Boleyne Jolly Miller† Mrs. Efﬁngton Stubbs Blue† Mrs. Potter† Aprahamian Leonard Mrs. Thingummy Brine Cutter† Old Cob† Belisarius Lord Mayor† Murray Bug† One-Eye† Best-Ingram Lory† Nathan Swain† Chyurda Pappa Doc† Cain Major Dover† Peter Teazle† Cotillion† Patience† Caroline Marie Antoinette Policar Morrel† Croaker† Plowman† Catherine Marshal Bertrand† President West† Curly† Poul Cato Matilda Carbury Queequeg Dadda Rand† Cervantes Matron† Rip Van Winkle† Dancing† Shalash Christine Miss Birch† Royce Domi Shrewd† Chuck Loyola† Miss Crump† Sawbones† Dow† Silent† Cleopatra Miss Hopkins† Semiramis Elam Dowtry Sirius† Connolly Norman† Miss King† Shep Elao Talenel Curly† Miss Saltire† Sir Carbury Fredor Talenelat Dante Miss Swindle† Skrimshander† Gart Ted Dave Mme. D’Artagnan Stamford Harold The Empress† Dives† Mollie Stigand Harvey Themos Tresting Dodo† Mouse† Sudeley Howard Theron Dr. Floss† Mr Stroll† Swubble Ien Threetrees Duck† Mr Thursgood The Director† Ilgrand Lender† Toffston Edgar Atheling† Mr. Beaufort† Tommy Barnes Ishar Verus Elmo Mr. Crisp† Unwin Ishi Walleye† Farmer Mitchell† Mr. Flowerdew Ursula Jim McGufﬁn† Weasel† Father Joseph† Mr. Lawrence Victor† Kerible the Enchanter† Willum Fury† Mr. Morris Vilkins Lilly† Wit Congar† Ginny Mrs Loveday Von Bischoff Henry VIII Mrs. Bates† Ysabel 39 out of 90 characters: 43% 30 out of 56 characters: 54% Table A1. Characters that were not identiﬁed by the system, supplied by the annotators. Characters whose names (partly) consist of a real word – such as ‘Curly’ or ‘Mercy’ – are Table A1. Characters that were not identiﬁed by the system, supplied by the annotators. Characters whose names (partly) consist of a real word – such as ‘Curly’ or ‘Mercy’ – are marked with a †. Checked against http://dictionary.com. Table A1. Characters that were not identiﬁed by the system, supplied by the annotators. Characters whose names (partly) consist of a real word – such as ‘Curly’ or ‘Mercy’ – are marked with a †. Checked against http://dictionary.com. Table A1. Characters that were not identiﬁed by the system, supplied by the annotators. Characters whose names (partly) consist of a real word – such as ‘Curly’ or ‘Mercy’ – are marked with a †. Checked against http://dictionary.com. 22/26 PeerJ Comput. Sci. reviewing PDF \| (CS-2018:08:30671:2:0:NEW 7 Mar 2019) Manuscript to be reviewed Classic Title Fraction of defaults Fraction of unidentiﬁed characters Average sentence length Average persons per sentence Fraction of sentences with a person Annotated sentences Unique characters Total character mentions 1984 0.55 0.00† 18.01 1.17 0.32 316 29 2162 A Study in Scarlet 0.83 0.50 18.99 1.17 0.18 193 34 837 Alice in Wonderland 0.26 0.56⋄ 20.99 1.23 0.79 316 17 656 Brave New World 0.35 0.17 15.87 1.06 0.25 299 51 1809 David Copperﬁeld 0.61 0.00† 22.79 1.08 0.49 261 157 9922 Dracula 0.93⋄ 0.00† 21.96 1.00† 0.06† 233 72 3369 Emma 0.43 0.10 22.38 1.38 0.81 224 78 6946 Frankenstein 0.86 0.22 25.80 1.19 0.17 300 29 658 Huckleberry Finn 0.59 0.14 23.46 1.20 0.40 215 82 1749 Jekyll and Hyde 0.67 0.29 26.19 1.17 0.34 120† 13† 523† Moby Dick 0.88 0.38 25.24 1.10 0.10 442 135 2454 Oliver Twist 0.36 0.33 21.64 1.23 0.68 303 69 4495 Pride and Prejudice 0.46 0.10 24.13 1.48 0.79 257 62 5104 The Call of the Wild 0.49 0.50 21.67 1.31 0.61 192 28 731 The Count of Monte Cristo 0.47 0.25 21.91 1.35 0.79 197 250 13562 The Lord of the Rings 0.47 0.48 16.30 1.20 0.46 769⋄ 134 5268 The Three Musketeers 0.60 0.36 19.19 1.13 0.49 265 115 4842 The Way We Live Now 0.57 0.46 18.93 1.14 0.47 341 147 13993⋄ Ulysses 0.57 0.33 13.35† 1.15 0.41 303 651⋄ 8510 Vanity Fair 0.24† 0.44 27.27⋄ 1.54⋄ 1.05⋄ 256 359 11503 Mean µ 0.56 0.28 21.30 1.21 0.48 290.10 125.60 4954.65 Standard Deviation σ 0.20 0.18 3.67 0.14 0.27 131.89 150.20 4403.32 Modern A Game of Thrones 0.29 0.00† 14.53 1.30 0.82⋄ 283 322⋄ 15839⋄ Assassin’s Apprentice 0.71 0.29 14.94 1.18 0.38 460 66 2857 Elantris 0.32 0.27 14.24 1.10 0.60 367 14† 226† Gardens of the Moon 0.75 0.44 12.20 1.03† 0.25 304 111 4479 Harry Potter 0.32 0.33 15.55 1.33 0.74 338 84 5114 Magician 0.49 0.17 14.78 1.16 0.45 310 115 4976 Mistborn 0.34 0.22 12.90 1.19 0.68 297 104 11672 Prince of Thorns 0.54 0.00† 12.33 1.14 0.38 107 79 2282 Storm Front 0.77 0.00† 14.02 1.05 0.18 211 43 2368 The Black Company 0.56 0.64⋄ 9.73† 1.07 0.26 305 42 1908 The Black Prism 0.50 0.14 13.19 1.04 0.40 380 88 10890 The Blade Itself 0.66 0.29 12.55 1.14 0.24 103 107 6769 The Colour of Magic 0.55 0.50 14.21 1.12 0.42 139 34 1454 The Gunslinger 0.78⋄ 0.25 13.43 1.11 0.17† 230 35 1159 The Lies of Locke Lamora 0.21† 0.09 16.90⋄ 1.38⋄ 0.77 305 105 6477 The Name of the Wind 0.45 0.10 12.98 1.14 0.45 310 137 6405 The Painted Man 0.30 0.28 14.67 1.29 0.70 301 137 9048 The Way of Kings 0.31 0.29 12.20 1.10 0.36 316 221 14696 The Wheel of Time 0.40 0.21 14.96 1.31 0.59 499⋄ 188 9426 Way of Shadows 0.32 0.13 13.53 1.32 0.56 88† 160 8721 Mean µ 0.48 0.23 13.69 1.17 0.47 282.65 109.60 6338.30 Standard Deviation σ 0.18 0.17 1.54 0.11 0.20 110.52 72.98 4535.60 µclassic −µmodern 0.08 0.05 7.61 0.04 0.01 7.45 16.00 -1383.65 Pooled σ 0.20 0.17 2.46 0.24 0.25 125 119 4473 p−value 0.21 0.39 ¿ 0.01 0.73 0.74 0.85 0.68 0.35 Signiﬁcant No No Yes No No No No No e A3. Computer Science Manuscript to be reviewed Manuscript to be reviewed Classic Title Author (Year) E-book No. / ISBN 1984 George Orwell (1949) 9780451518651 A Study in Scarlet Conan Doyle (1886) 244 Alice in Wonderland Lewis Carroll (1884) 19033 Brave New World Aldous Huxley (1865) 9780965185196 David Copperﬁeld Charles Dickins (1931) 766 Dracula Bram Stoker (1850) 345 Emma Jane Austen (1897) 158 Frankenstein Mary Shelley (1815) 84 Huckleberry Finn Mark Twain (1818) 76 Jekyll and Hyde Robert Stevenson (1851) 42 Moby Dick Herman Melville (1838) 2701 Oliver Twist Charles Dickins (1813) 730 Pride and Prejudice Jane Austen (1886) 1342 The Call of the Wild Jack London (1903) 215 The Count of Monte Cristo Alexandre Dumas (1844) 1184 The Fellowship of the Ring J. R. R. Tolkien (1954) 9780547952017 The Three Musketeers Alexandre Dumas (1844) 1257 The Way We Live Now Anthony Trollope (1875) 5231 Ulysses James Joyce (1922) 4300 Vanity Fair William Thackeray (1847) 599 Modern Title Author (Year) E-book No. / ISBN A Game of Thrones G.R.R. Martin (1996) 9780307292094 Assassin’s Apprentice Robin Hobb (1995) 9781400114344 Elantris Brandon Sanderson (2005) 9780765383105 Gardens of the Moon Steven Erikson (1999) 9788498003178 Harry Potter J.K. Rowling (1998) 9781781103685 Magician Raymond Feist (1982) 9780007466863 Mistborn Brandon Sanderson (2006) 9788374805537 Prince of Thorns Mark Lawrence (2011) 9786067192681 Storm Front Jim Butcher (2000) 9781101128657 The Black Company Glen Cook (1984) 9782841720743 The Black Prism Brent Weeks (2010) 9782352945260 The Blade Itself Joe Abercrombie (2006) 9781478935797 The Colour of Magic Terry Pratchett (1983) 9788374690973 The Gunslinger Steven King (1982) 9781501143519 The Lies of Locke Lamora Scott Lynch (2006) 9780575079755 The Name of the Wind Patrick Rothfuss (2007) 9782352949152 The Painted Man Peter Brett (2008) 9780007518616 The Way of Kings Brandon Sanderson (2010) 9780765326355 The Wheel of Time Robert Jordan (1990) 9781857230765 Way of Shadows Brent Weeks (2008) 9781607513513 Table A2. Classic and modern novels included in this study. The short E-book numbers are the catalog entry of novels obtained from Gutenberg. Novels obtained through online purchase are denoted by the longer ISBNs. 23/26 PeerJ Comput. Sci. reviewing PDF \| (CS-2018:08:30671:2:0:NEW 7 Mar 2019) Table A3. Overall statistics for classic and modern novels in our corpus. The highest scores in each column are highlighted with a ⋄, and the lowest scores with a †. The highest and lowest performing books for each class, in terms of F1score found in Tables 3 and 4, are marked with a grey ﬁll. Computer Science Computer Science Manuscript to be reviewed Manuscript to be reviewed Overall statistics for classic and modern novels in our corpus. The highest score column are highlighted with a ⋄, and the lowest scores with a †. The highest and lowes rming books for each class, in terms of F1score found in Tables 3 and 4, are marked wit ﬁll. Table A3. Overall statistics for classic and modern novels in our corpus. The highest scores in each column are highlighted with a ⋄, and the lowest scores with a †. The highest and lowest performing books for each class, in terms of F1score found in Tables 3 and 4, are marked with a grey ﬁll. 24/26 PeerJ Comput. Sci. reviewing PDF \| (CS-2018:08:30671:2:0:NEW 7 Mar 2019) Manuscript to be reviewed Classic Title Nodes Edges Average Degree Average Weighted Degree Network Diameter Graph Density Modularity Connected Components Average Clustering Coefﬁcient Average Path Length 1984 26 43 3.30 16.84 4 0.13 0.23 3 0.5 2.06 A Study in Scarlet 24 41 3.41 7.25 5 0.14 0.42 2 0.63 2.37 Alice in Wonderland 12 10† 1.66† 3.83† 3 0.15 0.15 2 0† 1.93 Brave New World 39 65 3.33 9.79 6 0.09 0.34 2 0.68 2.53 David Copperﬁeld 142 499 7.03 23.11 6 0.05 0.49 2 0.57 2.69 Dracula 55 124 4.51 18.29 6 0.08 0.12† 4 0.52 2.53 Emma 72 403 11.19 57.53⋄ 4 0.16 0.14 1 0.67 2.16 Frankenstein 20 38 3.80 10.60 5 0.20 0.51 2 0.75 2.41 Huckleberry Finn 62 121 3.90 8.42 7 0.06 0.52⋄ 4 0.60 3.30 Jekyll and Hyde 10† 21 4.20 14.60 2† 0.47⋄ 0.12 1 0.81⋄1.53† Moby Dick 90 169 3.76 7.38 8 0.04 0.44 8 0.59 3.33⋄ Oliver Twist 62 191 6.16 22.32 4 0.10 0.32 2 0.75 2.26 Pride and Prejudice 62 373 12.03 57.10 4 0.20 0.16 1 0.73 1.96 The Call of the Wild 23 44 3.83 10.00 6 0.17 0.46 1 0.62 2.46 The Count of Monte Cristo 228 799 7.01 24.05 7 0.03 0.40 3 0.56 2.88 The Fellowship of the Ring 105 260 4.95 11.51 6 0.05 0.29 2 0.63 2.73 The Three Musketeers 96 279 5.81 15.33 5 0.06 0.32 1 0.55 2.56 The Way We Live Now 135 630 9.33 39.17 5 0.07 0.36 3 0.69 2.43 Ulysses 522⋄ 4116⋄15.77⋄ 18.59 9⋄ 0.03 0.45 10⋄ 0.60 3.02 Vanity Fair 342 1349 7.89 22.73 7 0.02† 0.37 1 0.63 2.72 Mean µ 106 479 6.14 20 5.45 0.12 0.33 2.75 0.60 2.49 Standard Deviation σ 126.94 916.66 3.56 14.99 1.70 0.10 0.14 2.39 0.17 0.44 Modern A Game of Thrones 314⋄ 1648⋄10.50⋄ 22.46 6 0.03 0.48 1 0.54 2.81 Assassin’s Apprentice 55 110 4.00 9.09 6 0.07 0.34 2 0.49 2.65 Elantris 106 493 9.30 43.25⋄ 5 0.09 0.36 1 0.67 2.22† Gardens of the Moon 88 257 5.84 10.84 8 0.07 0.42 1 0.48 2.93 Harry Potter 67 198 5.9 19.37 5 0.09 0.15 1 0.68 2.23 Magician 84 209 4.98 10.76 6 0.06 0.43 2 0.58 2.83 Mistborn 89 255 5.73 33.89 6 0.07 0.04† 3 0.62 2.37 Prince of Thorns 59 111 3.76 6.98 6 0.07 0.37 2 0.42† 2.83 Storm Front 33 85 5.15 10.97 4† 0.16⋄ 0.31 1 0.64 2.26 The Black Company 30 45 3.00† 6.13† 6 0.10 0.20 3 0.561 2.43 The Black Prism 84 239 5.69 30.74 5 0.07 0.22 1 0.75⋄ 2.27 The Blade Itself 96 259 5.40 14.23 5 0.06 0.51 3 0.51 2.65 The Colour of Magic 27† 43† 3.19 7.93 6 0.12 0.38 1 0.50 2.67 The Gunslinger 31 69 4.45 8.52 7 0.15 0.41 1 0.43 2.87 The Lies of Locke Lamora 101 261 5.17 22.24 5 0.05 0.18 4 0.64 2.46 The Name of the Wind 109 197 3.62 8.99 9⋄ 0.03 0.67⋄ 5 0.46 4.06⋄ The Painted Man 132 444 6.73 23.15 7 0.05 0.53 1 0.63 2.70 The Way of Kings 172 448 5.21 20.79 6 0.03† 0.57 9⋄ 0.55 2.91 The Wheel of Time 167 545 6.53 16.66 7 0.04 0.35 3 0.55 2.84 Way of Shadows 145 441 6.08 22.14 6 0.04 0.46 4 0.61 2.71 Mean µ 99 317 5.50 17 6.05 0.07 0.36 2.45 0.56 2.68 Standard Deviation σ 66.37 348.92 1.85 10.05 1.15 0.04 0.15 1.99 0.09 0.4 µclassic −µmodern 7 162 0.64 3 -0.60 0.05 -0.03 0.30 0.04 -0.19 Pooled σ 101 695 2.83 12.83 1.45 0.08 0.15 2.18 0.13 0.43 p−value 0.83 0.47 0.49 0.55 0.20 0.09 0.42 0.67 0.37 0.17 Signiﬁcant No No No No No No No No No No A5. Computer Science Manuscript to be reviewed Manuscript to be reviewed Classic Modern Title Precision Recall F1score Title Precision Recall F1score 1984 77.33 72.87 75.03 A Game of Thrones 51.40 45.88 48.49 A Study in Scarlet⊙ 40.00 37.22 38.56 Assassin’s Apprentice⊙ 37.00 34.89 35.91 Alice in Wonderland 54.93 48.36 51.43 Elantris 72.33 73.75 73.03 Brave New World 55.00 53.57 54.28 Gardens of the Moon 12.67 14.00 13.30 David Copperﬁeld⊙ 38.52 37.82 38.16 Harry Potter 79.17⋄ 77.78⋄ 78.47⋄ Dracula⊙ 36.67 40.00 38.26 Magician 35.42 28.89 31.82 Emma 86.62⋄ 86.50⋄ 86.56⋄ Mistborn 61.99 60.62 61.30 Frankenstein⊙ 51.16 45.35 48.08 Prince of Thorns 69.44 70.83 70.13 Huckleberry Finn 82.38 82.82 82.60 Storm Front⊙ 40.54 39.19 39.85 Jekyll and Hyde 52.86 50.00 51.39 The Black Company⊙ 06.85† 05.71† 06.23† Moby Dick⊙ 60.98 57.72 59.31 The Black Prism 76.90 77.59 77.24 Oliver Twist 77.64 74.35 75.96 The Blade Itself 34.09 36.36 35.19 Pride and Prejudice 73.55 72.22 72.88 The Colour of Magic 30.77 27.56 29.08 The Call of the Wild 30.00 25.19 27.38 The Gunslinger 77.84 75.89 76.85 The Count of Monte Cristo 40.72 35.80 38.10 The Lies of Locke Lamora 62.77 59.16 60.91 The Fellowship of the Ring 63.23 60.61 61.90 The Name of the Wind 61.38 58.67 60.00 The Three Musketeers 13.91† 12.17† 12.99† The Painted Man 60.16 57.83 58.97 The Way We Live Now 66.07 66.79 66.43 The Way of Kings 65.87 64.42 65.14 Ulysses 66.67 66.98 66.82 The Wheel of Time 29.60 24.33 26.70 Vanity Fair 72.57 68.63 70.54 Way of Shadows 54.05 45.95 49.67 Mean µ 57.04 54.75 55.83 Mean µ 51.01 48.96 49.91 Standard Deviation σ 19.28 19.68 19.47 Standard Deviation σ 21.49 21.95 21.72 Table A4. Results of the complete BookNLP pipeline: Named entity recognition (Stanford NER), Character name clustering (e.g., “Tom”, “Tom Sawyer”, Sawyer”→TOM SAWYER) and Pronominal coreference resolution. The highest scores in each column are highlighted with a ⋄, and the lowest scores wit person are marked with a ⊙. 25/26 PeerJ Comput. Sci. reviewing PDF \| (CS-2018:08:30671:2:0:NEW 7 Mar 2019) Manuscript to be reviewed Social network measures for classic and modern novels. The highest scores in e mn are highlighted with a ⋄, and the lowest scores with a †. The highest and lowest ming books for each class, in terms of F1score found in Tables 3 and 4, are marked wi ﬁll. Table A5. Social network measures for classic and modern novels. The highest scores in each column are highlighted with a ⋄, and the lowest scores with a †. The highest and lowest performing books for each class, in terms of F1score found in Tables 3 and 4, are marked with a grey ﬁll. 26/26 PeerJ Comput. Sci. reviewing PDF \| (CS-2018:08:30671:2:0:NEW 7 Mar 2019)
https://openalex.org/W4291900453	https://journals.uis.no/index.php/JCSW/article/download/392/381	English	null	When crisis strikes	Comparative social work	2,022	cc-by-sa	8,641	When crisis strikes - Changes in work and professional identity among social workers in Norwegian Child Welfare Protection services during COVID-19 Keywords: Child Welfare Protection, Covid-19, Child Welfare Workers, professional identity, case prioritizing DOI: https://doi.org/10.31265/jcsw.v17.i1.392 Journal of Comparative Social Work 2022/1 Journal of Comparative Social Work 2022/1 Keywords: Child Welfare Protection, Covid-19, Child Welfare Workers, professional identity, case prioritizing DOI: https://doi.org/10.31265/jcsw.v17.i1.392 This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License. Abstract When the COVID-19 pandemic reached Norway in March 2020, comprehensive action was taken by the government, leading to the lockdown of welfare institutions, schools and kindergartens, and strict restrictions on physical meetings. This had severe consequences for Child Welfare Protection services (CWP). The restrictions stopped child welfare protection home visits and professionals in other welfare institutions, which usually observe children and notify child welfare protection services, were no longer able to identify children at risk. This article, drawing on interviews with 10 social workers, explores their experience during COVID-19. Applying Lipsky’s concept of street-level bureaucrats and theories of professional identity, the article documents how COVID-19 not only restricted, but also modified social workers jobs, and led to self-reflection on their professional identity. In turn, the findings suggest the potential for beneficial changes in practices in the aftermath of COVID-19. Keywords: Child Welfare Protection, Covid-19, Child Welfare Workers, professional identity, case prioritizing 154 Journal of Comparative Social Work 2022/1 Introduction When the COVID-19 pandemic hit Norway in March 2020, Norway went into lockdown, which had severe consequences for the municipal Child Welfare and Protection Service (CWP). Restrictions prohibited child welfare workers from carrying out their usual practice in investigating children’s care situations. CWP’s work during the pandemic was affected by infection control measures, the dominance of home- rather than office-based working, and primarily using digital communication. Partner organizations such as schools, health services and kindergartens were also affected, thereby disrupting cooperation routines (Bufdir, 2020). During the first weeks of the pandemic, the number of incoming ‘reports of concern’ was reduced. Even so, both professionals and governments concern for children at risk increased. Even though it was unclear how the pandemic would affect children, knowledge and experience from previous crises suggested that such restrictions could increase poor care situations and contribute to creating new ones (UNICEF, 2021). Both in Norway and internationally, concerns were raised about increasing the numbers of children at risk and a growing uncertainty about whether CWP could reach them (Bufdir, 2020; Welch & Haskins, 2020). A particular worry, both in Norway and internationally, was related to the increased risk of domestic abuse, both to a partner and towards children directly or as witnesses (Unicef, 2021; Tveito, 2021). Despite the pandemic, CWP’s remained committed to helping children at risk (Bufdir, 2020; FN, 1989). The research reported here explores how the changes in work conditions and infection control measures during the pandemic, as well as the emergence of new solutions and approaches, affected CWP work. A recent report suggests that CWPs changed their work routine priorities during the pandemic. However, the report also notes that there is a need to know more about how the pandemic affected frontline workers in CWP (Tveito, 2021). This article contributes to this gap in existing knowledge. The article reports on a qualitative study of the experiences of social workers in Norwegian child protection services during the COVID-19 pandemic, between March and June 2020. The main research question is therefore: 155 Journal of Comparative Social Work 2022/1 Journal of Comparative Social Work 2022/1 Journal of Comparative Social Work 2022/1 Journal of Comparative Social Work 2022/1 Journal of Comparative Social Work 2022/1 Previous research has explored Norwegian CWP leaders’ experiences during the first weeks of the pandemic (Tveito, 2021). This article explores how the social workers on the front-line, working with families and other welfare services, experienced changes in their investigation of cases during the first phase of the pandemic. Background Norway is known for its comprehensive welfare state, often referred to as the social democratic state. The state provides a wide range of fully tax-funded services to families and children, such as a publicly-funded school system, preventive and medical health services and child welfare and protection services (Kojan, 2011; Hollekim, Anderssen, & Daniel, 2016). Norwegian CWP’s work includes investigating and following-up children and families in a wide range of vulnerable life situations based on a set of core principles: support, prevention, early intervention and equality of opportunities (Kojan, 2011), focusing on the needs of the child in what has been named a service-and family-oriented perspective (Berrick, Dickens, Pösö, & Skivenes, 2016). In an international context, the wide mandate of Norwegian CWP differs from many other countries risk-oriented child welfare services (Berrick et al., 2016), in which child welfare services only act when child safety and protection is at risk (Kojan, 2011; Hollekim et al., 2016). The majority of notifications received by the Norwegian CWP about children potentially at risk come from other welfare institutions such as kindergartens, schools and health services (bufdir, n.d; Breimo et al., 2021). Home visits and meetings with children, families and professionals from other welfare institutions are an essential aspect of the CWP’s daily work. However, COVID-19 led to significant changes in the work of both the CWP and their collaboration partners in other welfare institutions. The first cases of coronavirus were confirmed in Norway in February 2020 (Government.no, n.d). The virus spread rapidly, and on March 12th the Government introduced a series of measures intended to stop the spread of the virus. Prime Minister Erna Solberg described the restrictions as “the strongest and most sweeping measures Norway has seen in peacetime” (Government.no., 2020, 24th March). Kindergartens and schools were closed for a month (Government.no, n.d,) and the CWP’s daily work changed dramatically. The work of the CWP shifted to an extensive use of home offices, digital meetings and a stricter prioritization of cases (Tveito, 2021). 156 Theoretical framework Our analytical framework is shaped by Lipsky’s notion of ‘street-level bureaucrats’ (1969), and considers the processes of developing and affirming professional identity. The concept of profession in this article includes those who work in the child welfare service, referred to as child welfare employees or social workers. For Lipsky, typical street-level bureaucrat work is characterized by a large number of cases relative to their responsibilities, therefore undermining the capacity to meet mandated responsibilities. Street-level bureaucrats often experience a sense of personal inadequacy or lack of competence, even though the inadequacy can be attributed to the nature of the job (Lipsky, 2010). Lipsky (2010, p. 31) notes, ‘Some jobs just cannot be done properly, given the ambiguity of goals and the technology of particular social services.’ Lipsky claims that the work of on-the-ground bureaucrats is characterized by a high work pressure due to a lack of resources, including time and a shortage of employees. The work of street-level bureaucrats is characterized by conflicting, ambiguous and constantly changing goals. The lack of clarity also makes it more difficult to provide feedback, and correct what works and what does not. This applies not only to employees working directly with clients, but also management's ability to exercise control over the policy the bureaucracy must pursue. Conflicts between, for example, client-oriented goals and organizational goals, can affect and determine social workers’ ability to treat clients as individuals. Similarly, resource limitations may constrain the possibility of home-based assistance that is considered best for the clients. Conflicting role expectations also contribute to ambiguity, and shape public expectations of social workers and how they view themselves (Lipsky, 2010). To deal with these work conditions, Lipsky describes several different coping strategies used by street-level bureaucrats (Lipsky, 2010). Modification is one coping strategy street-level bureaucrats use to cope with ambiguous goals. They develop 157 Journal of Comparative Social Work 2022/1 perceptions of their work and their clients that make it easier to achieve their goals; they modify their work by using welfare institution's routines, stereotypes and other mechanisms that simplify work tasks (Lipsky, 2010). A lack of resources and difficulties in achieving a goal can lead to the modification of goals to make them more feasible. Another coping mechanism to handle a demanding working condition is the introduction of routines to help ensure an equal treatment of users, and provide an effective way of handling cases (Lipsky, 2010). Theoretical framework Routines permit street-level bureaucrats to handle cases with flexibility according to each client’s individual needs, hence making their working conditions more manageable. Lipsky (2010) claims most public welfare institutions have their own procedures surrounding acute cases providing an alternative to standard routines. Also, when a case in CWP is considered acute, resources are redirected and there is a common understanding that the priority for other cases will be downgraded. According to Lipsky’s theoretical framework, child welfare workers can be defined as street-level bureaucrats, as their everyday work involves discretionary assessments, e.g., when the child welfare service investigates a child's care situation and assesses whether the care is ‘good enough’ (Lipsky, 2010, xiii; Bunkholdt & Kvaran, 2015, p. 119). Their common perceptions and coping strategies also form key aspects of a professional identity. Professional identity refers to an individual’s understanding of what it means to be and act as, for example, a social worker. It is a complicated concept that can have several meanings. Identity comes from the Latin word idem, which means ‘the same’ or identical (Molander & Terum, 2008, p. 17). However, the development of professional identity is not an individual process. Professional identity is developed through interactions with others, both in educational training and in everyday work as part of professional socialization (Almås, Vasset, & Ødegård, 2018). Heggen (2008) describes professional identity as a collective identity that involves a collective that ‘recognizes itself’ (p. 323), which then creates a collective basis for action. An example of a child welfare workers' collective basis for action is the five professional principles used as norms, and considered fundamental in all child welfare work (Bunkholdt & Kvaran, 2015, pp. 27-31): a child's best interest, attachment and relationship quality, biological ties, least invasive intervention and the child's 158 Journal of Comparative Social Work 2022/1 participation. A collective identity can be both internal and external. A collective inner definition is predicated on members identifying their own group and what they stand for, while an external definition is based on how others define the common features of a group to which they do not belong. Both these ways of defining a group identity lead to differentiation because they involve a natural distinction between ‘we’ and ‘them’ (Heggen, 2008, p. 323). Professional identity is not constant, but develops and changes with shifting situations and contexts. Theoretical framework Heggen (2008) particularly highlights the significance of professional experience and knowledge in situations of fluctuating working conditions, and how individuals apply their knowledge and experience to a new context. For the social workers in CWP, COVID-19 led to significant changes in their working conditions. We can see such changes as boundary-crossing (Heggen, 2008, p. 327) in to a new context. Through boundary-crossing, professional identity changes and develops. The collective ‘we’ develops dynamically as experience and knowledge are applied in new ways in new contexts. At the same time, professionals experience that ‘the others’ perception of them changes, in turn affecting their own perception of a ‘we’ (Heggen, 2008). Journal of Comparative Social Work 2022/1 Journal of Comparative Social Work 2022/1 was challenging, as 40 offices were contacted by e-mail, but only four participants responded. To prevent a long interval between recruiting informants, the second part of recruitment involved contacting 75 more offices, leading to a positive response from six additional participants. In total, 115 municipal CWP services were contacted, thus resulting in a sample of 10 social workers: five caseworkers and five professionals who had a leading position relating to the investigation of cases. The participants worked in both small and larger offices, and all were female full-time employees (see Table 1). Due to anonymization, age groups rather than the participant’s ages are used. Participant’s id Age group Years of experience Role Caseworker 1 37-46 8 Caseworker Caseworker 2 26-36 2 Caseworker Caseworker 3 37-46 7 Caseworker Team leader 4 37-46 15 Team leader for investigation cases Caseworker 5 26-36 1.5 Caseworker Team leader 6 57-66 22 Team leader for investigation cases Team leader 7 Unknown 2 Team leader for investigation cases Team leader 8 26-36 11 Team leader for investigation cases Team leader 9 47-56 12 Team leader for investigation cases Caseworker 10 26-36 4 Caseworker Table 1: Presentation of the participants Table 1: Presentation of the participants Table 1: Presentation of the participants The interviews took place between 27 April and 14 May 2020, and lasted between 29 and 44 minutes. The interviews were conducted by telephone and audio-recorded with the secure Nettskjema-dictaphone app (University of Oslo, n.d.), and were fully transcribed by the first author. All interviews started with consent for audio-recording and information about the study. The semi-structured interview guide was used to focus the interviews, provide comparability in data collection, engender trust and provide space for the informant to share their experience. Methodology Adopting a qualitative approach, data were collected through semi-structured in- depth interviews with 10 social workers working with investigating cases in the Norwegian child protection service. Interviews explored how measures to prevent the spread of coronavirus affected their investigations, and how staff handled these changes. Due to the pandemic restrictions, all interviews were conducted over the phone. The project was registered with the Norwegian Social Science Data Services (NSD) and approved on 17 April 2020. Participants were recruited by the first author, contacting municipal Child Welfare Protection Services in all regions of Norway, East, West, North and South, by e-mail. The request for participation was directed to each office manager with a request for permission to recruit employees. Information about, and the purpose of the study, were included in the request, which the managers were asked to pass on to employees interested in participating. The initial phase of the recruitment process 159 Data analysis This study employed a two-stage coding approach to analyse the data (Miles, Huberman, & Saldana, 2014). In the first stage, inductive codes were developed to provide an overview of the interviews (Nilssen, 2014). The codes used described and 160 Journal of Comparative Social Work 2022/1 summarized parts of the text. Both descriptive and in-vivo codes were also used during this stage (Miles et al., 2014). summarized parts of the text. Both descriptive and in-vivo codes were also used during this stage (Miles et al., 2014). The second stage was based on pattern coding to generate deductive codes. Pattern coding had several important functions in the analysis process; it organized larger units of data/codes into smaller analytical units that helped the researcher develop a more integrated understanding of both the events and interactions that shaped the experience of respondents (Miles et al., 2014). Findings evolved by relating topics from the interview guide to categories and themes relating to priorities and the caseworker role (Miles et al., 2014). Notes of thoughts, ideas, suggestions for categories and questions relating to the data were recorded during the analytical process. This was done deliberately to make sure that the analysis was actually searching for answers to the research question. In such a way, the analysis process went back and forth between inductive and deductive coding, thereby providing opportunities for further refining the codes and developing a more holistic understanding of the data (Nilssen, 2014). The codes were further developed to pattern codes that expressed the same theme or phenomenon (Miles et al., 2014) structured around two primary findings: (1) Improved case priorities and (2) Changed professional identity. Findings Improved case priorities child welfare work during the lockdown: Child Welfare Services may find themselves in situations where it is impossible to solve all the work tasks and challenges. Basic values regarding a child’s best interest and services are provided in accordance with sound professional standards that will be put as the test when a service whose mandate is to ensure children that the necessary help, care and protection has to prioritize between tasks which are all important and necessary. At the same time, consideration regarding the general infection control measures from the National Institute of Public Health must be taken, which can affect [Child Welfare Service] priorities. These prioritizations are no easy task to do. (The Ministry of Children, Youths and Families, 2020) Child Welfare Services may find themselves in situations where it is impossible to solve all the work tasks and challenges. Basic values regarding a child’s best interest and services are provided in accordance with sound professional standards that will be put as the test when a service whose mandate is to ensure children that the necessary help, care and protection has to prioritize between tasks which are all important and necessary. At the same time, consideration regarding the general infection control measures from the National Institute of Public Health must be taken, which can affect [Child Welfare Service] priorities. These prioritizations are no easy task to do. (The Ministry of Children, Youths and Families, 2020) All social workers in the study explained that prioritizing cases was also part of the daily work in CWP before the pandemic. The pandemic and infection control measures, however, forced them to prioritize to a far greater degree. Even though most cases were related to domestic violence and abuse, by definition cases of great concern, the prioritization resulted in only the most acute cases being handled at the beginning of the pandemic. One team leader described how this assessment process led to much more work in trying to assess whether there was a reason to believe that a child was at increased risk, prior to assigning a case a higher or lower priority: Only the acute cases were handled in the beginning (of the pandemic), the most serious and concerning cases where there might be exposure to violence and abuse. We spent an insane amount of time trying to contact schools, kindergartens and families [for information] to assess how to prioritize the individual cases. Improved case priorities The informants reported changes in the way they carried out investigations after the infection control measures were implemented, which in turn required them to be more selective and prioritize the cases they managed to a far greater extent. A quote from Caseworker 5 elaborates these changes: Limitation in physical meetings [because of national infection control measures] have forced us to prioritize differently [than pre-pandemic]. (..) How to work in the various cases are also a prioritization: for which cases is it enough that the first contact with the families is over the phone, and for which cases do we need [to meet physically] at the office? During the pandemic, we have had a system for risk assessment where we sorted cases in colour categories: red, yellow or green. Red coloured are cases where children’s health and life are at risk, yellow coloured are [children and families] in need of preventive measures, and green coloured represents cases which can wait for a bit longer. In each colour category, we also prioritize what needs to be handled first. For example, we may arrange fewer meetings than we would have done in a normal situation, but we still conduct the investigation. (Caseworker 5) 161 Journal of Comparative Social Work 2022/1 The quotation above represents what most informants experienced. The prioritization was something they were told to do. The Ministry of Children, Youths and Families (2020a) sent an information letter to all Child Welfare Services in Norway at the beginning of the pandemic, setting out how they should both prioritize and handle child welfare work during the lockdown: child welfare work during the lockdown: We had never worked like this before; it was a weird and stressful situation not knowing what’s going on in children’s home. (Team leader 9) To sift out the most serious cases, the caseworkers spent a lot of time contacting other public services for informal information, and also the families themselves. Information had to be obtained almost entirely by phone. Prior to the pandemic, caseworkers would have sought information by sending out written requests to public services, with a one-month deadline to respond in order to generate enough information for an assessment. But during the pandemic there was no time to wait for written information, and cases had to be assessed as quickly as possible. A stressful factor mentioned by some of the caseworkers related to other public services also being in lockdown, consequently making the usual informal sources limited by working from home. The respondents reported that collaborative public services had 162 Journal of Comparative Social Work 2022/1 It made child welfare workers assess each case and decide which cases they should handle first in greater detail than before the pandemic, often based on limited information: 163 Journal of Comparative Social Work 2022/1 Journal of Comparative Social Work 2022/1 The priorities have been the ages of the children, children's vulnerability, and of course the content of the message of concern. There have been several reports of concern in the recent past that have been serious. (Team leader 6) Nine out of 10informants explained how this led to new routines for prioritizing cases after an assessment of the severity of the report of concern. This required that as well as prioritizing how fast a case should be investigated further, a second type of prioritization in relation to which cases should be prioritized for physical meetings: It is a prioritization of which issues we must get into quickly and which cases can withstand waiting a bit. And how to work in the various cases is also a matter of priorities; for which cases is it “enough” that we carry out the first conversation as a conference call, and for which cases is there a need for a physical meeting with the parents. (Caseworker 5) Another consideration during this deliberation process was what information needed to be obtained and from whom, i.e., how extensive the investigation should be. The extension of a CWP investigation is regulated by the Child Welfare Act section 4-4, paragraph two, specifying that to help ensure confidentiality, the investigation shall not be more extensive than the purpose dictates (Barnevernloven, 1992). Given the limited informal information available, the child welfare workers noted that they had become very specific about the information they needed to obtain. Findings in this study show that most of the informants were more careful of where and how information was obtained, and how many meetings with the clients were required before making a decision. Journal of Comparative Social Work 2022/1 Journal of Comparative Social Work 2022/1 less access to their electronic journal systems while working from home, and that schools and kindergartens were closed and therefore unable to observe children’s everyday lives. This left caseworkers with a constant sense of uncertainty about what was actually going on in the lives of children who were possibly at risk, as well as making prioritizing cases harder for child welfare workers, because they had less information on which to base their assessments. Physical meetings, such as home visits, other meeting activities and travelling, were kept to a minimum or replaced with phone calls and digital meetings. The respondents reported that this made the workload feel heavier because they knew children were suffering due to the lockdown of society, and they had to work differently to assess which cases to handle quickly while working from their own homes. Work within the Norwegian Child Welfare Service altered in step with changes in government guidelines and restrictions between 12 March and 14 May 2020. According to the informants, the stressful period in the beginning of the pandemic was gradual as the restrictions changed, and physical meetings could once again take place and they returned to the office rather than working from home. Despite this process of normalization and the reintroduction of physical meetings, a more extensive prioritization of cases was still necessary. The prioritization of cases within the CWP is not new. Informants noted that they usually prioritized cases according to severity, and assessed whether each individual case was acute. Even so, the empirical findings show that the pandemic and infection control measures led to changes in the process and a more explicit prioritization of cases. Several of the informants referred to the guidelines provided by the Directorate for Children, Youth and Families for work during COVID-19 (Bufdir, 2020). The ‘report of concern’ governs how a case is to be conducted, but this has traditionally been prioritized based on different indicators of risk leading to a judgement of the ‘severity’ of the case. The difficulties in obtaining informal information about children and families from other public services also restricted due to the pandemic, resulted in caseworkers prioritizing cases based on the available information. Journal of Comparative Social Work 2022/1 As Caseworker 1 explained, ‘It is not like we have obtained information “blindly” in the past, but I think we make more accurate assessments of where and from whom we need to obtain information.’ One of the team leaders also noted that the pandemic led to improved prioritization: I think we have become better at prioritizing and assessing each case, because it is something that we have to do (...) and yes, to do what is good enough in each case. We have had to prioritize so that we do not do more than necessary (…) and I think that is something positive to take with us further. (Team leader 4) I think we have become better at prioritizing and assessing each case, because it is something that we have to do (...) and yes, to do what is good enough in each case. We have had to prioritize so that we do not do more than necessary (…) and I think that is something positive to take with us further. (Team leader 4) This suggests that previously, prioritization had not been good enough, and that investigations in some cases had been too extensive. While this may have implications for the assertion of pre-existing heavy case-loads and too few staff reported by caseworkers, there are still examples of situations where complex family situations required an extensive and comprehensive investigation. Team leader 7 said: 164 Journal of Comparative Social Work 2022/1 Journal of Comparative Social Work 2022/1 Journal of Comparative Social Work 2022/1 From my point of view, I can say that I have become better at prioritizing; what I should do first, and what can wait a little longer, and where information is to be obtained from and about what. I will use it after COVID-19 as well. This suggests that COVID-19 has led many CWP professionals to reconsider their approach to investigation. Both caseworkers and team leaders explained that the experience of investigating cases during the pandemic has led them to develop a more accurate approach that would continue after the pandemic. Journal of Comparative Social Work 2022/1 Journal of Comparative Social Work 2022/1 Journal of Comparative Social Work 2022/1 said she found it exciting to try new working methods, such as digital contact with clients. Digital communication became the main approach to maintaining contact with clients in requiring professionals to quickly adapt to new forms of engagement. Another informant described it as exciting to work during the pandemic, and emphasized that the restrictions led to positive changes in the way they worked: ‘I think this is an eye-opener for us and for digital communication [in child welfare work,] and have to use that resource in other ways than before’ (Caseworker 3). For several of the informants, digital communication was perceived as a working method that could be integrated into normal working practice after the pandemic. Digital communication contributed to an improved daily work routine that probably would never have been adopted without the pandemic. Working from home was emphasized as a change that, despite the disadvantage of being physically isolated from other colleagues, also required greater independent decision-making from the individual caseworker: For me, I think that it was ‘good timing that the pandemic appeared at this stage. I have been working for only two years. You will be forced to work even more independently despite a good follow-up from the leader. So in that way has the situation affected me; it has given me more confidence in myself. (Caseworker 2) For me, I think that it was ‘good timing that the pandemic appeared at this stage. I have been working for only two years. You will be forced to work even more independently despite a good follow-up from the leader. So in that way has the situation affected me; it has given me more confidence in myself. (Caseworker 2) Despite the limitations caused by the pandemic, such accounts illustrate how the infection control measures and changed working conditions also led to professional growth and a greater perception of themselves as a professional. The infection control measures restricted the child welfare workers work routines, but at the same time they also created opportunities for greater professional freedom and development. Such opportunities build self-confidence through a greater professional independence, and made it possible for them to be part of developing new working methods as the situation forced them to deviate from national guidelines. Changed professional identity The infection control measures imposed during the pandemic led to changes in working conditions for all the informants. A central topic in the interviews was how the changes had been handled by the caseworkers. Most of the informants reported a very rigid working framework before COVID-19. While there had been some variation in how case investigations were handled, the child welfare workers reported that the common framework based on national guidelines was quite rigid. Informants explained that during the pandemic this framework became less rigid, leading to both caseworkers and team leaders developing new approaches, and also developing themselves as social workers. The new ways of working, more often from home, also created new opportunities: It is not without challenges to be working from a home office, and you have to do investigation cases and child welfare work from home. But I also experienced that one finds new ways of doing the job one is supposed to. You get more creative. (...) It requires new ways of thinking that you may not be used to, such as digital communication. (…) In our job everything can show up, and you never know what you will meet. (Caseworker 5) Creativity was a concept that recurred across several of the interviews in relation to new ways of working during the pandemic. The term was also linked to finding new ways to conduct meetings when physical distance had to be maintained due to infection control measures, with one caseworker reporting a home visit conducted by live video. Such practices were totally unimaginable pre-pandemic. Caseworker 2 explained how CWP have realized it is possible to do child welfare work in different ways: ‘(..) There’s something with realizing it’s possible to do our work in other ways, like [having meetings over] video calls (..).’ Limitations in physical encounters with clients and working in special circumstances gave professionals more room to think differently than previously, and one informant 165 Journal of Comparative Social Work 2022/1 Journal of Comparative Social Work 2022/1 Journal of Comparative Social Work 2022/1 For the child welfare workers, this led to a sense of frustration. As one of the caseworkers explained, ‘I think we should have been on the list of critical social tasks from day one, but we were not. They did not even think of us’ (Caseworker 10). During the spring of 2020, professionals in CWP were redefined as socially critical, but this was too late, according to some informants. ‘There had to be a crisis before the Government saw that the child welfare service was important’ (Caseworker 3). Another informant said: We should have been on the list of socially critical occupations from the beginning, and I think that they [the government] may not quite see the importance of the child welfare service. Since they [the government] did not put us there in the beginning, they almost had to be told about us. (Caseworker 10) Informants also reported that the infection control measures that the government put in place, closing schools and kindergartens, had complicated their work. Informants reported that they believed that not only were child welfare service core competencies not seen as essential, but they were also not heard in relation to important decisions. The restrictions introduced during that pandemic had a significant impact on their work, both in making it more difficult, but also in potentially creating greater need. Despite these foreseeable consequences, CWP professionals were not considered in making such decisions: were not considered in making such decisions: I do not know which resource persons the Government has consulted in the decision, but it is clear that the children and families we work with are difficult to meet with such general measures. (…) I experience that we were neither consulted in such questions, nor heard. (Team leader 6) Even though several informants disagreed with the government restrictions, most informants reported following the guidelines. However, some informants did conduct home visits and travelled between municipalities, even though these were forbidden by the COVID-19 restrictions. On the one hand, informants had to adhere to the restrictions, but on the other hand they weighed this against their professional judgement and the needs of vulnerable families and children. Statements from both the team leaders and the caseworkers reveal that caseworkers hid their fears and put their health at risk just to do their jobs. Journal of Comparative Social Work 2022/1 For several of the child welfare workers, this created a greater sense of professional freedom, which they also experienced as inspiring. Another finding impacting on social workers’ professional identity was their experience of recognition from society and from the government. When Norway went into crisis mode, professionals in the CWP were not an occupational group listed as socially critical personnel (Regjeringen, 2020). The informants reported that the child welfare service was both ‘forgotten’ and not seen as important by central authorities. 166 Journal of Comparative Social Work 2022/1 Most of the caseworkers in this study also reported that they avoided using personal protective equipment, not because they were not afraid, but because they did not want to scare children and families. ‘I have not used personal protective equipment, I believe that would have scared the children. It would have been weird if a strange lady [caseworker] came with a face 167 Journal of Comparative Social Work 2022/1 Journal of Comparative Social Work 2022/1 mask’ (Caseworker 3). The perception of the importance of their work weighed heavily. To be able to build trust in order to conduct an investigation and make children feel comfortable, some caseworkers chose to expose themselves to potential infection. The informants’ frustrations related to their perception of the importance of their work and the lack of recognition prior to, and during, the initial phase of the pandemic. To be assigned to a revised list of socially critical occupations was therefore also an important recognition: I think it is crucial what they [the government] say about the child welfare service being important. (…) The child welfare service has been important throughout [the pandemic]. But I think that government (…) suddenly discovered that CWP was important. We experience now that we are actually seen as valuable for the job we do. (Caseworker 3) I think it is crucial what they [the government] say about the child welfare service being important. (…) The child welfare service has been important throughout [the pandemic]. But I think that government (…) suddenly discovered that CWP was important. We experience now that we are actually seen as valuable for the job we do. (Caseworker 3) Several of the child welfare workers interviewed highlighted the recognition of being put on the list of socially critical functions as an important reason for feeling more recognized as professionals than prior to the pandemic. Other welfare institutions also reached out to child welfare workers. Informants reported increased contact from schools during the pandemic, who contacted the Child Welfare Service due to concerns for children. The issues raised were both small and large, and the typical concern was about children that the schools had been concerned about for a long time, often pre-pandemic. Journal of Comparative Social Work 2022/1 The child welfare workers stated that before the pandemic they thought of their profession as important, but being recognized as important by the government and other welfare institutions strengthened their ‘we’ as a valuable professional group within the welfare system. Discussion Our findings highlight how CWPs faced changes in case prioritization, working conditions and complexity linked to the pandemic restrictions. The changed working environment and work conditions led to innovation in their professional work, and in turn also changed their perception of their collective professional identity, both as a professional ‘me and their professional ‘we’. Lipsky’s claims that one of the street-level bureaucrats coping strategies is to create routines that ensure an equal treatment of equal cases, and to promote efficiency 168 Journal of Comparative Social Work 2022/1 (Lipsky, 2010). This is a form of service rationing, and a strategy reported by the caseworkers and leaders in this study. By categorizing the cases, the informants adapted to the consequences of new routines, and thus made it possible to reallocate resources so that resources were deployed where the need was greatest: in acute cases. Only the most serious cases with greatest concern for children were prioritized. In terms of the CWP’s role and mandate, this can be seen as a transformation from a service- and family-oriented child welfare service, towards a more risk-oriented child welfare service (Berrick et al., 2016). Several of the informants also said that the guidelines governed their priorities. Applying Lipsky's concept of modification, this can be understood as a psychological withdrawal by the informants, in which they reduced their personal responsibility for the child welfare service performance during the pandemic while infection control measures were most stringent. For the informants, the guidelines that governed their priority-setting can be understood as such a modification, a way to protect themselves during demanding working conditions. By expressing that ‘we only follow the guidelines’, a certain distance between caseworkers responsibility for conducting child welfare work and the challenging situation (children at risk due to the pandemic) was created, justifying the new practice in contrast to the approach prior to the pandemic. However, several informants reported that they felt they had become better at conducting investigations and doing so less extensively than before the pandemic, a modification that resulted from the change in prioritization required by operating under the restriction of the pandemic. Lipsky (2010) claims that there is a contradiction in the working conditions for street-level bureaucrats, which means that they must both have the individual’s needs in focus, and also treat their clients with regard to the institution's routines, simplifying tasks by, for example, using standard routines. Journal of Comparative Social Work 2022/1 managing information became more accurate. In turn, the new procedures saved time and resources and made social workers better able to cope with the challenges of COVID-19, but also enabled them to look at their previous work in a more detached way. The Infection control measures and restrictions due to the pandemic ‘forced’ the informants to assess more precisely the sources and nature of information that needed to be obtained. These changes can be seen as modifying institutional routines, and in turn creating an opportunity for new modifications. Even though it takes less time to send out standard-written requests for information than to go deeper into each case and consider exactly what information is needed and from whom, this modification, for informants, resulting in them feeling they were more clearly addressing individual client’s needs. In turn, this created a changed institutional context, thereby enabling new and different modifications to their work. As well as creating new ways of working, the changed institutional context also created room for professional growth. Several informants talked about having greater confidence in themselves as professionals, and that working during the pandemic required greater independence than under ‘normal’ circumstances. One informant also spoke about the period as exciting because creativity and new ways of working became possible. It is conceivable that the informant's professional identity has developed during the pandemic. ‘Me’ as a professional social worker had developed as a result of what it constitutes. The context demanded new solutions. Here, it was the pandemic that constituted the context. The informants' internalized action- oriented guide on how to deal with the pandemic did not exist. They had to make a new guide for the situation, and it is the internal affairs of the individual that govern how some will face the situation. For that reason, professional identity must be understood normatively, and as constantly redefined in the individual's encounters with the field of practice (Heggen, 2008, p. 324). Professional identity should be understood on a more individual level than the professional identity related to an entire group, although these two understandings are closely linked to one another. The informants changed due to prioritization and assessments, and the way that these were ways of doing child welfare work differently and better can be seen in the light of personal identity development related to the execution of their professional role (Heggen, 2008). Discussion Street-level bureaucrats develop perceptions of their work that make daily work easier; hence, they modify their work so that they manage challenging working conditions (Lipsky, 2010). In adopting a better prioritization process, the informants can be understood as modifying a “taken for granted” institutional routine. By categorizing cases by colour codes to a far greater extent than pre-pandemic, a new routine for investigating cases during the pandemic was created. This led to a more standardized procedure for assessing and conducting the investigation cases. More importantly, the social workers also reported that the new approach to obtaining and 169 Journal of Comparative Social Work 2022/1 Journal of Comparative Social Work 2022/1 workers can be seen as boundary-crossers (Tuomi-Gröhn et al., in Heggen, 2008, p. 327). As Heggen explains, their professional identity, and their perception of what is characteristic for the identity of a child welfare worker, is constantly redefined through individual encounters in the practice field. During COVID-19, their perceptions of ‘me as a professional changed because the new situation required different solutions’, but also because ‘the others’ perception of social workers in CWP changed. As the findings of the study show, many of the social workers experienced for the first time in their career that what they did was considered important, and that they were critical personnel. External definition helps to create, maintain or change professional identity (Heggen, 2008). When child welfare workers were finally considered a socially critical group, the external signals reinforced a professional identity and self- definition that the child welfare service must be operational despite the pandemic, and was important. This external sign influenced the informants' professional identity, and made them deal with the situation accordingly, namely to find good solutions in a challenging situation. workers can be seen as boundary-crossers (Tuomi-Gröhn et al., in Heggen, 2008, p. 327). As Heggen explains, their professional identity, and their perception of what is characteristic for the identity of a child welfare worker, is constantly redefined through individual encounters in the practice field. During COVID-19, their perceptions of ‘me as a professional changed because the new situation required different solutions’, but also because ‘the others’ perception of social workers in CWP changed. As the findings of the study show, many of the social workers experienced for the first time in their career that what they did was considered important, and that they were critical personnel. External definition helps to create, maintain or change professional identity (Heggen, 2008). When child welfare workers were finally considered a socially critical group, the external signals reinforced a professional identity and self- definition that the child welfare service must be operational despite the pandemic, and was important. This external sign influenced the informants' professional identity, and made them deal with the situation accordingly, namely to find good solutions in a challenging situation. Journal of Comparative Social Work 2022/1 Entering the changed context of child welfare work, the social 170 Journal of Comparative Social Work 2022/1 Conclusion The COVID-19 pandemic appeared unexpectedly, and forced a massive change in the working practice of CWPs. As this article describes, the changes were not all negative. Despite the disadvantages the pandemic entailed, changes in the ways of working also created the context for changing practice creating a more precise and efficient investigation process, and for professional growth. In Norway, there is an ongoing debate about quality and the level of interventions carried out by the CWP, as well as their mandate and responsibilities. The findings of this study engage with this debate, and provide insights into how contextual changes can reveal the potential for changes in practice. Increased prioritizing due to the severity of the cases can be seen as a shift from service- and family oriented towards a more risk- oriented child welfare service, but it might as well also be seen as a step towards a greater focus on more accurate investigation processes. The contextual change propelled by the pandemic also created a possibility for applying knowledge and procedures in a new way, which in turn enabled the social workers to see their previous work more objectively, thus creating further possibilities for changes in practice in the future. As the pandemic is still ongoing, it remains unclear whether these changes will be integrated into mainstream professional practice. 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https://openalex.org/W2017129800	https://hal.science/hal-00803139/file/fpsyg-04-00119.pdf	English	null	Constraints on Letter-in-String Identification in Peripheral Vision: Effects of Number of Flankers and Deployment of Attention	Frontiers in psychology	2,013	cc-by	8,782	1Several studies have also examined effects of number of equidistant ﬂankers using various kinds of stimuli and with varying results (e.g., Wilkinson et al., 1997; Pelli et al., 2004; Põder and Wagemans, 2007; Põder, 2008). INTRODUCTION j g A handful of studies have tested for effects of non-adjacent ﬂankers on letter identiﬁcation in peripheral vision (Butler and Currie, 1986; Heller et al., 1995; Huckauf and Heller, 2002a,b)1. These studies compared the effects of two- vs. four-ﬂankers on Currie, 1986; Heller et al., 1995; Huckauf and Heller, 2002a,b) . These studies compared the effects of two- vs. four-ﬂankers on Myriam Chanceaux and Jonathan Grainger* gie Cognitive, Centre National de la Recherche Scientiﬁque, Aix-Marseille University, UMR 7290, Marseille, France Laboratoire de Psychologie Cognitive, Centre National de la Recherche Scientiﬁque, Aix-Marseille University, UMR 7290, M Reviewed by: Olaf Hauk, MRC Cognition and Brain Sciences Unit, UK Francesca Peressotti, University of Padova, Italy Edited by: Effects of non-adjacent ﬂanking elements on crowding of letter stimuli were examined in experiments manipulating the number of ﬂanking elements and the deployment of spatial attention.To this end, identiﬁcation accuracy of single letters was compared with identiﬁca- tion of letter targets surrounded by two, four, or six ﬂanking elements placed symmetrically left and right of the target. Target stimuli were presented left or right of a central ﬁxation, and appeared either unilaterally or with an equivalent number of characters in the contralat- eral visual ﬁeld (bilateral presentation). Experiment 1A tested letter targets with random letter ﬂankers, and Experiments 1B and 2 tested letter targets with Xs as ﬂanking stimuli. The results revealed a number of ﬂankers effect that extended beyond standard two-ﬂanker crowding. Flanker interference was stronger with random letter ﬂankers compared with homogeneous Xs, and performance was systematically better under unilateral presenta- tion conditions compared with bilateral presentation. Furthermore, the difference between the zero-ﬂanker and two-ﬂanker conditions was signiﬁcantly greater under bilateral presen- tation, whereas the difference between two-ﬂankers and four-ﬂankers did not differ across unilateral and bilateral presentation. The complete pattern of results can be captured by the independent contributions of excessive feature integration and deployment of spatial attention to letter-in-string visibility. Jay Rueckl, University of Connecticut, USA Jay Rueckl, University of Connecticut, USA INTRODUCTION targets (e.g., Bouma, 1970; Huckauf and Nazir, 2007; Grainger et al.,2010). More recently,these effects of lateral interference have been integrated within the wider perspective of crowding effects on the processing of various kinds of visual stimuli (e.g.,Pelli et al., 2004; Pelli and Tillman, 2008; see Levi, 2008; Whitney and Levi, 2011, for reviews). However, the vast majority of these studies have limited their investigation to the effects of stimuli that are the closest to the target, and have shown that target-ﬂanker separation determines target identiﬁcation accuracy. Much of this work can be summarized by Bouma’s law (Bouma, 1970; Pelli and Tillman, 2008), which states that critical spacing (i.e., the target-ﬂanker separation that allows target identiﬁcation at a criterion level of accuracy) is a linear function of target eccentricity. On the other hand, we currently know much less about how distractor stimuli that are not adjacent to the target, that is, are separated from the target by at least one other ﬂanking element,might inﬂuence target identiﬁcation. Yet natural cluttered environments in general, and printed text in particular, nearly always involve situations where there are non-adjacent ﬂanking elements. What factors inﬂuence our ability to identify a single letter pre- sented in a random string of letters? Answering this question is important because it will help develop our understanding of the very ﬁrst phase of the reading process during which visual fea- ture information is mapped in parallel onto position-coded letter identities in both central and peripheral vision (Grainger and van Heuven, 2003; Marzouki et al., 2013). The importance of under- standing the mechanisms involved has been nicely illustrated by the recent ﬁnding that increased letter spacing can facilitate read- ing in dyslexic children (Perea et al., 2012; Zorzi et al., 2012). Indeed, crowding is one factor known to have a large inﬂuence on letter-in-string perception, and manipulating inter-letter spacing modulates the amount of crowding. Crowding, or lateral masking, is the phenomenon whereby target identiﬁcation in peripheral vision is affected by the presence of nearby ﬂanking elements (Bouma, 1970; Pelli et al., 2004; Pelli and Tillman, 2008). In the present study we focus on one speciﬁc aspect of crowding effects – the contribution of non-adjacent ﬂanking elements – while examining possible interactions between crowding effects and the deployment of spatial attention. *Correspondence: Jonathan Grainger, Laboratoire de Psychologie Cognitive, Centre National de la Recherche Scientiﬁque, Aix-Marseille University, UMR 7290, Pôle 3C, Bâtiment 9 Case D, 3, Place Victor Hugo, 13331 Marseille Cedex 3, France. e-mail: jonathan.grainger@univ-amu.fr Keywords: letter perception, crowding, non-adjacent ﬂankers, number of ﬂankers, spatial attention Keywords: letter perception, crowding, non-adjacent ﬂankers, number of ﬂankers, spatial attention ORIGINAL RESEARCH ARTICLE bli h d 13 M h 2013 published: 13 March 2013 doi: 10.3389/fpsyg.2013.00119 Constraints on letter-in-string identiﬁcation in peripheral vision: effects of number of ﬂankers and deployment of attention www.frontiersin.org CROWDING AND NON-ADJACENT FLANKERS Effects of lateral interference on letter identiﬁcation is a well-studied phenomenon in experimental psychology, with resultsshowing that target letters ﬂanked by other letters to the left and to the right, are harder to identify than isolated letter March 2013 \| Volume 4 \| Article 119 \| 1 www.frontiersin.org Letter-in-string identiﬁcation Chanceaux and Grainger toward the fovea or not (Petrov and Meleshkevich, 2011). Further- more, Scolari et al. (2007) reported reduced critical spacing when targets were presented in a different color to ﬂankers, presum- ably because the different color helped attract attention to the target2. target letters located at the center of three-letter and ﬁve-letter strings that were presented left or right of a central ﬁxation. The results of these studies replicated the standard ﬁnding of a drop in performance with two ﬂanking stimuli compared with isolated targets (i.e., standard crowding), and demonstrated a further drop in performance from tow-ﬂankers to four-ﬂankers.A similar effect of number of ﬂankers was also reported for digit identiﬁcation by Strasburger et al. (1991). In order to provide a further test of attentional accounts of crowding driven by adjacent and non-adjacent ﬂankers, we included two manipulations expected to modify the deployment of spatial attention. First, we compared ﬂanking effects induced by random letter strings (Experiment 1A) with effects induced by homogeneous strings of Xs (Experiment 1B). The homoge- neous ﬂankers are expected to facilitate focusing of attention on the target as the odd man out (see Figure 1), and therefore to reduce ﬂanker interference compared with non-homogeneous ﬂankers (e.g., Scolari et al., 2007). Second, we presented stimuli either unilaterally (i.e., all stimuli grouped in one visual ﬁeld) or bilaterally, with an equivalent number of stimuli presented simultaneously in the contralateral ﬁeld (see Figure 1). Unilateral stimuli will enable rapid allocation of attention to target location (targets were always at the central position of strings), whereas bilateral stimuli will encourage a division of attention across the visual ﬁelds until processing of the post-cue that indicates the tar- get location. According to attentional accounts of crowding, we should see reduced ﬂanker interference under unilateral presen- tation conditions. More speciﬁcally, the combined manipulation of attentional deployment and number of ﬂankers will allow us to examine whether adjacent and non-adjacent ﬂankers are differ- entially affected by spatial attention. EXPERIMENT 1 Experiment 1 compared identiﬁcation of isolated letter targets, and targets embedded as the central letter in three-letter, and ﬁve- letter strings presented in the left and right visual ﬁeld under either unilateral or bilateral presentation conditions. In Experiment 1A, ﬂanking stimuli were other letters, and in Experiment 1B ﬂankers were homogeneous Xs (see Figure 1). EFFECTS OF DEPLOYMENT OF ATTENTION It is also possible that effects of non-adjacent ﬂankers reﬂect dif- ferences in the deployment of spatial attention as a function of string length. Strasburger et al. (1991) suggested that it might be harder to focus attention on the target location in longer strings, and this causes a cost in the processing of target identity as well as target position. This attentional account of effects of non- adjacent ﬂankers could be integrated within a general explanation of crowding according to which spatial attention determines the size of the crowding zone (e.g., Strasburger et al., 1991; Intriliga- tor and Cavanagh, 2001; Strasburger, 2005). According to these accounts it is possible to equate the empirically deﬁned crowd- ing zone (i.e., critical spacing) with the size of the spotlight of spatial attention. In line with this proposal, there is some evi- dence that crowding effects can be reduced by spatial cueing. This was shown most clearly by Yeshurun and Rashal (2010), who demonstrated a reduction in critical spacing for orientation identiﬁcation (target was a rotated T) with the prior presenta- tion of a spatial cue to target location. Spatial cueing has also been shown to affect the different impact of inward vs. out- ward ﬂankers as a function of whether or not attention is drawn CROWDING AND NON-ADJACENT FLANKERS To do so, we plan to test for partial interactions between a given attentional manipulation and the effects of adjacent ﬂankers (zero- vs. two-ﬂankers) on the one hand, and the effects of non-adjacent ﬂankers on the other. g These relatively understudied effects of non-adjacent ﬂank- ing stimuli are all the more interesting in that the non-adjacent ﬂanking stimuli typically fall outside of the critical spacing limits that determine standard crowding effects according to Bouma’s law. This has led a number of researchers to propose that dif- ferent mechanisms might be involved in non-adjacent ﬂanking effects compared with standard crowding (i.e., effects of adjacent ﬂankers). Whereas standard crowding would mostly reﬂect exces- sive feature integration (e.g., Pelli et al., 2004; Levi, 2008; Levi and Carney,2009),interference from non-adjacent ﬂankers would mainly reﬂect an increase in positional uncertainty with longer strings (e.g., Butler and Currie, 1986; Huckauf and Heller, 2002a). In other words, processing of target identity might not be harmed so much by non-adjacent ﬂankers, but performance would drop because of loss of information about target position. Given that all prior research has usedsimple identiﬁcation paradigms witheither full or partial report, positional uncertainty would induce incor- rect responses by participants reporting the identity of an item at the incorrect position. In the present study we test for effects of number of ﬂankers while controlling for positional uncertainty by using a two-alternative forced-choice (2AFC) procedure in which the alternative choice was not present in the display. That is, the alternative presented along with the target in 2AFC was never a let- ter present in the stimulus on that trial. If positional uncertainty is the key mechanism driving prior observations of a number of ﬂankers effect, then we ought to see a much reduced effect of this manipulation in the present study. Participants Students from the University of Provence were paid 5 C for their participation, 9 in Experiment 1A (8 women, mean age 22 years) and 17 in Experiment 1B (11 women, mean age 23.7 years). All participants were native speakers of French, and reported having normal or corrected to normal vision. Design and stimuli The stimuli consisted of 15 consonant letters presented in upper- case (B, C, D, F, G, H, J, K, L, M, N, P, R, S, and T). Three factors were manipulated as within-participants variables: visual 2We note that modulation of ﬂanker interference by spatial attention does not con- tradict accounts of crowding as resulting from pooling of information falling in the crowding zone, in as much as attention can boost the target’s capacity to resist the inhibitory action of ﬂanking stimuli (e.g., Grainger et al., 2010). Frontiers in Psychology \| Language Sciences March 2013 \| Volume 4 \| Article 119 \| 2 Chanceaux and Grainger Letter-in-string identiﬁcation FIGURE 1 \| Summary of the conditions tested in the present study in which number of ﬂankers (zero, two, four, six) and unilateral vs. bilateral presentation were manipulated. In all examples the target is the letter “H” presented in the LVF. Flankers in Experiment 1 are other letters. Flankers in Experiments 1A and 2 are Xs. Note that the conditions tested in Experiment 2 are the same as Experiment 1B, except that all stimuli in the contralateral ﬁeld of the bilateral condition are Xs, and with the zero-ﬂanker condition replaced by a six-ﬂanker condition. FIGURE 1 \| Summary of the conditions tested in the present study in which number of ﬂankers (zero, two, four, six) and unilateral vs. bilateral presentation were manipulated. In all examples the target is the letter “H” presented in the LVF. Flankers in Experiment 1 are other letters. Flankers in Experiments 1A and 2 are Xs. Note that the conditions tested in Experiment 2 are the same as Experiment 1B, except that all stimuli in the contralateral ﬁeld of the bilateral condition are Xs, and with the zero-ﬂanker condition replaced by a six-ﬂanker condition. ﬁeld (left or right of ﬁxation), number of ﬂankers (zero, two, or four), and laterality (unilateral or bilateral presentation). All 15 target letters were tested twice in each of the 12 experimen- tal conditions, leading to 360 trials (15 × 12 × 2)/participant. In Experiment 1A different consonants from the set of 15 were used as ﬂankers in the conditions with ﬂankers, and different combina- tions of ﬂankers were used for the different targets. In Experiment 1B all ﬂankers were the uppercase letter X. Procedure Participants were seated and asked to adjust a chinrest so that their eyes were level with the center of the computer screen. For the calibration phase, participants were asked to ﬁxate on dots presented at nine different points on the computer screen. The calibration was repeated after a familiarization phase and then every 30 trials. The sequence of events on a trial was as follows (see Figure 2). First, participants were asked to gaze on the ﬁxa- tion cross that was presented at the center of the computer screen. Participants’ ﬁxation had to ﬁrst stabilize on the cross for 200 ms. If the gaze moved during more than 50 ms outside of an area of 50 × 120 pixels around the ﬁxation cross (0.7˚ right or left), the cross remained on the screen. After 200 ms of continuous ﬁx- ation, the target and ﬂanker stimuli were presented for 200 ms. Stimuli were presented on one or both sides of the center of the screen aligned horizontally. Participants were seated at approxi- mately 80 cm from the monitor. At this distance a letter subtended about 0.6˚ in horizontal extent, and the center of the target was at an eccentricity of 2.7˚. The inter-letter spacing (center to cen- ter) was 0.6˚, such that the center of the most distant ﬂankers in the four-ﬂanker condition were located 3.9˚ from ﬁxation, at the limit of the theoretical extent of the target’s crowding zone (2.7˚ + 2.7˚/2 = 4.05˚; see Figure 2). Design and stimuli In the bilateral pre- sentation condition, the same combination of ﬂankers was used as in the unilateral condition for ﬂankers in the same visual ﬁeld as the target, and a different set of letters were shown in the contralateral ﬁeld. Stimuli were presented in random order with all experimental conditions mixed in a single block. The task was 2AFC, thus each target letter was paired with another letter from the set of 15 consonants, and that was not present in the stimulus display (target and alternative were presented after stimulus presentation). Each of the 15 letters served as the alternative choice on the same number of trials, and were distributed equally across the different conditions. All stimuli were presented in white 21-point Courier New font on a black background. Experiment 1A: different letter ﬂankers One of the participants was excluded from further analysis because of chance-level performance according to a Chi-square test (at 0.05 level). The data from the remaining participants were analyzed in a three-way by participants analysis of variance (ANOVA) with visual ﬁeld (LVF or RVF), number of ﬂankers (zero, two, or four), and laterality (unilateral or bilateral presentation). Percentage correct values averaged across these participants for each exper- imental condition are shown in Figure 4. The analysis revealed main effects of number of ﬂankers, F(2, 30) = 83.73, p < 0.001, and laterality, F(1, 15) = 97.97, p < 0.001, but no effect of visual ﬁeld, F(1, 15) = 0.07, p = 0.79. As in Experiment 1A, the main effect of number of ﬂankers was driven essentially by a large decrease in performance from zero- to two-ﬂankers,t(15) = 10.16, p < 0.001, but the difference between the two- and four-ﬂanker condition was also signiﬁcant, t(15) = 3.37, p < 0.01. There was a signiﬁcant interaction between number of ﬂankers and later- ality, F(2, 30) = 6.94, p < 0.01, with greater ﬂanker interference under bilateral presentation. Again, this interaction was driven mostly by the zero-ﬂanker and two-ﬂanker conditions, for which the partial interaction between number of ﬂankers and laterality Percentage correct values averaged across participants for each experimental condition are shown in Figure 3. We checked indi- vidual accuracy values to determine that all participants were above chance-level using a chi-square test. A repeated measures analysis of variance (ANOVA) was conducted on the mean per- centage correct/participant and condition. Number of ﬂankers (zero, two, four), laterality (unilateral vs. bilateral presentation), and visual ﬁeld (RVF vs. LVF) were within-participants variables. There was a clear effect of laterality with an advantage for unilat- eral presentation, F(1, 8) = 51, p < 0.001, and a strong main effect of number of ﬂankers, F(2, 16) = 149.86, p < 0.001, but no main effect of visual ﬁeld, F(1, 8) = 1.92, p = 0.20, and no signiﬁcant interactions with this factor (Number of ﬂankers ×Visual ﬁeld, p = 0.06, Laterality ×Visual ﬁeld, p = 0.89). Apparatus During stimulus presentation eye ﬁxation had to stay on the centerof thescreen,otherwisethetrialwascanceled.Afterstimulus presentation, a post-mask appeared on both sides of the ﬁxation cross, along with the two-alternative letter responses placed above and below the center of the screen. The mask consisted of two strings of ﬁve hash marks and remained on the screen until partic- ipants responded. The post-mask was accompanied by a post-cue to indicate which letter to identify,using two horizontal bars above and below one of the hash marks. The target letter was always the central letter of the string. Participants were instructed to respond An EyeLink 1000 eye tracker (SR Research Ltd.) was used to control for eye position. The eye tracker had a sampling rate of 1000 Hz and used an automatic saccadic detection algorithm based on a velocity threshold of 30˚/s and an acceleration thresh- old of 8000˚/s2, which corresponds to the cognitive conﬁguration for the EyeLink 1000. Stimuli were presented on a ViewSonic P227 monitor with a refresh rate of 100 Hz and a screen size of 1024 × 768 pixels. Stimulus presentation was controlled with Experiment Builder software (SR Research Ltd.). Eye movements were recorded from the right eye. March 2013 \| Volume 4 \| Article 119 \| 3 www.frontiersin.org Chanceaux and Grainger Letter-in-string identiﬁcation FIGURE 2 \| Sequence of events on a trial in the present study (top left), and distances used (bottom right). Fixation cross for 200 ms, followed by the stimulus display (target and any accompanying letters) for 200 ms, followed by a post-mask accompanied by two horizontal bars placed above and below the target location, plus the presentation of two-alternative letters for the 2AFC response. Inter-letter spacing, eccentricity of the closest letter and the central target letter, and theoretical extent of the crowding zone according to Bouma’s law (shaded ellipse) are shown bottom left. and below the target location, plus the presentation of two-alternative letters for the 2AFC response. Inter-letter spacing, eccentricity of the closest letter and the central target letter, and theoretical extent of the crowding zone according to Bouma’s law (shaded ellipse) are shown bottom left. FIGURE 2 \| Sequence of events on a trial in the present study (top left), and distances used (bottom right). Apparatus Fixation cross for 200 ms, followed by the stimulus display (target and any accompanying letters) for 200 ms, followed by a post-mask accompanied by two horizontal bars placed above p < 0.05. The partial interaction between number of ﬂankers and laterality was signiﬁcant for the zero-ﬂanker and two-ﬂanker con- ditions, F(1, 8) = 11.37, p < 0.01, with stronger effects of adjacent ﬂankers in the bilateral condition (31.7%) compared with the uni- lateral condition (23.8%). On the other hand, the interaction was only marginally signiﬁcant when limited to the two-ﬂanker and four-ﬂanker conditions, F(1, 8) = 5.04, p = 0.06, and was in the opposite direction. as accurately as possible by pressing either the upward arrow key (for the alternative above) or the downward arrow key (for the alternative below) to indicate which letter had appeared at the cued location. Participants had to respond on each trial even if they were unsure of the answer (forced-choice). An audio tone signaled a correct response. After participants’ response a blank screen appeared and the next trial began. The experiment lasted approximately 30 min. Experiment 1A: different letter ﬂankers The main effect of number of ﬂankers was driven mostly by a large decrease in per- formance from zero- to two-ﬂankers, t(8) = 18.54, p < 0.001, but the difference between the two- and four-ﬂanker condition was also signiﬁcant, t(8) = 3.9, p < 0.01.There was a signiﬁcant inter- action between number of ﬂankers and laterality, F(2, 16) = 4.72, March 2013 \| Volume 4 \| Article 119 \| 4 Frontiers in Psychology \| Language Sciences Chanceaux and Grainger Letter-in-string identiﬁcation FIGURE 3 \| Percent correct 2AFC to letter targets under unilateral and bilateral presentation conditions and as a function of the number of ﬂankers in Experiment 1A. Error bars are standard errors. FIGURE 3 \| Percent correct 2AFC to letter targets under unilateral and bilateral presentation conditions and as a function of the number of ﬂankers in Experiment 1A. Error bars are standard errors FIGURE 3 \| Percent correct 2AFC to letter targets under unilateral and bilateral presentation conditions and as a function of the number of ﬂankers in Experiment 1A. Error bars are standard errors. FIGURE 4 \| Percent correct 2AFC to letter targets under unilateral and bilateral presentation conditions and as a function of the number of ﬂankers (Xs) in Experiment 1B. Error bars are standard errors. FIGURE 4 \| Percent correct 2AFC to letter targets under unilateral and bilateral presentation conditions and as a function of the number of ﬂankers (Xs) in Experiment 1B. Error bars are standard errors. FIGURE 4 \| Percent correct 2AFC to letter targets under unilateral and bilateral presentation conditions and as a function of the number of ﬂankers (Xs) in Experiment 1B. Error bars are standard errors FIGURE 4 \| Percent correct 2AFC to letter targets under unilateral and bilateral presentation conditions and as a func (Xs) in Experiment 1B. Error bars are standard errors. AFC to letter targets under unilateral and bilateral presentation conditions and as a function of the number of ﬂankers ars are standard errors. was signiﬁcant, F(1, 15) = 12.59, p < 0.01, but was not signiﬁcant when examining only the two-ﬂanker and four-ﬂanker conditions, F(1, 15) = 0.54, p = 0.47. not signiﬁcant for non-adjacent ﬂankers (two- vs. four-ﬂankers), F(1, 23) = 2.10, p = 0.16. Combined analysis of Experiments 1A and 1B Experiment 1 revealed an effect of the number of ﬂanking letters on letter-in-string identiﬁcation, in conditions where effects of positional uncertainty were minimized by always presenting tar- gets at the center of strings, and using 2AFC. The decrement in performance from no ﬂankers to two-ﬂankers replicates the stan- dard effects of crowding on letter perception (e.g., Bouma, 1970; Huckauf and Nazir,2007; Grainger et al.,2010). The further decre- ment in performance from two-ﬂankers to four-ﬂankers replicates prior observations of an inﬂuence of non-adjacent ﬂankers (Butler In this joint analysis we examined the effect of type of ﬂanker (let- ters vs. Xs) and the effect of number of ﬂankers (see Figure 5). We found a signiﬁcant interaction between type of ﬂanker and number of ﬂankers, F(2, 46) = 5.06, p < 0.05. Flanker effects were greater when the ﬂankers were letters (Experiment 1A) than when they were Xs (Experiment 1B). The partial interaction between number of ﬂankers and type of ﬂanker was signiﬁcant for adjacent ﬂankers (zero- vs. two-ﬂankers), F(1, 23) = 4.73, p < 0.05, but was March 2013 \| Volume 4 \| Article 119 \| 5 www.frontiersin.org Chanceaux and Grainger Letter-in-string identiﬁcation FIGURE 5 \| Percent correct 2AFC to letter targets with letter ﬂankers compared with X ﬂankers as a function of the number of ﬂankers in Experiment 1, and averaging across unilateral and bilateral presentation and visual ﬁeld. Error bars are standard errors. FIGURE 5 \| Percent correct 2AFC to letter targets with letter ﬂankers compared with X ﬂankers as a function of the number of ﬂankers in Experiment 1, and averaging across unilateral and bilateral presentation and visual ﬁeld. Error bars are standard errors. to letter targets with letter ﬂankers compared with X ﬂankers as a function of the number of ﬂankers in Experiment l and bilateral presentation and visual ﬁeld. Error bars are standard errors. presentation favors divided attention across the visual ﬁelds (e.g., Chakravarthi and Cavanagh, 2009). In Experiment 1 the con- tralateral display always contained a distractor letter in the central position that was not the letter X, and this implies that partic- ipants had to process the post-cue in order to know where the target was. Experiment 2 examines to what extent the presence of a distractor letter in the contralateral ﬁeld determined the deployment of attention under bilateral presentation. Combined analysis of Experiments 1A and 1B To do so, ﬂankers were homogeneous Xs with no letter (other than Xs) in the contralateral ﬁeld in bilateral presentation conditions (see Figure 1). and Currie,1986; Strasburger et al.,1991; Heller et al.,1995; Huck- auf and Heller, 2002a,b). These effects of non-adjacent ﬂankers observed with the particular paradigm used in the present study helps rule out explanations of such effects as being due to an increase in positional uncertainty. Effects of number of ﬂankers interacted with the presence or not of a letter string in the contralateral visual ﬁeld. The two- ﬂanker condition showed signiﬁcantly stronger interference com- pared with the zero-ﬂanker condition under bilateral presentation conditions. This is in line with attentional accounts of crowd- ing (Strasburger et al., 1991; Intriligator and Cavanagh, 2001; Strasburger, 2005) according to which attentional deployment determines the extent of the crowding zone. However, contrary to this account, the difference between the two-ﬂanker and four- ﬂanker conditions actually diminished, albeit non-signiﬁcantly, in the bilateral condition, rather than increasing. Nevertheless, this could be due to ﬂoor effects affecting performance in the bilateral four-ﬂanker condition of Experiment 1 (see Figures 3 and 4). The same pattern was found with the type of ﬂanker manipulation (letters vs. Xs), which signiﬁcantly modulated the effects of adjacent ﬂankers (see Figure 5), while the difference between the two-ﬂanker and four-ﬂanker conditions was not sig- niﬁcantly affected by the type of ﬂanker. Again, ﬂoor effects in the four-ﬂanker condition could be driving this pattern. Experi- ment 2 therefore attempts to remove these ﬂoor effects in order to test whether crowding is systematically greater under bilateral presentation as the number of ﬂankers increases, as predicted by attentional accounts of crowding in general, and attentional accounts of effects of non-adjacent ﬂankers in particular. Finally, in Experiment 1 the maximum number of contiguous ﬂankers was four elements, with two to each side of the target. Given the stimulus size and spacing and the viewing distance employed in the present study, the most eccentric ﬂankers in the four-ﬂanker condition were at the limits of the critical spacing as deﬁned by Bouma’s law. It is therefore possible that the extra inter- ference generated by the four-ﬂanker condition compared with the two-ﬂanker condition could be driven by the mechanisms under- lying standard crowding. Experiment 2 therefore tests for effects of number of ﬂankers but this time including a six-ﬂanker condition replacing the no ﬂanker condition. Combined analysis of Experiments 1A and 1B In this condition, the center of the most distant ﬂankers were at 4.5˚ eccentricity, and hence fell outside of the crowding zone as deﬁned by Bouma’s law (see Figure 2). Frontiers in Psychology \| Language Sciences Participants Twenty-two students (6 men and 16 women, mean age 22.9 years) from the University of Provence volunteered to participate. All participants were native speakers of French and reported having normal or corrected to normal vision. Furthermore, in Experiment 1 there was a general advantage for unilateral presentation compared with bilateral presentation, most likely because unilateral presentation favors rapid alloca- tion of attention toward the target location, whereas bilateral March 2013 \| Volume 4 \| Article 119 \| 6 Frontiers in Psychology \| Language Sciences Chanceaux and Grainger Letter-in-string identiﬁcation RESULTS Two of the participants were excluded from further analysis because of chance-level performance according to a Chi-square test (at 0.05 level). The data from the remaining participants (shown in Figure 6) were analyzed in a three-way by participants’ analysis of variance (ANOVA) with visual ﬁeld (LVF or RVF), number of ﬂankers (two, four, or six), and laterality (unilateral or bilateral presentation) as variables. The analysis revealed main effects of number of ﬂankers, F(2, 38) = 6.28, p < 0.01, and later- ality, F(1, 19) = 15.50, p < 0.001, but no effect of visual ﬁeld, F(1, 19) = 0.14,p = 0.72. There was a signiﬁcant difference between the two-ﬂanker and four-ﬂanker conditions, F(1, 19) = 7.10, p < 0.05, and no signiﬁcant difference between the four-ﬂanker and six- ﬂanker conditions, F(1, 19) = 0.38, p = 0.54. Critically, there was no interaction between number of ﬂankers and laterality, F(2, 38) = 1.77, p = 0.47. Finally, there was no interaction between number of ﬂankers and laterality in Experiment 2, in conditions where there was no evidence that ﬂoor effects were affecting performance. Figure 6 shows very clearly that the difference between the two-ﬂanker and four-ﬂanker conditions was almost identical under unilateral and bilateral presentation conditions. This strongly suggests that the Design and stimuli suggests that effects of number of ﬂankers,manipulated here along the horizontal meridian, are at least partly determined by the extent of the crowding zone as determined by Bouma’s law. The design and stimuli were the same as in Experiment 1, with the exception that all distractors in both unilateral and bilateral con- ditions were the letter X, and the number of ﬂankers was two, four, or six. Thus,visual ﬁeld (LVF or RVF) was crossed with Number of Flankers (two, four, or six) and Laterality (bilateral or unilateral) in a 2 × 3 × 2 design. Each letter was the target in 24 trials, leading to 360 trials (15 × 24). Furthermore, the results of Experiment 2 revealed a signiﬁ- cant advantage for unilateral over bilateral presentation on letter- in-string identiﬁcation in peripheral vision, in conditions that arguably optimize target localization prior to presentation of the post-cue, in both the bilateral and unilateral conditions. When contrasted with the results obtained in the bilateral (two- and four- ﬂanker) conditions of Experiment 1B, the results of Experiment 2 conﬁrm that the presence of a distractor letter in the contralateral ﬁeld of the bilateral presentation condition in Experiment 1B was inducing a processing cost. This can be best attributed to the fact that in these conditions there was no cue to target location prior to presentation of the post-cue. This is akin to having two precues for target location as in the Chakravarthi and Cavanagh (2009) study. The relevant conditions in that study were the “Attend 1” and“Attend 2-Bilateral”conditions in an experiment where partic- ipants has to determine the orientation of a target letter T among a circular array of distractor Ts of varying orientation. Target location was indicated by a post-cue. In the “Attend 1” condi- tion, target location was also indicated by a precue, whereas in the “Attend 2-Bilateral” condition, target location was precued along with another location in the opposite hemiﬁeld of the circular dis- play. Chakravarthi and Cavanagh reported an 11% difference in performance between the single precue and dual bilateral precue conditions in their Experiment 1A, which is in line with the 12% difference found in Experiment 1B of the present study. These were the same as in the previous experiments. These were the same as in the previous experiments. GENERAL DISCUSSION In the present study participants had to identify target letters that appeared at a post-cued location under brief stimulus pre- sentations and pattern masking. Target letters could appear in isolation or as the central letter of three-letter, ﬁve-letter, or seven- letter strings, and the ﬂanking elements could either be composed of a set of different letters (Experiment 1A) or a homogenous string of Xs (Experiments 1B and 2). Target and ﬂankers were presented in peripheral vision, to the left or to the right of a cen- tral ﬁxation. Furthermore, the target and ﬂanking stimuli could be presented unilaterally, or bilaterally, with an equivalent num- ber of letters in the contralateral visual ﬁeld. The main ﬁndings of the present study can be summarized as follows: (1) num- ber of ﬂanking stimuli continued to affect letter identiﬁcation beyond the two-ﬂanker conditions of standard crowding experi- ments; (2) homogeneous × ﬂankers facilitated letter identiﬁcation compared with different letter ﬂankers; (3) unilateral presentation facilitated letter identiﬁcation compared with bilateral presenta- tion; and (4) the interfering effects of adjacent ﬂankers (zero- vs. two-ﬂankers) were reduced under unilateral compared with bilat- eral presentation and with X ﬂankers compared with different letter ﬂankers, whereas differences between the two-ﬂanker and four-ﬂanker conditions were not affected by these manipulations. FIGURE 7 \| Crowding zone determined by Bouma’s law without inward-outward asymmetry (upper panel) and with inward-outward asymmetry (bottom panel), following the model proposed by Nandy andTjan (2012). This demonstrates that the inward-outward asymmetry enables a complete non-adjacent letter to fall in the crowding zone in the four-ﬂanker condition of the present study. efﬁciency can be affected by ﬂanker interference and deployment of spatial attention. Here we discuss possible mechanisms under- lying ﬂanker interference effects, and in particular the number of ﬂankers effect found in the present study, before turning to discuss possible relations between crowding and spatial attention. The complete pattern of results reported in the present exper- iments can be captured by the combined inﬂuence of ﬂanking elements and spatial attention. In what follows we discuss possible mechanisms underlying the inﬂuence of each of these factors, and their possible interactions. One key result of the present study is that adding two additional ﬂankers to the standard two-ﬂanker condition, such that targets have two-ﬂankers on either side, causes a signiﬁcant decrease in targetidentiﬁcation,whereasperformancewasnotfurtheraffected by adding two additional ﬂankers (i.e., the six-ﬂanker condition tested in Experiment 2). 3Note that this is not the case in studies manipulating the number of equidistant ﬂankers (e.g., Wilkinson et al., 1997; Pelli et al., 2004; Põder and Wagemans, 2007; Põder, 2008). GENERAL DISCUSSION Given the target eccentricity tested in the present study (see Figure 2),it would appear that Bouma’s law pro- vides a good approximation of the extent of crowding observed with multiple aligned ﬂanking elements. Interference increases as more ﬂankers are added, as long as these ﬂankers fall with the crowding zone deﬁned by Bouma’s law. The fact that the most eccentric ﬂankers in the four-ﬂanker condition only partly fell within the crowding zone (see Figures 2 and 7), would imply that interference is determined by the number of features falling in that region, and not the number of complete ﬂankers. How- ever, when considering possible inward-outward asymmetries in crowding effects, it is possible to argue that it is in fact the number of complete ﬂanking elements that is critical here. DISCUSSION The results of Experiment 2 conﬁrmed the signiﬁcant decrement in performance with four-ﬂankers compared with two-ﬂankers seen in Experiment 1, while revealing no signiﬁcant difference between the four-ﬂanker and six-ﬂanker condition. This strongly FIGURE 6 \| Percent correct 2AFC to letter targets with X ﬂankers under unilateral and bilateral presentation conditions as a function of the number of ﬂankers in Experiment 2. Error bars are standard errors. Note the change in scale of the Y -axis given the reduced effect sizes in Experiment 2 due to the absence of a zero-ﬂanker condition. FIGURE 6 \| Percent correct 2AFC to letter targets with X ﬂankers under unilateral and bilateral presentation conditions as a function of the number of ﬂankers in Experiment 2. Error bars are standard errors. Note the change in scale of the Y -axis given the reduced effect sizes in Experiment 2 due to the absence of a zero-ﬂanker condition. standard errors. Note the change in scale of the Y -axis given the reduced effect sizes in Experiment 2 due to the absence of a zero-ﬂanker condition. FIGURE 6 \| Percent correct 2AFC to letter targets with X ﬂankers under unilateral and bilateral presentation conditions as a function of the number of ﬂankers in Experiment 2. Error bars are March 2013 \| Volume 4 \| Article 119 \| 7 www.frontiersin.org Letter-in-string identiﬁcation Chanceaux and Grainger FIGURE 7 \| Crowding zone determined by Bouma’s law without inward-outward asymmetry (upper panel) and with inward-outward asymmetry (bottom panel), following the model proposed by Nandy andTjan (2012). This demonstrates that the inward-outward asymmetry enables a complete non-adjacent letter to fall in the crowding zone in the four-ﬂanker condition of the present study. effects of non-adjacent ﬂankers were operating independently of spatial attention in the present study. effects of non-adjacent ﬂankers were operating independently of spatial attention in the present study. EFFECTS OF NUMBER OF FLANKERS The two additional ingredients in Nandy and Tjan’s account are the modiﬁcation of lateral connectivity in V1 at peripheral locations by attention allocated to these locations, and the temporal overlap between the duration that attention is allocated to a particular retinal location and the saccade to that location that is elicited by the attention shift. It is the fact that saccades are generally radial with respect to the fovea that causes the foveal-peripheral anisotropy (inward-outward asymmetry) of crowding. Without going into the ﬁner details of this theory, here we simply examined the extent to which parameters derived from Nandy and Tjan’s theoretical analysis could account for the effects of non-adjacent ﬂankers observed in the present study (see their Figure 3D). Given the letter size (0.6˚) and eccentricity (2.7˚) used in the present study, Nandy and Tjan’s parameters were found to predict a crowding zone that extended 1.62˚ inward and 4.32˚ outward.AsshowninFigure7,thisimpliesthatonecompletenon- adjacent letter falls in the crowding zone. In order to account for the effect of non-adjacent ﬂankers in the present study, it therefore would be the number of complete objects falling in the crowd- ing zone that would determine the amount of interference, rather than the number of visual features. Finally, the model correctly predicts no further decrement in performance when comparing the six-ﬂanker condition with the four-ﬂanker condition tested in Experiment 2. attention signiﬁcantly modulated the effects of non-adjacent ﬂankers (comparing zero- vs. two-ﬂankers) but did not signiﬁ- cantly modulate the difference between the two-ﬂanker and four- ﬂanker conditions. The results of Experiment 2 demonstrated that the latter ﬁnding was not due to ﬂoor effects. The absence of an interaction between number of ﬂankers and unilateral vs. bilateral presentation in Experiment 2, contradicts attentional accounts of such effects (Strasburger et al., 1991), and suggests that different mechanisms underlie the way non-adjacent ﬂankers and spatial attention can affect target identiﬁcation. On the other hand, effects of adjacent ﬂankers were signiﬁ- cantly reduced under unilateral compared with bilateral presenta- tion, and signiﬁcantly reduced when ﬂankers were homogeneous Xs compared with different letter ﬂankers. These ﬁndings are in line with prior research showing that that attracting atten- tion to the target location can reduce crowding (e.g., Yeshurun and Rashal, 2010). EFFECTS OF NUMBER OF FLANKERS The results of the present study conﬁrm prior observations of an effect of non-adjacent ﬂanking stimuli in letter identiﬁcation (Butler and Currie, 1986; Heller et al., 1995; Huckauf and Heller, 2002a,b) and digit identiﬁcation (Strasburger et al.,1991). Since all this prior work has used identiﬁcation paradigms with full or par- tial report, performance was driven by a combination of how well information about target identity and target position is processed. This led to the general consensus that the effects of non-adjacent ﬂanking stimuli would be mostly due to positional uncertainty increasing as a function of string length. This was thought to contrast with the effects of adjacent ﬂankers driven mainly by excessive feature integration (Pelli et al., 2004; Levi, 2008), since non-adjacent ﬂankers typically fall outside of the target’s crowding zone (determined by Bouma’s law) and therefore cannot generate interference in this way3. Bouma (1978) was the ﬁrst to propose that the receptive ﬁelds that determine the empirically observed crowding zone are elon- gated toward the periphery, and therefore to the left in the LVF and to the right in the RVF. More recently, Nandy and Tjan (2012) have proposed a theory of crowding that explains this inward- outward asymmetry. This asymmetry is thought to arise via a modiﬁcation of the receptive ﬁeld structure in V1 by the distor- tion of image statistics caused by saccadic eye movements. Nandy and Tjan argue that the inward-outward asymmetric nature of crowding is a consequence of the properties of lateral connectiv- ity in V1 (isotropic and independent of eccentricity) and the fact that the size of receptive ﬁelds of V1 neurons increase linearly with eccentricity (see van den Berg et al., 2010, for an alternative The use of a 2AFC procedure in the present study was designed to limit possible effects of positional uncertainty,since the alterna- tive choice was never present in the display. Therefore, efﬁciency in the processing of target identity is considered to be the main factor driving performance in the present experiments, and this March 2013 \| Volume 4 \| Article 119 \| 8 Frontiers in Psychology \| Language Sciences Letter-in-string identiﬁcation Chanceaux and Grainger account of inward-outward asymmetry based on the same proper- ties of cortical structure). EFFECTS OF NUMBER OF FLANKERS One way to accommodate such attentional inﬂuences within an account of crowding as excessive feature integration is via attention boosting the target’s capacity to resist ﬂanker interference (see e.g., the biased competition theory of spatial attention: Desimone and Duncan, 1995). The question then is why the difference between the two-ﬂanker and four- ﬂanker conditions was not affected by the same manipulations of spatial attention? Considering effects of bilateral vs. unilat- eral presentation, where a possible inﬂuence of ﬂoor effects was excluded in Experiment 2, one possibility is that while two- ﬂankers still enable focusing of attention on the target location, greater numbers of ﬂankers cause a signiﬁcant spread of atten- tion away from the target. In this way target identiﬁcation is still facilitated compared with bilateral presentation conditions, but since ﬂanking stimuli also beneﬁt from allocated attention, ﬂanker interference is not reduced. Our results would therefore reﬂect the combined inﬂuence of crowding and spatial attention on location-speciﬁc processing of letter identities (Marzouki et al., 2013). In support of this general approach, we have shown how apply- ing Nandy and Tjan’s (2012) model of inward-outward asymme- tries in crowding can help explain the pattern of serial position effects found for letter-in-string identiﬁcation in peripheral vision (Chanceaux and Grainger, 2012). However, the obvious critical test of this account of the non-adjacent ﬂanking effects seen in the present study will be to separately examine the effects of outward and inward ﬂankers. This is the object of on-going research. SPATIAL ATTENTION AND EFFECTS OF ADJACENT AND NON-ADJACENT FLANKERS Finally,no effects of visual ﬁeld were found in the present study, andnodifferencesinvisualﬁeldeffectswerefoundbetweenunilat- eral and bilateral presentation. This is in line with prior evidence that visual ﬁeld differences in letter-in-string identiﬁcation vary from being weak to absent, and that effects with random letter strings are systematically smaller than those obtained with words (e.g., Bouma, 1973; Jordan et al., 2003). This might well indi- cate that the very earliest phase of letter string processing involves neural structures (most likely middle occipital gyrus) located in both hemispheres. The results of the present study revealed a clear advantage for post-cued letter-in-string identiﬁcation under unilateral presenta- tion conditions compared with bilateral presentation.We attribute this advantage to the different deployment of spatial attention in these two conditions. In the unilateral condition, a single stim- ulus in one or the other visual ﬁeld will attract attention to the stimulus location, hence facilitating processing of the target. In the bilateral condition, attention will remained deployed across visual ﬁelds until target location has been identiﬁed and atten- tion can be directed to that location. Furthermore, the greater interference seen with different letter ﬂankers compared with homogeneous × ﬂankers, in Experiment 1, can also be explained in terms of differences in the deployment of spatial attention. Homogeneous ﬂankers would facilitate focusing of attention on the target as the odd man out, much like different colored targets were thought to help focus attention in the study by Scolari et al. (2007 see Whitney and Levi, 2011, for a discussion of perceptual grouping, popout, and crowding). REFERENCES Intriligator, J., and Cavanagh, P. (2001). The spatial resolution of visual attention. Cogn. Psychol. 43, 171–216. Chakravarthi, R., and Cavanagh, P. (2009). Bilateral ﬁeld advantage in visual crowding. Vision Res. 49, 1638–1646. Põder, E., and Wagemans, J. (2007). 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Zorzi,M.,Barbiero,C.,Facoetti,A.,Lon- ciari, L., Carrozzi, M., Montico, M., Butler, B. E., and Currie, A. (1986). On the nature of perceptual limits in vision: a new look at lateral masking. Psychol. Res. 48, 201–209. Põder, E. (2008). Crowding with detec- tion and coarse discrimination of simple visual features. J. Vis. 8, 24, 1–6. ACKNOWLEDGMENT This research was supported by ERC grant 230313. ACKNOWLEDGMENT This research was supported by ERC grant 230313. CONCLUSION The present study investigated the inﬂuence of number of ﬂanking elements and the bilateral vs. unilateral nature of the display in a post-cued letter-in-string identiﬁcation task. The results revealed a standard crowding effect in both unilateral and bilateral pre- sentation conditions, with target identiﬁcation being harder when targets were surrounded by two-ﬂankers compared with the no ﬂanker condition. Most important, however, is that a further increase in the number of ﬂankers (from two- to four-ﬂankers) caused a further decrement in performance. This interfering effect of number of ﬂankers was greater for all different letter One key result of the present study concerns the different way in which our two attentional manipulations modulated the effects of adjacent vs. non-adjacent ﬂankers. In Experiment 1, spatial March 2013 \| Volume 4 \| Article 119 \| 9 www.frontiersin.org www.frontiersin.org Chanceaux and Grainger Letter-in-string identiﬁcation more elements enter the crowding zone, plus the way spatial atten- tion, when sufﬁciently focused, boosts the target’s capacity to limit ﬂanker interference. ﬂankers compared with homogeneous × ﬂankers. Finally, ﬂanker interference was modulated by both the laterality manipulation and the type of ﬂanking stimulus when contrasting zero- and two-ﬂankers, but not when contrasting two- and four-ﬂankers. We argue that the most parsimonious account of the complete set of ﬁndings is in terms of ﬂanker interference increasing as ACKNOWLEDGMENT This research was supported by ERC grant 230313. Frontiers in Psychology \| Language Sciences REFERENCES Bonin (New York: Nova Science Publish- ers), 1–24. LanguageSciences,aspecialtyof Frontiers in Psychology. van den Berg, R., Roerdink, J. B. T. M., and Cornelissen, F. W. (2010). A neurophysiologically plausible pop- ulation code model for feature integration explains visual crowd- ing. PLoS Comput. Biol. 6:e1000646. doi:10.1371/journal.pcbi.1000646 Nandy, A. S., and Tjan, S. T. (2012). Saccade-confoundedimagestatistics explain visual crowding. Nat. Neu- rosci. 15, 463–469. Copyright © 2013 Chanceaux and Grainger. This is an open-access arti- cle distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are cred- ited and subject to any copyright notices concerning any third-party graphics etc. Heller, D., Huckauf, A., and Nazir, T. A. (1995). Interaktionseffekte im seitlichen Gesichtsfeld: Eine Erweiterung des Ansatzes von Bouma (1970) [Interaction effects in peripheral vision: an extension of Pelli, D. G., Palomares, M., and Majaj, N.J.(2004).Crowdingisunlikeordi- nary masking: distinguishing feature integration from detection. J. Vis. 4, 1136–1169. Whitney, D., and Levi, D. M. (2011). Visual crowding: a fundamental limit on conscious perception and March 2013 \| Volume 4 \| Article 119 \| 10 Frontiers in Psychology \| Language Sciences
https://openalex.org/W4391038454	https://sciendo.com/pdf/10.2478/amns.2023.2.01676	English	null	Innovation of Chinese Culture External Communication Based on Multivariate Statistical Analysis	Applied mathematics and nonlinear sciences	2,023	cc-by	9,314	Innovation of Chinese Culture External Communication Based on Multivariate Statistical Analysis Pengfei Fan1,†, Beibei Wang2 1. School of Marxism, Xi’an International Studies University, Xi’an, Shaanxi, 710128, China. 2. Chang’an No.8 Middle School of Xi’an, Xi’an, Shaanxi, 710106, China. , y, , , , 2. Chang’an No.8 Middle School of Xi’an, Xi’an, Shaanxi, 710106, China. Communicated by Z. Sabir Received January 22, 2023 Accepted July 7, 2023 Available online December 27, 2023 Communicated by Z. Sabir Received January 22, 2023 Accepted July 7, 2023 Available online December 27, 2023 Applied Mathematics and Nonlinear Sciences https://www.sciendo.com Innovation of Chinese Culture External Communication Based on Multivariate Statistical Analysis Applied Mathematics and Nonlinear Sciences, 9(1) (2024) 1-16 Applied Mathematics and Nonlinear Sciences, 9(1) (2024) 1-16 Keywords: Principal Component Analysis; Factor Analysis; Cultural Communication Model; Pearson Test; Chinese Culture. AMS 2010 codes: 00A73 Abstract With the development of economic globalization, the influence produced by cultural soft power has an irreplaceable role in the comprehensive strength of a country. In this paper, principal component analysis is used to classify the main factors affecting the effect of cultural communication, and factor analysis is combined to construct the effect dimensions of Chinese cultural communication. Pearson’s correlation is used to test the correlation between the effect of cultural communication and the four communication factors: degree of cultural satisfaction, degree of cultural cognition, development prospect and degree of publicity. From the perspective of social media information dissemination, a cultural communication model is constructed by combining social networks and used to analyze the effects of cultural communication. Combined with the effect of Chinese culture’s communication with the outside world, a communication path to optimize communication media to improve media influence rates is proposed. The results show that the correlation coefficient between the communication effect and the satisfaction degree factor is 0.348, and the correlation with the publicity factor is 0.317, of which the most relevant to the communication effect is the cultural cognition factor, with a correlation of 0.412. The number of cultural communicators first shows an increasing trend and reaches the maximum value at the moment of t=7, then gradually decreases from the maximum value and finally decreases to 0 at t=15. The influence of cultural communication in the network increases as the media influence rate increases. †Corresponding author. Email address: fanpengfei6@126.com ISSN 2444-8656 https://doi.org/10.2478/amns.2023.2.01676 © 2023 Pengfei Fan and Beibei Wang, published by Sciendo. This work is licensed under the Creative Commons Attribution alone 4.0 License. https://doi.org/10.2478/amns.2023.2.01676 1 Introduction With the rapid development of China, the dissemination of Chinese traditional culture to foreign countries is also in progress. The scale of the audience is gradually expanding, and more and more foreign friends have a strong interest in learning Chinese traditional culture [1-2]. Culture becomes an important part of comprehensive national power, which is a kind of soft power. The power of soft power comes from diffusion, and soft power is powerful only when a culture is widely spread [3-4]. It is precisely for this reason that, in today’s context where cultural soft power is the focus of competition among countries, it is necessary to focus on the external dissemination of Chinese culture if we want to rise in the forest of culture in the vast world [5]. In terms of the current development needs of China, China is doing a good job in the new era of Chinese outstanding culture of foreign dissemination has become an inevitable practice to show cultural confidence and improve China’s international influence [6]. Based on this, we should always insist on promoting the foreign dissemination of traditional Chinese culture so that Chinese culture can blossom with eye-catching luster and be recognized and loved by foreign audiences [7-8]. However, the promotion and dissemination of Chinese traditional culture has not been synchronized with the development of economic strength, and a variety of factors have made the dissemination effect unsatisfactory. Chinese culture is profound and far-reaching, with a history of 5,000 years up and down through all aspects of life, Xiao et al. believe that traditional Chinese architecture as a carrier of Chinese culture, from the cultural roots, fully explored the existence of traditional Chinese culture, in the guidance of the culture of ecological concepts into practice, planning to promote the organic development of the green city at the same time to promote the traditional culture of China [9]. Sun , L. and Sun, W. took the data of 31 provinces and cities in China in 2015 as an example and explored the impact of the cultural industry on regional economic growth from the perspective of spatial economic research, combining the spatial autocorrelation, spatial lag model, and spatial error model of spatial econometrics Moran’s index model [10]. Dai, Y. https://doi.org/10.2478/amns.2023.2.01676 Pengfei Fan, Beibei Wang. Applied Mathematics and Nonlinear Sciences, 9(1) (2024) 1-16 2 1 Introduction In order to promote the Chinese cultural heritage, utilizing new media and computer technology, Combining web design and three-dimensional model technology, a three-dimensional model-based visualization scheme is proposed to provide technical support for the promotion of traditional culture based on digital technology [11]. Culture as a country’s soft power, the degree of external communication to a certain extent on behalf of the country’s strength of influence, Zhao, F. on the basis of the integration of traditional media and new media, make full use of the characteristics of the new media technology, optimize the dissemination of traditional Chinese culture, the establishment of a new cultural dissemination system for the dissemination of traditional culture to provide a new opportunity for the better promotion and inheritance of Chinese culture [12]. A, Z. X. et al. designed two evolutionary operators inspired by the theory of cultural communication and proposed a new multi-objective evolutionary strategy based on the theory of cultural communication and solved the negative transmission problem by an adaptive information transmission strategy to solve the problem of multi-objective multi-task optimization of cultural communication [13]. Lena, S. D. and Panebianco, F. designed the incomplete information into the standard cultural communication framework to enhance the efficiency of cultural communication by providing a short discussion on the negative speculation of cultural leaders on the population and the negative impact of fostering culture when long-term cultural heterogeneity cannot be guaranteed [14]. This paper first analyzes the process of using principal components by combining multiple random variables into one or several composite indicators through appropriate linear combinations and replacing the original multiple variables with fewer composite indicators in an approximate way. Innovation of Chinese Culture External Communication Based on Multivariate Statistical Analysis 3 Combining the method of factor analysis to analyze the main communication factors in Chinese cultural communication, the cultural communication effect is divided into dimensions. Secondly, the communication mode of cultural information in social media is studied, and a cultural communication model is constructed based on social networks. The cultural communication model is applied to the analysis of the effects of Chinese cultural communication, and the relationship between the media influence rate and the time of communication is explored. Finally, by analyzing the main influencing factors of Chinese culture, the main factors of cultural diffusion can be obtained. 2.1.1 Principal component analysis In general, we often need to work with multiple variables in real-world problems: 1 2 , , , m   (1) (1) 1 2 , , , m   Make statistical inferences. In these problems, the number of variables is often large. With many variables, it is not easy to make decisions. Make statistical inferences. In these problems, the number of variables is often large. With many variables, it is not easy to make decisions. 1 Introduction The model is then used to analyze the effects of Chinese cultural communication on the Internet and to propose corresponding strategies for cultural communication. Combining the method of factor analysis to analyze the main communication factors in Chinese cultural communication, the cultural communication effect is divided into dimensions. Secondly, the communication mode of cultural information in social media is studied, and a cultural communication model is constructed based on social networks. The cultural communication model is applied to the analysis of the effects of Chinese cultural communication, and the relationship between the media influence rate and the time of communication is explored. Finally, by analyzing the main influencing factors of Chinese culture, the main factors of cultural diffusion can be obtained. The model is then used to analyze the effects of Chinese cultural communication on the Internet and to propose corresponding strategies for cultural communication. 1) The idea of principal component analysis 1) The idea of principal component analysis In order to make decisions easily, the original multiple random variables are combined in an appropriate linear fashion into one, or several composite indicators, and then the original multiple variables are approximated and replaced by a smaller number of composite indicators. This kind of composite index combined with the original multiple variables is called the principal component. The principle of principal component selection is: (1) The principal components are linear combinations of the original variables. (2) The principal components are not related to each other. (3) The selected principal components should reflect as much information about the original variables as possible. Sample correlation matrix and its characteristic decomposition Let equation (1) be a standardized random variable whose observed data matrix is: 11 12 1 21 22 2 1 2 m m ij n m n n nm y y y y y y Y y y y y          = =         (2) 11 12 1 21 22 2 1 2 m m ij n m n n nm y y y y y y Y y y y y          = =         (2) (2) Then the sample correlation array: Then the sample correlation array: Pengfei Fan, Beibei Wang. 1) The idea of principal component analysis Applied Mathematics and Nonlinear Sciences, 9(1) (2024) 1-16 4 11 12 1 21 22 2 1 2 1 m m T ij m m m m mm r r r r r r R Y Y r n r r r          = = =         (3) 11 12 1 21 22 2 1 2 1 m m T ij m m m m mm r r r r r r R Y Y r n r r r          = = =         (3) (3) is a symmetric matrix where the elements of the sample correlation array are: 1 1 n ij ki kj k r y y n = =  (4) (4) Assume that the full eigenvalues of the sample correlation array R are: Assume that the full eigenvalues of the sample correlation array R are: ( ) 1 2 1 2 , , , m m         (5) ( ) 1 2 1 2 , , , m m         (5) ( ) 1 2 1 2 , , , m m         ( ) 1 2 1 2 , , , m m         (5) The eigenvectors corresponding to its standard orthogonalization are: The eigenvectors corresponding to its standard orthogonalization are: (6) 1 2 , , , m u u u 1 2 , , , m u u u Construct the diagonal eigenvalue matrix: Construct the diagonal eigenvalue matrix: 1 2 m          =       1 2 m          =       (7) and orthogonal transformation matrices: and orthogonal transformation matrices:   11 12 1 21 22 2 1 2 1 2 , , , m m m m m mm u u u u u u U u u u u u u       = =         11 12 1 21 22 2 1 2 1 2 , , , m m m m m mm u u u u u u U u u u u u u       = =       (8) Then: (8) Then: (1) The random variable 1 2 , , , m   defined by equation (11) satisfies the following three properties: (2) (a) Random variable 1 2 , , , m   is a linear combination of 1 2 , , , m  . (3) (b) Random variable 1 2 , , , m   is uncorrelated two by two. (4) (c) The contribution of random variable 1 2 , , , m   to the original variable is greater from left to right, i.e.,: 1 1 2 1 2 ( 2,3, , ) i i m m i m         −  = + + + + + + (13) (13) Proof Firstly, the conclusion (a) of the theorem is known to be correct by equation (11), and the following is only necessary to prove the correctness of conclusion (b) and conclusion (c). Proof Firstly, the conclusion (a) of the theorem is known to be correct by equation (11), and the following is only necessary to prove the correctness of conclusion (b) and conclusion (c). Then: Then: T U RU =  (9) T U RU =  (9) (9) T U RU =  (9) and R can be decomposed into: and R can be decomposed into: and R can be decomposed into: and R can be decomposed into: T R U U =  (10) T R U U =  (10) (10) 3) Principal component construction 3) Principal component construction Let ( ) 1 2 , , , m   be the above normalized random vector whose matrix of sample observations is given by Eq. (11), notation: Let ( ) 1 2 , , , m   be the above normalized random vector whose matrix of sample observations is given by Eq. (11), notation: ( ) ( ) 1 2 1 2 , , , , , , m m U     = (11) (11) Innovation of Chinese Culture External Communication Based on Multivariate Statistical Analysis 5 Where U is the matrix given by Eq. (11) and Eq. (12) expanded and written as: Where U is the matrix given by Eq. (11) and Eq. (12) expanded and written as: 1 11 1 21 2 1 2 12 1 22 2 1 1 2 2 m m m m m m m mm m u u u u u u u u u             = + + +   = + + +    = + + +  (12) (12) Then 1 2 , , , m   is a linear combination of random variables 1 2 , , , m   . The properties satisfied by the random variable 1 2 , , , m   defined by equation (11) or equation (12) are given by the following theorem. Then 1 2 , , , m   is a linear combination of random variables 1 2 , , , m   . The properties satisfied by the random variable 1 2 , , , m   defined by equation (11) or equation (12) are given by the following theorem. Then: Meanwhile, the sample variance of random variable 1 2 , , , m   is known from (17) and (20), respectively: (20), ( ) 2 1 1 1 , 1,2, , n T ij j j j j i z z z z j m n n  = − = = =  (21) (21) 1 2 m       (22) (22) Therefore, the contribution of random variable 1 2 , , , m   to the original variable: Therefore, the contribution of random variable 1 2 , , , m   to the original variable: Therefore, the contribution of random variable 1 2 , , , m   to the original variable: 1 2 ( 1,2, , ) i m i m     = + + + (23) (23) That is, the contribution of 1 2 , , , m   to the original variable is larger than one from left to right, which shows that the conclusion (c) of the theorem holds. That is, the contribution of 1 2 , , , m   to the original variable is larger than one from left to right, which shows that the conclusion (c) of the theorem holds. Theorem (3) shows that the random variable 1 2 , , , s   constructed from Eq. (11) or (12) is exactly the principal component combined from the original random variable 1 2 , , , m  , where 1  is the first principal component with the largest contribution. Then: rom definition (11), the sample observation matrix of random vector ( ) 1 2 , , , m   is: From definition (11), the sample observation matrix of random vector ( ) 1 2 , , , m   is:   1 2 , , , m Z z z z YU = = (14) (14) Since Y is the observation matrix of the normalized random vector ( ) 1 2 , , , m   , the sum of the elements of the columns of Y are all 0, i.e: Since Y is the observation matrix of the normalized random vector ( ) 1 2 , , , m   , the sum of the elements of the columns of Y are all 0, i.e: 1 0 T T Y = (15) (15) Where 1 [1,1, ,1],0 [0,0, ,0] T T = = , so: Where 1 [1,1, ,1],0 [0,0, ,0] T T = = , so: Where 1 [1,1, ,1],0 [0,0, ,0] T T = = , so: ( ) 1 1 1 0 T T T T Z YU Y U = = = ( ) 1 1 1 0 T T T T Z YU Y U = = = (16) (16) This means that the sum of the elements of the columns of Z is also all 0, i.e: 1 2 0 m z z z = = = = 1 2 0 m z z z = = = = (17) 1 2 0 m z z z = = = = (17) 1 2 0 m z z z = = = = (17) Thus, the sample covariance matrix for random variable 1 2 , , , m   is: Thus, the sample covariance matrix for random variable 1 2 , , , m   is: Pengfei Fan, Beibei Wang. Then: Applied Mathematics and Nonlinear Sciences, 9(1) (2024) 1-16 6    1 2 1 2 1 1 , , , , , , T T m m R z z z z z z Z Z n n = = (18)    1 2 1 2 1 1 , , , , , , T T m m R z z z z z z Z Z n n = = (18)    1 2 1 2 1 1 , , , , , , T T m m R z z z z z z Z Z n n = = (18) Substituting (15), (6) and (13) in turn, we know (18): Substituting (15), (6) and (13) in turn, we know (18): Substituting (15), (6) and (13) in turn, we know (18): Substituting (15), (6) and (13) in turn, we know (18): 1 1 1 ( ) ( ) T T T T T R Z Z YU YU U Y YU U RU n n n = = = = =  (19) (19) n n n To wit: To wit: To wit: To wit: 1 2 1 T m R Z Z n           = = =       (20) (20) Equation (20) shows that the sample covariance of two random variables 1 2 , , , m   is 0, i.e., two random variables 1 2 , , , m   are uncorrelated, i.e., conclusion (b) of the theorem holds. Equation (20) shows that the sample covariance of two random variables 1 2 , , , m   is 0, i.e., two random variables 1 2 , , , m   are uncorrelated, i.e., conclusion (b) of the theorem holds. 2.1.2 Chinese cultural transmission factors Assuming that the number of Chinese cultural communication contents is n , each sample has p indicators, or 1 2 , , , p X X X . This is because the data of each cultural communication content is different and the dimensions are not consistent. Therefore, the collected data must be processed to meet the requirements. The goal is to have a better analysis with X representing the original and standardized variables,  being used to represent the special factors that only have an effect on X , Innovation of Chinese Culture External Communication Based on Multivariate Statistical Analysis 7 and 1 2 , , , ,( ) m F F F m p  being used to represent the standardized common factors. The steps of the factor model are as follows: and 1 2 , , , ,( ) m F F F m p  being used to represent the standardized common factors. The steps of the factor model are as follows: 1) 1 2 ( , , ) p x X X X  =  is the observed random vector with mean vector ( ) E X of 0 and covariance matrix ( ) COV X of , where covariance matrix  is equal to correlation matrix R . 2) 1 2 , , , m F F F F = , where ( ) m p  is the unobservable variables, their mean vector ( ) E F is 0 and covariance matrix ( ) COV F is 1, which means that the components of vector F are independent of each other. 2.1.2 Chinese cultural transmission factors 3) 1 2 ( , , , ,) P    =  is not related to F and the covariance matrix   of ( ) 0, E   = is a diagonal square matrix: 2 11 2 22 2 0 ( ) 0 pp CoV          = =       (24) (24) That is, the components of  are also independent of each other and then the model: That is, the components of  are also independent of each other and then the model: 1 11 1 12 2 1 1 2 21 1 22 2 2 2 1 1 2 2 m m m m p p p pm m p X a F a F a F X a F a F a F X a F a F a F    = + + + +   = + + + +    = + + + +  (25) (25) These steps mentioned above constitute the factor model. Following the above model, it is converted into the following matrix: These steps mentioned above constitute the factor model. Following the above model, it is converted into the following matrix: X AF  = + (26) where the matrix: 11 12 1 21 22 2 A 1 2 a a a m a a a m ap ap apm       =       (27) X AF  = + (26) where the matrix: X AF  = + (26) (26) where the matrix: where the matrix: 11 12 1 21 22 2 A 1 2 a a a m a a a m ap ap apm       =       (27) 11 12 1 21 22 2 A 1 2 a a a m a a a m ap ap apm       =       (27) (27) The above procedure is a factor-loading matrix. The factors are then analyzed to obtain the common factor that has the greatest degree of influence on each factor. This is done by: The above procedure is a factor-loading matrix. 2.1.2 Chinese cultural transmission factors The number of public factors should not be too many, not too few, not too many. If it is too small, it will result in the loss of information about the original variable information, so it is necessary to choose the appropriate number of public factors in factor analysis. 4) Use rotation to make factor variables easier to interpret. The use of rotation provides a better description of the factors and makes the factors interpretable by name. 4) Use rotation to make factor variables easier to interpret. The use of rotation provides a better description of the factors and makes the factors interpretable by name. 5) Naming the common factors. The naming of factors can be decided arbitrarily based on the experience of the researcher without going through strict rules. Note that the designated name must contain the full meaning of the initial variable to which it corresponds. 6) Calculation of factor scores. The parsing results were analyzed and evaluated in depth, the factors were scored comprehensively, and the relevant conclusions were obtained. 6) Calculation of factor scores. The parsing results were analyzed and evaluated in depth, the factors were scored comprehensively, and the relevant conclusions were obtained. 6) Calculation of factor scores. The parsing results were analyzed and evaluated in depth, the factors were scored comprehensively, and the relevant conclusions were obtained. 2.1.2 Chinese cultural transmission factors The factors are then analyzed to obtain the common factor that has the greatest degree of influence on each factor. This is done by: The above procedure is a factor-loading matrix. The factors are then analyzed to obtain the common factor that has the greatest degree of influence on each factor. This is done by: The above procedure is a factor-loading matrix. The factors are then analyzed to obtain the common factor that has the greatest degree of influence on each factor. This is done by: 1) Based on the problems identified, the variables available for analysis are selected, and the applicability of factor analysis to the variables being analyzed is tested. It is important to note that before conducting factor analysis, the researcher must first perform a correlation matrix on the raw data. When the correlation coefficient in the model is greater than 0.3, the model is not applicable to factor analysis. 1) Based on the problems identified, the variables available for analysis are selected, and the applicability of factor analysis to the variables being analyzed is tested. It is important to note that before conducting factor analysis, the researcher must first perform a correlation matrix on the raw data. When the correlation coefficient in the model is greater than 0.3, the model is not applicable to factor analysis. Pengfei Fan, Beibei Wang. Applied Mathematics and Nonlinear Sciences, 9(1) (2024) 1-16 8 2) Extraction of public factors. The main methods of public factor extraction using SPSS include principal component analysis, generalized least squares, generalized least squares and so on. In this paper, principal component analysis is used because this method is widely recognized. 3) Clarify the specific number of factors. The number of public factors should not be too many, not too few, not too many. If it is too small, it will result in the loss of information about the original variable information, so it is necessary to choose the appropriate number of public factors in factor analysis. 3) Clarify the specific number of factors. The number of public factors should not be too many, not too few, not too many. If it is too small, it will result in the loss of information about the original variable information, so it is necessary to choose the appropriate number of public factors in factor analysis. 3) Clarify the specific number of factors. 2.2.1 Modes of dissemination of cultural information The nature of the interaction relationship in social networks is actually a mapping of the real social relationship. According to the principle of six degrees of separation, each participant in social media has a dual identity, that is, the dissemination of information and the receiver. In the complete dissemination process, each participant will be subject to the constraints and influences of all kinds of external factors in the formation of different types of social cliques. These social cliques will gradually expand over time. These social groups will gradually expand, and eventually, multiple social groups interact together, constituting a large-scale social network. The information interaction framework in the social network, as shown in Figure 1, is the main network platforms, media, and recipients of these parts. From the perspective of social network intersection theory, the information dissemination framework of social media still belongs to a class of network information dissemination mode only due to the different roles existing in the network, the media’s specificity and the differentiation. Thus, social media still follow the information dissemination mode, with the characteristics of the dissemination of information, and in the study of government microblogging, it is still possible to follow the theoretical framework of the study. Innovation of Chinese Culture External Communication Based on Multivariate Statistical Analysis 9 Celebrities/ organizations (excluding media) Dissemination of online media Social Networking Platform General communicator/ audience General communicator/ audience In-Network Feedback Inflow of external information Figure 1. Social media and network information communication framework General communicator/ audience In-Network Feedback Dissemination of online media Inflow of external information Figure 1. Social media and network information communication framework 2.2.2 Modeling cultural communication Suppose there is an initial transmitter and m ignorant person. The total number of persons is then 1 N m = + . The probability of a direct transmission of culture by the initial transmitter is . The number of transmitters is then 1 N m = + . Consider multiple transmissions, i.e., a person whose culture is transmitted by the initial transmitter transmits it to others. For example, if initially there are m ignoramuses and in the secondary transmission, one person transmits the culture to another, this corresponds to the selection of two people out of m ignoramuses, both of whom believe in the culture. So, there are 2 2 m C  possibilities for this situation, yielding equation (28): 2 1 1 ( 1) 2 k k m m N m m m C     = + + − ++ ++ (28) (28) That is to say: (1 )m N  = + (1 )m N  = + (29) (29) When there is  initial disseminator, the number of people disseminating culture is: (1 )m N   = + (30) (1 )m N   = + (30) Assume that each initial propagator is able to propagate  person: Assume that each initial propagator is able to propagate  person: N  = (31) N  = (31) By using equations (30)(31), 1 1 m   = − can be obtained. By using equations (30)(31), 1 1 m   = − can be obtained. By using equations (30)(31), 1 1 m   = − can be obtained. Let  be the probability of believing in the culture at the first contact with the cultural communicator, and n be the number of people contacted by each cultural communicator. Then / ( 1) n N    = − , at this point, 1 = .  has different values when there are multiple communicators and they belong to different social groups.  determines the size of the fractal scale Pengfei Fan, Beibei Wang. Applied Mathematics and Nonlinear Sciences, 9(1) (2024) 1-16 10 and also the frequency of contact within or between social circles. He represents the number of groups with the same attributes in the population, e.g., there are multiple classes in a school, multiple schools and multiple companies in a region. 2.2.2 Modeling cultural communication Obviously, when the total number of people is fixed, the larger  is, the number of people within the social circle will relatively decrease, but the number of social circles will become larger. After the transformation, the number of people spread is: 1 1 ( 1) N n N N   −   = +   −   (32) (32) Where n  represents the number of people who believe in the culture after interacting with the culture communicator once. He is related to the number of ignorant people in the social group and the time the culture has existed. Let , n tm    = be the coefficient of proportionality. Obviously the larger the value of  , the longer the effect of cultural communication exists, and the larger the number of ignorant people, the more people will accept the culture. At this point, the number of culture transmitters becomes a time-dependent function: 1 ( ) 1 ( 1) N tm N t N   −   = +   −   (33) (33) The above is the case when there is only one transmitter transmitting culture, and when there are multiple cultural transmitters, it can be shown to have the same conclusion. The above is the case when there is only one transmitter transmitting culture, and when there are multiple cultural transmitters, it can be shown to have the same conclusion. In this study, the participants of the culture were divided into three categories: ignoramuses, transmitters, and immunizers. The initial number of immunizers is 0 because no one will be immune to the culture when it is not yet present. For larger social circles, / ( 1) n N  − has a similar value because most culture spreaders have the same social skills. At this point N can represent the number of smaller social circles. For 1 1/ (1 ) N q −= − , this will appear as an expression containing the exponent q similar to the Tsallis statistic, which is widely used in the study of chaos and fractals. The number of cultural communicators is 1/(1 ) 0 ( ) (1 (1 ) ( ) / ) q S t S q kI t t  − = + − , where 0 S is the number of initial communicators. 2.2.2 Modeling cultural communication In this study, ( ) ( ) I t tI t  , for ease of calculation, is made: 1 0 ( ) ( ) ( ) q q S ds t S t I t dt   − = (36) (36) Considering that a portion of the communicators will eventually become culturally immune, this leads to a system of differential equations for cultural communication: Considering that a portion of the communicators will eventually become culturally immune, this leads to a system of differential equations for cultural communication: novation of Chinese Culture External Communication Based on Multivariate Statistical Analysis Innovation of Chinese Culture External Communication Based on Multivariate Statistical Analysis 11 1 0 1 0 ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) q q q q S dI t S t I t dt S dS t S t I t S t dt dR t S t dt       − −  = −    = −    =   (37) 1 0 1 0 ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) q q q q S dI t S t I t dt S dS t S t I t S t dt dR t S t dt       − −  = −    = −    =   (37) (37) Where  is the immunization rate, i.e., the probability of a cultural transmitter becoming culturally immune. Unlike the SIR model of cultural diffusion, this model has an additional exponent q and a fractal scale . By varying the exponents q and , the model can be made to better simulate the trend of cultural diffusion. 2.2.2 Modeling cultural communication 1 0 ( ) ( ) ( ) ( ) 1 (1 ) (1 ) (1 ) 1 q q S ds t I t I t I t q t q q dt q       −      = + − − + −     −     (34)   1 0 ( ) ( ) ( ) ( ) q q S ds t S t I t tI t dt   − = +  (35) 1 0 ( ) ( ) ( ) ( ) 1 (1 ) (1 ) (1 ) 1 q q S ds t I t I t I t q t q q dt q       −      = + − − + −     −     (34) (34)   1 0 ( ) ( ) ( ) ( ) q q S ds t S t I t tI t dt   − = +  (35) (35) Where ( ) I t  is the derivative of ( ) I t . 3.2 Analysis of the Effectiveness of Chinese Culture Communication In order to better understand the effects of the cultural communication model, the population involved in the process of cultural communication is categorized into five categories: Chinese culture ignorance, Chinese culture beginner, Chinese culture transmitter, Chinese culture receiver, and Chinese culture immunizer. The population trends over time of users in these five states under cultural communication will be numerically simulated to verify the theoretical results in this section. In order to later explore the strategies for effective cultural diffusion in order to provide effective suggestions to relevant departments, we investigate how different parameters affect cultural diffusion. The total population of the system at the initial moment is set to be N(0) = 10000, the number of ignoramuses to be I(0) = 9999, the number of first-time acquaintances to be L(0) = 0, the number of cultural transmitters to be F(0) = 1, the number of receivers to be T(0) = 0, and the number of immunizers to be R(0) = 0, whose unit is the human being. The evolution of the number of users in each state under the cultural propagation model with the propagation time is shown in Fig. 2, which shows that from the moment t=0, the number of ignoramuses has been showing a decreasing trend, and finally, the number of ignoramuses decreases to a minimum value of 0 after t=10. The number of cultural transmitters first shows an increasing trend, reaches a maximum value at t=7, and then gradually decreases from the maximum value, and finally decreases to 0 at t=15. It can be seen that the trend is consistent with the real network. At the beginning of the Chinese culture gradually spread in the network after the emergence of Chinese culture. At this time, the number of users to spread the Chinese culture increased. With time, the culture of the heat gradually decreased, the attraction of the user gradually weakened, and the user gradually no longer paid attention to the culture. At this point, no more users are spreading the culture in the system, and the system is in a stable state. The number of users in the two states of Hesitant and Immunized is similar to that of Culture Propagators, both of which increase first and then decrease. The Ignorant and Culture Receiver experienced the most significant fluctuations in their numbers. 3.1 Analysis of Influencing Factors of Chinese Cultural Communication In this paper, we use the method of principal component analysis to analyze the influencing factors of Chinese culture dissemination, combining the relevant literature to select the four main factors of the degree of cultural satisfaction, the degree of cultural cognition, the pre-development factor, the degree of publicity, the variance interpretation of the factor is shown in Table 1, the eigenvalues of the four public factors are 3.014, 2.966, 1.651, 1.631, respectively, the eigenvalues of the initial factor greater than 1, so the first four public factors are extracted. There are 4, so the first 4 male factors are extracted. The variance contribution rates of the original indicators after the rotation of the 4 male factors are 34.507%, 19.242%, 12.914%, and 10.471%, respectively, and the cumulative contribution rate of the four factors is 77.134%, which indicates that the 4 male factors are able to explain 77.134% of the variance of all the variables, and the degree of explanation is good, and the method of factor analysis can be used to construct the dimensions of Chinese cultural communication effects. Table 1. The variance interpretation of the factor Ingredient Initial eigenvalue Sum of the rotating load squares Total Variance percentage Accumulate% Total Variance percentage Accumulate% 1 4.142 34.507 34.507 3.014 25.112 25.112 2 2.311 19.242 53.749 2.966 24.711 49.823 3 1.551 12.914 66.663 1.651 13.743 63.566 4 1.261 10.471 77.134 1.631 13.589 77.155 The Pearson correlation test of the correlation between the four dimensions of the cultural communication effect, the correlation test of the communication effect and the dimensions are shown in Table 2. The correlation between the four independent variables of the F1 satisfaction factor, F2 cultural cognition factor, F3 development prospect factor, and F4 propaganda effect factor are all less than 0.3, which also indicates that there is no covariance problem between the independent variables. The correlation coefficients between the four factors of F1 satisfaction factor, F2 cultural cognition factor, F3 development prospect factor and F4 propaganda effect factor and the overall effect on the dissemination of Chinese culture are significant p<0.05. The correlation coefficients of the dissemination effect with the satisfaction factor and the propaganda factor are 0.348 and 0.317, Pengfei Fan, Beibei Wang. Applied Mathematics and Nonlinear Sciences, 9(1) (2024) 1-16 12 among which the most correlated with the dissemination effect is the cultural cognition factor, with a correlation coefficient of 0.412. Table 2. 3.1 Analysis of Influencing Factors of Chinese Cultural Communication Correlation test of transmission effect and each dimension F1 Satisfaction factor F2 cultural cognitive factor F3 Development prospects factor F4 publicity effectors Communication effect F1 Satisfaction factor 1 F2 cultural cognitive factor 0.186 1 F3 Development prospects factor 0.231 0.253 1 F 4 publicity effectors 0.203 0.234 0.253 1 Communication effect 0.349 0.412 0.334 0.317 1 Note: ** at level 0.01 (double tail), the correlation was significant. 3.3 Effectiveness of Chinese culture dissemination in the extranet In order to reflect the effect of cultural diffusion of Kwon Chinese culture on the extranet, data from four networks were selected as the platform for analyzing cultural diffusion, and simulations were conducted in each of the following networks. Among them, Networks 1 and 2 are two simulation networks, i.e., BA scale-free network and SW small-world network. Networks 3 and 4 are two real networks, i.e., the social networks Facebook and Twitch, from the Stanford University complex network dataset, respectively. The relevant parameters of the four networks are shown in Table 3. The number of nodes of BA and SW is 5000, and the average clustering coefficients are 0.0214 and 0.2185, respectively. The number of nodes of Facebook and Twitch are 4039 and 9498, respectively, and the corresponding average clustering coefficients are 0.6056 and 0.2011. Table 3. Relevant parameters of the four networks Network name Number of nodes Side number Mean clustering coefficient Node Average The BA scale-free network 5000 59862 0.0214 23.9429 The SW Small World Network 5000 20000 0.2185 8.0 Facebook Network 4039 88236 0.6056 43.6909 Twitch Network 9498 153134 0.2011 32.2462 The spreading effect of Chinese culture on different online platforms is shown in Table 4. The peak spreading value of literature, sports, folklore, tea ceremony, food and dress in Chinese cultural connotation is different on different networks. Among them, in Facebook, the spreading effect of dress culture reaches the highest at t=11.02 until it dissipates at t of 21.26, with a duration of 10.24. In BA, the spreading effect of literature reaches the peak at time 8.98 until it ends at 14.52. Among the four networks, the longer duration of the time effect of Chinese culture dissemination is mainly concentrated in the Facebook and Twitch networks, indicating that dissemination in these two external networks is more effective. Table 4. 3.2 Analysis of the Effectiveness of Chinese Culture Communication The ignoramuses went from an initial 9500 to a final pair of 0, and the culture receivers went from an initial 0 to 8000. Innovation of Chinese Culture External Communication Based on Multivariate Statistical Analysis 13 Figure 2. The evolution of users of each state with the propagation time Figure 2. The evolution of users of each state with the propagation time In order to study the influence of media influence rate in the process of cultural dissemination, in this section, we will simulate the change of cultural communicators over time through experiments when the parameters related to media factors change. The peak value reached by the cultural communicators in the process of communication will be used to determine the influence of Chinese culture in the network and the scope of communication. The values of the parameters of media influence rate are set to 0.1, 0.3, 0.5, 0.7. The changes in the number of cultural communicators when the media influence rate changes are shown in Figure 3. The peak value of cultural communicators in the system is proportional to the media influence rate. The larger the value of media influence rate is, the larger the peak value of cultural communicators is, and vice versa. This indicates that as the rate of media influence increases, the influence of cultural communication in the network decreases. The increase in media influence results in an increase in the number of purveyors of Chinese cultural information in the system, which spreads Chinese cultural information. The results show that an increase in the media influence rate can effectively expand the influence of cultural communication and increase the influence of Chinese culture. Therefore, based on the above analysis, the government should make efforts to improve the credibility of the media in the minds of social network users, and the public should also improve their sense of social responsibility so as to increase the media influence rate, thus achieving the purpose of improving the dissemination of Chinese culture in the network. Figure 3. Changes in the number of cultural communicators Figure 3. Changes in the number of cultural communicators Pengfei Fan, Beibei Wang. Applied Mathematics and Nonlinear Sciences, 9(1) (2024) 1-16 14 3.3 Effectiveness of Chinese culture dissemination in the extranet The communication effect on different network platforms Network name Communication process indicators Literature Physical culture Folk custom Teaism Fine food Dress BA Arrive at peak time 8.98 5.04 4.91 6.24 5.16 5.9 Transmission dissipation time 14.52 13.32 12.41 21.79 9.68 15.67 SW Arrive at peak time 9.06 7.12 7.04 10.28 10.12 10.02 Transmission dissipation time 15.08 14.79 12.88 28.22 14.34 14.92 Facebook Arrive at peak time 14.06 11.98 12.24 10.12 10.16 11.02 Transmission dissipation time 22.21 21.08 18.86 32.24 18.24 21.26 Twitch Arrive at peak time 8.08 5.02 5.94 6.98 7.02 8.15 Transmission dissipation time 15.14 14.16 13.98 22.18 11.15 12.19 Table 4. The communication effect on different network platforms 3.4 Strategies for the External Communication of Chinese Culture 2) Formulate accurate cultural communication content with the help of cultural communication models 2) Formulate accurate cultural communication content with the help of cultural communication models With the help of the cultural communication model and related algorithms, we can formulate better cultural content according to the data on the communication effect of Chinese culture in the outside world. The most relevant factor for the communication effect is the cultural cognition factor, so we can improve the communication effect of Chinese culture by promoting the basic cognition of Chinese culture. 3.4 Strategies for the External Communication of Chinese Culture 1) Optimize communication media and improve media efficiency. 1) Optimize communication media and improve media efficiency. The media bridges the gap between China and the rest of the world, and is one of the necessary conditions in the process of spreading Chinese culture to the outside world. As the country’s demand for soft power enhancement deepens, the media should further strengthen the external communication of Chinese traditional culture represented by Chinese culture. Therefore, fine-tuning the operation of The media bridges the gap between China and the rest of the world, and is one of the necessary conditions in the process of spreading Chinese culture to the outside world. As the country’s demand for soft power enhancement deepens, the media should further strengthen the external communication of Chinese traditional culture represented by Chinese culture. Therefore, fine-tuning the operation of The media bridges the gap between China and the rest of the world, and is one of the necessary conditions in the process of spreading Chinese culture to the outside world. As the country’s demand for soft power enhancement deepens, the media should further strengthen the external communication of Chinese traditional culture represented by Chinese culture. Therefore, fine-tuning the operation of Innovation of Chinese Culture External Communication Based on Multivariate Statistical Analysis 15 overseas social media accounts is an effective way to solve the sloppy operation of new media at the national level and enhance the effect of tea culture dissemination. The Internet’s convenience in the era of new public diplomacy increases the possibility of collaboration between domestic and foreign cultural institutions and media organizations. National media should grasp the opportunity of the times, make good use of the communication resources of overseas media and market rules to jointly build ships, and realize the foreign dissemination of Chinese culture through flexible and diversified cooperation methods. The first step is to continue strengthening international cooperation with major Western media outlets. Enhance the acceptance of foreign audiences through localized narratives of local media, then use the rich resources of overseas communication channels of Western media to enhance the coverage of information, especially to strengthen the amount of investment in new media and the Internet, and finally attract the attention of foreign audiences with the help of internationalized marketing means to enhance the effective interaction on social media. 4 Conclusion Based on the multivariate statistical method, this paper constructs a cultural information dissemination model and uses it to analyze the effect of Chinese culture dissemination to foreign countries. The main conclusions are as follows: 1) The influence factors of Chinese culture dissemination are mainly reflected in the four factors of cultural satisfaction, cultural cognition, pre-development factor, and publicity degree. The variance contribution rates of the original indicators after the rotation of the four male factors are 34.507%, 19.242%, 12.914%, and 10.471%, respectively, and the cumulative contribution rate of the four factors is 77.134%. These four influencing factors can be used to construct the dimensions of Chinese cultural communication effects, as indicated. 2) Under the cultural communication model, the cultural communication effect eventually reaches the peak value positively proportional to the media influence rate. The larger the value of the media influence rate, the larger the peak value of the cultural communicator, and vice versa. Therefore, the media influence rate has a greater impact on the dissemination of Chinese culture. 3) Among the four extranet platforms, among the four networks, the time effect of Chinese culture dissemination maintained for a longer period of time mainly focuses on the Facebook and Twitch networks, which indicates that dissemination in these two extranet networks is more effective. In summary, it can be seen that the influencing factors of Chinese culture dissemination are mainly reflected in the influence rate of media and the dissemination of the extranet. Therefore, by improving the influence rate of national media and targeting cultural dissemination on the extranet, it can effectively improve the dissemination effect of Chinese culture. In summary, it can be seen that the influencing factors of Chinese culture dissemination are mainly reflected in the influence rate of media and the dissemination of the extranet. Therefore, by improving the influence rate of national media and targeting cultural dissemination on the extranet, it can effectively improve the dissemination effect of Chinese culture. Pengfei Fan, Beibei Wang. Applied Mathematics and Nonlinear Sciences, 9(1) (2024) 1-16 16 References [1] Zou, F. (2019). Research on the external communication and translation studies of traditional chinese medicine culture under the background of "one belt and one road ". Advances in Higher Education, 3(4), 116. [2] Dinkelberg, A., MacCarron, Pádraig, Maher, P. J., & Quayle, M. (2021). Homophily dynamics outweigh network topology in an extended axelrod’s cultural dissemination model. Physica A: Statistical Mechanics and its Applications, 578. [3] Boutsi, A. M., Verykokou, S., Soile, S., & Ioannidis, C. (2021). Α pattern-based augmented reality application for the dissemination of cultural heritage. Copernicus GmbH. [4] Yuqing, Z., & Yueming, Y. (2022). 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https://openalex.org/W1982435748	https://periodicos.uem.br/ojs/index.php/ActaSciEduc/article/download/11580/11580	Portuguese	null	Mercantilização do ensino superior: os desafios da universidade diante do atual cenário educacional	Acta Scientiarum. Education	2,011	cc-by	7,528	Mercantilização do ensino superior: os desafios da universidade diante do atual cenário educacional Diego Bechi Programa de Pós-graduação em Educação, Universidade de Passo Fundo, BR-285, 99052-900, Passo Fundo Sul, Brasil. E-mail: diego_bechi@yahoo.com.br RESUMO. O presente trabalho tem por propósito refletir sobre o atual contexto da Educação Superior no Brasil, propiciando o conhecimento de diferentes aspectos que comprovam a importância da ética na formação universitária e a necessidade de contrapô-la à tecnificação do Ensino Superior. Para isso, no primeiro momento, são analisadas as transformações socioculturais ocorridas nas últimas décadas. A apresentação dessas transformações possibilita conhecer as atuais tendências da Educação Superior a partir do processo de globalização, da urbanização, do predomínio do conhecimento e da internacionalização da educação. Com a passagem da “sociedade do trabalho” para a “sociedade do conhecimento”, influenciada pelo desenvolvimento das tecnologias da informação, iniciado em meados do século XX, o conhecimento tornou-se uma mercadoria altamente comercializável. A partir de então, os cursos superiores passaram a ser criados sob a ótica do mercado. A abertura do mercado educacional obteve respaldo na década de 1990, após a implementação de políticas educacionais de cunho neoliberal. Tais iniciativas proporcionaram expressivo aumento do número de IES particulares ou privado/mercantis. Ao compreender essa nova dinâmica que configura o mundo contemporâneo e a influência das políticas públicas no âmbito educacional, são apresentadas as consequências do processo de mercantilização do Ensino Superior e os desafios a serem enfrentados pela Universidade. Palavras-chave: globalização, expansão da Educação Superior, privatização, formação universitária. ABSTRACT. The commodification of higher education: the challenges of university education in face of the present scenario. The purpose of this work is to reflect on the current context of higher education in Brazil, providing knowledge of different aspects that prove the importance of ethics in university education and the need to contrast it to the technological development of higher education. For this, at first, we analyze the socio-cultural transformations that occurred in recent decades. The presentation of these transformations makes it possible to know the current trends in higher education from the process of globalization, urbanization, the prevalence of knowledge and internationalization of education. With the passage from the “society of work” to the “knowledge society”, influenced by the development of information technology, started in the mid-twentieth century, knowledge has become a highly marketable commodity. Since then, higher education majors began to be created from the perspective of the market. The opening of the educational market got support in the 1990s, after the implementation of neoliberal educational policies. DOI: 10.4025/actascieduc.v33i1.11580 DOI: 10.4025/actascieduc.v33i1.11580 Mercantilização do ensino superior: os desafios da universidade diante do atual cenário educacional These initiatives have provided a significant increase in the number of private or private/ commercial HEIs. By understanding this new dynamic that shapes the contemporary world and the influence of public policies in education, this work presents the consequences of the process of commodification of higher education and the challenges faced by universities. Keywords: globalization, expansion of higher education, privatization, university education. Acta Scientiarum. Education As transformações socioculturais na sociedade do conhecimento Ao analisar as transformações socioculturais ocorridas nas últimas décadas, nota-se que a Universidade vem sofrendo fortes interferências pelos anseios da sociedade globalizada. Para Panizzi (2006, p. 28), “vivemos em um tempo em que a universidade e a educação superior tornaram-se, elas próprias, objeto de interesse da própria globalização”. Por causa de seu caráter abrangente e complexo, o processo de globalização atinge todas as relações sociais. Isso acontece pelo fato de o conhecimento ter assumido uma nova dinâmica. Com a denominada revolução da informática, ele passa a ser produzido de forma rápida e cumulativa. Todos aqueles que têm acesso aos novos veículos de informações, seja em casa ou no trabalho, podem divulgar e popularizar seus conhecimentos. A internet diminuiu as distâncias entre as pessoas no que tange à disseminação de experiências e de concepções. Os avanços na comunicação, e a velocidade nas relações são evidentes: blogs, revistas eletrônicas e sites de relacionamentos e de pesquisas multiplicam, de forma explosiva, a exploração e a geração do conhecimento. Diante das modificações socioculturais que envolvem o mundo do trabalho, o predomínio e a valorização do conhecimento, e a própria educação, estão colocando novos desafios à Universidade. É preciso superar o modelo pedagógico, até então predominante, baseado na mera transmissão vertical de conhecimentos e das técnicas específicas ao exercício profissional. Para que os sujeitos sejam capazes de responder à complexidade e às exigências do mundo contemporâneo, a Universidade precisa expandir seu processo formativo para além das atividades limitadas à preparação para o mercado de trabalho. Segundo Panizzi (2006, p. 30), “não podemos mais formar os nossos estudantes como até agora fazíamos. Hoje, “ensinar”, “aprender”, “conhecer” e “ter competência” apresentam outros significados e valores, e não são a mesma coisa. Pensar não pode ser considerado mero diletantismo e abstração!”. g ç Essa nova dinâmica de produção e de divulgação do conhecimento é uma das características centrais do mundo contemporâneo. De acordo com Kesselring (2007, p. 23), “a chamada sociedade ‘pós- moderna’ é uma sociedade do conhecimento”. As tecnologias de informação, juntamente com os conhecimentos por ela difundidos, promoveram grandes transformações econômicas e sociais. Enquanto, na sociedade industrial, as ferramentas tecnológicas eram desenvolvidas com a exclusiva finalidade de facilitar o trabalho e de aumentar a produção, os conhecimentos atribuídos à técnica, atualmente, passaram a ser utilizados em prol da divulgação do próprio conhecimento. Introdução Maringá, v. 33, n. 1, p. 139-147, 2011 pelo ideário neoliberal, especialmente no campo das políticas educacionais, colocaram em cheque a natureza e o papel da Universidade. No mundo contemporâneo, com a denominada revolução da informática e os avanços no âmbito da comunicação, o conhecimento passou a ser altamente comercializável. Para poder explorar com mais intensidade o seu potencial lucrativo, na década de 90, foram estabelecidas novas leis nas quais Para compreendermos a problemática que envolve a mercantilização do Ensino Superior é necessário, primeiramente, refletir sobre algumas das transformações sócio-político-culturais ocorridas nos últimos anos. Os processos de globalização mediados pelo desenvolvimento das tecnologias da informação, somados aos objetivos e ações propostas Maringá, v. 33, n. 1, p. 139-147, 2011 Acta Scientiarum. Education 140 Bechi Bechi propiciaram expressivo aumento do número de instituições de Ensino Superior privado/mercantis. este estudo, são apresentadas as características que compõe o atual cenário educacional e as consequências provenientes do modelo de gestão empresarial e das alterações administrativas nas universidades. Por fim, são descritos os desafios e as reais funções da Universidade frente a uma sociedade complexa e dinâmica que exige dos sujeitos criticidade, flexibilidade e desenvolvimento da consciência ética, comprometida com o social. ç p p O atual estado de expansão e privatização do Ensino Superior no país vem transformando a Educação em mercadoria, ou seja, em unidade de negócios de cursos e atividades. Para que as instituições, gerenciadas por uma proposta de gestão empresarial, possam garantir sua sustentabilidade econômica e financeira, o produto ofertado deve ser flexível às exigências ditadas pelo mercado. Frente a este contexto, o desenvolvimento científico e cultural da comunidade acadêmica vem sendo prejudicado pela eliminação e pela reorganização das atividades que não agregam valor econômico (MÜHL, 2009). Com isso, as disciplinas de formação humana e social vêm sendo gradativamente extintas da estrutura curricular, a fim de tornar as mensalidades mais acessíveis aos alunos/clientes, o que possibilita uma saída para os cursos universitários se tornarem mais competitivos frente o atual cenário educacional. As disciplinas de ordem prática passam a ser valorizadas em detrimento das disciplinas teóricas ligadas à formação integral do ser humano. Essas transformações estão relacionadas ao surgimento de algumas contradições no seio da Universidade. Essas contradições deram origem ao que Boaventura de Souza Santos denominou de três grandes crises da universidade: “crise de hegemonia”, “crise de legitimidade” e a “crise institucional”. Acta Scientiarum. Education As transformações socioculturais na sociedade do conhecimento “Pela primeira vez na história da humanidade, a técnica não visa mais a resguardar, antes de tudo, o corpo do homem, mas ela deve, em primeiro lugar, expandir seu patrimônio intelectual” (KESSELRING, 2007, p. 23). A passagem da sociedade do trabalho para a Cabe agora compreender com maior exatidão a problemática até então posta de forma superficial. A primeira parte do texto tem por propósito analisar as modificações socioculturais pós-modernas que contribuíram para o atual processo de expansão e de mercantilizarão do Ensino Superior no país. Após Maringá, v. 33, n. 1, p. 139-147, 2011 Mercantilização do ensino superior 141 grandes metrópoles, onde há maior concentração e aprofundamento dos problemas sociais. sociedade do conhecimento trouxe consigo uma série de problemas que envolvem a vida cotidiana das pessoas. Essa nova onda tecnológica influenciou profundamente as relações sociais. Em tese Panizzi (2006, p. 25) salienta que, “já não somos mais os mesmos. Também não agimos mais da mesma forma”. No mundo contemporâneo, as grandes metrópoles formam “os espaços de excelência do fenômeno de expansão da economia globalizada” (PANIZZI, 2006, p. 19). Nessa segunda transformação da sociedade contemporânea, a crescente e descontrolada urbanização agrava os problemas socioeconômicos, aumentando os índices de desemprego e de violência. Além disso, é crescente o número de pessoas que vivem em favelas, lugar onde o espaço e as condições de vida são desumanos. Tendo ou não consciência dessa realidade, as pessoas continuam optando por morar nas grandes cidades. Por já se encontrarem em situações desfavoráveis em cidades menores ou no interior, muitas delas têm por principal propósito a conquista de um emprego e de uma melhor qualidade de vida. Outros se arriscam em busca de investimentos e demais vantagens encontradas exclusivamente nos grandes centros. Apesar dos problemas citados acima, esses locais constituem-se no espaço mais comum de instauração “das grandes corporações, das instituições financeiras, das redes de informações, dos sistemas de telefonia celular e de comunicação por cabo, dos bens de consumo sofisticados e dos serviços especializados” (PANIZZI, 2006, p. 19). Cria-se um emaranhado de relações e de trocas de bens e serviços, em que algumas pessoas conseguem conquistar sua autonomia financeira em meio ao mercado de trabalho, enquanto outras não conseguem sair de sua condição de miséria. As transformações socioculturais na sociedade do conhecimento Entre as mudanças ocorridas na sociedade e os problemas a ela atribuídos, estão a “globalização da vida e do trabalho”, a “crescente e desigual urbanização”, o “predomínio do conhecimento” e a “internacionalização da educação”1. Ao se adentrar no primeiro aspecto, nota-se que o mundo do trabalho assumiu novas atribuições na sociedade pós-industrial: as profissões estão passando por um processo de transformação, exigindo um novo perfil profissional e pessoal. “O novo perfil profissional passa a ser aquele que não se esgota na conclusão de um curso superior, devendo permanecer, portanto, constantemente atualizado, fato que exige formação permanente” (DALBOSCO, 2009, p. 59). Essas transformações têm origem na forma como o conhecimento passa a ser valorizado nessa fase de grande evolução tecnológica. Não basta ser diplomado, é necessário ser flexível diante das exigências do mercado. Uma das características do mundo contemporâneo é o aparecimento de novas e de diversas profissões para atender as demandas do mundo do trabalho. “As profissões deixaram de ser apenas aquelas clássicas que tínhamos, como Direito, Engenharia e Medicina” (PANIZZI, 2006, p. 16). Dessa forma, rompe-se a estabilidade das profissões relativas à era industrial. Hoje, o sujeito deve ser capaz de superar a fragmentação dos saberes próprios das profissões tradicionais. É necessária a interação com as diferentes áreas, indo além dos conhecimentos isolados. Tendo por base os conhecimentos específicos de cada área profissional, o sujeito deve criar novas formas de atuação, atitude pela qual lhe exigirá maior demanda de conhecimentos. Para isso, é imprescindível que o mesmo supere seu caráter individualista e passe a observar e a pensar coletivamente. Pensando na formação do novo profissional, Panizzi (2006, p. 16- 17) sustenta a ideia de que “uma boa formação foge de uma qualificação isolada e individual. Hoje, não temos mais uma formação individualizada. O sujeito pensante não se constrói mais se olhando sozinho, ao contrário, o olhar força para o coletivo”. Contudo, as disputas e as dificuldades de se inserir no mercado de trabalho se agravam ainda mais nas Em meio à vida globalizada, muitas pessoas conseguem estabelecer relações econômicas; porém, outros milhões são vítimas de uma sociedade excludente e sofrem os efeitos dos reais anseios de quem detém o poder. Outro problema que vem se agravando é o aumento da população de idosos nos países em desenvolvimento. 1 Sobre isso ver: PANIZZI, W. Universidade para quê? Porto Alegre: Libretos, 2006. Acta Scientiarum. Education Acta Scientiarum. Education 1 Sobre isso ver: PANIZZI, W. Universidade para quê? Porto Alegre: Libretos, 2006. As transformações socioculturais na sociedade do conhecimento Tendo em vista essa problemática, Dalbosco (2009, p. 61) faz algumas considerações acerca da postura pedagógica a ser assumida pelos educadores frente a esse novo cenário. De acordo com esse pesquisador, os estímulos cognitivos e estéticos resultantes da gigantesca circulação de informações “exigem dos educadores posturas pedagógicas inovadoras, capazes de se colocar muito além da relação pedagógica vertical baseada no binômio transmissão-assimilação do conhecimento, segundo o qual o professor ensina e o aluno aprende”. O ato de ensinar e de aprender deve superar o modo instrumental baseado na pura transmissão de saberes. Em síntese, a “nova pedagogia”, as experiências e o potencial investigativo do aluno devem ser explorados em sala de aula. Com o auxílio e com a mediação do professor, o aluno torna-se sujeito ativo na construção do seu conhecimento. A Educação deve ir ao encontro da formação da autonomia do homem contemporâneo. Panizzi (2006, p. 25) direciona toda essa problemática para o campo da universidade, uma vez que a pesquisadora entende que “uma nova pedagogia se impõe a partir dos diferentes papéis que são atribuídos aos professores e aos estudantes. Como a universidade reage a isso?”. Estando intimamente ligada a essas três transformações recém citadas, a internacionalização da Educação é mais um dos frutos do estado de globalização do mundo pós-moderno. Essa última transformação tem origem no intenso processo de cooperação internacional que há várias décadas vem se revelando importante para a atualização dos professores, dos pesquisadores e para melhorar as estruturas do sistema de Ensino Superior. Essa troca de experiências é possível porque o conhecimento é flexível nos diferentes contextos, circulando de um lugar ao outro sem perder seu valor. Entretanto, a exploração das fronteiras do conhecimento, por meio dos intercâmbios acadêmicos, vem trazendo graves implicações para os sistemas educacionais. Ao alertar sobre essa problemática, Panizzi (2006, p. 27) afirma que “é preciso estar atento para o significado dessas relações quando as grandes corporações de ensino se instalam e estabelecem uma forma única de pensar”. Diante dessa nova dinâmica que configura o mundo contemporâneo, o conhecimento parece estar enfrentando uma crise de identidade. Nunca, na história da humanidade, as pessoas tiveram acesso aos serviços de comunicação com a facilidade e a intensidade como vem ocorrendo atualmente. Porém, muitas delas possuem uma compreensão equivocada sobre o real significado da palavra conhecimento: confundem-no com a mera transmissão de informações. As transformações socioculturais na sociedade do conhecimento Há um novo padrão demográfico mundial “marcado pelos baixos níveis de fecundidade e de mortalidade, acompanhado de um crescente envelhecimento da população e de um aumento dos fluxos migratórios internacionais” (PANIZZI, 2006, p. 21). Nesse sentido, há constante redução de recursos humanos jovens e qualificados. Se não houver um planejamento capaz de superar essa deficiência, com o passar dos anos, agravar-se-ão ainda mais os problemas sociais hoje existentes. Para isso, é necessário que os políticos governantes assumam o desafio de qualificar e de promover oportunidades profissionais aos jovens. Isso ajudará a aumentar as suas expectativas e, consequentemente, haverá diminuição dos fluxos migratórios. Na acepção de Panizzi (2006, p. 22), os Maringá, v. 33, n. 1, p. 139-147, 2011 Acta Scientiarum. Education 142 Bechi que a pessoa que limita seus saberes a simples reportagens televisivas ou à leitura de pequenos fragmentos de conteúdos obtidos via internet, dificilmente terá capacidade intelectual e conhecimentos suficientes para avaliar criticamente a variedade de estímulos oriundos de sua relação com o meio social. dirigentes políticos têm o desafio de “propiciar aos nossos jovens não só uma formação educacional avançada em termos científicos e tecnológicos, mas também de promover oportunidades de sua absorção nos sistemas produtivos e respectivos projetos de desenvolvimento”. A terceira transformação sociocultural é atribuída à mercantilização do conhecimento. Com o grande desenvolvimento tecnológico iniciado em meados do século XX, o conhecimento e a Educação Superior obtiveram altas proporções econômicas. As tecnologias voltadas à comunicação transformaram o valor das coisas e dos bens. “As coisas passaram a ser diferentes e a ter valor diferente” (PANIZZI, 2006, p. 26). O conhecimento passou a ser visto como uma mercadoria altamente comercializável, indo ao encontro dos interesses da sociedade capitalista. E, para aguçá-los ainda mais, ao contrário do que acontece com os bens materiais, as ideias, ao serem vendidas, continuam em posse de quem as ofertou. Assim, uma única pessoa ou instituição poderá utilizá-las por inúmeras vezes, dependendo dos interesses da sociedade. Tendo por princípio essa lógica, a partir da década de 90, os cursos superiores passaram a ser criados sob a ótica do mercado. Conforme salienta Sguissardi (2008, p. 1013), essa tendência capitalista de transformar tudo em mercadoria, inclusive o conhecimento, abre a possibilidade de entender por que “os serviços educacionais, como um direito e um bem público, possam ser considerados como uma mercadoria, a educação-mercadoria, objeto de exploração de mais- valia ou de valorização”. Acta Scientiarum. Education Maringá, v. 33, n. 1, p. 139-147, 2011 Atual cenário educacional: a expansão do Ensino Superior e as crises enfrentadas pela Universidade Diante dessas quatro transformações gerais que caracterizam o “novo mundo”, espera-se que a universidade utilize suas experiências e seus conhecimentos em prol das expectativas e das necessidades da sociedade. É preciso romper o modelo acadêmico e pedagógico, predominante no Brasil e em toda a América Latina, voltado para atender apenas às necessidades do mundo do trabalho. A universidade não deve possuir como única função o licenciamento, por meio de diplomas e certificados, do exercício profissional. “No momento em que ela assume essa tarefa de autorizar o exercício da profissão, sem dúvida ela se engessa, se limita e, mais, se homogeneíza. Homogeneíza a si mesma – a universidade – e também o sistema educacional como um todo” (PANIZZI, 2006, p. 32). O ideal de universidade a ser construído deve ter o propósito de promover o desenvolvimento de competências e de aprendizagem nos alunos, tornando-os aptos a visualizar e a solucionar os problemas de forma independente e flexível. A prática pedagógica voltada à pura e simples transmissão de conhecimentos deve ser substituída por um modelo pedagógico embasado na aprendizagem de novas técnicas, novos métodos e novos processos capazes de integrar as pessoas à sociedade. Ao iniciar a análise sobre o modelo de expansão e de mercadorização da Educação Euperior no Brasil, é importante refletir sobre as seguintes questões: a Educação pode ser considerada um serviço comercial? As regras que valem para uma empresa podem ser literalmente transpostas para o Ensino Superior? A iniciativa privada em posse das IES terá por principal propósito a promoção de um ensino de qualidade? Que consequências a projeção cada vez maior do mercado educacional trará para o sistema universitário? Tendo em vista o atual cenário da educação superior, Sguissardi (2008, p. 995) afirma que a transformação da Educação em mercadoria é um fenômeno que “tenderá a abarcar todo o planeta, e, no caso, todo o espaço do sistema educacional”. No Brasil, a Lei de Diretrizes e Bases da Educação Nacional (LDB), aprovada em dezembro de 1996, possibilitou a edição de novos decretos a fim de normatizar a criação e a expansão das IES. A comercialização da Educação Superior teve amparo legal a partir da promulgação do Decreto nº 2.306, de 10 de agosto de 1997. 2 O Processo de Bolonha é resultado de um conjunto de resoluções assinadas em 1999 pelo Conselho de Ministros da União Europeia e fixadas na denominada “Declaração de Bolonha”, a fim de “melhorar a qualidade do ensino e da pesquisa através de uma intensificação da cooperação entre as universidades europeias” (KESSELRING, 2007, p.16). Mercantilização do ensino superior 143 mercantilização da Educação Superior, ocorrida nos últimos anos. Com a expansão das instituições de ensino, juntamente com o aumento da competitividade, a Universidade precisou assumir novas estratégias. Para garantir suas atividades no mercado educacional, muitas delas optaram por encurtar o tempo de duração dos cursos e passaram a incentivar a formação de tecnólogos. Outras, a fim obter maior lucratividade, aumentaram demasiadamente o número de alunos, pondo em risco a qualidade do ensino. Por esses e outros motivos, pode-se inferir que Universidade não está atendendo às demandas e às necessidades que a sociedade apresenta neste novo século. Na interpretação de Kesselring (2007, p. 28), o sistema universitário atual possui “poucas chances de futuro, já que uma grande parte de seus estudantes não chega a concluir os estudos ou não consegue se integrar ao mercado de trabalho”. internacionais vêm aumentando gradativamente seus investimentos, criando novos cursos e novos centros educacionais em todo o Brasil. Essa crescente competitividade em torno da Educação Superior põe a Universidade em sérios riscos, pois “uma das mudanças mais ‘perigosas’ para a atual estrutura da universidade é a possibilidade de que venha perder o seu privilégio nacional de expedidora de títulos acadêmicos” (PANIZZI, 2006, p. 27). Para a Universidade continuar garantindo sua legitimidade, é necessário que a mesma assuma, de modo crítico, a remodelagem das estruturas do Ensino Superior propostas por outros países, como é o caso do Processo de Bolonha na Europa2. A aceitação ou não de tais modelos deve passar por um processo de compreensão e de análise das referidas propostas, já que, conforme observa Panizzi (2006, p. 29), “(...) quando por nós apropriados, no entanto, devem ser considerados como resultados de uma experiência e não como modelo paradigmático a ser perseguido, já que tem como base suas respectivas realidades”. As transformações socioculturais na sociedade do conhecimento Ao contrário disso, o conhecimento está intimamente ligado à formação intelectual do homem. Essa, por sua vez, independe dos conteúdos decorrentes dos sistemas digitais. “O ‘conhecimento’ fruto da tecnologia é algo completamente diferente do conhecimento que tem como fonte o cérebro. ‘Conhecimento’ digital não é nada mais que informação” (KESSELRING, 2007, p. 24). Contudo, não se pode negar que há muitas informações relevantes ao se ter acesso ao mundo digital, mas esse contato deve ser realizado com cautela, pelo grande número de informações falsas oferecidas aos leitores. A partir disso, pode-se dizer Por movimentar bilhões de dólares, a Educação Superior passou a ser fortemente disputada. Com a finalidade de obter altos lucros, empresas nacionais e Maringá, v. 33, n. 1, p. 139-147, 2011 Acta Scientiarum. Education Mercantilização do ensino superior Acta Scientiarum. Education Bechi Bechi Segundo dados do INEP3, nos últimos anos houve um acelerado aumento do número de instituições e de matrículas no setor privado. No que tange o crescimento do número de IES, entre os anos 1994-2006 verificou-se um aumento de 13,7% das instituições públicas, muito pouco, se comparado ao aumento de 219,4% das instituições privadas. Essa desproporcionalidade apresenta-se também no processo de privatização das matrículas, ou seja, durante esses 12 anos, as públicas cresceram 75 contra 275, 2% das privadas. Assim o fenômeno que antes era próprio do mercado financeiro, industrial e comercial atingiu também o Ensino Superior. Com o processo de globalização, somados à valorização do conhecimento e às novas exigências da sociedade atual, o ensino tornou-se um mercado promissor, digno de grandes investimentos. Diante destas mudanças, “muito poucas diferenças parecem existir entre o modus faciendi dos protagonistas ou “consolidadores” do mercado educacional e dos empresários do mercado financeiro, industrial comercial ou outros” (SGUISSARDI 2008, p. 1015). Ao serem organizados de acordo com as perspectivas da economia de mercado, os Cursos Superiores começaram a sofrer inúmeras mudanças internas. Por estarem em consonância com o modelo empresarial, os principais agentes do processo de ensino e aprendizagem assumiram novos papéis: o aluno passou a ser considerado um cliente e o professor assumiu a função de prestador de serviços. Para atender aos desejos dos alunos/clientes os resultados a serem obtidos são de natureza imediata. As disciplinas de formação humana e sociais estão sendo extintas, restando apenas as disciplinas específicas referentes aos cursos oferecidos. Com a retirada dessas disciplinas, a duração dos cursos é encurtada, diminuindo os custos das mensalidades. Ao torná-las mais acessíveis à população, há aumento da competitividade da instituição no mercado educacional. “Competir e, no máximo, garantir a empregabilidade dos estudantes/clientes são as preocupações que afligem e ao mesmo tempo se põem como desafio para todos” (SGUISSARDI, 2008, p. 1015). Nesse sentido, ao mudarem os objetivos, mudaram-se também as atividades a serem realizadas. Em outras palavras, enquanto as finalidades da Educação forem reduzidas ao mero aperfeiçoamento profissional, as atividades assumirão apenas uma conotação prática, deixando os elementos teóricos em segundo plano. 3 Os dados do INEP utilizados no presente artigo são extraídos do texto: SG SS O ensino baseado somente na preparação para o mercado de trabalho não vem ao encontro das exigências do mundo contemporâneo. 3 Os dados do INEP utilizados no presente artigo são extraídos do texto: SGUISSARDI, V. Modelo de expansão da educação superior no Brasil: predomínio privado/mercantil e desafios para a regulação e a formação universitária. Educação e Sociedade, v. 29, n. 105, p. 991-1019, 2008. Atual cenário educacional: a expansão do Ensino Superior e as crises enfrentadas pela Universidade Ao obter esse reconhecimento, a educação superior tornou-se “objeto de lucro ou acumulação; uma mercadoria ou a “educação-mercadoria” de interesse dos empresários da educação, que viria se completar com seu par gêmeo de interesse de todos os empresários dos demais ramos industriais e comerciais, a “mercadoria-educação”” Porém, a realidade do Ensino Superior não condiz com esses anseios. A Universidade vem enfrentando vários problemas em decorrência da ç (RODRIGUES apud SGUISSARDI, 2008, p. 1000-1001). (RODRIGUES apud SGUISSARDI, 2008, p. 1000-1001). Maringá, v. 33, n. 1, p. 139-147, 2011 Acta Scientiarum. Education 144 Bechi “A universidade hoje, tal qual se apresenta, não responde às necessidades e as urgências do tempo presente” (PANIZZI, 2006, p. 46). O modelo acadêmico e pedagógico não está respondendo às demandas que a sociedade vem apresentando neste novo século. A defasagem na formação acadêmica, decorrentes dos novos interesses da Educação Superior, traz grandes prejuízos à carreira profissional dos graduandos. Os anseios de um futuro profissional promissor giram em torno apenas de promessas, principalmente a aqueles que esperam obter um bom retorno financeiro. No início do curso, tudo parece ser fácil e vantajoso; porém logo a seguir surgem as dificuldades impostas pela sociedade globalizada e os estudantes, despreparados, passam a enfrentar os desafios do exercício profissional e da vida em sociedade. Isso acontece porque o “nosso modelo acadêmico e pedagógico está ainda organizado para atender as necessidades e demandas de um mundo do trabalho que já não existe mais nos termos de um modelo de desenvolvimento baseado na era industrial clássica” (DALBOSCO, 2009, p. 65). Segundo dados do INEP3, nos últimos anos houve um acelerado aumento do número de instituições e de matrículas no setor privado. No que tange o crescimento do número de IES, entre os anos 1994-2006 verificou-se um aumento de 13,7% das instituições públicas, muito pouco, se comparado ao aumento de 219,4% das instituições privadas. Essa desproporcionalidade apresenta-se também no processo de privatização das matrículas, ou seja, durante esses 12 anos, as públicas cresceram 75 contra 275, 2% das privadas. Assim o fenômeno que antes era próprio do mercado financeiro, industrial e comercial atingiu também o Ensino Superior. Com o processo de globalização, somados à valorização do conhecimento e às novas exigências da sociedade atual, o ensino tornou-se um mercado promissor, digno de grandes investimentos. Diante destas mudanças, “muito poucas diferenças parecem existir entre o modus faciendi dos protagonistas ou “consolidadores” do mercado educacional e dos empresários do mercado financeiro, industrial comercial ou outros” (SGUISSARDI 2008, p. 1015). Ao serem organizados de acordo com as perspectivas da economia de mercado, os Cursos Superiores começaram a sofrer inúmeras mudanças internas. Por estarem em consonância com o modelo empresarial, os principais agentes do processo de ensino e aprendizagem assumiram novos papéis: o aluno passou a ser considerado um cliente e o professor assumiu a função de prestador de serviços. Para atender aos desejos dos alunos/clientes os resultados a serem obtidos são de natureza imediata. Acta Scientiarum. Education Bechi Essa dicotomia tornou-se mais radical em razão de a Universidade ter perdido a condição de única detentora do conhecimento. Na sociedade globalizada não existem fronteiras para o conhecimento. O desenvolvimento das tecnologias da informação, e o expressivo crescimento dos cursos superiores marcam a passagem do ensino de “elite” para o ensino de “massa”. Dados do INEP revelam que em 1999 o país obteve um total de 2.369.945 matrículas de Educação Superior. Passados sete anos, em 2006, o número de matrículas quase dobrou, totalizando 4.467.646. Isso significou um percentual de crescimento de 97,3% do número de matrículas de educação superior em menos de uma década. pelo aumento da concorrência e pelas exigências de produção de conhecimentos práticos. Para manter sua sobrevivência, surge a necessidade de responder aos interesses das classes populares. Inicia-se, então, a valorização da capacitação técnica e instrumental em detrimento aos conhecimentos eruditos, próprio do ensino elitizado. Ao submeter-se à produção de padrões culturais médios e de conhecimentos instrumentais, a universidade deixa de ser a única e principal produtora do conhecimento. A crise de legitimidade está intimamente ligada a essas mudanças. “Ela [a crise] manifesta-se, de modo especial, nas críticas daqueles que julgam estar ela [a universidade] alienando-se, progressiva e perigosamente, da realidade” (PANIZZI, 2006, p. 44). Essa crise surgiu na medida em que a Universidade deixou de ser imprescindível para uma grande parcela da sociedade. Por isso, afirma-se que a mesma não está atendendo às demandas da sociedade atual. “A universidade, infelizmente, se deslocou da realidade social, pois não reproduz os elementos que integram essa mesma realidade, na sua mobilidade” (PANIZZI, 2006 p. 45). Enquanto isso, os Cursos Superiores privado/mercantis vêm ganhando espaço no meio social por atender aqueles que não tiveram acesso à instituição pública ou a financiamentos. Os estudantes são motivados pela promessa de uma imediata inserção no mercado de trabalho e por serem mais acessíveis financeiramente. Ao analisar esse crescimento por categoria administrativa, tendo por base o mesmo período, o número de matrículas públicas obteve aumento de 45% (832.022 – 1.209.304), inferior ao das privadas que teve um avanço percentual de 75% (886.561 – 1.543.176). O setor privado/mercantil superou os demais com um explosivo aumento de 195% (651.362 – 1.924.166) do número de matrículas. Esses dados revelam o quanto a atual estrutura universitária vem deixando de contemplar aos interesses da população. impostas, contrapondo-se à agilidade administrativa das instituições mercantis. Para manter-se enquanto tal, a universidade precisa investir em programas de pós- graduação, pesquisa e possuir um número de professores com tempo integral superior aos previstos para os centros universitários. Além do mais, por ser uma universidade pública, de caráter não-estatal, a Universidade comunitária vem sendo tratada pelos governos e pela legislação como qualquer outra IES privado no país. Com o intuito de assegurar sua credibilidade frente à sociedade, a universidade comunitária precisa vencer os desafios internos e externos a ela impostos. Em observância a essa problemática, Paviani (2007, p. 32) salienta que “a universidade comunitária não sabe ainda se sua vocação institucional deve optar por ser uma universidade de massa ou uma universidade de excelência acadêmica ou ocupar um posto intermediário entre essas duas alternativas”. Bechi As disciplinas de formação humana e sociais estão sendo extintas, restando apenas as disciplinas específicas referentes aos cursos oferecidos. Com a retirada dessas disciplinas, a duração dos cursos é encurtada, diminuindo os custos das mensalidades. Ao torná-las mais acessíveis à população, há aumento da competitividade da instituição no mercado educacional. “Competir e, no máximo, garantir a empregabilidade dos estudantes/clientes são as preocupações que afligem e ao mesmo tempo se põem como desafio para todos” (SGUISSARDI, 2008, p. 1015). Nesse sentido, ao mudarem os objetivos, mudaram-se também as atividades a serem realizadas. Em outras palavras, enquanto as finalidades da Educação forem reduzidas ao mero aperfeiçoamento profissional, as atividades assumirão apenas uma conotação prática, deixando os elementos teóricos em segundo plano. ( p ) Para que o sujeito obtenha um bom desempenho profissional é preciso que ele estabeleça contato com conhecimentos gerais e interdisciplinares, já que, como justifica Paviani (2007, p. 41), “o especialista sem uma formação transversal, sem uma cultura geral, não pode ter nos dias atuais um bom desempenho”. A redução do tempo de duração dos cursos, somada ao aumento do número de estudantes por grupo, tem por resultado a diminuição da qualidade de ensino. Com o intuito de obter maior lucratividade, 92% das instituições têm dedicação exclusiva ao ensino. Nesse contexto, as aulas limitam-se à simples transmissão de conteúdos. Os alunos encontram-se submetidos aos saberes dos professores. A aprendizagem é apenas o resultado da memorização dos conteúdos por ele proferidos. Não há espaço para que os alunos pesquisem e interliguem seus saberes com outros conhecimentos afins. O modelo atual de ensino ignora “as tendências interdisciplinares e a necessidade de uma formação que desenvolva a criatividade, a capacidade de compreender a si e os outros, antes mesmo de entender isso ou aquilo no sentido técnico” (PAVIANI, 2007, p. 41). Maringá, v. 33, n. 1, p. 139-147, 2011 Com a expansão do Ensino Superior, juntamente com o predomínio e a valorização do conhecimento, a Universidade passou a ser vítima de diferentes contradições que deram origem ao que Santos (2008) denominou de tríplice crise: crise de Maringá, v. 33, n. 1, p. 139-147, 2011 Maringá, v. 33, n. 1, p. 139-147, 2011 Acta Scientiarum. Education Mercantilização do ensino superior 145 “hegemonia”; crise de “legitimidade”; e crise “institucional”. A primeira é resultado da contradição entre conhecimentos exemplares (formação das elites) e conhecimentos funcionais (formação dos trabalhadores). 4 Diante dessas mudanças, as universidades comunitárias vêm enfrentando dificuldades econômicas e financeiras pelas inúmeras exigências institucionais a elas Acta Scientiarum. Education Bechi Na tentativa de resgatar sua credibilidade diante dos diferentes grupos sociais, a crise hegemônica vem se agravando cada vez mais. Ela coloca a Universidade “em um mesmo nível de igualdade com outras tantas organizações, as quais, muitas vezes, se aventuram nessa missão, afastando- se, assim, da realidade, pela incapacidade de perceber e elaborar o conjunto das práticas sociais” (PANIZZI, 2006, p. 43). Resumindo, a crise de hegemonia é o resultado da “perda de poder” da Universidade com relação à sociedade, enquanto que a crise de legitimidade provém da “perda de crença” da sociedade sobre os objetivos e funções da Universidade (SANTOS apud RODRIGUES, 2001, p. 131). Em meio a essas contradições, entra em cena a crise institucional que vem rendendo muitas discussões e desafios para a Universidade. Atualmente são atribuídas fortes pressões sociais sobre essa instituição, a fim de que se inicie um processo de reestruturação do modelo educacional vigente. Santos (2008, p. 190) define essa terceira crise, da seguinte maneira: “a universidade sofre uma crise institucional na medida em que sua especificidade organizativa é posta em causa e se lhe pretende impor modelos organizativos Essa crise originou-se da contradição entre os conhecimentos exemplares próprios da concepção tradicional e os atuais anseios da sociedade capitalista. Apoiando-se no viés tradicional, cria-se um grau de exigência em função de garantir um ensino de excelência que prime pelo desenvolvimento do pensar crítico, pela divulgação dos conhecimentos científicos e humanísticos. Por outro lado, “o sistema universitário brasileiro vem crescendo com o objetivo de atender as necessidades da sociedade de uma maior competitividade e de fornecer formação superior para as novas formas de emprego e trabalho” (PAVIANI, 2007, p. 32). A Universidade4 passa a ser fortemente influenciada Maringá, v. 33, n. 1, p. 139-147, 2011 Acta Scientiarum. Education 146 Bechi Bechi vigentes noutras instituições tidas por mais eficientes”. Dessa forma, diante da expansão do Ensino Superior, em meio às crises por ela proporcionada, pode-se fazer o seguinte questionamento: quais são os principais desafios a serem enfrentados pela universidade no atual cenário educacional? transformar em agentes ativos do seu próprio processo de aprendizagem. Na visão de Panizzi (2006, p. 41), “pensar, ser sujeito ativo de sua construção pessoal e participar da vida social são – absolutamente – um importante, senão o mais significativo objetivo do processo educacional”. Essas interpretações aproximam-se da concepção de Kesselring (2007, p. Desafios da universidade numa perspectiva pós-moderna de emancipação A Universidade precisa definir novos objetivos e finalidades a fim de superar o modelo de ensino baseado exclusivamente na transmissão e inculcação nos alunos de informações e conhecimentos prontos. Isso porque, o conceito de formação universitária não se solidifica na mera aquisição e memorização de conhecimentos. A Educação Superior requer um processo formativo que prima pelo desenvolvimento de cidadãos críticos e responsáveis. Tendo em vista essa perspectiva, Paviani (2009, p. 49-50) afirma que a aprendizagem dos estudantes deverá ser orientada “visando ao desenvolvimento de habilidades e competências não apenas profissionais, mas também pessoais; não apenas imediata, mas também ao longo de suas vidas”. O desenvolvimento de tais habilidades ajudará o indivíduo a enfrentar as exigências da vida em sociedade e as transformações que vêm ocorrendo no mundo do trabalho. Por isso, não basta o sujeito ter o domínio de técnicas específicas ao exercício de determinadas profissões, é necessário que ele seja capaz de resolver, de forma independente e flexível, os desafios que poderão surgir ao longo de sua trajetória profissional. A função da universidade é uma função única e exclusiva. Não se trata somente de difundir conhecimentos. O livro também os difunde. Não se trata, somente, de conservar a experiência humana. O livro também a conserva. Não se trata, somente de preparar práticos ou profissionais, de ofícios ou artes. A aprendizagem direta os prepara, ou, em último caso, escolas muito mais singelas do que universidades. Trata-se de manter uma atmosfera de saber para se preparar o homem que o serve e o desenvolve. Trata-se de conservar o saber ativo e não morto nos livros ou no empirismo das práticas não intelectualizadas. Trata-se de formular intelectualmente a experiência humana, sempre renovada, para que a mesma se torne consciente e progressiva. Ter flexibilidade e se adaptar às exigências próprias da sociedade globalizada, em que os conhecimentos adquiridos tornam-se rapidamente inúteis, são competências intimamente ligadas ao desenvolvimento das habilidades do pensamento. Porém, “o desenvolvimento do pensamento, próprio da natureza humana, como conjunto das competências, não é ensinado stricto sensu, mas se desenvolve quando exercitado, vivido, provocado e passa a fazer parte da práxis cotidiana das pessoas” (PANIZZI, 2006, p. 41). Os alunos precisam participar de práticas que os instiguem a pensar e a refletir sobre questões sociais e éticas que envolvam a sua formação profissional. Bechi 21) sobre a Educação Superior ao afirmar que “a preocupação central do ensino universitário é o esclarecimento e, para usar um termo do século XX, a emancipação”. Esse forte apelo, ocorrido nos últimos anos, em prol de uma educação que prioriza o desenvolvimento intelectual e a moral dos indivíduos, é resultado da atual valorização da prática em detrimento das disciplinas humanas e sociais. O enxugamento e a flexibilização dos currículos, de acordo com as demandas do mercado educacional, vêm ameaçando a formação integral do ser humano. O fato é que, quando as instituições de Ensino Superior são administradas exclusivamente na perspectiva do lucro e do negócio, tornando a competência técnica a única referência na educação dos indivíduos, seus processos de ensino passam a inibir a formação crítica dos educandos. A estrutura universitária em elevação nos últimos anos, orientada por modelos de gestão empresarial, não vem formando pensadores. Anísio Teixeira (apud SGUISSARDI, 2008, p. 1009) coloca em dúvida esse novo sistema de Ensino Superior, quando escreve: Acta Scientiarum. Education License information: This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Referências DALBOSCO, C. A. A. Universidade, formação e indústria educacional. In: FÁVERO, A.; CENCI, A.; TROMBETTA, G. (Org.). Universidade, filosofia e cultura. Passo Fundo: UPF, 2009. p. 56-93. KESSELRING, T. Universidade, economia e sociedade: notas sobre o desenvolvimento atual das universidades europeias. Chronos, v. 34, n. 1, p. 15-29, 2007. MÜHL, E. H. Cultura empresarial e formação docente: os riscos da instrumentalização da educação superior. In: FÁVERO, A.; CENCI, A.; TROMBETTA, G. (Org.). Universidade, filosofia e cultura. Passo Fundo: UPF, 2009. p. 94-114. PANIZZI, W. Universidade para quê? Porto Alegre: Libretos, 2006. Mercantilização do ensino superior 147 conhecimentos, participando ativa e efetivamente da elaboração cultural e científica da comunidade acadêmica e da sociedade. lucratividade em meio à efervescência do mercado educacional, está impossibilitando o desenvolvimento de profissionais críticos e comprometidos com as causas sociais. Se isso não bastasse, a flexibilização e adaptação da estrutura curricular aos interesses do mercado vêm prejudicando o acesso e a produção livre do conhecimento. Em oposição ao modelo empresarial de ensino, a Universidade deve propiciar aos alunos, com o apoio de disciplinas como as humanas e as sociais, práticas pedagógicas que os levem a pensar de modo reflexivo sobre o homem e sobre sua relação com o mundo. Além disso, a aplicação de tais disciplinas poderia contribuir significativamente para a formação ética dos alunos. A sociedade necessita, portanto, de profissionais comprometidos com o social. Nesse sentido, pode-se afirmar que o apoio à pesquisa e à valorização das disciplinas humanas e sociais são fatores que contribuirão para a superação dos desafios colocados à Universidade, incentivando e fortalecendo o processo de emancipação dos sujeitos envolvidos. Nesse sentido, para que o trabalho docente não fique atrelado exclusivamente às demandas do mercado, a Universidade precisa criar condições que venham a incentivar e a fortalecer o desenvolvimento científico e cultural da comunidade acadêmica. Os cursos superiores com dedicação exclusiva ao ensino, em que a aprendizagem é o resultado do armazenamento e da memorização de conteúdos, não estão investindo significativamente no processo de produção científica. Em meio a um mercado educacional amplamente competitivo, a Universidade é, atualmente, a principal responsável pelo fortalecimento da pesquisa científica. Porém, o processo de produção e de socialização dos conhecimentos não pode ocorrer sem que, antes, sejam levados em conta os problemas relacionados à destruição do meio ambiente, à corrupção e a outras questões ligadas à qualidade de vida do ser humano. Esse processo formativo implica o surgimento de “um modelo de aplicação da ciência alternativo ao modelo de aplicação técnica, um modelo que subordine o know-how técnico ao know-how ético e comprometa a comunidade científica existencial ética e profissionalmente com o impacto da aplicação” (SANTOS, 2008, p. 224). Desafios da universidade numa perspectiva pós-moderna de emancipação O ensino estritamente técnico não forma sujeitos autônomos, dispostos a sustentar sua independência e sua integridade espiritual diante dos mecanismos massificadores e perante os ideários consumistas que permeiam a vida contemporânea. O desenvolvimento do raciocínio crítico não acontece somente pela imposição vertical de conhecimentos. Para isso, os alunos precisam se Para atingir tais anseios, a universidade deve estabelecer novas metas que venham a superar as transformações decorrentes dessa reorientação mercantilista da educação. Os altos índices de expansão da Educação Superior no Brasil e a transformação dos institutos e das faculdades em unidades de negócios são fatores responsáveis pela gradativa eliminação e marginalização das disciplinas humanas e sociais. O constante empobrecimento dos currículos, com o intuito de aumentar a Maringá, v. 33, n. 1, p. 139-147, 2011 Acta Scientiarum. Education Acta Scientiarum. Education Acta Scientiarum. Education Considerações finais PAVIANI, J. Os desafios da universidade comunitária. Chronos, v. 34, n. 1, p. 30-45, 2007. Acta Scientiarum. Education Esse artigo pretendeu suscitar o urgente debate sobre os rumos que a Universidade precisa tomar em um contexto marcado pela instrumentalização da Educação Superior. Tendo em vista os conhecimentos levantados durante a pesquisa, a universidade somente irá conquistar sua autonomia e sua credibilidade, frente ao atual estado de expansão e de privatização da Educação Superior, quando for valorizado o desenvolvimento intelectual e moral dos indivíduos. Para que isso aconteça, duas condições básicas se fazem necessárias: a intensificação do investimento em pesquisa e a valorização das disciplinas humanas e sociais. No que se refere ao primeiro aspecto, o envolvimento efetivo de professores e de alunos em atividades de pesquisa constitui um antídoto contra o estado de fragmentação do conhecimento, que foi fortalecido pelo atual processo de instrumentalização da Educação Superior. Em outras palavras, em vez de serem apenas receptores e divulgadores de informações, os agentes desse processo investigativo são levados a produzir PAVIANI, J. Ainda é possível a formação universitária? In: FÁVERO, A.; CENCI, A.; TROMBETTA, G. (Org.). PAVIANI, J. Ainda é possível a formação universitária? In: FÁVERO, A.; CENCI, A.; TROMBETTA, G. (Org.). Universidade, filosofia e cultura. Passo Fundo: UPF, 2009. p. 44-55. Universidade, filosofia e cultura. Passo Fundo: UPF, 2009. p. 44-55. RODRIGUES, M. M. A instabilidade da universidade vista a partir das contradições de suas crises: crise de hegemonia, crise de legitimidade e crise institucional. Educação e Filosofia, v. 15, n. 29, p. 129-140, 2001. SANTOS, B. S. Pela mão de Alice: o social e o político na pós-modernidade. São Paulo: Cortez, 2008. SGUISSARDI, V. Modelo de expansão da educação superior no Brasil: predomínio privado/mercantil e desafios para a regulação e a formação universitária. Educação e Sociedade, v. 29, n. 105, p. 991-1019, 2008. Received on October 30, 2010. Accepted on January 24, 2011. Maringá, v. 33, n. 1, p. 139-147, 2011
https://openalex.org/W1547689825	https://www.jneurosci.org/content/jneuro/23/19/7438.full.pdf	English	null	Topography of Interaural Temporal Disparity Coding in Projections of Medial Superior Olive to Inferior Colliculus	The journal of neuroscience/The Journal of neuroscience	2,003	cc-by	10,222	Received April 2, 2003; revised June 24, 2003; accepted June 24, 2003. This work was sponsored by National Institutes of Health (NIH) Grant R01-DC00189 (D.L.O.), National Science Foundation Grant IBN-9807872 (R.B.), NIH Grant F32-DC05737-01 (W.C.L.), and NIH Grant T32-DC00025 (W.C.L.). Correspondence should be addressed to Dr. Douglas L. Oliver, Department of Neuroscience, University of Con- necticut Health Center, 263 Farmington Avenue, Farmington, CT 06030-3401. E-mail: doliver@neuron.uchc.edu. G. E. Beckius’s present address: MS 8220-2238, Discovery Microscopy Laboratory, Pfizer Inc., Groton, CT 06340. R.Batra’spresentaddress:DepartmentofAnatomy,UniversityofMississippiMedicalCenter,Jackson,MS39216- 4505. Copyright © 2003 Society for Neuroscience 0270-6474/03/237438-12$15.00/0 Topography of Interaural Temporal Disparity Coding in Projections of Medial Superior Olive to Inferior Colliculus Douglas L. Oliver, Gretchen E. Beckius, Deborah C. Bishop, William C. Loftus, and Ranjan Batra Department of Neuroscience, University of Connecticut Health Center, Farmington, Connecticut 06030-3401 Douglas L. Oliver, Gretchen E. Beckius, Deborah C. Bishop, William C. Loftus, and Ranjan Batra Department of Neuroscience, University of Connecticut Health Center, Farmington, Connecticut 06030-3401 Neurons in the medial superior olive encode interaural temporal disparity, and their receptive fields indicate the location of a sound source in the azimuthal plane. It is often assumed that the projections of these neurons transmit the receptive field information about azimuth from point to point, much like the projections of the retina to the brain transmit the position of a visual stimulus. Yet this assumption has never been verified. Here, we use physiological and anatomical methods to examine the projections of the medial superiorolivetotheinferiorcolliculusforevidenceofaspatialtopographythatwouldsupporttransmissionofazimuthalreceptivefields. The results show that this projection does not follow a simple point-to-point topographical map of receptive field location. Thus, the representation of sound location along the azimuth in the inferior colliculus most likely relies on a complex, nonlinear map. Key words: auditory pathways; sound localization; binaural hearing; neural pathways; neuroanatomy methods; cat map of ITD in MSO to a similar map in the IC, the major auditory structure in the mammalian midbrain (see Fig. 1). Such a map of azimuth in the central nucleus of the IC (ICC) has been suggested in experiments using free-field stimulation (Aitkin et al., 1985). A rough map of interaural sound level differences, the cue for azi- muth at high frequencies, has also been reported in the IC (Irvine and Gago, 1990). In both the MSO and ICC, the ITD axis should be perpendicular to the frequency axis; however, it is unknown whether a topographical projection from MSO contributes to any map of space in the IC. In the present study, we used binaural physiology and anatomical methods to test the hypothesis that a rostrocaudal map of ITD receptive fields in MSO is transmitted to the ICC by point-to-point projections. 7438 • The Journal of Neuroscience, August 13, 2003 • 23(19):7438–7449 7438 • The Journal of Neuroscience, August 13, 2003 • 23(19):7438–7449 Behavioral/Systems/Cognitive Introduction The pathway from the medial superior olive (MSO) to the infe- rior colliculus (IC) conveys important information about the location of a sound in space. The MSO is a primary site for the neural computation of interaural temporal disparity (ITD) (Goldberg and Brown, 1969; Yin and Chan, 1990; Batra et al., 1997a,b; Brand et al., 2002; Cook et al., 2003), the cue for the position of low-frequency sounds in the horizontal or azimuthal direction (Hafter and Trahiotis, 1997). The MSO may be spatially organized by ITD as a prerequisite for topographical maps of azimuth in the higher auditory system. In the barn owl and chicken, neurons in nucleus laminaris (the ho- molog of MSO) are arranged in an orderly manner so that the ITD to which neurons are tuned increases along one axis of the nu- cleus (Sullivan and Konishi, 1986; Carr and Konishi, 1988; Over- holt et al., 1992). Whether a similar map of ITD is present in the mammalian MSO is not so clear. Morphological evidence sup- ports a delay line mechanism that could result in a rostrocaudal gradient of the preferred ITD (Smith et al., 1993; Beckius et al., 1999). A rough rostrocaudal map of ITD was found in this di- mension, with neurons tuned to ITDs near zero located rostrally and those tuned to ipsilateral delays located caudally (Yin and Chan, 1990). If spatial mapping of ITD is an important feature of the neural system subserving sound localization, then the ascend- ing projections of the MSO should be well organized. Point-to- point connections would efficiently convey information from a Fluorescent tracers i h h Af- ter 20 min in 0.5% H202 in neutral phosphate buffer (0.12 M, pH 7.4) and rinses in buffered Triton X-100, sections were incubated in the ABC complex (PK-4000; Vector Labs) overnight at 4°C in the presence of 0.5 M NaCl. After rinses, sections were incubated in diaminobenzidine (DAB) (D-5637; Sigma) with Co and Ni for 15 min and then incubated in a fresh volume of the same DAB solution with 0.005% H202 for 15 min. In many experiments, additional sections received this same treatment (without step 2) and were used for Nissl stains. Recordings were made with glass patch pipettes (2–20 m tip, resis- tance 0.5–5 M), and the same electrode was used for the injection. Electrodes were advanced with a microdrive (Burleigh Inchworm, Fish- ers, NY) mounted on the stereotaxic manipulator. For MSO injections, the electrode was initially positioned according to stereotaxic coordi- nates. An appropriate location for the IC or MSO injection was found by assessing neural responses to sound and making repeated penetrations. Acoustically driven responses of single or multiple units just above threshold were amplified with Dagan 2400 (Minneapolis, MN) and Princeton Applied Research (model 5113; Oak Ridge, TN) amplifiers. Action potentials were monitored by ear or discriminated by a BAK window discriminator and recorded with a unit event timer. p Step 2. Next, the sections underwent an immunohistochemical reac- tion to render the TMR dextran permanently visible. After rinsing in neutral buffer, sections were blocked in neutral buffered 10% horse se- rum (Invitrogen; 16050-015) containing 0.1% Triton X-100 for 2 hr and then incubated in anti-tetramethylrhodamine antisera made in rabbit (A-6397; Molecular Probes), 1:12,000 dilution in the blocking solution and 0.15 M NaCl overnight at 4°C. After rinses in Triton X-100 buffer, sections were incubated with an anti-rabbit, biotinylated secondary an- tisera (Jackson 711-065-152), 1:800 dilution, for 4 hr at 25°C. After rinses in Triton X-100 buffer, the sections were exposed to the ABC complex (PK-4000; Vector) in the same buffer overnight at 4°C. Finally, the avi- din–biotin–HRP complex was revealed with a DAB reaction without nickel or cobalt or, more often, a NovaRED reaction (SK-4800; Vector Labs), for 15 min. The sections were mounted from phosphate buffer onto subbed slides and cleared in Histoclear (HS-200; National Diagnos- tics) before coverslipping with Permount (SP15–500; Fisher). Earlier cases were treated with the freeze–thaw techniques to enhance immuno- staining as outlined in Beckius et al. Fluorescent tracers i h h In cases with the MSO injections of dextran, every third section, begin- ning with the first, was mounted onto slides and coverslipped with 10% 1,4-diazabicyclo (2.2.2) octane (Sigma) in glycerine and neutral phos- phate buffer. In most cases, sections were incubated first with Fluorescein Avidin DCS (A-2011; Vector Labs) at 1:1600. The fluorescein-labeled avidin binds with the BDA and adds to the signal emitted by the fluores- cein–dextran. Fluorescent sections containing retrogradely transported red or green latex microspheres were dried onto subbed slides and cov- erslipped with Krystalon (64969/71; EM Science, Gibbstown, NJ) be- cause the microspheres dissolve in glycerin-based mounting media. The spectral and ITD sensitivities of neurons and multiunit clusters at the injection site were assessed in both the MSO and IC. Units were tested for their best frequency (BF) with monaural and binaural pure tones. Sensitivity of the units to ITD was measured with a binaural-beat stim- ulus (Kuwada et al., 1979; Batra et al., 1997a). Low-frequency units (BF 2500 Hz) and some with high BF were stimulated with pure tones in each ear that differed by 1 Hz. These stimuli produced a continuous change in the interaural phase difference. In one case, a low-frequency unit was tested with sinusoidally amplitude-modulated (SAM) tones with the same modulation frequency at both ears but with carrier fre- quencies that differed by 1 Hz. The sensitivity of high-frequency units (BF 2500 Hz) to ITDs was tested with a stimulus that consisted of SAM tones to either ear that had the same carrier frequency but modulation frequencies that differed by 1 Hz (Batra et al., 1997a). SAM tones were modulated to a depth of 80%. Test stimuli were 5100 msec in duration, but the first 100 msec was not analyzed. The best ITD based on a com- posite response was calculated (Yin and Kuwada, 1983; Kuwada et al., 1987; Batra et al., 1997a). The composite response was generated by averaging the responses at all frequencies that displayed significant syn- chrony to the 1 Hz beat frequency (Rayleigh test of uniformity; p 0.001) (Mardia and Jupp, 1999). Conversion of fluorescent tracers to permanent, nonfluorescent reaction products p Every third section from MSO injection cases was used. Step 1. To render the biotinylated dextran visible, free-floating sections underwent avidin–biotin–HRP histochemistry (Oliver et al., 1994). Histology f g In early experiments, acoustic stimuli were produced by a digital stimu- lus system (Rhode, 1976) under the control of an LSI-11/73 computer (Digital Equipment, Nashua, NH). In later experiments, acoustic stimuli were generated by a TDT System 2 (Tucker Davis Technologies, Gaines- ville, FL) under the control of a PC computer. All sounds were delivered by the same Beyer earphones (DT-48, Hicksville, NY) via sealed enclo- sures. The sound delivery system was calibrated from 60 to 40,000 Hz. For injections of anterograde tracers in MSO, sounds were delivered through the hollow ear bars of the stereotaxic device, and calibration was performed at the end of the ear bar with a 1/8 inch microphone (Bru¨el & Kjaer). For injections of retrograde tracers in IC, tones were delivered through a hollow tube in custom silicone ear molds (PER-FORM silicone ear impression material) made for each individual cat, similar to those used in the unanesthetized rabbit (Kuwada et al., 1987; Batra and Fitz- patrick, 1997). The hollow tube incorporated a probe that extended 2 mm beyond the end of the tube. Calibrations were performed through this probe using a 1/2 inch microphone (Bru¨el & Kjaer). These calibra- tions were then corrected for the characteristic of the probe. gy After 7–10 d survival, animals were deeply anesthetized with the ket- amine/xylazine mixture and killed by cardiac perfusion with 50–75 ml of washout (2% sucrose and 0.05% lidocaine in 0.12 M phosphate buffer, pH 7.3–7.4) and 1000 ml of fixative (4% paraformaldehyde in 0.12 M phosphate buffer). Most brains with MSO injections (11 of 14) were cut in the frontal plane, and the remainder were cut in the horizontal plane perpendicular to it. Brains with IC injections were cut in the frontal plane (n 4) or in the sagittal plane (n 1). The tissue was cut on a freezing microtome into 50-m-thick sections, collected in 0.12 M phosphate buffer, and stored at 4°C. In general, the histology for all experiments was designed to preserve the fluorescence of the different tracers or to convert the tracer into permanent nonfluorescent reaction products in alternate sections. Materials and Methods Surgery h f There were two groups of experiments. In the first, anterograde tracers were injected into the right MSO, and in the second, retrograde tracers were injected into the right IC. Experiments were performed on 19 adult cats (Liberty Labs, Waverly, NY), and all procedures conformed to Na- tional Institutes of Health guidelines and protocols approved by the An- imal Care Committee of the University of Connecticut Health Center. In all experiments, the animal was anesthetized with a mixture of ketamine (33 mg/kg) and xylazine (1 mg/kg), intubated, and then maintained in an areflexive state with a mixture of isoflurane and medical grade oxygen. It was monitored for breathing rate and reflexive state, maintained at 37°C with a water blanket, and received intravenous saline or lactated Ringer’s solution during the procedure. The animal was placed in a double-walled sound attenuation chamber (IAC, Bronx, NY) and held in a stereotaxic device (Kopf, Tujunga, CA) for the MSO injections or in a custom head holder for the IC injections. For MSO injections, a craniotomy was per- formed over the cerebellum. For IC injections, a craniotomy was per- formed over the occipital cortex, and a small region of cortex was aspi- rated to permit visualization of the IC. After the recording and injections, the skin and muscles were sutured, the animal recovered in an intensive care unit incubator at 37°C until fully awake, and children’s aspirin (40 mg) was administered immediately postoperatively as an analgesic. All J. Neurosci., August 13, 2003 • 23(19):7438–7449 • 7439 Oliver et al. • Connections for ITD Coding in IC animals recovered from surgery without permanent neurological im- pairment and were able to eat and drink normally. animals recovered from surgery without permanent neurological im- pairment and were able to eat and drink normally. greenlatexmicrospheres(LumaFluor;1:1).Microsphereinjectionsrequired electrodes with 30–40 m tips and pressure injections of 300–700 nl with a Picospritzer (General Valve, Fairfield, NJ). greenlatexmicrospheres(LumaFluor;1:1).Microsphereinjectionsrequired electrodes with 30–40 m tips and pressure injections of 300–700 nl with a Picospritzer (General Valve, Fairfield, NJ). Acoustic stimulation and recordings Fluorescent tracers i h h (1999), but this later proved to be unnecessary. Analysis Experimentaldesignandhypothesis.Interauraltimedifferences(ITD)arehypoth- esized to be topographically organized along a rostrocaudal axis of the medial superior olive (MSO), within an isofrequency lamina. If so, the axons from MSO neurons are predicted to project to single laminas in the central nucleus of the inferior colliculus (IC) in a point-to-point manner. Injections of different labeled dextrans were made by iontophoresis in the same isofrequency plane to test this hypothesis. H, High frequency; L, low frequency; D, dorsal; V, ventral. The boutons plotted in the previous step were used to assess bouton gradients along the other axes perpendicular to the frequency axis (i.e., the dorsal-ventral axis and “oblique” axes between the dorsoventral and rostrocaudal axes). Three-dimensional serial reconstructions were made that allowed the sampled boutons to be viewed as a three-dimensional plane or contour, similar to the fibrodendritic lamina from which it was obtained. The three-dimensional field of boutons was projected onto two dimensions by rotating the contour until the planar surface was parallel to the viewing window (see Fig. 8C), and the differences in the Z coordi- nates were within the 200 m thickness of the lamina. The X and Y coordinates of the markers at this orientation were then rendered as a two-dimensional scatter plot (see Fig. 8D). To examine the density of boutons in the plane of the lamina, perpendicular to the frequency axis, we rotated the field of bouton markers in 10° increments. A histogram of bouton density perpendicular to the x-axis with 150–200 m bins was generated at each angle of rotation. BDA mixed with fluorescein–dextran in the caudal MSO (Fig. 2A) and an injection of TMR dextran in the same MSO at the rostral end (Fig. 2B). A single injection from another case is shown in Figure 2C, and its location at the dorsal aspect of MSO can be discerned from the cytoarchitecture of the superior olive in the adjacent Nissl-stained section (Fig. 2D). j g We identified the rostrocaudal position in the MSO of each injection site and its BF. Most of the injection sites were in the rostral half of the MSO. The locations of all injections are shown in Figure 3A, which depicts the MSO as a flat sheet, with low frequency at the top (dorsal in vivo) and high frequency at the bottom of the y-axis (ventral in vivo), whereas the rostrocaudal dimension of the MSO is the x-axis. Analysis y Microscopic analysis used low-magnification camera lucida drawings and high-magnification analysis with computer-assisted microscope sys- tems. Low-magnification, camera lucida drawings (20 magnification) were made with a Zeiss Axioskop microscope to show injection sites and dextran-filled axons. The axons and cell bodies labeled with fluorescent markers were viewed with epifluorescence microscopy [high numerical aperture (NA) 10/NA 0.5 or 25/NA 0.8 lenses], and microscopic data were collected with Neurolucida software (Microbrightfield, Colchester, VT) and an E-3200 Gateway computer. The microscopy system included a CCD video camera with gating capability for low-light conditions (CCD-72, Dage MTI, Michigan City, MI), a gating–integration control- ler (Instagator, model 105001, Dage MTI), a PC frame grabber–VGA card (FlashPoint Intrigue Lite, Integral Technologies, Indianapolis IN), a motorized stage controller (MC2000, Ludl Electronics Products, Haw- thorne, NY), and a shutter for the mercury lamp (D122; UniBlitz, Vin- cent Associates, Rochester, NY). Data from nonfluorescent sections was collected with bright-field optics on the same microscope system. Anal- ysis of the retrograde labeling in the MSO included three-dimensional (3D) reconstructions made with the Neurolucida system and displayed as solids (Solids Module, Microbrightfield, Inc.). Axonal bouton densities in laminas of the IC were estimated to deter- mine whether there were rostrocaudal gradients. In each case, we selected the most densely labeled lamina, and bouton counts in that lamina were made in serial nonfluorescent sections. For cases cut in the frontal or transverse plane (see Fig. 7A1), samples were obtained in regularly spaced sections (every third or sixth section). For the case cut in the horizontal plane (see Fig. 7B), every sixth horizontal section was sampled, and the lamina in each section was divided into seven regions along its rostro- caudal extent. Bouton counts within the individual samples were made with an optical fractionator (Stereo Investigator, Microbrightfield, Inc.). All of the boutons in the center half of the section thickness were counted within the sampled area. The number of boutons in a count was doubled to estimate the number of boutons in the entire section thickness. These complete reconstructions of all the boutons in the sample volume pro- duced more reliable counts than single optical dissector planes within the section. This method conforms to stereological counting methods (Ste- rio, 1984; Coggeshall and Lekan, 1996) because all boutons within the sample volume were counted (a serial reconstruction), and the profiles at the edge were not counted twice. Figure1. Analysis The center of each injection site is marked by a symbol, and its rostrocaudal extent is indicated by the length of the corresponding bar. The horizontal line on which each bar lies indicates the length of the MSO in that animal. The BF at each injection site is represented by the vertical position of the symbols. The BFs covered a broad range: 200 Hz to 9 kHz. In two animals, two injections were made in the same MSO, and both injection sites had similar best frequencies (300 Hz in case 31 and 1 kHz in case 33). Eight injections were made at low frequencies (2500 Hz). Of these, four were made at a BF of 1 kHz. Four single injections were made at higher frequency. In one Injections For injections of anterograde tracers in MSO, the electrodes were filled with one of two solutions of dextran in normal saline (Molecular Probes, Eugene, OR): (1) 10% tetramethyl-rhodamine (TMR) dextran (catalog #D-1817) or (2) a mixture containing 10% each of biotinylated dextran (BDA) (catalog #D-1956) and fluorescein–dextran (catalog #D-1820). Iontophoretic injections were made using a 51413 Precision Current Source (Stoelting, Wood Dale, IL) and currents of 2.0 to 3.5 A (7 sec pulses, 50% duty cycle, 5–23 min total duration). For injections of retro- grade tracers in IC, several types of injection solutions were used, all mixed in normal saline: (1) 10% TMR dextran; (2) 6% Fluorogold (FG) (Fluoro- chrome, Inc., Denver, CO); (3) a mixture containing 3% FG and 5% BDA; (4)redlatexmicrospheres(LumaFluor,Inc.,Naples,FL)diluted1:1;and(5) To render fluorescent FG permanently visible in a nonfluorescent form, an immunohistochemical method similar to that for anterograde transport of TMR dextran (step 2) was used on every third section in IC injection cases. After H202 treatment, buffer rinse, and blocking with horse serum, the sections were incubated in a primary anti-FG antisera made in rabbit (Chemicon AB153) 1:8000 overnight at 4°C. The same biotinylated anti-rabbit antisera followed by ABC reaction was used as in 7440 • J. Neurosci., August 13, 2003 • 23(19):7438–7449 Oliver et al. • Connections for ITD Coding in IC Figure1. Experimentaldesignandhypothesis.Interauraltimedifferences(ITD)arehypoth- esized to be topographically organized along a rostrocaudal axis of the medial superior olive (MSO), within an isofrequency lamina. If so, the axons from MSO neurons are predicted to project to single laminas in the central nucleus of the inferior colliculus (IC) in a point-to-point manner. Injections of different labeled dextrans were made by iontophoresis in the same isofrequency plane to test this hypothesis. H, High frequency; L, low frequency; D, dorsal; V, ventral. step 2 above. After the ABC reaction, a DAB incubation with nickel and cobalt was used, 4 min without H202 and 4 min with 0.0005% H202. step 2 above. After the ABC reaction, a DAB incubation with nickel and cobalt was used, 4 min without H202 and 4 min with 0.0005% H202. Termination pattern of MSO axons in IC There was no clear relationship between the best ITD and the rostrocaudal location of the injection sites in the MSO. Figure 3B shows the location of all injection sites along the rostrocaudal axis of the MSO relative to the best ITD recorded at that site. (High- frequency units that were sensitive to ITDs in envelopes are shown as squares.) The units at the injection sites were excited by inputs to either ear and generally exhibited ITD sensitivity similar to that seen in previous studies of the anesthetized cat (Yin and Chan, 1990). Units had predominantly “peak-type” responses in that they discharged maximally at the same ITD at all frequencies with which they were tested. Most units in our sample were tuned to ITDs within the free-field range of the cat (approximately 325 sec) (Roth et al., 1980), and most characteristic delays (7 of 10) and best ITDs (11 of 12) were within this range. Neverthe- less, individual units recorded at different locations in the same MSO could show responses at variance with the rough rostrocau- dal gradient of best ITD demonstrated by Yin and Chan (1990) (Fig. 3B, regression line). Recordings at BF 300 Hz (Figs. 2A,B, 3A, triangles with X) are shown in Figure 4. Here, the best ITD at the rostral site (Fig. 4, top) was 86 sec, whereas at the more Axons that terminated in IC were distinguished by the presence of terminal boutons (Fig. 5C, micrographs 176 and inset 171 ), that is, the swelling at the Node of Ranvier or end of the axonal branch that indicates presynaptic specializations and the location of synaptic contacts (Oliver et al., 1995). Only axons with bou- tons are considered in this analysis. After the two injections in the same MSO in case 31, the axons from each injection were labeled with a different color and could be easily distinguished within the same section. In fluorescent sections, axons labeled with TMR were red, whereas axons labeled with the BDA–fluorescein dex- tran mixture were green (Fig. 5C, micrograph 176). In nonfluo- rescent sections processed for immunocytochemistry, boutons labeled with TMR were red, and those labeled with BDA were black (Fig. 5C, micrograph inset 171 ). Axons from a single point in MSO terminated along the entire rostrocaudal extent of the laminas in the ICC. These terminal fields were continuous in the rostrocaudal direction throughout the central nucleus (Fig. Injections of dextran into MSO Because the goal of the study was to examine the topography of the projection from the MSO to the IC (Fig. 1), we made small injections of dextran into the MSO that resulted in axonal trans- port to the IC. The injections were confined to the MSO or ex- tended beyond the margins of the MSO, but they did not invade the lateral superior olive (LSO). Figure 2 shows an injection of J. Neurosci., August 13, 2003 • 23(19):7438–7449 • 7441 Oliver et al. • Connections for ITD Coding in IC Figure2. InjectionsofdextraninMSOseenintransversesectionsafterconversiontononfluorescentreactionproduct.A,AninjectionofBDAinthecaudalMSOincase31ataBFof300Hz.B,An injectionofTMRdextraninrostralMSOinthesamecaseasA.C,AninjectionofBDAindorsalMSOincase55.D,ANissl-stainedsectionadjacenttothatinCshowingthecytoarchitectureofMSO.LSO, Lateral superior olivary nucleus. Scale bar, 200 m. Figure2. InjectionsofdextraninMSOseenintransversesectionsafterconversiontononfluorescentreactionproduct.A,AninjectionofBDAinthecaudalMSOincase31ataBFof300Hz.B,An injectionofTMRdextraninrostralMSOinthesamecaseasA.C,AninjectionofBDAindorsalMSOincase55.D,ANissl-stainedsectionadjacenttothatinCshowingthecytoarchitectureofMSO.LSO, Lateral superior olivary nucleus. Scale bar, 200 m. caudal site it was 94 sec. As in the previous study, the best ITD of the recording did not predict its location in the MSO. animal, the injection site was very ventral and caudal in MSO, and recordings were made at this site from two separate units with BFs of 5.3 and 9.2 kHz (Fig. 3A) (plotted at 5 kHz). Termination pattern of MSO axons in IC 5B, ICC) and stopped dorsally 0.5 mm short of the border with the dorsal cortex (Fig. 5B, DC). Small 7442 • J. Neurosci., August 13, 2003 • 23(19):7438–7449 Oliver et al. • Connections for ITD Coding in IC Figure3. LocationsandphysiologicalcharacteristicsofinjectionsitesinMSO.A,Bestfrequencies androstrocaudallocationsofinjectionsitesinMSO.Insomecases(triangleswithX,stars),twoinjec- tionsmatchedinfrequencyweremadeinthesameMSOattwodifferentrostrocaudallocations.Note thatrecordingsofbestITDwerenotavailable(NA,stars)atallinjectionsites.B,RelationshipofbestITD at injection site to the rostrocaudal position in MSO. Positive ITDs indicate contralateral delays and correspondtosoundsemanatingfromtheipsilateralhemifield.Linesfittoalldataaresimilartothatof Yin and Chan (1990) with units near zero ITD tending to be more rostral. Closed symbols denote single-unitrecordings;opensymbolsaremultiunitrecordings. 5, two small injections (Figs. 2A,B, 5A, sections 81 and 117) were madeinthesameMSOatthesameBF(300Hz).TMR-labeledaxons (red) from the rostral injection (Fig. 5A, section 117) overlapped fluorescein–dextran-labeled axons (green) from the caudal injec- tion (Fig. 5A, section 81) in the dorsolateral corner of the central nucleus (Fig. 5B). The zone of overlap was at least 200 m wide and was seen in all sections through the central nucleus (Fig. 5C). g g The labeling in the ICC from the two injections differed slightly in that the TMR axons were located somewhat more ven- tromedially (Fig. 5C, red axons, sections 165–183). Although the most lateral of this labeling was continuous rostrocaudally, sep- arate bands of label were seen medially. The most medial labeling consisted of narrow, 200 m bands of labeled axons separated by wider gaps. In contrast, the BDA-labeled axons from the caudal injection (Fig. 5A, section 81) were more dorsolateral in the cen- tral nucleus. The slight offset in the labeling may have been a result of spread of the tracer in MSO to sections coding somewhat different frequencies, although both injections were centered at the same BF. Another example of how a single point in MSO sends axons along the entire rostrocaudal length of the ICC is shown in Figure 6. This case also compares the results of two injections made at different rostrocaudal positions in the same MSO (Fig. 6A). Most axons terminated in a single lamina that can be seen by recon- structing the entire IC in 3D and rotating the reconstruction 40° (Fig. 6B). Because the sections in this case were cut in the hori- zontal plane (section 225, inset), the distribution of the axons along the laminas is seen as labeling from caudolateral to rostro- medial (Fig. 6C, sections 219–237) (the dorsal-most sections have the highest numbers). Each panel shows the MSO projec- tions in three adjacent sections that were processed with different histological methods. Termination pattern of MSO axons in IC In each section, terminals from both injec- tions contribute to labeling along the length of the lamina except in the dorsal-most section, where blood vessels interrupt the lamina. Figure3. LocationsandphysiologicalcharacteristicsofinjectionsitesinMSO.A,Bestfrequencies androstrocaudallocationsofinjectionsitesinMSO.Insomecases(triangleswithX,stars),twoinjec- tionsmatchedinfrequencyweremadeinthesameMSOattwodifferentrostrocaudallocations.Note thatrecordingsofbestITDwerenotavailable(NA,stars)atallinjectionsites.B,RelationshipofbestITD at injection site to the rostrocaudal position in MSO. Positive ITDs indicate contralateral delays and correspondtosoundsemanatingfromtheipsilateralhemifield.Linesfittoalldataaresimilartothatof Yin and Chan (1990) with units near zero ITD tending to be more rostral. Closed symbols denote single-unitrecordings;opensymbolsaremultiunitrecordings. numbers of collaterals extended into the rostral pole nucleus (Fig. 5B, section 195, RP) at the level of the superior colliculus. p Inputs from two points in the MSO to the ICC overlapped along the rostrocaudal dimension. In the case depicted in Figure Distribution of synaptic boutons from MSO axons in ICC laminas Figure 4. Neuronal recordings in the same MSO (case 31, BF 300 Hz) could be tuned to ITDs runningcountertothepostulatedITDtopography.A,RostralMSOsingle-unitrecording(3mm anterior to the caudal end of MSO). B, Caudal MSO multiunit recording (1 mm from caudal end of MSO). Left panels, Response as a function of ITD at different frequencies constructed from responsestobinaural-beatstimuli.Rightpanels,Compositedelaycurvescalculatedbyaverag- ing the responses shown in the left panel. The best ITD is the peak of the curve. TMR, Tetra- methylrhodamine dextran; BDA, biotinylated dextran amine. Point-to-point connections between MSO and IC isofrequency laminas appear to be absent on the basis of results of the small MSO injections presented above. If there is a gradient in the representation of ITD in the MSO perpendicular to the frequency axis, however, it could be transmitted to the IC effectively by a gradient in the density of the synapses, even if there are no point- to-point connections. If such a gradient were present in the ros- trocaudal direction, for example, neurons in the caudal MSO might make a higher density of synaptic contacts in the caudal part of the isofrequency laminas in ICC, whereas rostral MSO neurons might terminate more densely in the rostral IC. To ex- amine this possibility, axonal boutons were counted in a single lamina in the ICC of five animals (Fig. 7). Electron microscopy shows that boutons are the sites of synaptic contacts made by axons from MSO (Oliver et al., 1995). The boutons can be iden- tified clearly and analyzed at the light microscopic level (Fig. 5C, sections 176, 171 ). It was relatively easy to identify the same lamina in sections cut in the transverse plane (Fig. 7A1) or hori- zontal plane (Fig. 7B), and we could compare the density of bou- tons in each sample. Boutons were counted in the central 100 m of the 200-m-thick lamina (Fig. 7A1, inset, A2). Figure 4. Neuronal recordings in the same MSO (case 31, BF 300 Hz) could be tuned to ITDs runningcountertothepostulatedITDtopography.A,RostralMSOsingle-unitrecording(3mm anterior to the caudal end of MSO). B, Caudal MSO multiunit recording (1 mm from caudal end of MSO). Left panels, Response as a function of ITD at different frequencies constructed from responsestobinaural-beatstimuli.Rightpanels,Compositedelaycurvescalculatedbyaverag- ing the responses shown in the left panel. The best ITD is the peak of the curve. TMR, Tetra- methylrhodamine dextran; BDA, biotinylated dextran amine. The first analysis was bouton density measured in serial sec- tions through the IC (Fig. 7C1–C3). Distribution of synaptic boutons from MSO axons in ICC laminas B, Serial 3D reconstruction made from individual plots of sectionsthroughtheIC.The40°rotationaroundthey-axisshowsthelaminainwhichtheaxons terminate at its narrowest, on-edge view. Almost all of the labeling is confined to this one lamina. C, Higher-magnification plots to show the projections in the IC labeled from both MSO injection sites. Arrowheads indicate the rostromedial end of the lamina. Each drawing shows the combined labeling from three adjacent horizontal sections (no rotation) prepared with y The second analysis of bouton density was made after a three- dimensional reconstruction of the lamina from which the bou- tons were counted. Three cases cut in the transverse plane are shown for comparison. In one case the BF was 300 Hz (Fig. 8A), and in the other two cases the BFs were 1 and 5 kHz (Fig. 8B) (cases 85 and 56, respectively). The boutons in a single lamina were rotated to essentially a two-dimensional surface in the plane of the page (Fig. 8C). In this way, the density of the boutons could be observed along all possible axes perpendicular to the frequency axis, i.e., the axis orthogonal to the two-dimensional surface (Fig. 8D). In two-dimensional scatter plots of the boutons, it was evi- dent that the two inputs labeled in the same case were generally coextensive in the lamina (Fig. 8D, black and gray spheres), and there was not a systematic separation of inputs as has been seen in the projections of lateral superior olive and dorsal cochlear nu- cleus to the same lamina (Oliver et al., 1997); however, the den- sity of the inputs was not homogeneous, and there were patches of several hundred micrometers in diameter in which one input was more prevalent than the other. There was little evidence for a linear gradient of bouton density in any direction. When histo- grams of bouton density were made at different angles of orien- tation, the density of boutons varied most dramatically when it was measured parallel to the longest axis of the laminar plane (Fig. 8E), similarly equivalent to measurements made for the sectional analysis (Fig. 7), and least dramatically when perpen- dicular to the longest axis (Fig. 8F). Other axes were similar to one of these two extremes. The density of termination along most axes showed one or more prominent peaks, but these were not related to the location of the MSO injection (Fig. 8E,G,H). Distribution of synaptic boutons from MSO axons in ICC laminas Boutons were not distributed in a consistent way. Because boutons were counted from the caudal-most section of the IC, none of the samples revealed a J. Neurosci., August 13, 2003 • 23(19):7438–7449 • 7443 Oliver et al. • Connections for ITD Coding in IC Figure5. MSOinjectionsandlaminarICprojectionsincase31.A,TransversesectionstoshowMSOinjectionsat300HzBFattwodifferentrostrocaudalpositions.BDAinjection(green,caudal) TMRdextraninjection(red,rostral).Highernumberedsectionsareatmorerostrallevels.B,TransversesectionsthatshowaxonsfromMSOinjectionsprojectingtocentralnucleusofIC(ICC).Axons fromtherostralinjection(red)overlapthosefromthecaudalinjection(green),andbothtypesarefoundthroughouttherostrocaudalextentofthecentralnucleus.C,Highermagnificationplotsand micrographs show the boutons of axons labeled with the two tracers. Bouton fields partially overlap. Boutons with the two colors are easily distinguished in digital micrographs of the axons with fluorescentlabelingandafterconversionofthedextrantononfluorescentform.Insection176,twodigital,monochromeimagesofthesamefieldweretakenwithrhodamineandfluoresceinfilter Figure5. MSOinjectionsandlaminarICprojectionsincase31.A,TransversesectionstoshowMSOinjectionsat300HzBFattwodifferentrostrocaudalpositions.BDAinjection(green,caudal); TMRdextraninjection(red,rostral).Highernumberedsectionsareatmorerostrallevels.B,TransversesectionsthatshowaxonsfromMSOinjectionsprojectingtocentralnucleusofIC(ICC).Axons fromtherostralinjection(red)overlapthosefromthecaudalinjection(green),andbothtypesarefoundthroughouttherostrocaudalextentofthecentralnucleus.C,Highermagnificationplotsand micrographs show the boutons of axons labeled with the two tracers. Bouton fields partially overlap. Boutons with the two colors are easily distinguished in digital micrographs of the axons with fluorescentlabelingandafterconversionofthedextrantononfluorescentform.Insection176,twodigital,monochromeimagesofthesamefieldweretakenwithrhodamineandfluoresceinfilter setsanda10/NA0.5lens,andthencombinedtomakeared–green–blueimage.Insection171 ,boutonsareimagedwithacolordigitalcameraand40/NA1.3lens.Allimageswereadjusted forcolorlevel,contrast,andbrightness.BIC,BrachiumofIC;CG,centralgray;CM,commissureofIC;CN,cochlearnucleus;DC,dorsalcortex;LL,laterallemniscus;MNTB,medialnucleusofthetrapezoid body; RP, rostral pole nucleus; SC, superior colliculus; VL, ventrolateral nucleus. Figure5. MSOinjectionsandlaminarICprojectionsincase31.A,TransversesectionstoshowMSOinjectionsat300HzBFattwodifferentrostrocaudalpositions.BDAinjection(green,caudal); TMRdextraninjection(red,rostral).Highernumberedsectionsareatmorerostrallevels.B,TransversesectionsthatshowaxonsfromMSOinjectionsprojectingtocentralnucleusofIC(ICC).Axons fromtherostralinjection(red)overlapthosefromthecaudalinjection(green),andbothtypesarefoundthroughouttherostrocaudalextentofthecentralnucleus.C,Highermagnificationplotsand micrographs show the boutons of axons labeled with the two tracers. Bouton fields partially overlap. Boutons with the two colors are easily distinguished in digital micrographs of the axons with fluorescentlabelingandafterconversionofthedextrantononfluorescentform.Insection176,twodigital,monochromeimagesofthesamefieldweretakenwithrhodamineandfluoresceinfilter setsanda10/NA0.5lens,andthencombinedtomakeared–green–blueimage.Insection171 ,boutonsareimagedwithacolordigitalcameraand40/NA1.3lens.Allimageswereadjusted forcolorlevel,contrast,andbrightness.BIC,BrachiumofIC;CG,centralgray;CM,commissureofIC;CN,cochlearnucleus;DC,dorsalcortex;LL,laterallemniscus;MNTB,medialnucleusofthetrapezoid body; RP, rostral pole nucleus; SC, superior colliculus; VL, ventrolateral nucleus. 7444 • J. Neurosci., August 13, 2003 • 23(19):7438–7449 Oliver et al. • Connections for ITD Coding in IC systematic increase or decrease in the bouton density that covar- ied with the rostrocaudal position of the injection in the MSO (Fig. 7C1). However, different points along a lamina received different amounts of input. In the three cases shown in Figure 7C1, the maximum bouton density was located near the middle of each of the ICC laminas, but the peaks are staggered along the x-axis. Because these cases differed in the rostrocaudal length of the IC lamina and the BF, this appearance may be a byproduct of laminas that began at different positions relative to the caudal- most section through the IC. More informative were the two cases in which two injections were made at the same BF at differ- ent rostrocaudal positions in the MSO (Figs. 5, 6). Case 33 (Fig. 7C2) showed a maximum bouton density at the rostral end of the ICC laminas for both injections. Case 31 (Fig. 7C3) had the max- imum density from rostral MSO in the middle of the IC lamina, whereas the caudal injection had peaks of high density rostrally and caudally in the ICC. Figure6. MSOaxonsconvergeonasingleICClaminaincase33.A,InjectionsinMSOattwo recording sites with 1000 Hz BF. Distribution of synaptic boutons from MSO axons in ICC laminas What is clear from both the sectional and three-dimensional analysis is the lack of a uniform bouton density or linear gradient along any dimension of the ICC laminas. Moreover, the propor- tion of synaptic inputs from different parts of MSO, as repre- sented by boutons, varies almost randomly along a lamina and in a way that was not systematically related to the location of the injection within MSO. Injections in IC and retrograde labeling of MSO neurons different histological methods to reveal BDA, TMR, or BDA TMR. Because of a technical problem,thedistinctionofTMRfromBDAinonesectionwasnotdefinitive.Thissectiondesig- nated “BDA TMR” does not imply that the same axons were labeled by both tracers. LTB, Lateral trapezoid body. Injections in IC and retrograde labeling of MSO neurons The experiments above revealed little evidence for transmission of a point-to-point map of azimuth from MSO to IC and sug- Figure6. MSOaxonsconvergeonasingleICClaminaincase33.A,InjectionsinMSOattwo recording sites with 1000 Hz BF. B, Serial 3D reconstruction made from individual plots of sectionsthroughtheIC.The40°rotationaroundthey-axisshowsthelaminainwhichtheaxons terminate at its narrowest, on-edge view. Almost all of the labeling is confined to this one lamina. C, Higher-magnification plots to show the projections in the IC labeled from both MSO injection sites. Arrowheads indicate the rostromedial end of the lamina. Each drawing shows the combined labeling from three adjacent horizontal sections (no rotation) prepared with Figure6. MSOaxonsconvergeonasingleICClaminaincase33.A,InjectionsinMSOattwo recording sites with 1000 Hz BF. B, Serial 3D reconstruction made from individual plots of sectionsthroughtheIC.The40°rotationaroundthey-axisshowsthelaminainwhichtheaxons terminate at its narrowest, on-edge view. Almost all of the labeling is confined to this one lamina. C, Higher-magnification plots to show the projections in the IC labeled from both MSO injection sites. Arrowheads indicate the rostromedial end of the lamina. Each drawing shows the combined labeling from three adjacent horizontal sections (no rotation) prepared with J. Neurosci., August 13, 2003 • 23(19):7438–7449 • 7445 Oliver et al. • Connections for ITD Coding in IC Figure7 ThedensityofboutonsinsingleICClaminas A Intransversesections(A1) bouton Oliver et al. • Connections for ITD Coding in IC g gested a divergence of projections from MSO neurons. Some MSO neurons with axons projecting to an entire IC lamina might be intermingled with neurons with axons that have more re- stricted projections. In the case of this “mixed divergence,” we would predict that a focal injection of retrograde tracer in the IC might produce denser labeling in some parts of the MSO than in others, and this pattern might vary with the rostrocaudal position of the injection site. On the other hand, if all axons diverged completely, we would expect a uniform distribution of retro- gradely labeled cells, and this pattern would not vary dramatically with the rostrocaudal position of the IC injection. The data from retrograde transport is consistent with MSO neurons that have completely divergent projections. In all cases, labeled neurons were distributed evenly along the rostrocaudal extent of MSO, regardless of the location of the injection site in the IC. Injections of retrograde tracers were made in the low- frequency IC and were relatively restricted in their rostrocaudal spread along the laminas of the central nucleus (Fig. 9). Two injections were in the mid ICC at frequencies 400 Hz (Fig. Injections in IC and retrograde labeling of MSO neurons 9A,B, gray, IC/Horizontal View), and the caudal part of the in- jection site was at the same level as the caudal-most commissure of the IC. A third injection (Fig. 9C, gray, IC/Horizontal View) was in the rostral ICC at a similar BF (350 Hz). All three injections produced a similar pattern of labeling in MSO (Fig. 9A–C, gray spheres in MSO/Lateral View) with labeled neurons at the dorsal edge of MSO at all rostrocaudal levels. Injections at the same BF in the dorsal cortex of IC rostrally (Fig. 9A, DC, black, in IC/ Horizontal View) or caudal cortex (Fig. 9B, CC, black, in IC/ Horizontal View) produced either no or few labeled cells (Fig. 9B, black spheres in MSO), respectively, at the same locations. In contrast, an injection at 1000 Hz in rostral central nucleus (Fig. 9C, black, IC/Horizontal View) resulted in a continuous band of labeled cells just below the labeled cells from the lower-frequency site (Fig. 9C, black spheres in MSO). Figure t The density of the labeled neurons along the rostrocaudal axis of MSO was not obviously related to the location of the injection site. Although the two cases at mid IC had nearly the same loca- tion, these small injections produced small numbers of labeled cells in each section and with slightly fewer labeled cells at the rostral end in one case and the caudal end in the other case (Fig. 9A,B). The somewhat larger injection in the rostral IC (Fig. 9C, black) produced a denser but nearly continuous band of labeled neurons. The most obvious discontinuities in Figure 9C are re- lated to the 3D reconstructions from sagittal sections and not the location of the injection site. Coarse mapping of ITD in IC? The present data suggest that the individual MSO axons termi- nate along the entire rostrocaudal length of the fibrodendritic laminas in the central nucleus of the IC rather than in discrete point-to-point connections. It is unlikely that this rostrocaudally extensive projection to the ICC is caused by the diffusion of tracer along the rostrocaudal axis of MSO because the present injections were restricted in their rostrocaudal spread. Moreover, our re- sults are unlikely to be caused by fibers of passage in the injection sites. Although we cannot absolutely rule out fibers of passage, their contribution seems minimal. If fibers of passage were a problem, they should be seen in the two cases with two injections in MSO. We would expect the rostral injections to produce more labeling in the IC than the caudal injections because MSO effer- ents run parallel to the nucleus to exit rostrally. Plus, axons with both fluorescent labels should be observed but were not. A final argument that minimizes the contribution of fibers of passage is that the results of the anterograde experiments are fully consis- tent with the findings from retrograde experiments. Topographical maps relevant to ITD processing were not a focus of previous anatomical studies of MSO projections to the IC. Early studies with lesion methods (Van Noort, 1969) and Figure 8. Measurements of bouton gradients along arbitrary axes perpendicular to the fre- quency axis. A, Three-dimensional reconstruction of sampled boutons in case 31 (BF 300 Hz). The sampled area in each section is enclosed in a rectangle. B, Reconstructions of sampled boutonsincase85(BF1kHz)andcase56(BF5kHz).C,Case31afterrotationsothatthesurface of the lamina enclosing the boutons is parallel to the plane of view. The viewing angle is indicated by the arrow in A. D, The boutons in C have been flattened onto the x-y plane and rotated so that the longest axis is parallel to the x-axis of the graph. Only 300 TMR (black) and BDA (gray) randomly selected boutons terminals are shown, and the BDA bouton terminals have been offset from the TMR terminals by 3% to the right for the purpose of illustration. All bouton terminals were included in the analyses shown in E–H. E, The number of boutons, as a percentage of all the boutons of the same type, are plotted as a function of x-axis position (E, Figure 8. Measurements of bouton gradients along arbitrary axes perpendicular to the fre- quency axis. Topography of ITD coding in MSO Our data provide no support for the idea that the azimuth is encoded by MSO neurons arranged rostrocaudally according to the best ITD, but they do not rule out this possibility. Indeed, the recordings at injection sites indicate that any topography that is present for ITD must be weak. Yin and Chan (1990) provided evidence for such a topography by merging data from several animals. Although our sample of recordings from low-frequency MSO neurons was weighted toward the rostral half, the two widely separated recordings in the same MSO had best ITDs inconsistent with the assumed topographical arrangement. Both the individual and paired recordings suggest a large degree of variability even within the same animal. In our study, some of the variability may have been caused by our use of multiunit record- ings, but similar variability is present in the data of Yin and Chan (1990). Some neurons at a single rostrocaudal position had very different best ITDs, and neurons with similar best ITDs were found at very different rostrocaudal locations (Yin and Chan, 1990, their Fig. 13). Thus, any map of ITD in the MSO must be coarse, at best. Discussion The present results imply that a simple, linear system of fine- grain, point-to-point connections does not convey a spatial map of ITD, or any other stimulus parameter, from the MSO to the IC. Small groups of adjacent neurons labeled with anterograde trac- areasalongthelaminaineachsection.Thesesamesampleareaswereanalyzedineachsection and summed, and the sum corresponds to the bouton density from a single section in the transverseplane.C1,Histogramsofboutondensityinserialsectionsfromthreeseparatecases, eachwithitsMSOinjectioninadifferentrostrocaudallocation.Thepeaksofmaximumbouton density are staggered in different sections along the rostrocaudal dimension of the IC. C2, Bouton density in serial sections after two injections in MSO at 1000 Hz BF at different rostro- caudalpositions.MaximumboutondensityisattherostralendoftheICforbothinjections.C3, BoutondensityinserialsectionsaftertwoinjectionsinMSOat300HzBFatdifferentrostrocau- dalpositions.MaximumboutondensityfromtherostralMSOisinthemiddleoftheIC,whereas the caudal injection has peaks of high density rostrally and caudally in the IC. areasalongthelaminaineachsection.Thesesamesampleareaswereanalyzedineachsection and summed, and the sum corresponds to the bouton density from a single section in the transverseplane.C1,Histogramsofboutondensityinserialsectionsfromthreeseparatecases, eachwithitsMSOinjectioninadifferentrostrocaudallocation.Thepeaksofmaximumbouton density are staggered in different sections along the rostrocaudal dimension of the IC. C2, Bouton density in serial sections after two injections in MSO at 1000 Hz BF at different rostro- caudalpositions.MaximumboutondensityisattherostralendoftheICforbothinjections.C3, BoutondensityinserialsectionsaftertwoinjectionsinMSOat300HzBFatdifferentrostrocau- dalpositions.MaximumboutondensityfromtherostralMSOisinthemiddleoftheIC,whereas the caudal injection has peaks of high density rostrally and caudally in the IC. Figure7. ThedensityofboutonsinsingleICClaminas.A,Intransversesections(A1),bouton counts were made from within a single lamina, such as that shown in higher magnification to the right of the section. The two types of dextran-labeled axons in this case are shown in gray andblack.Thesamelaminawasidentifiedandcountedineachserialsection.Acountingframe (A2) was moved down the middle of the lamina to control the sampled area (the center of the rectangleinthehighermagnificationofthelamina).Boutonsthatintersectedwiththetwogray edgesofthecountingframewerenotincluded.B,Horizontalsectionshowingthesevensample Oliver et al. • Connections for ITD Coding in IC 7446 • J. Neurosci., August 13, 2003 • 23(19):7438–7449 ers send their axons along the entire length of laminas in the central nucleus of the IC. Quantitative analysis of the boutons from these axons shows that the synapses from these axons do not exhibit a gradient in density related to their connections to the MSO. The arrangement of retrogradely labeled neurons in MSO also supports the absence of a gradient because points in the IC laminas receive convergent inputs from neurons that are evenly distributed along the rostrocaudal extent of the MSO. These data suggest that information about the azimuthal location of a sound source in the IC does not depend on a simple map transmitted from the MSO. parallel to the long axis) and y-axis position (F, perpendicular to the long axis). The same bin widthisusedinbothhistograms.Thedistributionofboutonsalongotheraxeswasalsochecked by rotating the data in 10° steps and replotting the histograms (data not shown). G, Bouton distributionincase56,paralleltothelongestaxis(plottedalongthex-axis)andperpendicular tothatplottedalongthey-axis.H,Boutondistributionincase85.C,Caudal;D,dorsal;M,medial; L, lateral; V, ventral. Coarse mapping of ITD in IC? A, Three-dimensional reconstruction of sampled boutons in case 31 (BF 300 Hz). The sampled area in each section is enclosed in a rectangle. B, Reconstructions of sampled boutonsincase85(BF1kHz)andcase56(BF5kHz).C,Case31afterrotationsothatthesurface of the lamina enclosing the boutons is parallel to the plane of view. The viewing angle is indicated by the arrow in A. D, The boutons in C have been flattened onto the x-y plane and rotated so that the longest axis is parallel to the x-axis of the graph. Only 300 TMR (black) and BDA (gray) randomly selected boutons terminals are shown, and the BDA bouton terminals have been offset from the TMR terminals by 3% to the right for the purpose of illustration. All bouton terminals were included in the analyses shown in E–H. E, The number of boutons, as a percentage of all the boutons of the same type, are plotted as a function of x-axis position (E, parallel to the long axis) and y-axis position (F, perpendicular to the long axis). The same bin widthisusedinbothhistograms.Thedistributionofboutonsalongotheraxeswasalsochecked by rotating the data in 10° steps and replotting the histograms (data not shown). G, Bouton distributionincase56,paralleltothelongestaxis(plottedalongthex-axis)andperpendicular tothatplottedalongthey-axis.H,Boutondistributionincase85.C,Caudal;D,dorsal;M,medial; L, lateral; V, ventral. J. Neurosci., August 13, 2003 • 23(19):7438–7449 • 7447 Oliver et al. • Connections for ITD Coding in IC retrograde methods (Roth et al., 1978; Adams, 1979; Brunso- Bechtold et al., 1981; Aitkin and Schuck, 1985; Maffi and Aitkin, 1987) emphasized the laterality of the projections and the con- vergence of inputs from different sources. The tonotopic organi- zation of these midbrain projections was described in the cat and in nucleus laminaris of the barn owl in studies using autoradio- graphic tracing methods (Henkel and Spangler, 1983; Takahashi and Konishi, 1988). In these studies, there was extensive rostro- caudal labeling in IC; however, those studies could not distin- guish labeled axons from labeled synaptic boutons, and the size of the injections relative to the size of the MSO or nucleus laminaris was larger than in the present studies. Figure9. RetrogradelabelinginMSOafterinjectionsinIC.A,Injectionsofgreen(gray)and red(black)latexmicrospheresinICofcase66andretrogradetransporttoMSO.Stacksoffrontal (transverse)sectionsshowtheICinjection(left).Asinglesectionfromthebrainstematthelevel ofthesuperioroliveshowsMSO(arrow).The3Dsolidsreconstructionoftheinjectionsitesinthe IC shows a horizontal view as seen from the dorsal surface of the brain (top right). Coarse mapping of ITD in IC? The IC is transparentsothatthelocationsoftheinjectionsitescanbeseen.The3Dsolidsreconstruction ofMSOshowsretrogradelylabeledneurons(bottomright,grayspheres).TheMSOinthelateral viewisrotated45°aroundtherostrocaudalaxissothatitappearsasaflatsurfaceparalleltothe page.ThisviewofMSOisasiftheeyewereatthelocationindicatedbythearrow.B,Injections of green (gray) and red (black) microspheres in frontal sections of case 7 and retrogradely labeled neurons in MSO (gray and black spheres respectively) (details are in A) C Despite rostrocaudally projecting axons along an isofre- quency lamina, could these axons convey a coarse map of ITD? One method to convey a coarse map from different MSO sites would be a coarse shift in the entire projection to the ICC that varies systematically from one site to another. Shifts in labeling were not reported in previous studies of the cat (reference cita- tions above), but those studies did not specifically look for such a shift; however, a rostrocaudal shift in the projections to IC was reported in the barn owl (Takahashi and Konishi, 1988). Injec- tions at different positions along the ITD axis were made in nu- cleus laminaris in different owls. In the present data, reproducible shifts in the boutons were not found in any dimensions of the ICC perpendicular to the frequency axis; however, a coarse map of ITD could be conveyed by an uneven distribution of synaptic inputs along a lamina. Our findings suggest that MSO axons may distribute their synaptic inputs heterogeneously as they travel along the ICC lamina. Injectionsoffluorogold(gray)at350HzBFandredlatexmicrospheres(black)at1kHzBF.Stacks of sagittal sections are at top left. Horizontal view of IC (right) and lateral view of MSO (left bottom) show retrogradely labeled neurons in MSO (gray and black spheres) as in A and B. D, Dorsal; R, rostral; L, lateral; M, medial; V, ventral. Scale bars, 1 mm. The encoding of ITD in the IC This would create ICC neurons with different ITDs distributed almost randomly within a lamina. Carr CE, Konishi M (1988) Axonal delay lines for time measurement in the owl’s brainstem. Proc Natl Acad Sci USA 85:8311–8315. Coggeshall RE, Lekan HA (1996) Methods for determining numbers of cells and synapses: a case for more uniform standards of review. J Comp Neu- rol 364:6–15. Cook DL, Schwindt PC, Grande LA, Spain WJ (2003) Synaptic depression in the localization of sound. Nature 421:66–70. ITD-sensitive inputs from other sources might converge with MSO inputs to contribute to an irregular spatial distribution of ITD sensitivity in ICC. Both anatomical (Oliver, 2000) and phys- iological (Stanford et al., 1992; Batra et al., 1993; McAlpine et al., 1998) data support convergence in the IC. Neurons sensitive to ITDs are present in the LSO (Finlayson and Caspary, 1991; Joris and Yin, 1995; Batra et al., 1997a,b) and in the dorsal nucleus of the lateral lemniscus (DNLL) (Brugge et al., 1970), and both of these nuclei project to the IC where they may converge with the inputs from the MSO. Because the ipsilateral LSO and DNLL provide inhibition to the IC and may terminate on separate parts of a lamina (Oliver, 2000), the particular pattern of convergence may produce zones with different functionality and a nonlinear, distributed arrangement of ITD sensitivity. Finlayson PG, Caspary DM (1991) Low-frequency neurons in the lateral superior olive exhibit phase-sensitive binaural inhibition. J Neurophysiol 65:598–605. Fitzpatrick DC, Batra R, Stanford TR, Kuwada S (1997) A neuronal popu- lation code for sound localization. Nature 388:871–874. Goldberg JM, Brown PB (1969) Response properties of binaural neurons of dog superior olivary complex to dichotic tonal stimuli: some physiologi- cal mechanisms of sound localization. J Neurophysiol 32:613–636. Hafter ER, Trahiotis C (1997) Functions of the binaural system. In: Ency- clopedia of acoustics (Crocker MJ, ed), pp 1461–1480. New York: Wiley. Henkel CK, Spangler KM (1983) Organization of the efferent projections of the medial superior olivary nucleus in the cat as revealed by HRP and autoradiographic tracing methods. J Comp Neurol 221:416–428. Irvine DR, Gago G (1990) Binaural interaction in high-frequency neurons in inferior colliculus of the cat: effects of variations in sound pressure level on sensitivity to interaural intensity differences. J Neurophysiol 63:570–591. Cellular mechanisms are also likely to shape the network that codes sound location in the IC. References d ( McAlpine D, Jiang D, Shackleton TM, Palmer AR (1998) Convergent input from brainstem coincidence detectors onto delay-sensitive neurons in the inferior colliculus. J Neurosci 18:6026–6039. Adams JC (1979) Ascending projections to the inferior colliculus. J Comp Neurol 183:519–538. Aitkin L, Schuck D (1985) Low frequency neurons in the lateral central nucleus of the cat inferior colliculus receive their input predominantly from the medial superior olive. Hear Res 17:87–93. McAlpine D, Jiang D, Palmer AR (2001) A neural code for low-frequency sound localization in mammals. Nat Neurosci 4:396–401. Oliver DL (2000) Ascending efferent projections of the superior olivary complex. Microsc Res Tech 51:355–363. Aitkin LM, Pettigrew JD, Calford MB, Phillips SC, Wise LZ (1985) Repre- sentation of stimulus azimuth by low-frequency neurons in inferior col- liculus of the cat. J Neurophysiol 53:43–59. Oliver DL, Morest DK (1984) The central nucleus of the inferior colliculus in the cat. J Comp Neurol 222:237–264. Batra R, Fitzpatrick DC (1997) Neurons sensitive to interaural temporal disparities in the medial part of the ventral nucleus of the lateral lemnis- cus. J Neurophysiol 78:511–515. Oliver DL, Kuwada S, Yin TC, Haberly LB, Henkel CK (1991) Dendritic and axonal morphology of HRP-injected neurons in the inferior colliculus of the cat. J Comp Neurol 303:75–100. the cat. J Comp Neurol 303:75–100. Batra R, Kuwada S, Stanford TR (1993) High-frequency neurons in the in- ferior colliculus that are sensitive to interaural delays of amplitude- modulated tones: evidence for dual binaural influences. J Neurophysiol 70:64–80. Oliver DL, Beckius GE, Ostapoff EM (1994) Connectivity of neurons in identified auditory circuits studied with transport of dextran and micro- spheres plus intracellular injection of lucifer yellow. J Neurosci Methods 53:23–27. Batra R, Kuwada S, Fitzpatrick DC (1997a) Sensitivity to interaural tempo- ral disparities of low- and high-frequency neurons in the superior olivary complex: I. Heterogeneity of responses. J Neurophysiol 78:1222–1236. Oliver DL, Beckius GE, Shneiderman A (1995) Axonal projections from the lateral and medial superior olive to the inferior colliculus of the cat: a study using electron microscopic autoradiography. J Comp Neurol 360:17–32. Batra R, Kuwada S, Fitzpatrick DC (1997b) Sensitivity to interaural tempo- ral disparities of low- and high-frequency neurons in the superior olivary complex: II. Coincidence detection. J Neurophysiol 78:1237–1247. Oliver DL, Beckius GE, Bishop DC, Kuwada S (1997) Simultaneous antero- grade labeling of axonal layers from lateral superior olive and dorsal cochlear nucleus in the inferior colliculus of cat. The encoding of ITD in the IC Our results make it unlikely that any map of ITD in the MSO is transmitted by point-to-point topography to the IC. Thus, if there is a topographical organization of stimulus azimuth in the IC, as suggested by free-field recordings (Aitkin et al., 1985), it is not a reflection of a spatially mapped projection from the MSO. The question therefore arises: how is ITD encoded in the IC? Two broad systems are possible. The first is that the IC encodes only an average ITD, signaling a sound in the contralateral hemifield (McAlpine et al., 2001). The true ITD is decoded at a higher level by weighing the relative activities of neurons in the left and right IC. In this view, the pattern of innervation in IC reflects the averaging of ITD infor- mation from the MSO of one side. Such averaging would not completely obliterate sensitivity to ITD, because most neurons in the MSO are tuned to ITDs corresponding to sounds in the con- tralateral hemifield; however, such a system would predict tuning in the IC that is broader than that in the MSO. Exactly the oppo- site has been reported: tuning to ITDs appears to be sharper in the IC than in the MSO (Yin and Chan, 1990; Fitzpatrick et al., 1997). Thus, the system appears to require encoding of ITDs on a rela- tively fine scale. An alternative possibility is that ITD could be encoded by groups of neurons sensitive to particular ITDs, but these neurons may be organized in a nonlinear map. Neurons with different best ITDs may be located in different regions within a lamina in Figure9. RetrogradelabelinginMSOafterinjectionsinIC.A,Injectionsofgreen(gray)and red(black)latexmicrospheresinICofcase66andretrogradetransporttoMSO.Stacksoffrontal (transverse)sectionsshowtheICinjection(left).Asinglesectionfromthebrainstematthelevel ofthesuperioroliveshowsMSO(arrow).The3Dsolidsreconstructionoftheinjectionsitesinthe IC shows a horizontal view as seen from the dorsal surface of the brain (top right). The IC is transparentsothatthelocationsoftheinjectionsitescanbeseen.The3Dsolidsreconstruction ofMSOshowsretrogradelylabeledneurons(bottomright,grayspheres).TheMSOinthelateral viewisrotated45°aroundtherostrocaudalaxissothatitappearsasaflatsurfaceparalleltothe page.ThisviewofMSOisasiftheeyewereatthelocationindicatedbythearrow.B,Injections of green (gray) and red (black) microspheres in frontal sections of case 7 and retrogradely labeled neurons in MSO (gray and black spheres, respectively) (details are in A). C, 7448 • J. Neurosci., August 13, 2003 • 23(19):7438–7449 Oliver et al. • Connections for ITD Coding in IC the ICC. Our finding that the MSO boutons are distributed with an uneven density within a single lamina suggests that an ICC neuron at one point in the lamina may receive a heavier input from one point in MSO than from another. The encoding of ITD in the IC There is a heterogeneous popu- lation of neurons in the IC (Peruzzi et al., 2000; Sivaramakrish- nan and Oliver, 2001), some of which may receive more MSO inputs than others. Local interconnections within the IC (Oliver and Morest, 1984; Oliver et al., 1991) may be important to com- bine ITD, interaural level, and spectral information. Most excit- ing is the recent discovery that IC neurons show long-term po- tentiation (Wu et al., 2002) and plasticity (Ma and Suga, 2001). This suggests that local synaptic mechanisms may be involved in coding sound location in the ICC. Joris PX, Yin TCT (1995) Envelope coding in the lateral superior olive. I. S i i i i l i diff J N h i l 6 Joris PX, Yin TCT (1995) Envelope coding in the lateral superior olive. I. Sensitivity to interaural time differences. J Neurophysiol 73:1043–1062. Joris PX, Yin TCT (1995) Envelope coding in the lateral superior olive. I. Sensitivity to interaural time differences. 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Beckius GE, Batra R, Oliver DL (1999) Axons from anteroventral cochlear nucleus that terminate in medial superior olive of cat: observations re- lated to delay lines. J Neurosci 19:3146–3161. n the inferior colliculus of cat. J Comp Neurol 382:215–229. Overholt EM, Rubel EW, Hyson RL (1992) A circuit for coding interaural time differences in the chick brainstem. J Neurosci 12:1698–1708. Brand A, Behrend O, Marquardt T, McAlpine D, Grothe B (2002) Precise inhibition is essential for microsecond interaural time difference coding. Nature 417:543–547. Peruzzi D, Sivaramakrishnan S, Oliver DL (2000) Identification of cell types in brain slices of the inferior colliculus. Neuroscience 101:403–416. Rhode WS (1976) A digital system for auditory neurophysiological re- search. In: Current computer technology in neurobiology (Brown P, ed), pp 543–567. Washington, DC: Hemisphere Press. Brugge JF, Anderson DJ, Aitkin LM (1970) Responses of neurons in the dorsal nucleus of the lateral lemniscus of cat to binaural tonal stimulation. J Neurophysiol 33:441–458. Brunso-Bechtold JK, Thompson GC, Masterton RB (1981) HRP study of the organization of auditory afferents ascending to central nucleus of inferior colliculus in cat. J Comp Neurol 197:705–722. Roth GL, Aitkin LM, Andersen RA, Merzenich MM (1978) Some features of the spatial organization of the central nucleus of the inferior colliculus of the cat. J Comp Neurol 182:661–680. Oliver et al. • Connections for ITD Coding in IC Roth GL, Kochhar R, Hind JE (1980) Interaural time differences: implica- tions regarding the neurophysiology of sound localization. J Acoust Soc Am 68:1643–1651. Sullivan WE, Konishi M (1986) Neural map of interaural phase difference in the owl’s brainstem. Proc Natl Acad Sci USA 83:8400–8404. 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https://openalex.org/W2794582383	https://europepmc.org/articles/pmc5916305?pdf=render	English	null	Long‐term persistence of horse fecal <scp>DNA</scp> in the environment makes equids particularly good candidates for noninvasive sampling	Ecology and evolution	2,018	cc-by	9,275	Received: 19 October 2017 \| Revised: 30 January 2018 \| Accepted: 5 February 2018 Received: 19 October 2017 \| Revised: 30 January 2018 \| Accepted: 5 February 2018 Received: 19 October 2017 \| Revised: 30 January 2018 \| Accepted: 5 February 2018 DOI: 10.1002/ece3.3956 Long-term persistence of horse fecal DNA in the environment makes equids particularly good candidates for noninvasive sampling Sarah R. B. King1 \| Kathryn A. Schoenecker2 \| Jennifer A. Fike2 \| Sara J. Oyler-McCance2 1Natural Resource Ecology Laboratory, Department of Ecosystem Science and Sustainability, Colorado State University, Fort Collins, CO, USA 2United States Geological Survey, Fort Collins Science Center, Fort Collins, CO, USA 1Natural Resource Ecology Laboratory, Department of Ecosystem Science and Sustainability, Colorado State University, Fort Collins, CO, USA 2United States Geological Survey, Fort Collins Science Center, Fort Collins, CO, USA Abstract Fecal DNA collected noninvasively can provide valuable information about genetic and ecological characteristics. This approach has rarely been used for equids, despite the need for conservation of endangered species and management of abundant feral populations. We examined factors affecting the efficacy of using equid fecal samples for conservation genetics. First, we evaluated two fecal collection methods (paper bag vs. ethanol). Then, we investigated how time since deposition and month of col- lection impacted microsatellite amplification success and genotyping errors. Between May and November 2014, we collected feral horse fecal samples of known age each month in a feral horse Herd Management Area in western Colorado and documented deterioration in the field with photographs. Samples collected and dried in paper bags had significantly higher amplification rates than those collected and stored in ethanol. There was little difference in the number of loci that amplified per sample between fresh fecal piles and those that had been exposed to the environment for up to 2 months (in samples collected in paper bags). After 2 months of exposure, ampli- fication success declined. When comparing fresh (0–2 months) and old (3–6 months) fecal piles, samples from fresh piles had more matching genotypes across samples, better amplification success and less allelic dropout. Samples defecated during the summer and collected within 2 months of deposition had highest number of geno- types matching among samples, and lowest rates of amplification failure and allelic dropout. Due to the digestive system and amount of fecal material produced by equids, as well as their occurrence in arid ecosystems, we suggest that they are particularly good candidates for noninvasive sampling using fecal DNA. Ecology and Evolution. 2018;8:4053–4064. \| 4053 www.ecolevol.org This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2018 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. 4054 \| 1 \| INTRODUCTION The promise of these noninvasive approaches has yet to be fully realized and extends to a wide variety of species for which demographic and other ecological information is needed. In addition to variation in amplification effectiveness by spe- cies or collection method, successful amplification and genotyping of fecal DNA depends on time since deposition and environmen- tal factors. Amplification success was reported to be higher in the colder temperatures of winter for several studies (Harris et al., 2010; Hettinga et al., 2012; Liu et al., 2014b; Lucchini et al., 2002; Maudet, Luikart, Dubray, Von Hardenberg, & Taberlet, 2004). In general, fecal pellets persist longer in dry environments or where they are shel- tered from rain (Harestad & Bunnell, 1987). While aridity may pre- serve DNA, UV light will degrade it (Ravanat, Douki, & Cadet, 2001), thus fecal samples that have had prolonged exposure to sunlight may be less likely to yield amplifiable DNA. In addition, amplification suc- cess is affected by time since deposition and ambient temperature, as well as dew point (Murphy et al., 2007). Collecting fresh fecal samples reduces the impacts of these environmental effects, with several studies demonstrating less successful amplification of older feces (Foran, Crooks, & Minta, 1997; Lucchini et al., 2002; Maudet et al., 2004; Piggott, 2004; Poole et al., 2011). Desiccation rates of feces can vary during different times of the year, making assess- ment of age in the field difficult (Stenglein, Waits, Ausband, Zager, & Mack, 2010). Collecting very fresh fecal samples can be time- consuming, as it requires knowledge of where the animals are likely to have been or even direct observation of the individual. Sampling only areas of known use by a target species may bias population es- timates. Therefore, if older feces can yield amplifiable DNA, then a greater number of samples can potentially be collected from a wider or more randomly chosen area. Five of the seven extant equid species are threatened with ex- tinction (Moehlman, King, & Kebede, 2016). Conversely, the two domesticated equids (horses, Equus ferus caballus, and donkeys, E. africanus asinus) are abundant in feral populations to the point of being considered nuisance species in some places (Garrott & Oli, 2013; Woolnough et al., 2012). For both rare and common equids, conservation and management can be greatly enhanced by under- standing genetic characteristics of populations and individuals. 4054 \| 1 \| INTRODUCTION 4054 KING et al. KING et al. noninvasive sampling for genetic analysis. Ideally, such sampling methods could be explained to personnel with no scientific train- ing, using supplies that are readily available. Equids produce large amounts of fecal material that can be found easily, making this source of DNA easier to collect than hair. Fecal samples are typically placed in ethanol, with some studies reporting that this method of preservation produced better amplification results than when col- lected in DMSO/EDTA/Tris/salt (DETs) buffer (e.g., Panasci et al., 2011). Additional sample collection methods (other than ethanol and DETs buffer) include freezing samples in plastic bags or dry- ing them with silica either immediately or after initial collection in alcohol (Frantzen, Silk, Ferguson, Wayne, & Kohn, 1998; Hettinga et al., 2012; Murphy et al., 2002; Nsubuga et al., 2004; Panasci et al., 2011; Wasser, Houston, Koehler, Cadd, & Fain, 1997). The results of studies have been mixed in terms of which sampling methods pro- duce the best results (Renan et al., 2012), and there may be a spe- cies- or genus-specific relationship between collection method and DNA amplification success. Noninvasive methods such as the use of DNA extracted from fecal samples are increasingly being used to examine occupancy, popula- tion size, diet, and even hormones of a wide range of species (Bowser, Diamond, & Addison, 2013; Ernest, Penedo, May, Syvanen, & Boyce, 2000; Macandza, Owen-Smith, & Le Roux, 2014; Oyler- McCance et al., 2018; Powell & Monfort, 2001; Prugh, Ritland, Arthur, & Krebs, 2005; Schoenecker et al., 2014). Noninvasive sampling of fecal DNA has been used for conservation goals as varied as estimat- ing population sizes of forest elephants (Loxodonta cyclosis, Eggert, Eggert, & Woodruff, 2003; Eggert et al., 2013), identifying hybrids in a reintroduced population of red wolves (Canis rufus, Adams, Kelly, & Waits, 2003), intra-community relationships of bonobos (Pan pa- niscus, Gerloff, Hartung, Fruth, Hohmann, & Tautz, 1999), and use of resources by individual Sonoran pronghorn (Antilocapra americana sonoriensis, Woodruff, Lukacs, Christianson, & Waits, 2016). Several studies have highlighted the advantages of noninvasive sampling compared to traditional methods (Beja-Pereira, Oliveira, Alves, Schwartz, & Luikart, 2009; Kohn & Wayne, 1997; Luikart, Ryman, Tallmon, Schwartz, & Allendorf, 2010), and in some cases they have been shown to be at least as effective at monitoring various param- eters, if not more so, than traditional methods. Abstract Correspondence Sarah R. B. King, Natural Resource Ecology Laboratory, Department of Ecosystem Science and Sustainability, Colorado State University, Fort Collins, CO, USA. Email: sarah.king@colostate.edu Funding information U.S. Geological Survey Fort Collins Science Center; Bureau of Land Management Wild Horse and Burro Program conservation, Equus, genotype, horse, noninvasive sampling \| 4053 www.ecolevol.org Ecology and Evolution. 2018;8:4053–4064. 4054 \| 1 \| INTRODUCTION Unlike ruminants, equids are bulk feeders which consume high quan- tities of relatively low-quality forage to meet their nutritional needs (Schoenecker, King, Nordquist, Nandintsetseg, & Cao, 2016). This results in production of large quantities of fecal matter rich in ep- ithelial cells from passage through the gut, making them potentially very good candidates for fecal analyses. Fecal DNA has been used to inform the conservation of some endangered equid species: the African wild ass (E. africanus, Rosenbom, Costa, Steck, Moehlman, & Beja-Pereira, 2011), Przewalski’s horse (E. ferus przewalskii, Liu et al., 2014a), and Grevy’s zebra, (E. grevyi, Kebede et al., 2014). However, noninvasive sampling has been more commonly used for carnivores than herbivores (Harris et al., 2010; Poole, Reynolds, Mowat, & Paetkau, 2011), and techniques have mostly been tested on carni- vores (e.g., Murphy, Kendall, Robinson, & Waits, 2007). Thus, sample collection techniques for herbivores need to be tested and opti- mized, as factors affecting amplification success of fecal DNA may be different in this group. We conducted research to assess whether it would be feasible to use fecal DNA to examine various parameters of feral horse ecology and population biology. The objectives of this study were to compare two techniques for collecting feral horse fecal samples (paper bags and ethanol vials); investigate the relationship between fecal pile age and genotyping success, and the optimal time of year to collect sam- ples (through both amplification success and genotyping errors); and develop a visual guide documenting horse feces deterioration over For conservation and management of wildlife and feral animals there, is a need for consistent, inexpensive, and simple methods of \| 4055 KING et al. FI G U R E 1 Weather data from the Pine Ridge National Oceanic and Atmospheric Administration (NOAA) Remote Automatic Weather Station (RAWS), located 1 km east of Little Book Cliffs Herd Management Area, Colorado, USA. Gray bars show the total precipitation (cm) each month during the 2014 study period. Mean temperature (°C) and relative humidity (%) for each month are shown by solid circles; mean maximum and minimum values are shown by triangles KING et al. 4055 FI G U R E 1 Weather data from the Pine Ridge National Oceanic and Atmospheric Administration (NOAA) Remote Automatic Weather Station (RAWS), located 1 km east of Little Book Cliffs Herd Management Area, Colorado, USA. 4054 \| 1 \| INTRODUCTION Gray bars show the total precipitation (cm) each month during the 2014 study period. Mean temperature (°C) and relative humidity (%) for each month are shown by solid circles; mean maximum and minimum values are shown by triangles coordinates, and took samples from a random 10 of the 20 piles. In June, we re-visited and photographed the 20 piles marked in May and sampled a random 10 of the May piles. We marked twenty fresh piles from June as in May, and sampled a random 10 of the June piles. This was repeated every month until November: each month 20 fresh piles were marked with a stake, photographed, and GPS location re- corded with a random 10 sampled; plus all of the 20 piles marked in every previous month were visited and photographed, with samples taken randomly from 10. New fecal piles close to a marked pile could be detected by their relative freshness; if they were directly adjacent to a marked pile we sampled from the opposite side. Our sampling strategy (sampling a random 10 piles of the 20 marked piles) ensured that a sufficient amount of the pile would remain at the end of the study; thus, not all piles were sampled every month. In most months, it was possible to find 20 fresh piles within the same day. However in May, June, and October it was not possible to find 20 fresh piles on the same day, so some piles from these months were separated in age by one to 5 days (mean 30 ± 3 days SD between sample collec- tion points overall). Each pile was fresh on the day it was first sam- pled, photographed, and marked. Deterioration of fecal piles over time was documented with digital photographs. time in the environment to aid in field collection. We expected to see some difference in amplification success and genotyping errors between the collection methods, that as samples aged there would be a rapid decline in amplification success, and that the optimal time to collect samples would be in cool or dry times of the year. 2.1 \| Study area We conducted our study at the 146 km2 Little Book Cliffs Wild Horse Herd Management Area, near Grand Junction, Colorado, USA, between May and November 2014. The area mostly consists of mesa top covered in pinyon pine (Pinus edulis) and juniper (Juniperus spp.) interspersed with sagebrush (Artemisia spp.) and grass mead- ows. The area is dissected by deep canyons, some of which have grass and water along the bottom. Water sources for horses and wildlife are located throughout the area as natural creeks or main- tained in tanks. Data from the Pine Ridge National Oceanic and Atmospheric Administration (NOAA) Remote Automatic Weather Station (RAWS), located on a mesa adjacent to the study site, showed that during our study period conditions were mostly dry, with the exception of 8 cm of precipitation falling in both August and September (Figure 1). Fecal samples were collected and stored in two ways: paper bags, and vials of 95% ethanol. We avoided contamination of sam- ples using nitrile gloves or sterile tongue depressors to manipulate the fecal bolus. For paper bag samples, we placed a whole fecal bolus in a paper lunch bag with details of the sample written on it (sam- ple number, collector, area, date). We then placed these paper bag samples in large cotton bags to hang and dry during the sampling session. Three to 5 days after collection, we transferred samples to a drying oven to desiccate at 40°C for 3 days. We collected samples in ethanol from July onwards: we placed approximately 2 cm3 of a bolus from the same pile as the paper bag sample in to a vial of etha- nol (estimating a ratio of 1:3 to 1:4 feces to alcohol within each vial). 2.3 \| Molecular methods We cut a small amount of feces (~5 mm3, or enough to fill about 1 ml of a 1.7 ml tube) from the outer layer of a sample for analyses. We retained the rest of the sample for other tests. Genomic DNA was isolated from horse feces following the animal tissue protocol of the DNeasy96 Blood and Tissue kit (Qiagen Inc., Valencia, CA) with the following modifications: (1) samples were incubated overnight at 56°C in 900 μl Buffer ATL with 20 μl proteinase K and 20 μl 1M DTT; and (2) DNA was eluted in 80 μl Buffer AE. Extraction negatives were included with every set of extractions. Samples were amplified across eight variable microsatellite loci (AHT4: called HMB4 in Binns, Holmes, Holliman, and Scott (1995), HMS1: Guérin, Bertaud, and Amigues (1994), HTG4 and HTG6: Ellegren, Johansson, Sandberg, and Andersson (1992), and HMS3, HMS6, HMS7, ASB2: redesigned primers from Eggert et al. (2010)), which were chosen because they were highly polymorphic, worked well with fecal DNA, and were easy to score. These markers were tested to ensure that they had a low probability of identity (i.e., the probability that two individuals would have the same genotype (Waits, Luikart, & Taberlet, 2001): using the GenAlex Excel add-in (Peakall & Smouse, 2006, 2012) we determined that this was true at five microsatellites that were least polymorphic (p < .001). The eight microsatellites were amplified using the preamplification method described by Piggott, Bellemain, Taberlet, and Taylor (2004). The preamplification method is a two- step procedure that involved an initial PCR using a pool of unlabeled primer pairs for all eight loci. This initial step was performed follow- ing the conditions outlined in Piggott et al. (2004) with the excep- tion of using 10 μl fecal DNA as the template for the 50-μl reaction. The second step used 3 μl of the PCR product produced in the first step as template for 12.5 μl reactions containing 0.2 mmol/L of each dNTP, 1× GoTaq Flexi Buffer (Promega), 1.5 mmol/L MgCl2, 1× BSA, 0.5 ⋅mol/L of each primer (dye-labeled forward), and 1 U of Taq DNA polymerase (Promega). 2.4.1 \| Comparison of collection method To compare paper bag and ethanol samples, we only used fecal sam- ples from the same fecal pile that that were collected and stored using both methods. We examined amplification success in several ways. First, within each collection method, we counted the number of samples that had successful amplification at all eight loci. Further, since we knew that we needed amplification at a minimum of five loci for unique identification, we determined amplification rates for only those samples with genotypes at five or more loci. Finally, we counted the number of samples that did not amplify at any loci and compared those results between collection methods. Paired Student’s t-tests were used to determine statistical significance of results. 2.3 \| Molecular methods The amplification conditions for the second step were as follows: 94°C for 2 min, then 94°C for 1 min, annealing temperature (55°C: ASB2, HTG4, HTG6; 59°C: HMS6; 60°C: AHT4, HMS3, HMS7, HMS1) for 1 min, 72°C for 1 min for 40 cycles, fol- lowed by 72°C for 10 min, and a final extension at 60°C for 45 min. We ran positive and negative controls during all PCR amplifications. PCR products were multi-loaded based on product size and primer label, combined with GeneScan LIZ 600 internal lane size stand- ard (Applied Biosystems), and electrophoresed through a capillary gel matrix using an AB3500 Automated DNA Sequencer (Applied Biosystems). Allele sizes were determined for each locus using GeneMapper v5 software (Applied Biosystems). Fecal samples can have lower quantities of DNA that is often of lower quality, both factors that increase the probability of 4056 \| Vials of ethanol were kept at room temperature and stored upright to prevent leakage. 4056 \| Vials of ethanol were kept at room temperature and stored upright to prevent leakage. compared to identify and quantify errors within that sample. In addition, multiple samples from the same fecal pile sampled at different times as deposition can be amplified and genotypes compared to determine whether errors are more prevalent in older samples. Potential errors can include false alleles (where the genotype obtained is incorrect due to PCR errors, problems with electrophoresis, or human error) or allelic dropout (where one allele of a heterozygote does not amplify) and comparisons among amplifications within a sample can help identify these errors and determine the correct genotype (Broquet & Petit, 2004). To minimize error for each DNA extraction, each sample was PCR-amplified multiple times (at least twice but often three, four, or five times) at every microsatellite locus and we compared genotypes across these multiple amplifications. If a sample had matching genotypes in at least two amplifications, that match- ing genotype was retained. Samples that did not have matching genotypes after multiple amplifications or that amplified only once were treated as missing data at that locus. Genotypes were scored by two independent observers. If a sample failed to am- plify or was scored differently between the two attempts, it was re-amplified until two matching genotypes were obtained or the DNA was depleted. Vials of ethanol were kept at room temperature and stored upright to prevent leakage. 2.2 \| Sample collection We took samples from fecal piles deposited in meadows in the north of the study area (an area known as North Soda) because of accessi- bility and to retain consistency. In May 2014, we found 20 fecal piles that were either observed being defecated or were determined to be very fresh (still moist on the outside and attracting flies). We marked each pile with a numbered stake, took a photograph, recorded GPS 4056 KING et al. KING et al. amplified at all eight loci compared to only 17.2% of ethanol sam- ples. When we consider only samples with genotypes for at least five loci (the number of loci needed to uniquely identify individu- als), 90% of the samples collected in paper bags were usable com- pared to only 55.2% of those that were collected in ethanol. The complete failure to amplify at any locus was higher (17.6%) for ethanol samples compared to only 2.8% of samples collected in paper bags. 3.3 Using the full data set from paper bag samples (May to November, n = 280 samples), we found little difference in the number of loci that amplified per sample between samples from fresh piles and those that had been exposed to the environment for up to 2 months. After this, there was a decline in amplification success (ANOVA F1,278 = 22.49, p = <.0001; Figure 2), but even the relatively small number of samples (n = 10) from fecal piles that were 6 months old amplified at six of the eight loci on average. As our samples came from unknown individuals we could not definitively assess genotyp- ing success (i.e., we did not know the true genotype), but one would expect multiple samples collected from the same fecal pile over time to have the same genotype. When looking at repeated samples from the same fecal pile, samples from fresher piles (0–2 months old) had more matching genotypes across samples (t = 3.546, df = 39.816, p = .001), better amplification success (i.e., less missing data at a locus; t = −3.6881, df = 31.141, p = .0009), and less allelic dropout (t = −2.5935, df = 33.029, p = .01405) than samples from older piles (3–6 months old; Figure 3). The number of false alleles were similar between samples from fresh and old piles (t = −0.41715, df = 55.966, p = .6782). 2.4.3 \| Comparing genotyping error between fresh and old samples To investigate whether genotyping error was higher in older sam- ples, we again used only samples collected in paper bags, and re- moved any samples that had genotypes at fewer than five loci. Most marked fecal piles had multiple samples taken through time (the piles were of various age). For each fecal pile, we separated the data into two groups: fresh (0–2 months old) and old (3 – 6 months old). We then compared the genotypes for the eight loci among the two or three samples within each group (fresh vs. old) for each fecal pile. We counted instances of allelic dropout (where one sample was a heterozygote and one or more samples were homozygous for one of the two matching alleles), missing data (failure to amplify), false alleles (three or more alleles present), and matching genotypes. Differences were examined using Student’s t-tests. 4 \| DISCUSSION As only a few studies have been conducted on equids using nonin- vasively collected fecal DNA (Kebede et al., 2014; Liu et al., 2014a; Rosenbom et al., 2011), our study to assess the feasibility of using such methods along with an optimization of collection methods and quantification of genotyping errors could be used as a guide for subsequent efforts. Most published noninvasive studies of mam- mals collected and stored fecal samples in ethanol (Adams & Waits, 2006; Brinkman, Schwartz, Person, Pilgrim, & Hundertmark, 2009; 3.1 \| Subjective description of deterioration of fecal piles There was a noticeable difference in color and consistency between fecal piles deposited in May, June, and July, and those deposited in September, October, and November (Table 1). In the spring/early summer, fecal boli were green and less formed than those deposited later in the year. This appears to have affected how they deterio- rated, as the boli that were less formed when deposited turned white and desiccated over 3 months, whereas boli that were more formed when deposited remained solid and a darker color for longer. 2.4.4 \| Comparison of error rates of fresh samples deposited in different months of the year As temperature and precipitation vary considerably from May to November in our study area, and because the diet of horses shifts throughout the summer, we tested whether genotyping error rates differed between fecal piles deposited in different months. To ex- amine this, we used only those samples collected in paper bags that were fresh (0–2 months old), removing any samples that had genotypes at fewer than five loci. We compared the genotypes for the eight loci among the two or three samples from each fecal pile and tested differences among months in genotyping error (allelic dropout, missing data, false alleles) and matching genotypes using ANOVA. We used these repeated samples from fresh piles (0–2 months old) to assess which month was best for equid fecal sample collec- tion. Samples deposited in June, July, and August tended to perform best on the four metrics we tested (Figure 4). May and September had the highest rates of missing data (F5,46 = 6.492, p = .000121), with May also having high rates of allelic dropout (F5,46 = 3.109, p = .01686) and lowest number of genotypes matching among sam- ples (F5,46 = 2.617, p = .03648). Rates of false alleles were low, with no statistical difference among months (F5,46 = 0.7195, p = .6121), but they appear greater in samples from May (Figure 4). 2.4.2 \| Comparison of amplification success through time Because samples collected in paper bags had higher amplification success (see Results section) and because sample collection in etha- nol did not span the entire summer, we examined the relationship between amplification success and age of fecal pile using only sam- ples collected in paper bags. For this analysis, we sorted samples by age of fecal pile (months since time of deposition) when the sample was collected. We then calculated number of loci that successfully amplified for each sample in each fecal age group. We tested effect of age on amplification success using ANOVA with fecal pile age as a discrete variable. Fecal samples can have lower quantities of DNA that is often of lower quality, both factors that increase the probability of genotyping error (Taberlet, Waits, & Luikart, 1999). To account for such errors, DNA extracted from a fecal sample can be am- plified multiple times and genotypes from those amplifications KING et al. 4057 3.2 \| Comparison of collection method Between July and November 2014, 250 samples were collected from 120 fecal piles in both paper bags and ethanol vials. Samples collected in paper bags had far greater amplification success than those collected in ethanol vials (Student’s t = 11.896, df = 249, p = <.0001; Table 2). Over half (55.6%) of paper bag samples 4058 KING et al. (Continues) ription of fecal pile appearance and deterioration at Little Book Cliffs Wild Horse Herd Management Area, Colorado, USA between May and November. e deterioration of the same pile over time 1 th 2 th 3 th 4 th 5 th 6 th TA B LE 1 Subjective description of fecal pile appearance and deterioration at Little Book Cliffs Wild Horse Herd Management Area, Colorado, USA between May and November. Photographs demonstrate the deterioration of the same pile over time Month Fresh 1 month 2 months 3 months 4 months 5 months 6 months May Often loose (some or all boli not Brownish white, intact, boli often Whitish brown, intact, often boli Mostly white, intact White, intact White, beginning to decay White, decaying, often scattered Wild Horse Herd Management Area, Colorado, USA between May and November. TA B LE 1 Subjective description of fecal pile appearance and deterioration at Little Book Cliffs Wild Horse Herd Management Area, Colorado, USA between May and November. De Barba et al., 2010; Eggert et al., 2003; Gerloff et al., 1999; Harris et al., 2010; Maudet et al., 2004; Valiere et al., 2006). Some com- parison studies found ethanol to be the most effective method for preserving fecal DNA (Murphy et al., 2002; Panasci et al., 2011), al- though their comparison tests did not include collecting and dry- ing samples in paper bags. Liu et al. (2014c) found ethanol to be the most effective preservative of Przewalski’s horse fecal DNA, in comparison with drying and freezing the fecal sample. We were therefore somewhat surprised to find that for feral horses, collecting and storing fecal samples in paper bags was much more successful than ethanol. Paper bags have been used to gather fecal samples for genetic data in a few other studies (Piggott, 2004; Piggott & Taylor, 2003; Poole et al., 2011), including for equids (Kebede et al., 2014; Rosenbom et al., 2011), but to our knowledge this is the first study to compare the amplification of DNA from fecal samples collected in ethanol to those collected in paper bags. Renan et al. (2012) found an interaction between the collection and preservation method and the method used to extract DNA from Asiatic wild ass (Equus hemio- nus) fecal samples, and also found ethanol (and subsequent drying with silica) to be least effective. TA B LE 2 Amplification success of samples collected from horse fecal piles in both paper bags and vials of ethanol at Little Book Cliffs Herd Management Area, Colorado, USA. 3.2 \| Comparison of collection method Number of samples that amplified at ≥5 loci is given as the probability of identity (PID) was ≤0.001 when at least five loci were included for both collection methods Paper bags (N = 250) Ethanol vials (N = 250) Amplified at 8 loci (%) 139 (55.6) 43 (17.2) Amplified at ≥5 loci (%) 225 (90) 138 (55.2) Did not amplify at any loci (%) 7 (2.8) 44 (17.6) 3.2 \| Comparison of collection method Photographs demonstrate the deterioration of the same pile over time Month Fresh 1 month 2 months 3 months 4 months 5 months 6 months May Often loose (some or all boli not formed), bright greenish brown Brownish white, intact, boli often stuck together Whitish brown, intact, often boli are stuck together Mostly white, intact White, intact White, beginning to decay White, decay often scatte June Sometimes loose, bright greenish brown to bright brown Brown, intact, boli often stuck together Brownish white, intact, boli often stuck together Brownish white to whitish brown, intact Whitish brown, intact, sometimes scattered Whitish brown to white, beginning to decay July Occasionally loose, bright brownish green Brown, intact Dull brown with white patches Brown to whitish brown with some brown patches Whitish brown to white, may be scattered August Bright brown Brown, intact Dull brown with white patches Brown to whitish brown with some brown patches TA B LE 1 Subjective description of fecal pile appearance and deterioration at Little Book Cliffs Wild Horse Herd Management Area, Colorado, USA between May and N Photographs demonstrate the deterioration of the same pile over time 5 months to 3 months Brown, intact August July KING et al. 4059 Month Fresh 1 month 2 months 3 months 4 months 5 months 6 months September Bright brown Brown, intact Dull brown with white patches October Dark brown Dried dark brown, intact November Dark brown TA B LE 1 (Continued) 3 months 2 months Dried dark brown, Dried dark 1 month intact Dark brown Dark brown November October KING et al. KING et al. 4060 4060 \| FI G U R E 2 Boxplot showing the number of loci that amplified from samples collected in paper bags from feral horse fecal piles at Little Book Cliffs Herd Management Area, Colorado, USA when they were fresh (0 months) to 6 months old TA B LE 2 Amplification success of samples collected from horse fecal piles in both paper bags and vials of ethanol at Little Book Cliffs Herd Management Area, Colorado, USA. FI G U R E 2 Boxplot showing the number of loci that amplified from samples collected in paper bags from feral horse fecal piles at Little Book Cliffs Herd Management Area, Colorado, USA when they were fresh (0 months) to 6 months old FI G U R E 2 Boxplot showing the number of loci that amplified from samples collected in paper bags from feral horse fecal piles at Little Book Cliffs Herd Management Area, Colorado, USA when they were fresh (0 months) to 6 months old FI G U R E 3 Difference in genotyping errors between samples from fresh fecal piles (0–2 months old) and old fecal piles (3–6 months old) collected in paper bags from feral horse fecal piles at Little Book Cliffs Herd Management Area, Colorado, USA. Loci matching is the number of loci that matched (out of a total of eight loci) in a genotype of multiple samples taken from the same fecal pile Most authors have advocated for fecal samples to be collected as soon after deposition as possible in order to prevent degradation of DNA due to weather and ultraviolet radiation (e.g., Foran et al., 1997). There was a significant increase in DNA amplification errors five to 7 days after defecation in three species where it was tested (coyote (C. latrans), Panasci et al., 2011; snowshoe hares (Lepus americanus), Cheng, Hodges, Sollmann, & Mills, 2017; Sitka black-tailed deer (Odocoileus hemionus sitkinensis), Brinkman et al., 2009). Although DNA was amplified from brown bear (Ursos arctos) scat after being exposed to the environment for 60 days, Murphy et al. (2007) found the most significant decline in amplification success was after the first 2 days. In marked contrast to these studies, horse feces had good amplification success (amplification at >90% of loci) and low error rates until it had been in the environment for longer than 2 months. This indicates that FI G U R E 3 Difference in genotyping errors between samples from fresh fecal piles (0–2 months old) and old fecal piles (3–6 months old) collected in paper bags from feral horse fecal piles at Little Book Cliffs Herd Management Area, Colorado, USA. Number of samples that amplified at ≥5 loci is given as the probability of identity (PID) was ≤0.001 when at least five loci were included for both collection methods Paper bags (N = 250) Ethanol vials (N = 250) Amplified at 8 loci (%) 139 (55.6) 43 (17.2) Amplified at ≥5 loci (%) 225 (90) 138 (55.2) Did not amplify at any loci (%) 7 (2.8) 44 (17.6) FI G U R E 2 Boxplot showing the number of loci that amplified from samples collected in paper bags from feral horse fecal piles at Little Book Cliffs Herd Management Area, Colorado, USA when they were fresh (0 months) to 6 months old loci (%) Paper bags are less expensive than alcohol and vials: at the time of our study (2014) paper bags cost about $0.04 per sample, whereas the cost of 25 ml of 95% ethanol plus a 50-ml conical tube was about $1.89 per sample. While paper bag samples needed careful drying, they only required space for storage. In contrast, ethanol should be stored in a flammable cabinet and there are vari- ous regulations controlling shipping of it. Further disadvantages of collecting samples in ethanol are the risks of spills erasing sample information from the tube, and the weight of carrying it in the field. The only potential complication of collecting samples in paper bags is that very fresh samples may need to be double-bagged to avoid seepage and contamination of other samples. Paper bags not only resulted in better genetic data than vials of ethanol, but are sim- pler and less expensive for collection of equine fecal samples. As such, this method is ideal for field staff both in remote locations in Asia and Africa where wild equids occur, and for managers of feral horse and burro populations who may not have access to lab- oratory supplies, or who spend extensive periods in remote field settings. Removing the constraint of collecting fresh feces has ramifications for sampling design, as surveys for collecting equid fecal samples can be carried out using transects or a random- ized design to avoid detection bias, rather than focusing around water holes, trails, or other areas where animals are known to congregate. During our study, there was variation in rainfall and temperature among the months, with the climate being mostly dry. We expected the best genetic results to be from samples deposited in the cold- est or driest months, as in other studies (Liu et al., 2014b; Lucchini et al., 2002; Maudet et al., 2004; Nsubuga et al., 2004). Our results indicated that samples from fecal piles deposited from June to August and collected when fresh (≤2 months old) provided the best genetic data. Although August was one of the wettest months of our study it was also one of the warmest. Thus, in the arid western United States, we would recommend collecting fecal samples that are less than 2 months old in late summer, and in other locations during a hot dry season. Fecal piles deposited in May had a high incidence of mold, both in the field and in collected samples. This could potentially explain why samples deposited in May had higher rates of amplification failure and genotyping error. Qualitative eval- uation of fecal piles also indicated that sample collection is likely to be most efficient during mid to late summer, as feces are more formed on deposition and remain a brown color for longer. It is therefore possible that a combination of a transition to a drier diet from fresh spring grass, and warmer weather potentially drying the fecal sample quickly despite rain, contributed to better preserva- tion of fecal DNA. The results from our study, that paper bags were more success- ful than ethanol vials for sample collection, that DNA from horse fecal samples amplifies well even 2 months after deposition, and that the best time to collect feces is during summer, suggest that there may be something about equid feces that predisposes it to be suitable for noninvasive sampling. As hind-gut fermenters, equids spend about half their time feeding (King, Asa, Pluháček, Houpt, & Ransom, 2016; Schoenecker et al., 2016), and thus have an almost constant stream of food moving through their digestive system. Loci matching is the number of loci that matched (out of a total of eight loci) in a genotype of multiple samples taken from the same fecal pile FI G U R E 3 Difference in genotyping errors between samples from fresh fecal piles (0–2 months old) and old fecal piles (3–6 months old) collected in paper bags from feral horse fecal piles at Little Book Cliffs Herd Management Area, Colorado, USA. Loci matching is the number of loci that matched (out of a total of eight loci) in a genotype of multiple samples taken from the same fecal pile \| 4061 KING et al. FI G U R E 4 Comparison of error rate in samples deposited in different months of the year (2014) and collected in paper bags from feral horse fecal piles at Little Book Cliffs Herd Management Area, Colorado, USA. November is excluded as there were no repeat samples from piles in that month \| 4061 KING et al. 4061 FI G U R E 4 Comparison of error rate in samples deposited in different months of the year (2014) and collected in paper bags from feral horse fecal piles at Little Book Cliffs Herd Management Area, Colorado, USA. November is excluded as there were no repeat samples from piles in that month to rainfall hastening decay rates (Brinkman et al., 2009; Lehmkuhl et al., 1994). Perhaps because most studies used only fresh samples, few report how they qualify estimates of fecal age in the field. 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Conservation Biology, 28, 107–118. https://doi.org/10.1111/cobi.12161 None declared. ACKNOWLEDGMENTS Brinkman, T. J., Schwartz, M. K., Person, D. K., Pilgrim, K. L., & Hundertmark, K. J. (2009). Effects of time and rainfall on PCR success using DNA extracted from deer fecal pellets. Conservation Genetics, 11, 1547–1552. https://doi.org/10.1007/ s10592-009-9928-7 We would like to thank Jim Dollerschell, Rangeland Management Specialist at the Bureau of Land Management, and members of the Friends of the Mustangs volunteer group for their assistance with this study. We would also like to thank the fecal sampling volunteers for their fieldwork assistance, and Tracy Susan for her help in the lab. This study was jointly funded by the U.S. Geological Survey Fort Collins Science Center and the Bureau of Land Management Wild Horse and Burro Program. Any use of trade, firm, or product names is for descriptive purposes only and does not imply endorsement by the U.S. Government. Broquet, T., & Petit, E. (2004). Quantifying genotyping errors in noninva- sive population genetics. Molecular Ecology, 13, 3601–3608. https:// doi.org/10.1111/j.1365-294X.2004.02352.x Cheng, E., Hodges, K. E., Sollmann, R., & Mills, L. S. (2017). Genetic sampling for estimating density of common species. Ecology and Evolution, 7, 6210–6219. https://doi.org/10.1002/ece3.3137 De Barba, M., Waits, L. P., Genovesi, P., Randi, E., Chirichella, R., & Cetto, E. (2010). Comparing opportunistic and systematic sampling methods for non-invasive genetic monitoring of a small translocated brown bear population. Journal of Applied Ecology, 47, 172–181. https://doi. org/10.1111/j.1365-2664.2009.01752.x Sarah R. B. King http://orcid.org/0000-0002-9316-7488 Foran, D. R., Crooks, K. R., & Minta, S. C. (1997). Species identification from scat: An unambiguous genetic method. Wildlife Society Bulletin, 25, 835–839. AUTHOR CONTRIBUTIONS Eggert, L. S., Eggert, J. A., & Woodruff, D. S. (2003). Estimating pop- ulation sizes for elusive animals: The forest elephants of Kakum National Park, Ghana. Molecular Ecology, 12, 1389–1402. https://doi. org/10.1046/j.1365-294X.2003.01822.x Sarah King, Kate Schoenecker, and Sara Oyler-McCance conceived the ideas and designed the methodology; Sarah King collected the data; Jenny Fike and Sara Oyler-McCance conducted the genetic analyses; Sarah King and Sara Oyler-McCance analyzed the data; Sarah King, Kate Schoenecker, Jenny Fike, and Sara Oyler-McCance contributed to the drafting and revision of the manuscript and gave final approval for publication. Eggert, L. S., Powell, D. M., Ballou, J. D., Malo, A. F., Turner, A., Kumer, J., Maldonado, J. E. (2010). Pedigrees and the study of the wild horse population of Assateague Island National Seashore. Journal of Wildlife Management, 75, 963–973. https://doi.org/10.2193/2009-231 Ellegren, H., Johansson, M., Sandberg, K., & Andersson, L. (1992). Cloning of highly polymorphic microsatellites in the horse. Animal Genetics, 23, 133–142. Ernest, H. B., Penedo, M. C. T., May, B. P., Syvanen, M., & Boyce, W. M. (2000). Molecular tracking of mountain lions in the Yosemite valley region in California: Genetic analysis using microsatel- lites and faecal DNA. Molecular Ecology, 9, 433–441. https://doi. org/10.1046/j.1365-294x.2000.00890.x This results in heavily mucosal feces providing an abundance of epithe- lial cells surrounding and/or throughout the fecal sample, such that even after some environmental degradation sufficient DNA remains for successful amplification. The size of a bolus of equid feces (nor- mally at least 3 cm in diameter) and the fact that they are found in a clumped pile may act to shelter fecal DNA from degradation by UV light and precipitation. Furthermore, most equids are found in arid and semi-arid ecosystems, which could promote faster drying rates and persistence of fecal boli. 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https://openalex.org/W2620933887	https://www.duo.uio.no/bitstream/10852/62312/1/Sagedal-2017-The%2beffect%2bof%2ba%2bprenatal%2blifestyl.pdf	English	null	The effect of a prenatal lifestyle intervention on glucose metabolism: results of the Norwegian Fit for Delivery randomized controlled trial	BMC pregnancy and childbirth	2,017	cc-by	9,086	The effect of a prenatal lifestyle intervention on glucose metabolism: results of the Norwegian Fit for Delivery randomized controlled trial Linda R. Sagedal1* , Ingvild Vistad1, Nina C. Øverby2, Elling Bere2, Monica K. Torstveit2, Hilde Lohne-Seiler2, Elisabet R. Hillesund2, Are Pripp3 and Tore Henriksen4 * Correspondence: linda.sagedal@sshf.no 1Department of Obstetrics and Gynecology/Department of Research, Sørlandet Hospital, Postbox 416, 4604 Kristiansand, Norway Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background: The effectiveness of prenatal lifestyle intervention to prevent gestational diabetes and improve maternal glucose metabolism remains to be established. The Norwegian Fit for Delivery (NFFD) randomized, controlled trial studied the effect of a combined lifestyle intervention provided to a general population, and found significantly lower gestational weight gain among intervention participants but no improvement in obstetrical outcomes or the proportion of large infants. The aim of the present study is to examine the effect of the NFFD intervention on glucose metabolism, including an assessment of the subgroups of normal-weight and overweight/obese participants. Methods: Healthy, non-diabetic women expecting their first child, with pre-pregnancy body mass index (BMI) ≥19 kg/m2, age ≥18 years and a singleton pregnancy of ≤20 gestational-weeks were enrolled from healthcare clinics in southern Norway. Gestational weight gain was the primary endpoint. Participants (n = 606) were individually randomized to intervention (two dietary consultations and access to twice-weekly exercise groups) or control group (routine prenatal care). The effect of intervention on glucose metabolism was a secondary endpoint, measuring glucose (fasting and 2-h following 75-g glucose load), insulin, homeostatic assessment of insulin resistance (HOMA-IR) and leptin levels at gestational-week 30. Results: Blood samples from 557 (91.9%) women were analyzed. For the total group, intervention resulted in reduced insulin (adj. Mean diff −0.91 mU/l, p = 0.045) and leptin levels (adj. Mean diff -207 pmol/l, p = 0.021) compared to routine care, while glucose levels were unchanged. However, the effect of intervention on both fasting and 2-h glucose was modified by pre-pregnancy BMI (interaction p = 0.030 and p = 0.039, respectively). For overweight/obese women (n = 158), intervention was associated with increased risk of at least one glucose measurement exceeding International Association of Pregnancy and Diabetes Study Group thresholds (33.7% vs. 13.9%, adj. OR 3.89, p = 0.004). Conclusions: The Norwegian Fit for Delivery intervention lowered neither glucose levels nor GDM incidence, despite reductions in insulin and leptin. Prenatal combined lifestyle interventions designed for a general population may be unsuited to reduce GDM risk, particularly among overweight/obese women, who may require earlier and more targeted interventions. Trial registration: ClinicalTrials.gov ID NCT01001689, registered July 2, 2009, confirmed completed October 26, 2009 (retrospectively registered). Keywords: Gestational diabetes, Intervention, Lifestyle, Overweight, Obesity © The Author(s). Sagedal et al. BMC Pregnancy and Childbirth (2017) 17:167 DOI 10.1186/s12884-017-1340-6 Sagedal et al. BMC Pregnancy and Childbirth (2017) 17:167 DOI 10.1186/s12884-017-1340-6 Open Access Intervention l f Details of NFFD’s dietary and physical activity compo- nents and the rationale behind them are previously pub- lished [17, 19]. The dietary component was based on ten recommendations designed to increase awareness of food choices, with advice to increase intake of water, vegetables and fruit and reduce snack food consumption. Abstract 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Sagedal et al. BMC Pregnancy and Childbirth (2017) 17:167 Page 2 of 12 encompassing the cities of Kristiansand and Mandal and the more rural surrounding areas. The protocol for the trial is previously published [17]. Midwives at eight healthcare clinics enrolled participants between September 2009 and February 2013. Women were eli- gible if they were nulliparous, with a singleton preg- nancy of ≤20 gestational weeks, had a pre-pregnancy body mass index (BMI) ≥19 kg/m2, were literate in Norwegian or English, and provided signed, informed consent. Exclusion criteria were pre-existing diabetes, disabilities precluding participation in a physical fit- ness program (based on national and international rec- ommendations) [18], on-going substance abuse, or planned relocation outside the study area before deliv- ery. The first 20 participants comprised a feasibility study. The protocol was modified to include a lower age limit of 18 years and to allow randomisation after initial questionnaires and blood tests were completed, in order to assure that participants were sufficiently motivated and avoid missing data. Participating clinics documented attendance of 4245 women during the inclusion period, of whom we estimate that 1610 were nulliparous (Fig. 1) [16]. Ethics, consent and permissions The trial was performed in accordance with the Declar- ation of Helsinki. The present study is a planned second- ary analysis of the NFFD trial, and was included in the consent and ethical approval of the trial. The Norwegian Regional Committee for Medical Research Ethics South- East-C approved the trial and modifications (REK reference 2009/429), including specific approval for the storage and analysis of frozen serum in Research Biobank Notification no. 2594. Signed, informed consent was obtained from all participants. The Norwegian Fit for Delivery (NFFD) randomized controlled trial tested the effect of a prenatal lifestyle intervention consisting of dietary counseling and su- pervised exercise groups on a general population in- cluding normal-weight, overweight and obese women. We have previously reported that the NFFD interven- tion resulted in a significant reduction in gestational weight gain (GWG) of 1.3 kg from pre-pregnancy to term but showed no significant effect of intervention on the incidence of GDM based on 2006 World Health Organization (WHO) criteria or on the proportion of large newborns [16]. The aim of the present paper is to examine the effect of intervention on levels of glucose, insulin, homeostatic assessment of insulin resistance (HOMA-IR) and leptin measured at gestational-week 30, including an assessment of intervention effect on the subgroups of normal-weight and overweight/obese women. Randomisation and blinding After receiving signed consent forms and confirming that blood tests and questionnaires were completed, a research nurse assigned participants consecutively to the intervention or control arm of the study utilizing a computer-generated list with 1:1 allocation ratio and blocks of 20. All examinations, blood test evaluations and scoring of questionnaires were performed by asses- sors blinded to group allocation. Background Maternal glucose regulation appears to be fundamentally important for fetal growth and pregnancy health. Ob- servational studies demonstrate a linear relationship be- tween maternal glucose levels and adverse obstetrical outcomes, particularly fetal macrosomia [1, 2], and ran- domized trials demonstrate that treatment of mild hyperglycemia reduces the incidence of these same out- comes [3, 4]. Gestational diabetes mellitus (GDM) is defined as hyperglycemia first detected during preg- nancy, ultimately due to insufficient insulin production relative to the physiologic insulin resistance of preg- nancy [5]. The level of maternal glucose that consti- tutes a risk for mother and fetus is much debated, and there is currently no international consensus on glucose thresholds for the diagnosis of GDM [6–8]. Effective antenatal lifestyle intervention to improve maternal glucose metabolism and reduce GDM risk is in high de- mand [9]. Trials published to date indicate that prenatal interventions combining diet and exercise reduce gesta- tional weight gain but not GDM risk [10–13]. Few trials of antenatal diet and exercise have reported levels of glucose, insulin and insulin resistance [8]. These levels may give information about alterations in maternal metabolism that are not disclosed by simply reporting the incidence of GDM. Women who are overweight or obese often enter pregnancy with increased insulin re- sistance, and examination of glucose metabolism for this subgroup of women is therefore of particular inter- est [5, 14]. Leptin levels are also relevant to interven- tions affecting weight gain, as this adipocyte appears to play a role in glucose regulation [15]. Measurements h immunoassay (Roche Diagnostics), with coefficient of vari- ance of 4%. Leptin was analyzed using competitive radio- immunoassay (Millipore), with coefficient of variance of 7%. HOMA-IR was calculated as: (insulin(mU/l) x fasting glucose(mmol/l))/22.5. Leptin, insulin and HOMA-IR were missing for eight participants (3 intervention, 5 con- trol), due to errors in freezing or transport. All missing values were considered missing completely at random. Three insulin and HOMA-IR values (1 intervention, 2 control) were excluded from analysis as outliers. The primary aims of the NFFD trial were to examine if intervention resulted in differences in GWG, birth weight of term infants, the proportion of term infants >4000 g, maternal fat percent at 36 gestational-weeks, and the incidence of operative deliveries. Maternal glucose levels at 30 gestational-weeks was a primary endpoint, while the proportion of women with elevated 2-h glucose tolerance tests and measurement of hormones related to glucose metabolism were secondary endpoints of the trial. Assessment of the subgroup of overweight/obese women was specified in the trial protocol. Methods BMC Pregnancy and Childbirth (2017) 17:167 Page 4 of 12 Sample size We predicted a 20% prevalence of newborns with birth- weight > 4000 g in the control group based on 2005 sta- tistics from the Norwegian birth registry [24], and deter- mined empirically that a reduction to 10% in the intervention group would be clinically relevant. We cal- culated that 198 women were required in each study arm to demonstrate statistical significance with a power of 80%. We also expected a 10% incidence of GDM (based on 2-h glucose ≥7.8 mmol/l) [22, 23] in the con- trol group, and determined that a reduction to 3% in the intervention group would be clinically significant. We cal- culated that we would have 80% power to detect a statisti- cally significant difference between groups with 200 participants in each arm. To allow for participant drop- out and premature deliveries and to allow for analysis of subgroups, we planned to randomize 600 participants. Pre-pregnancy weight was self-reported. Participants were weighed at their healthcare clinic at inclusion, and at Sørlandet Hospital at 30 gestational-weeks (Tanita BC 418, Tokyo, Japan). Feasibility study participants re- ported their height; later participants were measured using a stadiometer (Seca Leicester, Hamburg, Germany). Pre-pregnancy BMI was calculated based on self-reported pre-pregnancy weight and measured height when avail- able. Participants were weighed on admission to the delivery ward. If missing, last weight in the prenatal record was recorded with corresponding date. GWG at term was calculated for women delivering at ≥37 gestational-weeks with weight available within 2 weeks of admission. Participants completed questionnaires at trial inclusion and at gestational-week 36, either electronically or in print. No questionnaires were completed at gestational-week 30. Diet was assessed by 43 food-frequency questions, analyzed using a pre-determined score (range 0–10, with higher score denoting healthier eating behavior). The score is pre- viously described in detail, and has demonstrated acceptable test-retest reliability [19]. Physical activity was assessed with the International Physical Activity Questionnaire (IPAQ) short version, scored using IPAQ analysis algorithms. The IPAQ is validated in a Scandinavian population [21]. Methods NFFD is a randomized, blinded, controlled trial with two parallel groups performed in southern Norway, Sagedal et al. BMC Pregnancy and Childbirth (2017) 17:167 Page 3 of 12 Fig. 1 Trial profile for analysis of glucose metabolism, Norwegian Fit for Delivery trial. Blood tests were collected after fasting and two hours after glucose challenge at 30 weeks of gestation. Of 606 women randomized, 557 (91.9%) provided blood samples for analysis. An intention to treat (ITT) analysis of pregnancy outcomes included 591 women, excluding 13 from trial participation as described above and excluding two of 31 who withdrew from trial participation, due to lack of consent for use of data Fig. 1 Trial profile for analysis of glucose metabolism, Norwegian Fit for Delivery trial. Blood tests were collected after fasting and two hours after glucose challenge at 30 weeks of gestation. Of 606 women randomized, 557 (91.9%) provided blood samples for analysis. An intention to treat (ITT) analysis of pregnancy outcomes included 591 women, excluding 13 from trial participation as described above and excluding two of 31 who withdrew from trial participation, due to lack of consent for use of data access to a NFFD internet site, and invitation to one cooking class and one evening meeting with informa- tion on the NFFD trial and the value of regular exercise and healthy diet in pregnancy. There was no calorie restriction or specific limitation of fats or carbohydrates. Counselling was performed twice, by phone, with a four to six week interval. Counsellors were either experienced clinical dieticians or graduate students in public health, trained and supervised by the NFFD team. The physical activity component consisted of access to twice-weekly exercise classes at a local gym facility, led by physical therapists or students in sports science, trained and quality-controlled by the NFFD team. Attendance was recorded. Participants were en- couraged to engage in 30 min of moderate-intensity physical activity on three additional days per week. Life- style recommendations were reinforced with booklets, Participants in the control group received routine pre- natal care following Norwegian standard: eight prenatal appointments, including one second-trimester ultrasound examination, with additional care as needed, provided free of charge. Routine care includes a booklet with advice on prenatal nutrition, physical activity and recommendations for weight gain based on current Institute of Medicine (IOM) guidelines (normal-weight: 11.5–16 kg, overweight: 7–11.5 kg, obese: 5–9 kg) [20]. Sagedal et al. Results d t t d b l HOMA IR ( dj M diff 0 21 Table 1 Baseline characteristics of participants Intervention (n = 281) Control (n = 276) Mean SD Mean SD Age at trial inclusion (years) 28.0 4.2 28.0 4.5 Gestational age at inclusion (weeks) 15.4 2.7 15.6 2.5 Pre-pregnancy weight (kg) 67.5 11.9 67.3 12.4 Height (cm) 168.7 5.6 168.9 6.7 Pre-pregnancy BMI (kg/m2) 23.7 4.0 23.6 3.8 Inclusion weight (kg)a 69.8 12.2 70.1 12.6 Glucose, fasting (mmol/l) 4.43 0.38 4.45 0.40 C-Reactive Protein (mg/l) 4.38 4.26 4.36 4.04 N % N % BMI category, pre-pregnancy Underweight 2 0.7 3 1.1 Normal-weight 193 68.7 201 72.8 Overweight 64 22.8 52 18.8 Obese 22 7.8 20 7.2 Education levelb 12 years or less 84 30.0 89 32.4 < 4 years of higher education 101 36.1 84 30.5 ≥4 years of higher education 95 35.5 102 37.1 Occupationc Employed outside the home 230 81.9 239 86.9 Student 33 11.7 16 5.8 Unemployed 7 2.5 13 4.7 Long-term sick leave 6 2.1 4 1.4 Homemaker 5 1.8 3 1.1 Cohabitationc Husband/boyfriend/partner 274 97.5 263 95.6 Other 7 2.5 12 4.4 Household Income (NKR)d ≤400,000 89 31.7 84 30.8 401–700,000 79 28.1 76 27.8 > 700,000 99 35.2 93 34.1 Refrained from response 14 5.0 20 7.3 Smoking statusc Smoker 8 2.8 13 4.7 Non-smoker 273 97.2 262 95.3 aWeight at inclusion was missing for 8 (2 control and 6 intervention) participants bEducation information missing for 1 intervention and 1 control participant cInformation on occupation, cohabitation and smoking missing for 1 control participant dIncome information missing for 3 control participants The 606 women included in the NFFD trial were equally distributed into intervention and control groups (Fig. 1), of which 591 (97.5%) were included in a previously- published intention to treat (ITT) analysis of interven- tion effect on obstetrical outcomes [16]. An additional 34 women withdrew or were excluded from participation (Fig. 1) such that 557 (91.9%) women were included in the present analysis. Compared to the ITT analysis, missing participants in the intervention group (15/296, 5.1%) were younger (24.9 vs. 28.0 years, p = 0.005), more often without higher education (71.4% vs. 30.0%, p = 0.004) and reported lower income (p = 0.034), but had a similar distribution of occupations, pre-pregnancy BMI categories, and healthcare clinics compared with intervention participants who were tested. Statistics Unadjusted comparison of intervention and control groups was performed using student t-test or chi-square test as appropriate. Difference between the randomized groups for continuous or binary variables was assessed using multiple linear or logistic regression models ad- justed for age, education, income level and smoking at inclusion, pre-pregnancy BMI category and gestational age at measurement. Variables included in the adjusted analysis were chosen based on clinical relevance (pre- pregnancy BMI category and smoking) and/or measured differences between intervention and control group (ges- tational age at measurement) and/or measured differ- ences between included and missing participants (age, education and income). Effect modification between ran- domized groups and patient characteristics on continu- ous outcomes was assessed by an interaction term in the multiple linear regression models. For binary outcomes, effect modification was assessed by the Breslow-Day test of homogeneity of odds ratios. No further adjustment for BMI category was performed when analysis was stratified according to pre-pregnancy BMI. P-values <0.05 were considered statistically significant. All tests were two-sided. We used SPSS for Windows version 21.0 for all statistical analyses. Prior to randomisation, fasting blood tests were assessed for evidence of pre-existing diabetes (defined as glucose ≥7.0 mmol/l) [22]. No participants were ex- cluded on this basis. At gestational-week 30, plasma glu- cose was measured after overnight fast and again at 2-h after 75 g glucose load. All tests were performed at Sørlandet Hospital using a Cobas 6000 c501 chemistry analyzer (Roche Diagnostics). Glucose levels ≥7.0 mmol/l at fasting and/or ≥7.8 mmol/l at 2-h were classified as ele- vated, based on contemporary national [23] and WHO 2006 criteria [22], and participants and their primary care physicians were informed. Glucose at 2-h was missing for 12 participants (9 intervention, 3 control), primarily due to vomiting. Fasting serum samples were frozen and stored at −80 °C. Frozen samples were analyzed at Aker Hor- mone laboratory using a Modular E170 analyzer (Roche), batched to decrease interassay variation. Insulin was ana- lyzed using non-competitive electrochemoluminescence Page 5 of 12 Page 5 of 12 Sagedal et al. BMC Pregnancy and Childbirth (2017) 17:167 Table 1 Baseline characteristics of participants Results 95% CI p-value Gestational weight gain to term (kg) From pre-pregnancy a 14.41 6.26 15.66 5.54 −1.25 −2.28, −0.22 0.017 −1.2 −2.2, −0.2 0.021 From trial inclusionb 12.11 5.17 12.99 4.68 −0.89 −1.75, −0.02 0.044 −1.0 −1.8, −0.1 0.025 Gestational weight gain prior to glucose testing (kg) From pre-pregnancyc 9.22 4.67 9.86 4.37 −0.64 −0.11, 1.40 0.096 −0.52 −1.28, 0.20 0.170 From trial inclusiond 6.96 3.24 7.18 2.96 −0.22 −0.72, 0.30 0.407 −0.24 −0.74, 0.27 0.359 Gestational weight gain analyzed as continuous outcome variable using Student’s t-test for unadjusted comparison of intervention and control groups, and multiple regression analysis including age, smoking status, educational level and income at trial inclusion. Analysis of weight gain prior to glucose testing also included gestational length at time of measurement (analysis from pre-pregnancy) or interval between measurements (analysis from trial inclusion) aGestational weight gain to term missing for 47 participants: 31 who delivered at <37 gestational-weeks (16 intervention, 15 control) and 16 without measured weight at or within 2 weeks of delivery (9 intervention, 7 control) bAn additional 6 participants were without measured weight at trial inclusion (5 intervention, 1 control) cWeight gain prior to glucose testing missing for 1 participant (intervention) without weight measured at glucose testing dAn additional 8 participants were without weight measured at inclusion (6 intervention, 2 control) Table 2 Gestational weight gain, NFFD population Table 3 Effect of NFFD intervention on glucose regulation Table 3 Effect of NFFD intervention on glucose regulation Intervention Control Intervention effect (n = 281) (n = 276) Unadjusted Adjusted Mean SD Mean SD Mean diff. 95% CI p-value Adj. Mean diff. Results Missing par- ticipants in the control group (19/295, 6.4%) were not significantly different from those who were tested. g y Among women in the intervention arm, 253/281 (90.0%) received both dietary consultations, 25/281 (8.9%) received one, and 3/281 (1.1%) received none. All received access to exercise classes and 267/281 (95.0%) attended at least one class. The number of classes attended prior to glucose-testing varied from 0 to 24, with median 10. The baseline characteristics of the 557 participants included in the present analysis were similar in the two groups (Table 1). Participants were predominantly white, of European descent. The majority of women in both groups were normal-weight pre-pregnancy. Five participants with pre- pregnancy BMI ≤18.5 kg/m2 (inclusion failures; 2 interven- tion and 3 control participants) were included in the normal-weight BMI category for statistical analyses. There was a similar proportion of control and intervention par- ticipants from each clinic (p = 0.196). Glucose-testing was performed slightly earlier in the intervention group (29.9 vs. 30.1 gestational-weeks, p = 0.036), such that gestational length at glucose-testing was included in adjusted analyses. The intervention group showed a statistically significant reduction of GWG to term compared to controls, but GWG prior to glucose-testing was not significantly differ- ent between intervention and control groups (Table 2). There was no modification of intervention effect on GWG based on pre-pregnancy BMI category. The effect of NFFD intervention on biochemical ele- ments of glucose metabolism was assessed for the whole population and for the subgroups of normal-weight (BMI < 25, n = 399) and overweight/obese participants (BMI ≥25 kg/m2, n = 158), see Table 3. The NFFD intervention resulted in lower insulin levels for the intervention group vs. the control group (Table 3) and a strong trend toward lower insulin levels among normal-weight women (adj. Mean diff. -0.91 mU/l, (95%CI -1.86, 0.04), p = 0.056). Normal-weight women also had a trend toward reduced insulin resistance as demonstrated by lower HOMA-IR (adj. Mean diff. -0.21, (95%CI -0.041, 0.01), p = 0.056). Further, the interven- tion was associated with a significant reduction of leptin for both the whole intervention population and the Sagedal et al. BMC Pregnancy and Childbirth (2017) 17:167 Page 6 of 12 Table 2 Gestational weight gain, NFFD population Intervention Control Intervention effect (n = 281) (n = 276) Unadjusted Adjusted Mean SD Mean SD Mean diff. 95% CI p-value Adj. Mean diff. Results 95% CI p-value Glucose, fasting (mmol/L) Whole population 4.66 0.40 4.65 0.34 0.01 −0.05, 0.07 0.724 −0.00 −0.06, 0.06 0.912 BMI < 25 kg/m2 a 4.56 0.34 4.61 0.32 −0.05 −0.11, 0.02 0.142 −0.04 −0.10, 0.03 0.239 BMI ≥25 kg/m2 b 4.87 0.45 4.74 0.37 0.13 −0.01, 0.26 0.059 0.11 −0.02, 0.25 0.094 Glucose, 2 h (mmol/L) Whole population 6.07 1.34 6.08 1.16 −0.01 −0.22, 0.21 0.964 0.030 −0.18, 0.24 0.776 BMI < 25 kg/m2 5.84 1.15 6.03 1.09 −0.19 −0.42, 0.03 0.089 −0.16 −0.38, 0.07 0.175 BMI ≥25 kg/m2 6.59 1.57 6.20 1.32 0.39 −0.07, 0.85 0.099 0.30 −0.18, 0.77 0.217 Insulin (mU/l) Whole population 11.06 5.54 11.69 6.19 −0.63 −1.62, 0.36 0.210 −0.91 −1.79,-0.02 0.045 BMI < 25 kg/m2 9.37 4.20 10.28 5.25 −0.91 −1.86, 0.04 0.060 −0.93 −0.03, 1.88 0.056 BMI ≥25 kg/m2 14.81 6.28 15.62 6.91 −0.80 −2.89, 1.28 0.446 −0.83 −2.97, 1.31 0.468 HOMA-IRc Whole population 2.34 1.30 2.45 1.41 −0.11 −0.34, 0.11 0.332 −0.18 −0.38, 0.03 0.089 BMI < 25 kg/m2 1.92 0.94 2.13 1.35 −0.20 −0.41, 0.01 0.056 −0.21 −0.41, 0.01 0.056 BMI ≥25 kg/m2 3.25 1.50 3.35 1.70 −0.09 −0.60, 0.41 0.712 −0.11 −0.63, 0.42 0.692 Leptin (pmol/l) Whole population 2471.1 1254.1 2606.7 1215.1 −135.6 −342.7, 71.5 0.199 −207.8 −383.4, −32.1 0.021 BMI < 25 kg/m2 2048.0 982.9 2251.7 971.4 −203.7 −398.0, −9.3 0.040 −201.7 −395.4, −7.9 0.041 BMI ≥25 kg/m2 3415.7 1283.7 3587.9 1286.0 −172.3 −577.7, 233.1 0.403 −256.9 −662.2, 148.4 0.212 Hormone levels and HOMA-IR analyzed as continuous outcome variables using Student’s t-test for unadjusted comparison of intervention and control groups, and multiple regression analysis including age, smoking status, educational level and income at trial inclusion, and gestational length at time of testing aSubpopulation with pre-pregnancy BMI < 25 kg/m2: intervention group n = 195, control group n = 204 bSubpopulation with pre-pregnancy BMI ≥25 kg/m2: intervention group n = 86, control group n = 72 cHOMA-IR calculated as (insulin x fasting glucose)/22.5 Sagedal et al. BMC Pregnancy and Childbirth (2017) 17:167 Page 7 of 12 challenge (Table 3). However, analysis showed a significant interaction (effect modification) between pre-pregnancy BMI category and intervention effect on glucose levels at both time points (p = 0.030 for fasting glucose, p = 0.039 for 2-h glucose), which is illustrated in Fig. 2. Among overweight/obese women, there was a trend toward subgroup of normal-weight women. Results For the smaller sub- group of overweight/obese women, there was no signifi- cant reduction in leptin, insulin or HOMA-IR levels as a result of intervention. The intervention had no effect on glucose levels for the group as a whole, either fasting or 2-h after glucose Fig. 2 Interaction of NFFD intervention and pre-pregnancy BMI on glucose levels. Glucose measured after fasting and 2-h after 75 g glucose load Fig. 2 Interaction of NFFD intervention and pre-pregnancy BMI on glucose levels. Glucose measured after fasting and 2-h after 75 g glucose l Fig. 2 Interaction of NFFD intervention and pre-pregnancy BMI on glucose levels. Glucose measured after fasting and 2-h after 75 g glucose load Sagedal et al. BMC Pregnancy and Childbirth (2017) 17:167 Page 8 of 12 slightly higher fasting glucose levels for those receiving intervention compared to controls. both when measured from pre-pregnancy and from trial inclusion, and attended a similar number of exercise clas- ses (median 8 vs. nine classes, p = 0.283) compared to those who had lower glucose levels. There was no associ- ation between dietary score or IPAQ score at inclusion and risk of exceeding IADPSG thresholds at gestational- week 30 (p > 0.05), for either the intervention or control group. Glucose levels at trial inclusion were strongly asso- ciated with exceeding IADPSG thresholds at gestational- week 30 for both intervention and control groups, also after adjusting for pre-pregnancy BMI category, and age, income and educational level (p < 0.001). However, over- weight/obese intervention participants had increased risk of exceeding IADPG thresholds also after controlling for glucose levels at inclusion in the adjusted analysis (adj. OR 4.24, p = 0.004). As previously reported, there was no significant dif- ference between intervention and control groups in the proportion of glucose values exceeding 2006 WHO thresholds for GDM, which are still in use in Norway. Applying proposed-revised Norwegian thresholds (fasting glucose ≥5.3 mmol/l and/or 2-h glucose ≥9.0 mmol/l), there was a trend toward a greater proportion of inter- vention participants with elevated glucose (8.8% vs 4.8%, adj. OR 2.01, 95%CI 0.95, 4.26, p = 0.069). Using thresh- olds recommended by the International Association of Diabetes and Pregnancy Study Groups (IADPSG) (fasting glucose ≥5.1 mmol/l and/or 2-h glucose ≥8.5 mmol/l), the intervention group had a significantly larger proportion of women with one or more elevated glucose levels com- pared to the control group (17.4 vs. 10.5%, adj. Results OR 1.8 (95%CI 1.1, 3.0) p = 0.029). Despite the increased proportion of intervention group women with glucose levels exceeding IADPSG thresholds, the intervention group showed no significant increase in newborn birth-weight or the proportion of large newborns, either for the group as a whole [16] or for the overweight/obese subgroup (Table 4). Assessing risk of exceeding IADPSG thresholds showed a significant modification of intervention effect by pre-pregnancy BMI category, stratified as normal- weight and overweight/obese (interaction p = 0.048). While the proportion of normal-weight women with glucose levels exceeding IADPSG thresholds was similar in the intervention and control groups (10.3% and 9.3% respectively, adj. OR 1.1 (95%CI 0.6, 2.2) p = 0.71), among overweight/obese women there was a significantly larger proportion of intervention partici- pants with elevated glucose levels (33.7% vs. 13.9% for intervention and control group respectively, adj. OR 3.9 (95%CI 1.6, 9.7) p = 0.004). g y g g ) az-score and percentile determined according to population-specific assessment of birth weight according to sex and gestational age [40] bAnalysis not performed due to small numbers adjusted analysis by Student t-test for continuous values and chi-square for binary outcomes. Adjusted analysis with additional variables of ag el, income and smoking status at inclusion, child’s sex and gestational length at delivery (sex and gestational length not included in z-score an gth not included in analysis of gestational age at birth) y y q y j y g come and smoking status at inclusion, child’s sex and gestational length at delivery (sex and gestational length not included in z-score an not included in analysis of gestational age at birth) length not included in analysis of gestational age at birth) az-score and percentile determined according to population-specific assessment of birth weight according to sex and gestational age [40] bA l i f d d ll b y Student t-test for continuous values and chi-square for binary outcomes. Adjusted analysis with additional variables of age, educational oking status at inclusion, child’s sex and gestational length at delivery (sex and gestational length not included in z-score analysis, gestational n analysis of gestational age at birth) Interpretation There is little information available to date on the effect of prenatal combined lifestyle interventions on glucose, insu- lin and leptin levels, as most trials report the effect of intervention on GDM incidence rather than biochemical parameters. The effect of NFFD intervention on the glu- cose metabolism of normal-weight women is consistent with the findings of Vinter et al., who reported significantly lower insulin and HOMA-IR levels at gestational-week 28–30 following lifestyle intervention, but no significant differences in glucose levels or the incidence of GDM, albeit in an exclusively overweight/obese population [25]. Among non-pregnant individuals, exercise is well docu- mented to improve glycemic control through improved insulin sensitivity [26]. It is plausible that a combination of exercise and diet can lessen insulin resistance, without being of sufficient intensity and/or duration to change plasma glucose levels. In the NFFD intervention group, women attended a median of 10 exercise classes (9 for overweight/obese participants) over a mean of 14 weeks between inclusion and testing, while the intended attend- ance was twice per week. Although we lack information about total physical activity level during this period of pregnancy, it is reasonable to suppose that greater compli- ance might have resulted in greater intervention effect. However, varying compliance is a hallmark of clinical prac- tice, such that the present results likely reflect the effect of providing the NFFD intervention in a general population. For overweight/obese women participating in the NFFD trial, assignment to exercise classes may have in- advertently discouraged further leisure-time physical ac- tivity, particularly among sedentary women. Exercise routines were designed to adjust to varied fitness levels, possibly allowing larger women to limit their exertion. Larger women may also have been intimidated by classes where normal-weight women were in the majority, per- haps explaining why overweight/obese women had lower attendance than normal-weight participants. In addition, NFFD dietary recommendations were not specifically de- signed to reduce GDM risk and contained no advice on restriction of calories, carbohydrates or fat. Our finding of an increased proportion of elevated glucose levels among intervention participants compared to controls was unexpected, and its significance is unclear. Reassuringly, we found no increase in large newborns among intervention participants, an outcome that is closely associated with elevated maternal glucose. Discussion Main findings Overall, there was little beneficial effect of the NFFD lifestyle intervention on participant glucose levels, al- though there was a small but significant reduction of insulin and leptin levels. The intervention appeared to have divergent effect on glucose metabolism dependent on participants’ pre-pregnancy BMI status. For normal- weight women, the intervention had a weak positive effect on glucose metabolism, as evidenced by a trend (p < 0.1) toward reduced insulin and insulin-resistance Focusing on overweight/obese women in the interven- tion group showed that those with glucose levels exceed- ing IADPSG thresholds had similar GWG prior to testing, Table 4 Neonatal outcomes for overweight/obese NFFD participants Table 4 Neonatal outcomes for overweight/obese NFFD participants Intervention Control Intervention effect (n = 86) (n = 72) Unadjusted Adjusted Mean SD Mean SD Mean diff. 95% CI p-value Adj. Mean diff. 95% CI p-value Gestational age at birth (weeks) 39.48 1.64 39.29 1.90 0.18 −0.37, 0.74 0.51 0.23 −0.33, 0.80 0.41 Birth weight (g) 3485 484 3466 506 19 −134, 172 0.81 −24 −138, 91 0.68 Birth weight z-scorea −0.16 0.82 −0.10 0.72 −0.06 −0.30, 0.18 0.63 −0.03 −0.27, 0.22 0.83 Length at birth (cm) 50.2 2.1 50.0 2.5 0.2 −0.5, 0.9 0.57 −0.2 −0.7, 0.3 0.51 Ponderal index 2.76 0.20 2.77 0.23 −0.01 −0.07, 0.06 0.86 0.01 −0.06, 0.08 0.83 N % N % OR 95%CI p-value Adj. OR 95%CI p-value >4 kg at term 15 18.3 9 13.6 1.41 0.58, 4.48 0.44 1.30 0.46, 3.70 0.61 >4.5 kg at term 0 0 1 1.2 b b 0.88 b b 1.00 Birth weight > 90th percentilea 4 4.9 3 4.5 b b 0.95 1.58 0.31, 8.13 0.58 az-score and percentile determined according to population-specific assessment of birth weight according to sex and gestational age [40] bAnalysis not performed due to small numbers Sagedal et al. BMC Pregnancy and Childbirth (2017) 17:167 Page 9 of 12 and significantly lower leptin values, although there was no change in mean glucose levels or the proportion ex- ceeding thresholds for GDM diagnosis. For overweight and obese women this picture was different, with a trend towards higher fasting glucose, but without any change in the other metabolic parameters. The effect of these trends on the prevalence of GDM varied depending on criteria used. When IADPSG thresholds were employed an increase in GDM was observed, whereas when using older WHO criteria there was no difference between groups. Discussion Main findings sensitive to interventions affecting energy metabolism and may precede changes in glucose levels or clinical endpoints. p The divergent effect of lifestyle intervention on glucose metabolism based on pre-pregnancy BMI has, to our knowledge, not previously been reported. However, earlier trials have shown that women with higher pre-pregnancy BMI demonstrate resistance to intervention effect. Polley et al. reported that behavioural intervention reduced ex- cessive GWG among normal-weight women, while over- weight and obese women had a trend in the opposite direction [30]. Hui et al. [31] and Phelan et al. [32] both reported that a lifestyle intervention performed in a mixed population only reduced GWG among normal-weight women, and Phelan also reported a significant treatment- by-weight interaction for gestational hypertension [32]. The BMI-modified effects of lifestyle intervention have several possible explanations, which may be synergistic. Larger women may differ from normal-weight women in their understanding of and compliance with intervention. Additionally, overweight and obese women may enter trials with a metabolic state that is less sensitive to intervention than that of normal-weight women. Interpretation Several meta-analyses have concluded that combined lifestyle interventions in pregnancy have no effect on risk of GDM, with approximately half of the included trials demonstrating a non-significant increase in risk of GDM using varied criteria [12, 13]. The recently published RADIEL study is one of only two trials, to our knowledge, to report a significant reduction in the incidence of GDM following a combined lifestyle intervention [11, 33]. While results from individual trials must be assessed with cau- tion, comparison may provide some insight. In contrast to the NFFD trial, RADIEL participants were included pre-gestation or in early pregnancy, which may be of g g The temporal sequence of changes in the biochemical and clinical parameters following lifestyle intervention in pregnancy are not well known. In the current study, the reduction of leptin found in the total NFFD intervention group may indicate that adipokines are sensitive to inter- ventions affecting energy metabolism. Leptin is essential in energy regulation and glucose metabolism [27, 28], and is secreted by both maternal adipocytes and placental tro- phoblasts during pregnancy [15]. Others have found that lower mid-pregnancy leptin levels are associated with reduced insulin resistance [28]. For the child, there is evidence that maternal mid-pregnancy leptin may be an indicator of fetal growth, with lower levels associated with reduced birth weight adjusted for gestational age [29]. Adi- pokines such as leptin may therefore be particularly Sagedal et al. BMC Pregnancy and Childbirth (2017) 17:167 Page 10 of 12 critical importance. There is evidence that disposition for GDM is determined prior to pregnancy, with subclinical metabolic dysfunction before conception [14, 34]. RADIEL participants were also presumably highly motivated, as they were included in the trial based on their high-risk status. In contrast, including overweight/obese women with a normal-weight popula- tion, as was done in the NFFD trial, may have under- mined the potentially greater importance of lifestyle changes for this more high-risk group. exercise, which is proposed to lower insulin resistance at least in part through up-regulation of skeletal muscle glucose transporter protein GLUT4 [39]. Information regarding lifestyle at the time of glucose-testing is not available, limiting our assessment of the impact of diet and physical activity on biochemical results. In addition, lack of information on participants’ ethnic background and family history, both of which can affect glucose me- tabolism, may contribute to residual confounding. Strengths and limitations The major strengths of the NFFD trial are its randomized, controlled design and the large size of the population studied, with relatively few missing values. Measured weight at inclusion and at the time of testing make it possible to accurately assess GWG and its association with metabolic findings. A major limitation of the current analysis is that, although examination of the sub- group of overweight/obese women was detailed in the trial protocol, <30% of participants were overweight/ obese and the trial was not adequately powered to detect changes in smaller subgroups. Intervention effect of equivalent size may therefore be more easily detectable in the large subgroup of normal-weight women, as in the analysis of leptin. Another limitation is that due to individual randomization, women living in close proxim- ity and attending the same clinic were often in different trial groups; it is possible that control participants were influenced by both intervention participants and clinic personnel who were informed of the purpose of the trial. While cluster randomization of clinics would have re- duced such “contamination”, it would have introduced within-clinic correlations such as familial/genetic distri- bution, and likely required larger sample size in order to demonstrate intervention effect [35]. Due to practical and financial constraints, insulin resistance was assessed using HOMA-IR, which has shown significant correlation in pregnancy with the gold standard of the euglycemic in- sulin clamp [36], although an index incorporating multiple insulin measurements during glucose-testing might more accurately reflect skeletal muscle insulin resistance [37, 38]. This may be relevant in an intervention including Conclusions The findings of the NFFD trial contribute to the growing evidence that GDM is difficult to prevent using combined lifestyle interventions administered during the second and third trimesters of pregnancy. Interventions aimed at a general population may miss the mark, particularly for overweight and obese women. Future research should focus on the efficacy of early intervention, preferably starting pre-pregnancy, and on methods for increasing participant motivation and compliance. Authors’ contributions LRS and IV conceived the idea for the NFFD trial. LRS, NØ, EB, HLS, TH and IV wrote the protocol. NØ, EB, MT, HLS, and ERH supervised the intervention. LRS, ERH, MT and IV supervised participant follow-up and data collection. LRS, TH, and AHP performed the data analysis. The article was written by LRS, with substantial contributions from all co-authors. All authors read and approved the final version. Abbreviations d BMI: Body mass index; GDM: Gestational diabetes mellitus; GWG: Gestational weight gain; HOMA-IR: Homeostatic assessment of insulin resistance; IADPSG: International Association of Diabetes and Pregnancy Study Groups; IOM: Institute of Medicine; IPAQ: International Physical Activity Questionnaire; IPD: Individual patient data; ITT: Intention to treat; NFFD: Norwegian Fit for Delivery; WHO: World Health Organization Availability of data and materials The dataset generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Funding The NFFD trial was funded by the Norwegian South-Eastern Regional Health Authority, with additional funding from the municipalities of Aust Agder and Vest Agder and the department of Research of Sørlandet Hospital. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Interpretation Also important, NFFD trial participants were predominantly white, European and highly educated, which may limit the external validity of results. Acknowledging that the effect of intervention may vary significantly among groups and individuals is im- portant in planning future studies. Also important, in the current analysis, the effect of intervention on GDM risk was dependent on the thresholds used. This finding illustrates the difficulty of assessing trials that employ varying criteria for GDM diagnosis, and suggests that systematic review of individual patient data (IPD ana- lysis) may be more suitable than standard meta-analysis for exploring the effect of prenatal interventions on glu- cose metabolism and gestational diabetes risk. Acknowledgements We thank all the women who participated in the NFFD study. Thanks to participating healthcare clinics for data collection; the departments of Research, Medical Biochemistry and Obstetrics and Gynecology of Sørlandet Hospital for facilitating the study; Nils Tryland for coordinating sample analysis; Spicheren Fitness Centre, Trivsel Health and Fitness Centre, Klinikken, and Trend Trim Fitness Centre for the use of their gym facilities; and our NFFD fitness instructors and diet counselors. CONSORT guidelines h f The reporting of NFFD trial results in the present paper adheres to CONSORT guidelines for reporting of randomized controlled trials. 18. ACOG Committee opinion. Number 267, January 2002: Exercise during pregnancy and the postpartum period. Obstet Gynecol. 2002;99(1):171–3. 19. Overby NC, Hillesund ER, Sagedal LR, Vistad I, Bere E. The Fit for Delivery study: rationale for the recommendations and test-retest reliability of a dietary score measuring adherence to 10 specific recommendations for prevention of excessive weight gain during pregnancy. Matern Child Nutr. 2015;11(1):20–32. Consent for publication Not applicable. 12. Bain E, Crane M, Tieu J, Han S, Crowther CA, Middleton P. Diet and exercise interventions for preventing gestational diabetes mellitus. Cochrane Database Syst Rev. 2015;4:Cd010443. 13. Simmons D. Prevention of gestational diabetes mellitus: where are we now? Diabetes Obes Metab. 2015;17(9):824–34. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Received: 19 October 2016 Accepted: 23 May 2017 23. Tore Henriksen HT, Torun C. Veileder i Fødselshjelp 2008 In: Veileder i Fødselshjelp 2008. Edited by Norsk Gynekologisk Forening (Norwegian Society of Gynecology and Obstetrics); 2008. p. 112. 24. Hånes H. Birth weight in Norway– fact sheet with statistics. Norwegian Institute of Public Health; 2005. References 1. Metzger BE, Lowe LP, Dyer AR, Trimble ER, Chaovarindr U, Coustan DR, et al. Hyperglycemia and adverse pregnancy outcomes. N Engl J Med. 2008; 358(19):1991–2002. 1. Metzger BE, Lowe LP, Dyer AR, Trimble ER, Chaovarindr U, Coustan DR, et al. Hyperglycemia and adverse pregnancy outcomes. N Engl J Med. 2008; 358(19):1991–2002. 25. Vinter CA, Jorgensen JS, Ovesen P, Beck-Nielsen H, Skytthe A, Jensen DM. Metabolic effects of lifestyle intervention in obese pregnant women. Results from the randomized controlled trial 'Lifestyle in Pregnancy' (LiP). Diabet Med. 2014;31(11):1323–30. 2. Landon MB, Mele L, Spong CY, Carpenter MW, Ramin SM, Casey B, et al. The relationship between maternal glycemia and perinatal outcome. Obstet Gynecol. 2011;117(2 Pt 1):218–24. 2. Landon MB, Mele L, Spong CY, Carpenter MW, Ramin SM, Casey B, et al. The relationship between maternal glycemia and perinatal outcome. Obstet Gynecol. 2011;117(2 Pt 1):218–24. 26. Borghouts LB, Keizer HA. Exercise and insulin sensitivity: a review. Int J Sports Med. 2000;21(1):1–12. 3. Landon MB, Spong CY, Thom E, Carpenter MW, Ramin SM, Casey B, et al. A multicenter, randomized trial of treatment for mild gestational diabetes. N Engl J Med. 2009;361(14):1339–48. 3. Landon MB, Spong CY, Thom E, Carpenter MW, Ramin SM, Casey B, et al. A multicenter, randomized trial of treatment for mild gestational diabetes. N Engl J Med. 2009;361(14):1339–48. 27. Chehab FF. 20 years of leptin: leptin and reproduction: past milestones, present undertakings, and future endeavors. J Endocrinol. 2014;223(1): T37–48. 4. Crowther CA, Hiller JE, Moss JR, McPhee AJ, Jeffries WS, Robinson JS. Effect of treatment of gestational diabetes mellitus on pregnancy outcomes. N Engl J Med. 2005;352(24):2477–86. 28. McIntyre HD, Chang AM, Callaway LK, Cowley DM, Dyer AR, Radaelli T, et al. Hormonal and metabolic factors associated with variations in insulin sensitivity in human pregnancy. Diabetes Care. 2010;33(2):356–60. 5. Lain KY, Catalano PM. Metabolic changes in pregnancy. Clin Obstet Gynecol. 2007;50(4):938–48. 29. Shroff MR, Holzman C, Tian Y, Evans RW, Sikorskii A. Mid-pregnancy maternal leptin levels, birthweight for gestational age and preterm delivery. Clin Endocrinol. 2013;78(4):607–13. 6. Metzger BE, Gabbe SG, Persson B, Buchanan TA, Catalano PA, Damm P, et al. International association of diabetes and pregnancy study groups recommendations on the diagnosis and classification of hyperglycemia in pregnancy. Diabetes Care. 2010;33(3):676–82. 30. Polley BA, Wing RR, Sims CJ. Randomized controlled trial to prevent excessive weight gain in pregnant women. Int J Obes Relat Metab Disord. 2002;26(11):1494–502. 7. Ethics approval and consent to participate The Norwegian Regional Committee for Medical Research Ethics (REK) South-East-C approved the NFFD trial protocol and modifications (REK reference 2009/429). REK South-East is affiliated with the Faculty of Medicine of the University of Oslo, and responsible for all research conducted in the South-Eastern region of Norway. The C-group of REK South-East provided approval for the NFFD trial, including Sørlandet Hospital and all involved healthcare clinics. Initial approval December 8, 2008 and July 28, 2009; approval of modification August 23, 2010. All participants provided informed written consent to participation. 14. Catalano P, de Mouzon SH. Maternal obesity and metabolic risk to the offspring: why lifestyle interventions may have not achieved the desired outcomes. Int J Obes. 2015;39(4):642–9. 15. Fasshauer M, Bluher M, Stumvoll M. Adipokines in gestational diabetes. Lancet Diab Endocrinol. 2014;2(6):488–99. 16. Sagedal LR, Overby NC, Bere E, Torstveit MK, Lohne-Seiler H, Smastuen M, et al. Lifestyle intervention to limit gestational weight gain: the Norwegian Fit for Delivery randomised controlled trial. BJOG. 2017;124(1):97–109. Epub Jan 2016. 17. Sagedal LR, Overby NC, Lohne-Seiler H, Bere E, Torstveit MK, Henriksen T, et al. Study protocol: fit for delivery - can a lifestyle intervention in pregnancy result in measurable health benefits for mothers and newborns? A randomized controlled trial. BMC Public Health. 2013;13:132. Competing interests LRS received a research grant from the South-Eastern Norway Regional Health Authority in order to perform the NFFD trial. The authors declare that they have no competing interests. Page 11 of 12 Page 11 of 12 Page 11 of 12 Sagedal et al. BMC Pregnancy and Childbirth (2017) 17:167 Author details 1 20. Institute of Medicine and National Research Council. Weight gain during pregnancy: Reexamining the guidelines. Washington, D.C.: National Academies Press; 2009. 1Department of Obstetrics and Gynecology/Department of Research, Sørlandet Hospital, Postbox 416, 4604 Kristiansand, Norway. 2Department of Public Health, Sports and Nutrition, University of Agder, Postbox 422, 4604 Kristiansand, Norway. 3Department of Biostatistics and Epidemiology, Oslo U i it H it l P tb 4950 N d l 0424 O l N 4S ti f 1Department of Obstetrics and Gynecology/Department of Research, Sørlandet Hospital, Postbox 416, 4604 Kristiansand, Norway. 2Department of Public Health, Sports and Nutrition, University of Agder, Postbox 422, 4604 Kristiansand, Norway. 3Department of Biostatistics and Epidemiology, Oslo University Hospital, Postbox 4950, Nydalen, 0424 Oslo, Norway. 4Section of Obstetrics, Women and Children’s Division, Oslo University Hospital and University of Oslo, Postbox 4950, Nydalen, 0424 Oslo, Norway. 21. Craig CL, Marshall AL, Sjostrom M, Bauman AE, Booth ML, Ainsworth BE, et al. International physical activity questionnaire: 12-country reliability and validity. Med Sci Sports Exerc. 2003;35(8):1381–95. 22. World Health Organization. Definition and Diagnosis of Diabetes Mellitus and Intermediate Hyperglycaemia: Report of a WHO/IDF Consultation. Geneva: World Health Organization; 2006. Received: 19 October 2016 Accepted: 23 May 2017 Received: 19 October 2016 Accepted: 23 May 2017 References McIntyre HD, Colagiuri S, Roglic G, Hod M. Diagnosis of GDM: a suggested consensus. Best Pract Res Clin Obstet Gynaecol. 2015;29(2):194–205. 31. Hui AL, Back L, Ludwig S, Gardiner P, Sevenhuysen G, Dean HJ, et al. Effects of lifestyle intervention on dietary intake, physical activity level, and gestational weight gain in pregnant women with different pre-pregnancy body mass index in a randomized control trial. BMC Pregnancy Childbirth. 2014;14:331. 8. Agha-Jaffar R, Oliver N, Johnston D, Robinson S. Gestational diabetes mellitus: does an effective prevention strategy exist? Nat Rev Endocrinol. 2016;12(9):533–46. 8. Agha-Jaffar R, Oliver N, Johnston D, Robinson S. Gestational diabetes mellitus: does an effective prevention strategy exist? Nat Rev Endocrinol. 2016;12(9):533–46. 32. Phelan S, Phipps MG, Abrams B, Darroch F, Schaffner A, Wing RR. Randomized trial of a behavioral intervention to prevent excessive gestational weight gain: the Fit for Delivery Study. Am J Clin Nutr. 2011;93(4):772–9. 9. Phelan S. Windows of opportunity for lifestyle interventions to prevent gestational diabetes mellitus. Am J Perinatol. 2016;33(13):1291–9. 9. Phelan S. Windows of opportunity for lifestyle interventions to prevent gestational diabetes mellitus. Am J Perinatol. 2016;33(13):1291–9. 10. Thangaratinam S, Rogozinska E, Jolly K, Glinkowski S, Roseboom T, Tomlinson JW, Kunz R, Mol BW, Coomarasamy A, Khan KS. Effects of interventions in pregnancy on maternal weight and obstetric outcomes: meta-analysis of randomised evidence. BMJ (Clinical research ed). 2012;344:e2088. 33. Petrella E, Malavolti M, Bertarini V, Pignatti L, Neri I, Battistini NC, et al. Gestational weight gain in overweight and obese women enrolled in a healthy lifestyle and eating habits program. J Matern Fetal Neonatal Med. 2014;27(13):1348–52. 34. Hedderson MM, Darbinian J, Havel PJ, Quesenberry CP, Sridhar S, Ehrlich S, et al. Low Prepregnancy Adiponectin concentrations are associated with a marked increase in risk for development of gestational diabetes mellitus. Diabetes Care. 2013;36(12):3930–7. 11. Koivusalo SB, Rono K, Klemetti MM, Roine RP, Lindstrom J, Erkkola M, et al. Gestational diabetes mellitus can be prevented by lifestyle intervention: the Finnish gestational diabetes prevention study (RADIEL): a randomized controlled trial. Diabetes Care. 2016;39(1):24–30. Sagedal et al. BMC Pregnancy and Childbirth (2017) 17:167 35. Sanson-Fisher RW, Bonevski B, Green LW, D'Este C. Limitations of the randomized controlled trial in evaluating population-based health interventions. Am J Prev Med. 2007;33(2):155–61. 36. Kirwan JP, Huston-Presley L, Kalhan SC, Catalano PM. Clinically useful estimates of insulin sensitivity during pregnancy: validation studies in women with normal glucose tolerance and gestational diabetes mellitus. Diabetes Care. 2001;24(9):1602–7. 37. Matsuda M, DeFronzo RA. Insulin sensitivity indices obtained from oral glucose tolerance testing: comparison with the euglycemic insulin clamp. Diabetes Care. 1999;22(9):1462–70. 38. Abdul-Ghani MA, Matsuda M, Balas B, DeFronzo RA. Muscle and liver insulin resistance indexes derived from the oral glucose tolerance test. Diabetes Care. 2007;30(1):89–94. 39. Mottola MF, Artal R. Fetal and maternal metabolic responses to exercise during pregnancy. Early Hum Dev. 2016;94:33–41. 40. Skjærven R, Gjessing HK, Bakketeig LS. Birthweight by gestational age in Norway Acta Obstet Gynecol Scand 2000;79(6):440–9 Sagedal et al. BMC Pregnancy and Childbirth (2017) 17:167 Page 12 of 12 Page 12 of 12 35. Sanson-Fisher RW, Bonevski B, Green LW, D'Este C. Limitations of the randomized controlled trial in evaluating population-based health interventions. Am J Prev Med. 2007;33(2):155–61. 36. Kirwan JP, Huston-Presley L, Kalhan SC, Catalano PM. Clinically useful estimates of insulin sensitivity during pregnancy: validation studies in women with normal glucose tolerance and gestational diabetes mellitus. Diabetes Care. 2001;24(9):1602–7. 37. Matsuda M, DeFronzo RA. Insulin sensitivity indices obtained from oral glucose tolerance testing: comparison with the euglycemic insulin clamp. Diabetes Care. 1999;22(9):1462–70. 38. Abdul-Ghani MA, Matsuda M, Balas B, DeFronzo RA. Muscle and liver insulin resistance indexes derived from the oral glucose tolerance test. Diabetes Care. 2007;30(1):89–94. 39. Mottola MF, Artal R. Fetal and maternal metabolic responses to exercise during pregnancy. Early Hum Dev. 2016;94:33–41. 40. Skjærven R, Gjessing HK, Bakketeig LS. Birthweight by gestational age in Norway. Acta Obstet Gynecol Scand. 2000;79(6):440–9. 35. Sanson-Fisher RW, Bonevski B, Green LW, D'Este C. Limitations of the randomized controlled trial in evaluating population-based health interventions. Am J Prev Med. 2007;33(2):155–61. 36. Kirwan JP, Huston-Presley L, Kalhan SC, Catalano PM. Clinically useful estimates of insulin sensitivity during pregnancy: validation studies in women with normal glucose tolerance and gestational diabetes mellitus Diabetes Care. 2001;24(9):1602–7. 36. Kirwan JP, Huston-Presley L, Kalhan SC, Catalano PM. Clinically useful estimates of insulin sensitivity during pregnancy: validation studies in women with normal glucose tolerance and gestational diabetes mellitus. Diabetes Care. 2001;24(9):1602–7. 37. Matsuda M, DeFronzo RA. Insulin sensitivity indices obtained from oral glucose tolerance testing: comparison with the euglycemic insulin clamp. Diabetes Care. 1999;22(9):1462–70. 37. Matsuda M, DeFronzo RA. Insulin sensitivity indices obtained from oral glucose tolerance testing: comparison with the euglycemic insulin clamp. Diabetes Care. 1999;22(9):1462–70. 38. Abdul-Ghani MA, Matsuda M, Balas B, DeFronzo RA. Muscle and liver insulin resistance indexes derived from the oral glucose tolerance test. Diabetes Care. 2007;30(1):89–94. 38. Abdul-Ghani MA, Matsuda M, Balas B, DeFronzo RA. Muscle and liver insulin resistance indexes derived from the oral glucose tolerance test. Diabetes Care. 2007;30(1):89–94. 39. Mottola MF, Artal R. Fetal and maternal metabolic responses to exercise during pregnancy. Early Hum Dev. 2016;94:33–41. 40. Skjærven R, Gjessing HK, Bakketeig LS. Birthweight by gestational age in Norway. Acta Obstet Gynecol Scand. 2000;79(6):440–9. 40. Skjærven R, Gjessing HK, Bakketeig LS. Birthweight by gestational age in Norway. Acta Obstet Gynecol Scand. 2000;79(6):440–9. Sagedal et al. 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https://openalex.org/W4377823756	https://peerj.com/articles/15315v0.2/submission	English	null	Peer Review #3 of "Hyperoside alleviates doxorubicin-induced myocardial cells apoptosis by inhibiting the apoptosis signal-regulating kinase 1/p38 pathway (v0.1)"	null	2,023	cc-by	14,611	Manuscript to be reviewed Manuscript to be reviewed 12 Abstract 26 Results: Hyperoside ameliorated DOX-induced oxidative stress in HL-1 cells, up-regulated GSH, 27 SOD and CAT activity, reduced ROS production and inhibited MDA overproduction. Moreover, 28 in addition to promoting HL-1 cell apoptosis, DOX administration also increased B-cell 29 lymphoma (Bcl)-2-associated X-protein and cleaved caspase-3 protein levels and decreased Bcl- 30 2 protein level. Hyperoside therapy, however, significantly reversed the impact of DOX on the 31 cardiomyocytes. Mechanically, DOX treatment increased the phosphorylation of the ASK1/p38 32 axis whereas hyperoside treatment attenuated those changes. In a further step, hyperoside 33 synergizes with DOX to kill MDA-MB-231 cells. 34 Conclusions: Hyperoside protects HL-1 cells from DOX-induced cardiotoxicity by inhibiting the 35 ASK1/p38 signaling pathway. Meanwhile, hyperoside maintained the toxicity of DOX in MDA- 36 MB-231 cells. 34 Conclusions: Hyperoside protects HL-1 cells from DOX-induced cardiotoxicity by inhibiting the 35 ASK1/p38 signaling pathway. Meanwhile, hyperoside maintained the toxicity of DOX in MDA- 36 MB-231 cells. Hyperoside alleviates doxorubicin-induced myocardial cells apoptosis by inhibiting the apoptosis signal-regulating kinase 1 /p38 pathway Lingxia Chen 1, 2 , Zhi Qin 1, 2 , Zhong-bao Ruan Corresp. 1, 2 1 Department of Cardiology, Nanjing University of Traditional Chinese Medicine, Nanjing, Jiangsu, China 2 Department of Cardiology, Taizhou People’s Hospital, Taizhou, Jiangsu, China Corresponding Author: Zhong-bao Ruan Email address: tzcardiac@163.com Background: Cardiotoxicity is a side eﬀect of the anthracycline broad-spectrum anti- tumor agent, doxorubicin (DOX). Hyperoside, a ﬂavonoid glycoside extracted from many herbs, has anti-apoptotic and anticancer properties. However, its impact on the alleviation of DOX-induced apoptosis in cardiomyocytes remains elusive. Methods: The HL-1 cell line was treated with 100 μM hyperoside for 1 h prior to treatment with 100 μM hyperoside and 1 μM DOX for 24 h. Cell counting kit-8 (CCK-8) assay was used to detect cell viability; DCFH-DA ﬂuorescent probe was used to detect (reactive oxygen species) ROS; biochemical methods was used to detect the activity of glutathione (GSH), catalase (CAT), superoxide dismutase (SOD), malondialdehyde (MDA); the degree of apoptosis following DOX insult was assessed using immunoﬂuorescence staining and terminal deoxynucleotidyl transferase mediated deoxy uridine triphosphate nick end labeling (TUNEL) assay; the change in protein expression of apoptosis signal-regulating kinase 1 (ASK1), p38, and apoptosis markers was determined using western blot. Results: Hyperoside ameliorated DOX-induced oxidative stress in HL-1 cells, up-regulated GSH, SOD and CAT activity, reduced ROS production and inhibited MDA overproduction. Moreover, in addition to promoting HL-1 cell apoptosis, DOX administration also increased B-cell lymphoma (Bcl)-2- associated X-protein and cleaved caspase-3 protein levels and decreased Bcl-2 protein level. Hyperoside therapy, however, signiﬁcantly reversed the impact of DOX on the cardiomyocytes. Mechanically, DOX treatment increased the phosphorylation of the ASK1/p38 axis whereas hyperoside treatment attenuated those changes. In a further step, hyperoside synergizes with DOX to kill MDA-MB-231 cells. Conclusions: Hyperoside protects HL-1 cells from DOX-induced cardiotoxicity by inhibiting the ASK1/p38 signaling pathway. Meanwhile, hyperoside maintained the cytotoxicity of DOX in MDA-MB-231 cells. PeerJ reviewing PDF \| (2022:10:78807:1:0:CHECK 26 Mar 2023) Manuscript to be reviewed 1 Hyperoside alleviates doxorubicin-induced myocardial cells apoptosis by inhibiting the apoptosis 2 signal-regulating kinase 1/p38 pathway 3 4 Lingxia Chen1,2, Zhi Qin1,2 , Zhong-bao Ruan1, 2 5 6 1 Department of Cardiology, Nanjing University of Traditional Chinese Medicine, Nanjing, 7 Jiangsu, China 8 2 Department of Cardiology, Taizhou People’s Hospital, Taizhou, Jiangsu, China 9 10 Correspondence E-mail: tzcardiac@163.com (Zhong-bao Ruan) 11 12 Abstract 13 Background: Cardiotoxicity is a side effect of the anthracycline broad-spectrum anti-tumor 14 agent, doxorubicin (DOX). Hyperoside, a flavonoid glycoside extracted from many herbs, has 15 anti-apoptotic and anticancer properties. However, its impact on the alleviation of DOX-induced 16 apoptosis in cardiomyocytes remains elusive. 17 Methods: The HL-1 cell line was treated with 100 μM hyperoside for 1 h prior to treatment with 18 100 μM hyperoside and 1 μM DOX for 24 h. Cell counting kit-8 (CCK-8) assay was used to 19 detect cell viability; DCFH-DA fluorescent probe was used to detect (reactive oxygen species) 20 ROS; biochemical methods was used to detect the activity of glutathione (GSH), catalase (CAT), 21 superoxide dismutase (SOD), malondialdehyde (MDA); the degree of apoptosis following DOX 22 insult was assessed using immunofluorescence staining and terminal deoxynucleotidyl 23 transferase mediated deoxy uridine triphosphate nick end labeling (TUNEL) assay; the change in 24 protein expression of apoptosis signal-regulating kinase 1 (ASK1), p38, and apoptosis markers 25 was determined using western blot. 26 Results: Hyperoside ameliorated DOX-induced oxidative stress in HL-1 cells, up-regulated GSH, 27 SOD and CAT activity, reduced ROS production and inhibited MDA overproduction. Moreover, 28 in addition to promoting HL-1 cell apoptosis, DOX administration also increased B-cell 29 lymphoma (Bcl)-2-associated X-protein and cleaved caspase-3 protein levels and decreased Bcl- 30 2 protein level. Hyperoside therapy, however, significantly reversed the impact of DOX on the 31 cardiomyocytes. Mechanically, DOX treatment increased the phosphorylation of the ASK1/p38 32 axis whereas hyperoside treatment attenuated those changes. In a further step, hyperoside 33 synergizes with DOX to kill MDA-MB-231 cells. 34 Conclusions: Hyperoside protects HL-1 cells from DOX-induced cardiotoxicity by inhibiting the 35 ASK1/p38 signaling pathway. Meanwhile, hyperoside maintained the toxicity of DOX in MDA- 36 MB-231 cells. 37 Manuscript to be reviewed 49 Apoptosis signal-regulated kinase 1 (ASK1) is a type of reactive oxygen species (ROS)-sensitiv 50 mitogen-activated protein kinase (MAP3K) that activates p38 and c-Jun N-terminal kinase (JNK 51 pathways via a cascade connection mediated by MAP kinase kinases to cause cell apoptosis. 52 Evidence has reported that the ROS-ASK1-p38 pathway is significant for the treatment of 53 cardiomyocyte apoptosis using DOX (Jiang L et al.,2020). Additionally, Kun et al. reported tha 54 DOX-induced oxidative stress caused the phosphorylation of ASK1 and p38, podocyte damage 55 and ultimately, renal injury in murine podocytes (Gao K et al., 2014). Inhibition of the ROS- 56 ASK1-p38 pathway decreases apoptosis in conditions such as subarachnoid hemorrhage (Gao X 57 et al., 2022), non-alcoholic steatohepatitis (Lan T et al., 2022), and diabetic cardiomyopathy 58 (Ding W et al., 2021). Hyperoside, a flavonoid glycoside extracted from various herbs, such as 59 Hypericaceae and Rosaceae, has several cytoprotective properties, including anti-apoptosis, 60 antioxidation, and anticancer property (Wang Q et al., 2022; Ferenczyova K, Kalocayova B & 61 Bartekova M, 2020). Hyperoside reduces mitochondrial dysfunction in podocytes during DOX- 62 induced kidney injury (Chen Z et al.,2017). Additionally, hyperoside has been reported to reduc 63 damage and oxidative stress in human keratinocyte cells by negatively regulating the p38 64 signaling pathway (Charachit N et al., 2022). However, whether hyperoside can protect 65 cardiomyocytes from DOX-induced apoptosis and its underlying mechanism have not yet been 66 fully elucidated. 67 In our work, we explored the impact of hyperoside on DIC in an HL-1 cardiomyocyte cell 68 line. 69 70 Materials & Methods 71 2.1 Materials 72 Hyperoside was purchased from MedChemExpress (USA) as a crystalline powder with a 73 purity of 99.56%. It was dissolved in DMSO and prepared into a mother liquor of 100mM. DOX 74 was purchased from MedChemExpress as a crystalline powder with a purity of 99.48%. It was 75 dissolved in DMSO and prepared as a 10 mM mother liquor. The were diluted to the desired 76 concentration when using. One-step terminal deoxynucleotidyl transferase deoxyuridine 77 triphosphate nick end labeling (TUNEL) apoptosis assay kit, reactive oxygen species (ROS) 78 assay kit and malondialdehyde (MDA) content assay kit purchased from Beyotime Biotech Inc 79 (Shanghai, China). 38 Introduction 39 Doxorubicin (DOX) is an anthracycline antibiotic used in clinical practice for tumor 40 chemotherapy (Herrmann J, 2020; Sawicki KT et al., 2021). Research has revealed that DOX has PeerJ reviewing PDF \| (2022:10:78807:1:0:CHECK 26 Mar 2023) Manuscript to be reviewed 45 There are various mechanisms of DIC, among which oxidative stress, mitochondrial dysfunction, 46 and apoptosis have been most widely reported (Wallace KB, Sardão VA & Oliveira PJ, 2020; 47 Wenningmann N et al., 2019). Therefore, pharmacological interventions are required in these 48 patients to attenuate DIC. 49 Apoptosis signal-regulated kinase 1 (ASK1) is a type of reactive oxygen species (ROS)-sensitive 50 mitogen-activated protein kinase (MAP3K) that activates p38 and c-Jun N-terminal kinase (JNK) 51 pathways via a cascade connection mediated by MAP kinase kinases to cause cell apoptosis. 52 Evidence has reported that the ROS-ASK1-p38 pathway is significant for the treatment of 53 cardiomyocyte apoptosis using DOX (Jiang L et al.,2020). Additionally, Kun et al. reported that 54 DOX-induced oxidative stress caused the phosphorylation of ASK1 and p38, podocyte damage, 55 and ultimately, renal injury in murine podocytes (Gao K et al., 2014). Inhibition of the ROS- 56 ASK1-p38 pathway decreases apoptosis in conditions such as subarachnoid hemorrhage (Gao X 57 et al., 2022), non-alcoholic steatohepatitis (Lan T et al., 2022), and diabetic cardiomyopathy 58 (Ding W et al., 2021). Hyperoside, a flavonoid glycoside extracted from various herbs, such as 59 Hypericaceae and Rosaceae, has several cytoprotective properties, including anti-apoptosis, 60 antioxidation, and anticancer property (Wang Q et al., 2022; Ferenczyova K, Kalocayova B & 61 Bartekova M, 2020). Hyperoside reduces mitochondrial dysfunction in podocytes during DOX- 62 induced kidney injury (Chen Z et al.,2017). Additionally, hyperoside has been reported to reduce 63 damage and oxidative stress in human keratinocyte cells by negatively regulating the p38 64 signaling pathway (Charachit N et al., 2022). However, whether hyperoside can protect 65 cardiomyocytes from DOX-induced apoptosis and its underlying mechanism have not yet been 66 fully elucidated. 43 quarter of patients undergo chemotherapy-induced DIC in a dose-dependent manner, which 44 severely restricts the clinical application of DOX (Swain SM, Whaley FS & Ewer MS, 2003). 45 There are various mechanisms of DIC, among which oxidative stress, mitochondrial dysfunctio 46 and apoptosis have been most widely reported (Wallace KB, Sardão VA & Oliveira PJ, 2020; 47 Wenningmann N et al., 2019). Therefore, pharmacological interventions are required in these 48 patients to attenuate DIC. Manuscript to be reviewed 41 a strong binding affinity for topoisomerase and accumulates in cardiomyocytes as an isomer, 42 causing severe DOX-induced cardiotoxicity (DIC) (Sangweni NF et al.,2022). Clinically, one- 43 quarter of patients undergo chemotherapy-induced DIC in a dose-dependent manner, which 44 severely restricts the clinical application of DOX (Swain SM, Whaley FS & Ewer MS, 2003). 45 There are various mechanisms of DIC, among which oxidative stress, mitochondrial dysfunction, 46 and apoptosis have been most widely reported (Wallace KB, Sardão VA & Oliveira PJ, 2020; 47 Wenningmann N et al., 2019). Therefore, pharmacological interventions are required in these 48 patients to attenuate DIC. 49 Apoptosis signal-regulated kinase 1 (ASK1) is a type of reactive oxygen species (ROS)-sensitive 50 mitogen-activated protein kinase (MAP3K) that activates p38 and c-Jun N-terminal kinase (JNK) 51 pathways via a cascade connection mediated by MAP kinase kinases to cause cell apoptosis. 52 Evidence has reported that the ROS-ASK1-p38 pathway is significant for the treatment of 53 cardiomyocyte apoptosis using DOX (Jiang L et al.,2020). Additionally, Kun et al. reported that 54 DOX-induced oxidative stress caused the phosphorylation of ASK1 and p38, podocyte damage, 55 and ultimately, renal injury in murine podocytes (Gao K et al., 2014). Inhibition of the ROS- 56 ASK1-p38 pathway decreases apoptosis in conditions such as subarachnoid hemorrhage (Gao X 57 et al., 2022), non-alcoholic steatohepatitis (Lan T et al., 2022), and diabetic cardiomyopathy 58 (Ding W et al., 2021). Hyperoside, a flavonoid glycoside extracted from various herbs, such as 59 Hypericaceae and Rosaceae, has several cytoprotective properties, including anti-apoptosis, 60 antioxidation, and anticancer property (Wang Q et al., 2022; Ferenczyova K, Kalocayova B & 61 Bartekova M, 2020). Hyperoside reduces mitochondrial dysfunction in podocytes during DOX- 62 induced kidney injury (Chen Z et al.,2017). Additionally, hyperoside has been reported to reduce 63 damage and oxidative stress in human keratinocyte cells by negatively regulating the p38 64 signaling pathway (Charachit N et al., 2022). However, whether hyperoside can protect 65 cardiomyocytes from DOX-induced apoptosis and its underlying mechanism have not yet been 66 fully elucidated. 41 a strong binding affinity for topoisomerase and accumulates in cardiomyocytes as an isomer, 42 causing severe DOX-induced cardiotoxicity (DIC) (Sangweni NF et al.,2022). Clinically, one- 43 quarter of patients undergo chemotherapy-induced DIC in a dose-dependent manner, which 44 severely restricts the clinical application of DOX (Swain SM, Whaley FS & Ewer MS, 2003). 86 2.2 Cell culture and treatments 98 CCK-8 test was used to detecte cell viability. To determine the optimal concentration of DOX 99 that can lead to a survival rate close to but not exceeding, 50%, we used DOX of different 100 concentrations (0 μM, 0.1 μM, 0.5 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, and 10 μM) to treat the 101 HL-1 cells. To determine the safe contentration range of hyperoside that will not cause severe 102 cell toxicity, we treated HL-1 cells with hyperoside at concentrations of 0−500 μM (0 μM, 5 μM, 103 10 μM, 50 μM, 100 μM, 200 μM, 300 μM, 400 μM and 500 μM). Further, hyperoside of final 104 concentrations (0 μM, 25 μM, 50 μM, 100 μM, 125 μM, 150 μM, and 175 μM) was used to 105 examine whether it will inhibit DOX-induced cell injuring in HL1 cells. After incubation for 24 106 hours, ten microliters of CCK-8 reagent were added into each well of a 96-well plate and then 107 incubated at 37 ℃for two hours. The absorbance of optical density (OD) value at 450 nm was 108 measured using a microplate reader (Bio Tek, Vermont, USA). Each treatment was performed in 109 triplicate. The percentage of cell viability for each group was calculated as follows: cell viability 110 rate = OD of the experimental group/OD of the control group ×100. 111 Manuscript to be reviewed Manuscript to be reviewed 81 Technology Co., Ltd. (Beijing, China).Cell counting kit-8 (CCK-8) was purchased from Apexbio 82 (USA). Fetal bovine serum (Bovine serum) was purchased from Sigma (Santa Clara, CA, USA). 83 HL-1 cells and MDA-MB-231 cells were obtained from Shanghai Fuheng Biotechnology Co., 84 Ltd. (Shanghai, China). 85 86 2.2 Cell culture and treatments 87 HL-1 cells were cultured in the Claycomb media (Sigma, USA) containing 10% fetal bovine 88 serum, 2 mM L-glutamine (Solarbio, China), 100 U/mL penicillin (Gibco, USA) and 100 μg/mL 89 streptomycin (Gibco, USA) at 37 ℃. Cells were randomly allocated into four groups, i.e., the 90 control, hyperoside, DOX and DOX + hyperoside groups. Cells in the control group were 91 cultured in the medium for 24 h; cells in the hyperoside group were cultured in a medium 92 supplemented with 100 μM hyperoside for 24 h; cells in the DOX group were cultured in a 93 medium supplemented with 1 μM DOX for 24 h; cells in the DOX + hyperoside group were 94 pretreated with 100 μM hyperoside for 1 h and then treated with 100 μM hyperoside and 1 μM 95 DOX together for 24 hours. 96 97 2.3 Test for cell viability 98 CCK-8 test was used to detecte cell viability. To determine the optimal concentration of DOX 99 that can lead to a survival rate close to but not exceeding, 50%, we used DOX of different 100 concentrations (0 μM, 0.1 μM, 0.5 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, and 10 μM) to treat the 101 HL-1 cells. To determine the safe contentration range of hyperoside that will not cause severe 102 cell toxicity, we treated HL-1 cells with hyperoside at concentrations of 0−500 μM (0 μM, 5 μM, 103 10 μM, 50 μM, 100 μM, 200 μM, 300 μM, 400 μM and 500 μM). Further, hyperoside of final 104 concentrations (0 μM, 25 μM, 50 μM, 100 μM, 125 μM, 150 μM, and 175 μM) was used to 105 examine whether it will inhibit DOX-induced cell injuring in HL1 cells. After incubation for 24 106 hours, ten microliters of CCK-8 reagent were added into each well of a 96-well plate and then 107 incubated at 37 ℃for two hours. The absorbance of optical density (OD) value at 450 nm was 108 measured using a microplate reader (Bio Tek, Vermont, USA). Manuscript to be reviewed Catalase (CAT) activity assay kit, superoxide dismutase (SOD) activity assa 80 kit and reduced glutathione (GSH) content assay kit purchased from Solarbio Science & 67 In our work, we explored the impact of hyperoside on DIC in an HL-1 cardiomyocyte cell 68 line. 69 72 Hyperoside was purchased from MedChemExpress (USA) as a crystalline powder with a 73 purity of 99.56%. It was dissolved in DMSO and prepared into a mother liquor of 100mM. DOX 74 was purchased from MedChemExpress as a crystalline powder with a purity of 99.48%. It was 75 dissolved in DMSO and prepared as a 10 mM mother liquor. The were diluted to the desired 76 concentration when using. One-step terminal deoxynucleotidyl transferase deoxyuridine 77 triphosphate nick end labeling (TUNEL) apoptosis assay kit, reactive oxygen species (ROS) 78 assay kit and malondialdehyde (MDA) content assay kit purchased from Beyotime Biotech Inc 79 (Shanghai, China). Catalase (CAT) activity assay kit, superoxide dismutase (SOD) activity assay 80 kit and reduced glutathione (GSH) content assay kit purchased from Solarbio Science & PeerJ reviewing PDF \| (2022:10:78807:1:0:CHECK 26 Mar 2023) Manuscript to be reviewed Each treatment was performed in 109 triplicate. The percentage of cell viability for each group was calculated as follows: cell viability 110 rate = OD of the experimental group/OD of the control group ×100. 111 112 2.4 Immunofluorescence (IF) staining 113 C ll fi d ith 4% f ld h d (S l bi Chi ) f 15 i d f th bl k d 81 Technology Co., Ltd. (Beijing, China).Cell c 82 (USA). Fetal bovine serum (Bovine serum) 83 HL-1 cells and MDA-MB-231 cells were ob 84 Ltd. (Shanghai, China). 85 86 2.2 Cell culture and treatments 87 HL-1 cells were cultured in the Claycomb 88 serum, 2 mM L-glutamine (Solarbio, China) 89 streptomycin (Gibco, USA) at 37 ℃. Cells w 90 control, hyperoside, DOX and DOX + hyper 91 cultured in the medium for 24 h; cells in the 92 supplemented with 100 μM hyperoside for 2 93 medium supplemented with 1 μM DOX for 94 pretreated with 100 μM hyperoside for 1 h a 95 DOX together for 24 hours. 96 97 2.3 Test for cell viability 98 CCK-8 test was used to detecte cell viabil 99 that can lead to a survival rate close to but n 100 concentrations (0 μM, 0.1 μM, 0.5 μM, 1 μM 101 HL-1 cells. To determine the safe contentrat 102 cell toxicity, we treated HL-1 cells with hyp 103 10 μM, 50 μM, 100 μM, 200 μM, 300 μM, 4 104 concentrations (0 μM, 25 μM, 50 μM, 100 μ 105 examine whether it will inhibit DOX-induce 106 hours, ten microliters of CCK-8 reagent wer 107 incubated at 37 ℃for two hours. The absorb 108 measured using a microplate reader (Bio Tek 109 triplicate. The percentage of cell viability fo 110 rate = OD of the experimental group/OD of 111 112 2.4 Immunofluorescence (IF) staining 113 Cells were fixed with 4% paraformaldehy 114 with an immunofluorescent blocking solutio 81 Technology Co., Ltd. (Beijing, China).Cell counting kit-8 (CCK-8) was purchased from Apexbio 82 (USA). Fetal bovine serum (Bovine serum) was purchased from Sigma (Santa Clara, CA, USA). 83 HL-1 cells and MDA-MB-231 cells were obtained from Shanghai Fuheng Biotechnology Co., 84 Ltd. (Shanghai, China). Manuscript to be reviewed 120 (DAPI) (Solarbio, China). Images were captured using a fluorescence microscope (Leica, Oskar, 121 Germany), and fluorescence intensity was quantified using the ImageJ software. 122 123 2.5 TUNEL staining 124 According to the manufacturer’s protocol, a one-step TUNEL apoptosis assay kit (Beyotime, 125 China) was used to assess apoptosis in HL-1 cells and MDA-MB-231 cells. Cells were fixed 126 with 4% paraformaldehyde for 30 min and further permeabilized in 0.3% Triton X-100 for 5 min 127 at 15−25 ℃. And then the cells were incubated with TUNEL reagent for 1 hour at 37 ℃ in the 128 dark. The samples were examined with a fluorescence microscope after being counterstained 129 with DAPI. The fluorescence intensity was further analyzed using ImageJ software. The ratio of 130 TUNEL-positive nuclei to total DAPI-stained nuclei was calculated to assess the apoptotic rate. 131 132 2.6 Detection of ROS level 133 According to the manufacturer’s protocol, a ROS assay kit was used to assess ROS level in 134 HL-1 cells. In short, the cells were incubated with culture medium containing DCFH-DA 135 fluorescent probe and hoechst 33342 staining solution for live cells (Beyotime, China) reagent 136 for 30 min at 37 ℃ in the dark. The samples were examined with a fluorescence microscope. 137 Then the fluorescence intensity was further analyzed using ImageJ software. 138 139 2.7 Measurement of SOD, CAT, GSH and MDA levels 140 MDA content assay kit, CAT activity assay kit, SOD activity assay kit and GSH content assay 141 were used to measure GSH, CAT, SOD, MDA levels. All operations are in accordance with the 142 manufacturer’s protocol. In short, after extracting GSH, MDA, CAT and SOD, respectively, the 143 absorbance of OD value were measured using a microplate reader. And the GSH, MDA, CAT 144 and SOD levels were calculated according to the formulas provided in the instructions. 145 146 2.8 Western blot (WB) analysis 147 Total protein was extracted from HL1 cells using radio immunoprecipitation assay lysis buffer 148 (Beyotime, China) containing phenyl methane sulfonyl fluoride (PMSF, MCE, USA), 149 phosphatase inhibitor. Then an enhanced bicinchoninic acid protein assay kit (Beyotime, China) 150 was used to quantified the concentration of total protein. Manuscript to be reviewed In short, after extracting GSH, MDA, CAT and SOD, respectively, the 143 absorbance of OD value were measured using a microplate reader. And the GSH, MDA, CAT 144 and SOD levels were calculated according to the formulas provided in the instructions. 145 146 2.8 Western blot (WB) analysis 147 Total protein was extracted from HL1 cells using radio immunoprecipitation assay lysis buffer 148 (Beyotime, China) containing phenyl methane sulfonyl fluoride (PMSF, MCE, USA), 149 h h t i hibit Th h d bi i h i i id t i kit (B ti Chi ) 120 (DAPI) (Solarbio, China). Images were captured using a fluorescence microscope (Leica, Oskar, 121 Germany), and fluorescence intensity was quantified using the ImageJ software. 132 2.6 Detection of ROS level 133 According to the manufacturer’s protocol, a ROS assay kit was used to assess ROS level in 134 HL-1 cells. In short, the cells were incubated with culture medium containing DCFH-DA 135 fluorescent probe and hoechst 33342 staining solution for live cells (Beyotime, China) reagent 136 for 30 min at 37 ℃ in the dark. The samples were examined with a fluorescence microscope. 137 Then the fluorescence intensity was further analyzed using ImageJ software. 138 139 2.7 Measurement of SOD, CAT, GSH and MDA levels 140 MDA content assay kit, CAT activity assay kit, SOD activity assay kit and GSH content assay 141 were used to measure GSH, CAT, SOD, MDA levels. All operations are in accordance with the 142 manufacturer’s protocol. In short, after extracting GSH, MDA, CAT and SOD, respectively, the 143 absorbance of OD value were measured using a microplate reader. And the GSH, MDA, CAT 144 and SOD levels were calculated according to the formulas provided in the instructions. 145 146 2.8 Western blot (WB) analysis 147 Total protein was extracted from HL1 cells using radio immunoprecipitation assay lysis buffer 148 (Beyotime, China) containing phenyl methane sulfonyl fluoride (PMSF, MCE, USA), 149 phosphatase inhibitor. Then an enhanced bicinchoninic acid protein assay kit (Beyotime, China) 150 was used to quantified the concentration of total protein. Equal amounts of protein were 151 separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), 152 transferred to polyvinylidene fluoride membranes (Millipore, USA), and blocked with NcmBlot 153 blocking buffer (NCM Biotech Co., Ltd, China) at 15−25 ℃ for 10 min. 112 2.4 Immunofluorescence (IF) staining ( ) g 113 Cells were fixed with 4% paraformaldehyde (Solarbio, China) for 15 min and further blocked 114 with an immunofluorescent blocking solution (Beyotime Biotech Inc, China) for 1 h at 15−25 115 ℃. Next, the cells were incubated with anti-cleaved caspase-3 (1:100, AF7022, Affinity) at 4 ℃ 116 overnight. And the anti-cleaved caspase-3 was diluted by antibody dilution (NCM Biotech Co., 117 Ltd, China). Samples were incubated with goat anti-rabbit secondary antibody (immunoglobulin 118 G) (diluted by PBS, 1:500, A23420, Abbine) for 1 h at room temperature in the dark, followed 119 by sealing with the Antifade Mounting Medium containing 4ʹ,6-diamidino-2-phenylindole 113 Cells were fixed with 4% paraformaldehyde (Solarbio, China) for 15 min and further blocked 114 with an immunofluorescent blocking solution (Beyotime Biotech Inc, China) for 1 h at 15−25 115 ℃. Next, the cells were incubated with anti-cleaved caspase-3 (1:100, AF7022, Affinity) at 4 ℃ 116 overnight. And the anti-cleaved caspase-3 was diluted by antibody dilution (NCM Biotech Co., 117 Ltd, China). Samples were incubated with goat anti-rabbit secondary antibody (immunoglobulin 118 G) (diluted by PBS, 1:500, A23420, Abbine) for 1 h at room temperature in the dark, followed 119 by sealing with the Antifade Mounting Medium containing 4ʹ,6-diamidino-2-phenylindole 113 Cells were fixed with 4% paraformaldehyde (Solarbio, China) for 15 min and further blocked 114 with an immunofluorescent blocking solution (Beyotime Biotech Inc, China) for 1 h at 15−25 117 Ltd, China). Samples were incubated with goat anti-rabbit secondary antibody (immunoglobulin 118 G) (diluted by PBS, 1:500, A23420, Abbine) for 1 h at room temperature in the dark, followed b li i h h A if d M i M di i i 4ʹ 6 di idi 2 h li d l PeerJ reviewing PDF \| (2022:10:78807:1:0:CHECK 26 Mar 2023) Manuscript to be reviewed Equal amounts of protein were 151 separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), 152 transferred to polyvinylidene fluoride membranes (Millipore, USA), and blocked with NcmBlot 153 blocking buffer (NCM Biotech Co., Ltd, China) at 15−25 ℃ for 10 min. The membranes were 154 incubated with the primary antibody at 4 ℃ overnight and probed with the secondary antibody at 155 room temperature for 1 h. Protein bands were observed under a gel documentation system 156 (SYNGENE, Cambridge, UK). Quantitative analysis of the proteins was conducted using the 157 ImageJ software. The primary and secondary antibodies used in WB analyses were as follows: 158 anti-t-ASK1 (1:1000, ab45178, Abcam), anti-p-ASK1 (1:10000, ab278547, Abcam), anti-t-p38 159 (1:2000, ab170099, Abcam), anti-p-p38 (1:1000, ab195049, Abcam), anti-B-cell lymphoma p 120 (DAPI) (Solarbio, China). Images were captured using a fluorescence microscope (Leica, Oskar, 121 Germany), and fluorescence intensity was quantified using the ImageJ software. 122 123 2.5 TUNEL staining 124 According to the manufacturer’s protocol, a one-step TUNEL apoptosis assay kit (Beyotime, 125 China) was used to assess apoptosis in HL-1 cells and MDA-MB-231 cells. Cells were fixed 126 with 4% paraformaldehyde for 30 min and further permeabilized in 0.3% Triton X-100 for 5 min 127 at 15−25 ℃. And then the cells were incubated with TUNEL reagent for 1 hour at 37 ℃ in the 128 dark. The samples were examined with a fluorescence microscope after being counterstained 129 with DAPI. The fluorescence intensity was further analyzed using ImageJ software. The ratio of 130 TUNEL-positive nuclei to total DAPI-stained nuclei was calculated to assess the apoptotic rate. 131 132 2.6 Detection of ROS level 133 According to the manufacturer’s protocol, a ROS assay kit was used to assess ROS level in 134 HL-1 cells. In short, the cells were incubated with culture medium containing DCFH-DA 135 fluorescent probe and hoechst 33342 staining solution for live cells (Beyotime, China) reagent 136 for 30 min at 37 ℃ in the dark. The samples were examined with a fluorescence microscope. 137 Then the fluorescence intensity was further analyzed using ImageJ software. 138 139 2.7 Measurement of SOD, CAT, GSH and MDA levels 140 MDA content assay kit, CAT activity assay kit, SOD activity assay kit and GSH content assay 141 were used to measure GSH, CAT, SOD, MDA levels. All operations are in accordance with the 142 manufacturer’s protocol. Manuscript to be reviewed 160 (Bcl)-2-associated X-protein (Bax) (1:5000, ab32503, Abcam), anti-Bcl-2 (1:10000, ab182858, 161 Abcam), anti-cleaved caspase-3 (1:1000, 9664,cell signaling technology), anti-p53 (1:1000, 162 2524, cell signaling technology) and anti-β-actin (1:10000, AF7018, Affinity) antibodies, and 163 horseradish peroxidase goat-anti-rabbit secondary antibody (1:10,000, YFSA02, YiFeiXue 164 Biotechnology). All the primary antibodies used were diluted by antibody dilution (NCM 165 Biotech Co., Ltd, China) and the secondary antibody was diluted by 1× tris-buffered saline tween 166 (TBST) buffer (Solarbio, China). β-actin levels were taken as the control. 160 (Bcl)-2-associated X-protein (Bax) (1:5000, ab32503, Abcam), anti-Bcl-2 (1:10000, ab182858, 161 Abcam), anti-cleaved caspase-3 (1:1000, 9664,cell signaling technology), anti-p53 (1:1000, 162 2524, cell signaling technology) and anti-β-actin (1:10000, AF7018, Affinity) antibodies, and 163 horseradish peroxidase goat-anti-rabbit secondary antibody (1:10,000, YFSA02, YiFeiXue 164 Biotechnology). All the primary antibodies used were diluted by antibody dilution (NCM 165 Biotech Co., Ltd, China) and the secondary antibody was diluted by 1× tris-buffered saline tween 166 (TBST) buffer (Solarbio, China). β-actin levels were taken as the control. 166 (TBST) buffer (Solarbio, China). β-actin levels were taken as the control. 167 168 2.9 Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) 169 Total RNA from HL-1 cells was extracted using the TRIzol Universal Reagent (TIANGEN, 170 Beijing, China) following the manufacturer’s instructions. The total RNA concentration of each 171 sample was then measured using nanodrop 2000 (Thermo Scientific, America). Next, the 172 complementary DNA (cDNA) was synthesized by using the All-In-One 5X RT MasterMix 173 (ABM, G490, USA). The procedure was conducted following the manufacturer’s protocol. In 174 short, the reaction was executed at 25 ℃ for 10 min and then 42 ℃ for 15 min, followed by 5 175 min at 85 ℃. Quantitative analysis of RNA was accomplished using the Roche LightCycler 480 176 (Roche, Basel, Switzerland) with BlasTaq™ 2X qPCR MasterMix (ABM, G891, USA). The 177 reaction conditions were as follows: The reaction was executed at 95 ℃ for 3 min, followed by 178 40 cycles of 15 s at 95 ℃ and 60 s at 60 ℃. The relative quantification of the target genes was 179 normalized by GAPDH levels and calculated using the 2−ΔΔCT method. The primer sequences 180 were presented in table1. 181 182 2.10 Statistical analysis 183 GraphPad Prism 8 was used to analyse the data from at least three independent trials, which 184 were indicated as mean ± standard error of mean. Manuscript to be reviewed The membranes were 154 incubated with the primary antibody at 4 ℃ overnight and probed with the secondary antibody at 155 room temperature for 1 h. Protein bands were observed under a gel documentation system 156 (SYNGENE, Cambridge, UK). Quantitative analysis of the proteins was conducted using the 157 ImageJ software. The primary and secondary antibodies used in WB analyses were as follows: 158 anti-t-ASK1 (1:1000, ab45178, Abcam), anti-p-ASK1 (1:10000, ab278547, Abcam), anti-t-p38 159 (1:2000, ab170099, Abcam), anti-p-p38 (1:1000, ab195049, Abcam), anti-B-cell lymphoma PeerJ reviewing PDF \| (2022:10:78807:1:0:CHECK 26 Mar 2023) Manuscript to be reviewed Multiple groups comparisons were evaluated 185 using one-way analysis of variance, and comparisons between two groups were evaluated using 186 Student’s t-test. Significance of statistics was defined as P < 0.05. * P < 0.001, P < 0.01, * 187 P < 0.05. 188 189 Results 190 3.1 Optimal concentration of DOX and hyperoside for HL-1 cells 191 Cell survival rate was used to ascertain the optimal concentration of DOX and hyperoside. 192 The CCK-8 result revealed that HL-1 cell survival rate steadily reduced with increasing DOX 193 concentrations. Specifically, the cell viability was 63.12% at the concentration of 1 μM DOX, 194 which was significant different compared to that when the concentration of DOX was 0 μM 195 (Fig.1A). Additionally, we used IF staining to examine the expression level of cleaved caspase-3 196 in HL-1 cells after they were treated with DOX of different doses. The results showed that 197 increasing DOX concentrations gradually raised cleaved-caspase 3 level. Specifically, when the 198 concentration of DOX was 1 μM, the average densitometric value of cleaved caspase-3 was 199 39.98%, which was significant different compared to that when the concentration of DOX was 0 168 2.9 Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) 168 2.9 Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) 169 Total RNA from HL-1 cells was extracted using the TRIzol Universal Reagent (TIANGEN, 170 Beijing, China) following the manufacturer’s instructions. The total RNA concentration of each 171 sample was then measured using nanodrop 2000 (Thermo Scientific, America). Next, the 172 complementary DNA (cDNA) was synthesized by using the All-In-One 5X RT MasterMix 173 (ABM, G490, USA). The procedure was conducted following the manufacturer’s protocol. In 174 short, the reaction was executed at 25 ℃ for 10 min and then 42 ℃ for 15 min, followed by 5 175 min at 85 ℃. Quantitative analysis of RNA was accomplished using the Roche LightCycler 480 176 (Roche, Basel, Switzerland) with BlasTaq™ 2X qPCR MasterMix (ABM, G891, USA). The 177 reaction conditions were as follows: The reaction was executed at 95 ℃ for 3 min, followed by 178 40 cycles of 15 s at 95 ℃ and 60 s at 60 ℃. The relative quantification of the target genes was 179 normalized by GAPDH levels and calculated using the 2−ΔΔCT method. The primer sequences 180 were presented in table1. 168 2.9 Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) 169 Total RNA from HL-1 cells was extracted using the TRIzol Universal Reagent (TIANGEN, 170 Beijing, China) following the manufacturer’s instructions. The total RNA concentration of each 171 sample was then measured using nanodrop 2000 (Thermo Scientific, America). Next, the 172 complementary DNA (cDNA) was synthesized by using the All-In-One 5X RT MasterMix 173 (ABM, G490, USA). The procedure was conducted following the manufacturer’s protocol. In 174 short, the reaction was executed at 25 ℃ for 10 min and then 42 ℃ for 15 min, followed by 5 175 min at 85 ℃. Quantitative analysis of RNA was accomplished using the Roche LightCycler 480 176 (Roche, Basel, Switzerland) with BlasTaq™ 2X qPCR MasterMix (ABM, G891, USA). The 177 reaction conditions were as follows: The reaction was executed at 95 ℃ for 3 min, followed by 178 40 cycles of 15 s at 95 ℃ and 60 s at 60 ℃. The relative quantification of the target genes was 179 normalized by GAPDH levels and calculated using the 2−ΔΔCT method. The primer sequences 180 were presented in table1. 3.1 Optimal concentration of DOX and hyperoside for HL-1 cells 191 Cell survival rate was used to ascertain the optimal concentration of DOX and hyperoside. 192 The CCK-8 result revealed that HL-1 cell survival rate steadily reduced with increasing DOX 193 concentrations. Specifically, the cell viability was 63.12% at the concentration of 1 μM DOX, 194 which was significant different compared to that when the concentration of DOX was 0 μM 195 (Fig.1A). Additionally, we used IF staining to examine the expression level of cleaved caspase-3 196 in HL-1 cells after they were treated with DOX of different doses. The results showed that 197 increasing DOX concentrations gradually raised cleaved-caspase 3 level. Specifically, when the 198 concentration of DOX was 1 μM, the average densitometric value of cleaved caspase-3 was 199 39.98%, which was significant different compared to that when the concentration of DOX was 0 PeerJ reviewing PDF \| (2022:10:78807:1:0:CHECK 26 Mar 2023) Manuscript to be reviewed 209 210 3.2 Hyperoside diminished DOX-induced oxidative stress damage in HL-1 cells 211 To explore the potential mechanism of cardiomyocyte protection associated with hyperoside, 212 markers related to oxidative stress injury were examined. DCFH-DA fluorescent probe was used 213 to measure expression level of ROS, and the results suggested that hyperoside significantly 214 inhibited DOX-induced ROS production in HL-1 cardiomyocytes (Fig. 2A, B). Further, The 215 results of biochemical assays showed that DOX remarkably decreased the activity of antioxidant 216 enzymes (GSH, CAT, SOD) in HL-1 cells, which rebounded after hyperoside pretreatment (Fig. 217 2C, D and E); MDA was overproduced in the DOX group and diminished in the DOX + 218 hyperoside group (Fig. 2F). The above outcomes revealed that hyperoside can reduce ROS 219 production, increase the activity of antioxidant enzymes, and consequently attenuate DOX- 220 induced oxidative stress damage in HL-1 cardiomyocytes. 221 222 3.3 Hyperoside attenuated DOX-induced apoptosis in HL-1 cells 223 DOX-induced apoptosis was visualized by the TUNEL staining of HL-1 cells. The 224 fluorescence microscopic images and data analysis revealed that the number of TUNEL-positive 225 cells expressing the green tunneling magnetoresistance signal was markedly increased in the 226 DOX group versus the control group. The results revealed a remarkable increase in apoptosis. 227 Whether hyperoside treatment attenuated DOX-induced apoptosis was further explored. The 228 results revealed that hyperoside treatment significantly reduced DOX-induced apoptosis in HL-1 229 cardiomyocytes, which was indicated by a remarkable decrease in TUNEL-positive cells (Fig.3A 230 and B). IF staining was used to detect cleaved caspase-3. The results revealed that cleaved 231 caspase-3 expression was markedly increased following DOX treatment, and the effect was 232 attenuated by hyperoside treatment (Fig.3C and D). 233 234 3.4 Hyperoside down-regulated apoptosis-related proteins in DOX-treated HL-1 cells 235 To investigate mechanisms of apoptosis following DOX treatment, qRT-PCR was used to 236 identify Bax/Bcl-2 and cleaved caspase-3 mRNA levels of cells in each group. The results 237 confirmed that hyperoside administration significantly reduced DOX-induced increased levels of 238 Bax/Bcl-2 and cleaved caspase-3 (Fig. 4A and B). Further, we examined the levels of apoptosis- 239 related proteins, and the WB results revealed significantly increased levels of Bax/Bcl-2 yperoside diminished DOX-induced oxidative stress damage in HL-1 cells 211 To explore the potential mechanism of cardiomyocyte protection associated with hyperoside, 212 markers related to oxidative stress injury were examined. DCFH-DA fluorescent probe was used 213 to measure expression level of ROS, and the results suggested that hyperoside significantly 214 inhibited DOX-induced ROS production in HL-1 cardiomyocytes (Fig. 2A, B). Further, The 215 results of biochemical assays showed that DOX remarkably decreased the activity of antioxidant 216 enzymes (GSH, CAT, SOD) in HL-1 cells, which rebounded after hyperoside pretreatment (Fig. 217 2C, D and E); MDA was overproduced in the DOX group and diminished in the DOX + 218 hyperoside group (Fig. 2F). The above outcomes revealed that hyperoside can reduce ROS 219 production, increase the activity of antioxidant enzymes, and consequently attenuate DOX- 220 induced oxidative stress damage in HL-1 cardiomyocytes. Manuscript to be reviewed The results revealed that cleaved 231 caspase-3 expression was markedly increased following DOX treatment, and the effect was 232 attenuated by hyperoside treatment (Fig.3C and D). 233 234 3.4 Hyperoside down-regulated apoptosis-related proteins in DOX-treated HL-1 cells 235 To investigate mechanisms of apoptosis following DOX treatment, qRT-PCR was used to 236 identify Bax/Bcl-2 and cleaved caspase-3 mRNA levels of cells in each group. The results 237 confirmed that hyperoside administration significantly reduced DOX-induced increased levels of 238 Bax/Bcl-2 and cleaved caspase-3 (Fig. 4A and B). Further, we examined the levels of apoptosis- 239 related proteins, and the WB results revealed significantly increased levels of Bax/Bcl-2 200 μM (Fig.1D and E). Therefore, we selected 1 μM as the ideal concentration of DOX to induce 201 HL-1 cell damage. Further, we treated HL-1 cells with hyperoside at concentrations of 0−500 202 μM, and the CCK-8 result revealed that hyperoside at concentrations equal to or higher than 200 203 μM could cause significant changes in the survival rates of these HL-1 cells (Fig.1B). Thus, we 204 choose hyperoside at concentrations of 0-175 μM for further optimization. The result revealed 205 that hyperoside improved the survival rate of HL-1 cells within a specific range from 50 μM to 206 100 μM; however, hyperoside treatment did not significantly increase cell viability with the 207 further increases in its concentrations (Fig.1C). Thus, 100 μM was selected as the optimal 208 concentration of hyperoside for subsequent experiments in this study. 200 μM (Fig.1D and E). Therefore, we selected 1 μM as the ideal concentration of DOX to induce 201 HL-1 cell damage. Further, we treated HL-1 cells with hyperoside at concentrations of 0−500 202 μM, and the CCK-8 result revealed that hyperoside at concentrations equal to or higher than 200 203 μM could cause significant changes in the survival rates of these HL-1 cells (Fig.1B). Thus, we 204 choose hyperoside at concentrations of 0-175 μM for further optimization. The result revealed 205 that hyperoside improved the survival rate of HL-1 cells within a specific range from 50 μM to 206 100 μM; however, hyperoside treatment did not significantly increase cell viability with the 207 further increases in its concentrations (Fig.1C). Thus, 100 μM was selected as the optimal 208 concentration of hyperoside for subsequent experiments in this study. Manuscript to be reviewed Manuscript to be reviewed 200 μM (Fig.1D and E). Therefore, we selected 1 μM as the ideal concentration of DOX to induce 201 HL-1 cell damage. Further, we treated HL-1 cells with hyperoside at concentrations of 0−500 202 μM, and the CCK-8 result revealed that hyperoside at concentrations equal to or higher than 200 203 μM could cause significant changes in the survival rates of these HL-1 cells (Fig.1B). Thus, we 204 choose hyperoside at concentrations of 0-175 μM for further optimization. The result revealed 205 that hyperoside improved the survival rate of HL-1 cells within a specific range from 50 μM to 206 100 μM; however, hyperoside treatment did not significantly increase cell viability with the 207 further increases in its concentrations (Fig.1C). Thus, 100 μM was selected as the optimal 208 concentration of hyperoside for subsequent experiments in this study. 209 210 3.2 Hyperoside diminished DOX-induced oxidative stress damage in HL-1 cells 211 To explore the potential mechanism of cardiomyocyte protection associated with hyperoside, 212 markers related to oxidative stress injury were examined. DCFH-DA fluorescent probe was used 213 to measure expression level of ROS, and the results suggested that hyperoside significantly 214 inhibited DOX-induced ROS production in HL-1 cardiomyocytes (Fig. 2A, B). Further, The 215 results of biochemical assays showed that DOX remarkably decreased the activity of antioxidant 216 enzymes (GSH, CAT, SOD) in HL-1 cells, which rebounded after hyperoside pretreatment (Fig. 217 2C, D and E); MDA was overproduced in the DOX group and diminished in the DOX + 218 hyperoside group (Fig. 2F). The above outcomes revealed that hyperoside can reduce ROS 219 production, increase the activity of antioxidant enzymes, and consequently attenuate DOX- 220 induced oxidative stress damage in HL-1 cardiomyocytes. 221 222 3.3 Hyperoside attenuated DOX-induced apoptosis in HL-1 cells 223 DOX-induced apoptosis was visualized by the TUNEL staining of HL-1 cells. The 224 fluorescence microscopic images and data analysis revealed that the number of TUNEL-positive 225 cells expressing the green tunneling magnetoresistance signal was markedly increased in the 226 DOX group versus the control group. The results revealed a remarkable increase in apoptosis. 227 Whether hyperoside treatment attenuated DOX-induced apoptosis was further explored. The 228 results revealed that hyperoside treatment significantly reduced DOX-induced apoptosis in HL-1 229 cardiomyocytes, which was indicated by a remarkable decrease in TUNEL-positive cells (Fig.3A 230 and B). IF staining was used to detect cleaved caspase-3. Manuscript to be reviewed 240 following DOX treatment whereas hyperoside treatment reversed the effect (Fig.4C and D). 241 Meanwhile, the administration of hyperoside significantly inhibited DOX-induced high 242 expression of the cleaved caspase-3 protein (Fig.4C and E). In addition, level of p53 was 243 markedly increased in DOX-treated HL-1 cardiomyocytes but falled back after hyperoside 244 pretreatment (Fig. 4C and F). 245 246 3.5 Hyperoside inhibited ASK1/p38 signaling pathway in DOX-treated HL-1 cells 247 To explore the possible signaling pathways involved in the above apoptosis pathway. We 248 explored the ASK1/p38 signaling pathway and the role of hyperoside in regulating this pathway. 249 According to WB analyses, the phosphorylation levels of ASK1 and p38 were considerably 250 elevated following DOX treatment; however, the administration of hyperoside markedly 251 reversed the effect of this treatment (Fig. 5A-C). 252 253 3.6 Hyperoside promotes the anticancer property of DOX 254 Human breast cancer MDA-MB-231 cell was used to detect the impact of hyperoside on 255 DOX-treated cancer cells. As the CCK-8 result revealed that DOX can cause dose-dependent 256 damage to MDA-MB-231 cells, the higher the concentration of DOX, the worse the cell viability 257 (Figure 6A). Of interest, hyperoside displayed a synergistic cytotoxic response to DOX when 258 treated with MDA-MB-231 cells (Figure 6B). Further, TUNEL staining results showed that 259 hyperoside accentuated DOX-induced apoptosis in MDA-MB-231 cells (Figure 6C, D). The 260 findings above suggested that hyperoside alleviated DIC while promoting the cytotoxicity of 261 DOX on cancer cells. 262 263 Discussion 264 In our study, we identified the importance of hyperoside in inhibiting DOX-induced HL-1 265 cell apoptosis by restraining the activation of the ASK1/p38 signaling pathway. Doxorubicin is a 266 commonly used clinical antitumor drug. However, due to its low selectivity of therapeutic 267 targets, DOX can cause toxic side effects on liver (Al-Qahtani WH et al., 2022), kidney (Wu Q et 268 al., 2023), nerve (Orabi MAA et al., 2021), etc. in patients while treating tumors, the most 269 serious of which is cardiotoxicity (Chen Y, Shi S & Dai Y, 2022). DOX can cause acute, 270 subacute, and chronic irreversible cardiotoxicity, eventually leading to heart failure (Sheibani M 271 et al., 2022). After stimulation with 1 μM DOX for 24 h, we found decreased survival rate and 272 increased apoptosis of HL-1 cells, indicating that DOX induced cardiotoxicity. Zhang et al. 3.3 Hyperoside attenuated DOX-induced apoptosis in HL-1 cells 222 3.3 Hyperoside attenuated DOX-induced apoptosis in HL-1 cells 223 DOX-induced apoptosis was visualized by the TUNEL staining of HL-1 cells. The 224 fluorescence microscopic images and data analysis revealed that the number of TUNEL-positive 225 cells expressing the green tunneling magnetoresistance signal was markedly increased in the 226 DOX group versus the control group. The results revealed a remarkable increase in apoptosis. 227 Whether hyperoside treatment attenuated DOX-induced apoptosis was further explored. The 228 results revealed that hyperoside treatment significantly reduced DOX-induced apoptosis in HL-1 229 cardiomyocytes, which was indicated by a remarkable decrease in TUNEL-positive cells (Fig.3A 230 and B). IF staining was used to detect cleaved caspase-3. The results revealed that cleaved 231 caspase-3 expression was markedly increased following DOX treatment, and the effect was 232 attenuated by hyperoside treatment (Fig.3C and D). 234 3.4 Hyperoside down-regulated apoptosis-related proteins in DOX-treated HL-1 cells 235 To investigate mechanisms of apoptosis following DOX treatment, qRT-PCR was used to 236 identify Bax/Bcl-2 and cleaved caspase-3 mRNA levels of cells in each group. The results 237 confirmed that hyperoside administration significantly reduced DOX-induced increased levels of 238 Bax/Bcl-2 and cleaved caspase-3 (Fig. 4A and B). Further, we examined the levels of apoptosis- 239 related proteins, and the WB results revealed significantly increased levels of Bax/Bcl-2 PeerJ reviewing PDF \| (2022:10:78807:1:0:CHECK 26 Mar 2023) Manuscript to be reviewed 273 reported that hyperoside alleviated LPS-induced cardiomyocyte death (Zhang J, Liu Y & Liu L, 274 2021), and similarly in the present study, survival rate and apoptosis rate of HL-1 cells was 240 following DOX treatment whereas hyperoside treatment reversed the effect 240 following DOX treatment whereas hyperoside treatment reversed the effect (Fig.4C and D). 241 Meanwhile, the administration of hyperoside significantly inhibited DOX-induced high 242 expression of the cleaved caspase-3 protein (Fig.4C and E). In addition, level of p53 was 243 markedly increased in DOX-treated HL-1 cardiomyocytes but falled back after hyperoside 244 pretreatment (Fig. 4C and F). 245 246 3.5 Hyperoside inhibited ASK1/p38 signaling pathway in DOX-treated HL-1 cells 247 To explore the possible signaling pathways involved in the above apoptosis pathway. We 248 explored the ASK1/p38 signaling pathway and the role of hyperoside in regulating this pathway. 249 According to WB analyses, the phosphorylation levels of ASK1 and p38 were considerably 250 elevated following DOX treatment; however, the administration of hyperoside markedly 251 reversed the effect of this treatment (Fig. 5A-C). 252 253 3.6 Hyperoside promotes the anticancer property of DOX 254 Human breast cancer MDA-MB-231 cell was used to detect the impact of hyperoside on 255 DOX-treated cancer cells. As the CCK-8 result revealed that DOX can cause dose-dependent 256 damage to MDA-MB-231 cells, the higher the concentration of DOX, the worse the cell viability 257 (Figure 6A). Of interest, hyperoside displayed a synergistic cytotoxic response to DOX when 258 treated with MDA-MB-231 cells (Figure 6B). Further, TUNEL staining results showed that 259 hyperoside accentuated DOX-induced apoptosis in MDA-MB-231 cells (Figure 6C, D). The 260 findings above suggested that hyperoside alleviated DIC while promoting the cytotoxicity of 261 DOX on cancer cells. 262 263 Discussion 264 In our study, we identified the importance of hyperoside in inhibiting DOX-induced HL-1 265 cell apoptosis by restraining the activation of the ASK1/p38 signaling pathway. Doxorubicin is a 266 commonly used clinical antitumor drug. However, due to its low selectivity of therapeutic 267 targets, DOX can cause toxic side effects on liver (Al-Qahtani WH et al., 2022), kidney (Wu Q et 268 al., 2023), nerve (Orabi MAA et al., 2021), etc. in patients while treating tumors, the most 269 serious of which is cardiotoxicity (Chen Y, Shi S & Dai Y, 2022). Manuscript to be reviewed DOX can cause acute, 270 subacute, and chronic irreversible cardiotoxicity, eventually leading to heart failure (Sheibani M 271 et al., 2022). After stimulation with 1 μM DOX for 24 h, we found decreased survival rate and 272 increased apoptosis of HL-1 cells, indicating that DOX induced cardiotoxicity. Zhang et al. 273 reported that hyperoside alleviated LPS-induced cardiomyocyte death (Zhang J, Liu Y & Liu L, 274 2021), and similarly in the present study, survival rate and apoptosis rate of HL-1 cells was 275 decreased after hyperoside pretreatment, tentatively demonstrating its cardioprotective effect. 276 Former reports have revealed that DOX is converted to semi-quinone DOX (SQ-DOX) by 277 the action of uncoupled nitric oxide synthase, NADPH oxidase and etc. SQ-DOX readily reduces 278 oxygen molecules (O2) to superoxide anions (O2-), which are further transformed to hydrogen 279 peroxide (H2O2) with the assistance of SOD (Octavia Y et al., 2012). H2O2 can generate highly 240 following DOX treatment whereas hyperoside treatment reversed the effect (Fig.4C and D). 241 Meanwhile, the administration of hyperoside significantly inhibited DOX-induced high 242 expression of the cleaved caspase-3 protein (Fig.4C and E). In addition, level of p53 was 243 markedly increased in DOX-treated HL-1 cardiomyocytes but falled back after hyperoside 244 pretreatment (Fig. 4C and F). 241 Meanwhile, the administration of hyperoside significantly inhibited DOX-induced high 242 expression of the cleaved caspase-3 protein (Fig.4C and E). In addition, level of p53 was 243 markedly increased in DOX-treated HL-1 cardiomyocytes but falled back after hyperoside 244 pretreatment (Fig. 4C and F). 246 3.5 Hyperoside inhibited ASK1/p38 signaling pathway in DOX-treated HL-1 cells 247 To explore the possible signaling pathways involved in the above apoptosis pathway. We 248 explored the ASK1/p38 signaling pathway and the role of hyperoside in regulating this pathway. 249 According to WB analyses, the phosphorylation levels of ASK1 and p38 were considerably 250 elevated following DOX treatment; however, the administration of hyperoside markedly 251 reversed the effect of this treatment (Fig. 5A-C). Manuscript to be reviewed 280 reactive hydroxyl radicals (OH) catalyzed by ferrous ions (Kajarabille N & Latunde-Dada GO, 281 2019). Moreover, the aforementioned ROS will oxidize with DNA, RNA, proteins, and lipids in 282 cells, damaging the subcellular structure and causing apoptosis and damage to cardiomyocytes. 283 SOD, CAT and GSH are important endogenous antioxidant enzymes that reflect the antioxidant 284 ability of an organism (Jing M et al., 2020; Xiang Q et al., 2022). MDA is the final product, also 285 an indirect biomark, of lipid peroxidation damage (Zhang J et al.,2011). Previous studies have 286 reported that DOX-treated cardiomyocytes produce excessive ROS, accumulate MDA, and cause 287 a decrease in endogenous cardiac antioxidant enzymes (Wang X et al., 2022; Pharoah BM et al., 288 2023; Sun L et al., 2022). Thus, excessive production of ROS and decayed activity of antioxidant 289 enzymes are essential pathological characteristics of DIC. Similar to these findings, we detected 290 a large production of ROS, a significant increase in MDA content, and a downregulation of 291 SOD, GSH, and CAT in DOX-treated HL-1 cells, indicating that DOX caused oxidative stress 292 injury in cardiomyocytes. Interestingly, the changes in ROS and MDA were attenuated after 293 hyperoside pretreatment, while the levels of SOD, GSH, and CAT also rebounded. It is 294 suggested that hyperoside enhanced the antioxidant capacity of cardiomyocytes, thus alleviating 295 DOX-induced cardiotoxicity. He et al. found that hyperoside down-regulated MDA levels and 296 increased SOD activity in an in vivo hypoxia model in mice (He S et al., 2021). Shah et al. found 297 that DOX treatment attenuated GSH and SOD activities and up-regulated lipid peroxidative 298 value level in in vivo experiments of rats. And hyperoside markedly reversed these changes and 299 enhanced the antioxidant capacity of rat hearts (Shah S et al., 2013). These studies demonstrated 300 that hyperoside also has significant antioxidant property in vivo in animals. yp g p p y 301 Myocardial apoptosis is a crucial mechanism of DIC. The Bcl-2 family plays a significant 302 role in caspase activation and apoptosis (Ma W et al., 2020; Childs AC et al., 2002). There are 303 two categories of Bcl-2 family: anti-apoptotic proteins Bcl-2, Bcl-xL, etc. and pro-apoptotic 304 proteins Bax, Noxa, etc (Rosa N et al., 2022). Manuscript to be reviewed As an important marker of apoptosis, caspase-3 is 305 identified to be a significant regulatory molecule of the apoptosis pathway (Li L, Wang S & Zhou 306 W, 2022). Different stimulation signals initiate apoptosis via various pathways, however, the 307 ultimate common effect is to activate caspase-3. DOX administration causes excessive oxidative 308 stress and further activates the intrinsic apoptotic process, by upregulating Bax, caspase-3 and 309 downregulating Bcl-2, eventually causing cardiomyocyte death (Wenningmann N et al., 2019; Li 310 P et al., 2021). More specifically, our study discovered that DOX treatment can increase 311 expression of cleaved caspase-3 and Bax, while decreasing expression of Bcl-2, and these 312 changes can be reversed by hyperoside. The result suggests that hyperoside can reduce 313 cardiomyocyte apoptosis, further suggesting a protective property of hyperoside on DIC. Xiao et 314 al. found that hyperoside decreased the expression of Bax, cleaved caspase-3, and upregulated 315 the level of Bcl-2 in a hypoxia/reoxygenation model of rat cardiomyocytes, demonstrating its 316 anti-apoptotic property, which is in accordance with our findings (Xiao R et al., 2017). A 317 previous research has reported that DOX can upregulate tumor suppressor gene p53 level, which 318 can regulate Bcl-2 family proteins (e.g., Noxa, Bax) (Yu J & Zhang L, 2005), change the 319 Bax/Bcl-2 ratio, causing mitochondria to permeabilize and release cytochrome c and caspase, 301 Myocardial apoptosis is a crucial mechanism of DIC. The Bcl-2 family plays a significant 302 role in caspase activation and apoptosis (Ma W et al., 2020; Childs AC et al., 2002). There are 303 two categories of Bcl-2 family: anti-apoptotic proteins Bcl-2, Bcl-xL, etc. and pro-apoptotic 304 proteins Bax, Noxa, etc (Rosa N et al., 2022). As an important marker of apoptosis, caspase-3 is 305 identified to be a significant regulatory molecule of the apoptosis pathway (Li L, Wang S & Zhou 306 W, 2022). Different stimulation signals initiate apoptosis via various pathways, however, the 307 ultimate common effect is to activate caspase-3. DOX administration causes excessive oxidative 308 stress and further activates the intrinsic apoptotic process, by upregulating Bax, caspase-3 and 309 downregulating Bcl-2, eventually causing cardiomyocyte death (Wenningmann N et al., 2019; Li 310 P et al., 2021). More specifically, our study discovered that DOX treatment can increase 311 expression of cleaved caspase-3 and Bax, while decreasing expression of Bcl-2, and these 312 changes can be reversed by hyperoside. 253 3.6 Hyperoside promotes the anticancer property of DOX 264 In our study, we identified the importance of hyperoside in inhibiting DOX-induced HL-1 265 cell apoptosis by restraining the activation of the ASK1/p38 signaling pathway. Doxorubicin is a 266 commonly used clinical antitumor drug. However, due to its low selectivity of therapeutic 267 targets, DOX can cause toxic side effects on liver (Al-Qahtani WH et al., 2022), kidney (Wu Q et 268 al., 2023), nerve (Orabi MAA et al., 2021), etc. in patients while treating tumors, the most 269 serious of which is cardiotoxicity (Chen Y, Shi S & Dai Y, 2022). DOX can cause acute, 270 subacute, and chronic irreversible cardiotoxicity, eventually leading to heart failure (Sheibani M 271 et al., 2022). After stimulation with 1 μM DOX for 24 h, we found decreased survival rate and 272 increased apoptosis of HL-1 cells, indicating that DOX induced cardiotoxicity. Zhang et al. 273 reported that hyperoside alleviated LPS-induced cardiomyocyte death (Zhang J, Liu Y & Liu L, 274 2021), and similarly in the present study, survival rate and apoptosis rate of HL-1 cells was 275 decreased after hyperoside pretreatment, tentatively demonstrating its cardioprotective effect. 276 Former reports have revealed that DOX is converted to semi-quinone DOX (SQ-DOX) by 277 the action of uncoupled nitric oxide synthase, NADPH oxidase and etc. SQ-DOX readily reduces 278 oxygen molecules (O2) to superoxide anions (O2-), which are further transformed to hydrogen 279 peroxide (H2O2) with the assistance of SOD (Octavia Y et al., 2012). H2O2 can generate highly PeerJ reviewing PDF \| (2022:10:78807:1:0:CHECK 26 Mar 2023) Manuscript to be reviewed The result suggests that hyperoside can reduce 313 cardiomyocyte apoptosis, further suggesting a protective property of hyperoside on DIC. Xiao et 314 al. found that hyperoside decreased the expression of Bax, cleaved caspase-3, and upregulated 315 the level of Bcl-2 in a hypoxia/reoxygenation model of rat cardiomyocytes, demonstrating its 316 anti-apoptotic property, which is in accordance with our findings (Xiao R et al., 2017). A 317 previous research has reported that DOX can upregulate tumor suppressor gene p53 level, which 318 can regulate Bcl-2 family proteins (e.g., Noxa, Bax) (Yu J & Zhang L, 2005), change the 319 Bax/Bcl-2 ratio, causing mitochondria to permeabilize and release cytochrome c and caspase, PeerJ reviewing PDF \| (2022:10:78807:1:0:CHECK 26 Mar 2023) Manuscript to be reviewed 320 leading to cardiomyocyte apoptosis (Zhang C et al., 2011; Zhang S et al., 2012). Consistent with 321 the above literatures, we determined that DOX treatment can increase expression of p53, and 322 hyperoside can significantly reversed the change. 320 leading to cardiomyocyte apoptosis (Zhang C et al., 2011; Zhang S et al., 2012). Consistent with 321 the above literatures, we determined that DOX treatment can increase expression of p53, and 322 hyperoside can significantly reversed the change. 320 leading to cardiomyocyte apoptosis (Zhang C et al., 2011; Zhang S et al., 2012). Consistent with 321 the above literatures, we determined that DOX treatment can increase expression of p53, and 322 hyperoside can significantly reversed the change. 323 Mitogen activated protein kinase (MAPK) is considered to be a crucial target molecule that 324 induce apoptosis (Das J et al., 2011). The MAPK pathway has 32 main branching pathways, of 325 which the p38 MAPK cascade is mainly engaged in the response of cells to stress (Keshet Y & 326 Seger R, 2010). Numerous studies have validated the close relationship between the p38 MAPK 327 cascade and DIC. Activated p38 can induce cardiomyocyte apoptosis by activating the 328 downstream molecules Bax, Bcl-2, and p53 (Thandavarayan RA et al., 2010; Guo R et al., 2013; 329 Spallarossa P et al., 2006). ASK1, a key regulatory factor in the MAPK cascade pathway, is 330 sensitive to oxidation and reduction. It can be activated under oxidative stress conditions and 331 then triggers apoptosis via phosphorylation of JNK and P38 (Jalmi SK & Sinha AK, 2015; Dai 332 WL et al., 2020). Jiang et al. have demonstrated that DOX can promote p38 phosphorylation 333 levels by activating ASK1 signaling pathway, leading to cardiomyocyte apoptosis (Jiang L et al., 334 2020). Several reports have validated that the inhibition of the ASK1/p38 signaling pathway 335 alleviated the pathological damage caused by apoptosis (Li H et al., 2022; Wu Y et al., 2018). In 336 this study, hyperoside dramatically reduced dox-induced phosphorylation of ASK1 and p38, in 337 keeping with the above reports. The result suggested that hyperoside is a regulator engaged in the 338 negative control of ASK1/p38 activation and that DOX-induced myocardial apoptosis may be 339 related to ASK1/p38. , p p , 322 hyperoside can significantly reversed the change. Manuscript to be reviewed 323 Mitogen activated protein kinase (MAPK) is considered to be a crucial target molecule that 324 induce apoptosis (Das J et al., 2011). The MAPK pathway has 32 main branching pathways, of 325 which the p38 MAPK cascade is mainly engaged in the response of cells to stress (Keshet Y & 326 Seger R, 2010). Numerous studies have validated the close relationship between the p38 MAPK 327 cascade and DIC. Activated p38 can induce cardiomyocyte apoptosis by activating the 328 downstream molecules Bax, Bcl-2, and p53 (Thandavarayan RA et al., 2010; Guo R et al., 2013; 329 Spallarossa P et al., 2006). ASK1, a key regulatory factor in the MAPK cascade pathway, is 330 sensitive to oxidation and reduction. It can be activated under oxidative stress conditions and 331 then triggers apoptosis via phosphorylation of JNK and P38 (Jalmi SK & Sinha AK, 2015; Dai 332 WL et al., 2020). Jiang et al. have demonstrated that DOX can promote p38 phosphorylation 333 levels by activating ASK1 signaling pathway, leading to cardiomyocyte apoptosis (Jiang L et al., 334 2020). Several reports have validated that the inhibition of the ASK1/p38 signaling pathway 335 alleviated the pathological damage caused by apoptosis (Li H et al., 2022; Wu Y et al., 2018). In 336 this study, hyperoside dramatically reduced dox-induced phosphorylation of ASK1 and p38, in 337 keeping with the above reports. The result suggested that hyperoside is a regulator engaged in the 338 negative control of ASK1/p38 activation and that DOX-induced myocardial apoptosis may be 339 related to ASK1/p38. 340 Hyperoside is a kind of flavonol glycosides with antitumor property. On lung cancer cells in 341 vitro, hyperoside downregulated Bcl-2 level, upregulated Bax level, and increased the expression 342 of anti-tumor factors such as p53. In addition, in vivo experiments in mice have also 343 demonstrated its inhibitory effect on tumor angiogenesis (Liu YH et al., 2016). Fu et al. revealed 344 that hyperoside exerted anti-cancer function by inducing autophagy in lung cancer cells. And 345 further, this might be related to the inhibition of Akt/mTOR/p70S6K signaling pathway and the 346 activation of ERK1/2 pathway (Fu T et al., 2016). Also in breast cancer, hyperoside inhibited the 347 activity and migratory capacity of breast cancer cells, and activated mitochondrial apoptosis 348 pathway by inhibiting ROS/NF-κB signaling pathway, which in turn induced breast cancer cell 349 apoptosis (Qiu J et al., 2019). 372 References 373 [1] Herrmann J. Adverse cardiac effects of cancer therapies: cardiotoxicity and arrhythmia. Nat 374 Rev Cardiol. 2020 Aug;17(8):474-502. doi: 10.1038/s41569-020-0348-1. 373 [1] Herrmann J. Adverse cardiac effects of cancer therapies: cardiotoxicity and arrhythmia. Nat 374 Rev Cardiol 2020 Aug;17(8):474-502 doi: 10 1038/s41569-020-0348-1 373 [1] Herrmann J. Adverse cardiac effects of cancer therapies: cardiotoxicity and arrhythmia. Nat 374 Rev Cardiol. 2020 Aug;17(8):474-502. doi: 10.1038/s41569-020-0348-1. 375 [2] Sawicki KT, Sala V, Prever L, Hirsch E, Ardehali H, Ghigo A. Preventing and Treating 376 Anthracycline Cardiotoxicity: New Insights. Annu Rev Pharmacol Toxicol. 2021 Jan 377 6;61:309-332. doi: 10.1146/annurev-pharmtox-030620-104842. 375 [2] Sawicki KT, Sala V, Prever L, Hirsch E, Ardehali H, Ghigo A. Preventing and Treating 376 Anthracycline Cardiotoxicity: New Insights. Annu Rev Pharmacol Toxicol. 2021 Jan 377 6;61:309-332. doi: 10.1146/annurev-pharmtox-030620-104842. 378 [3] Sangweni NF, Gabuza K, Huisamen B, Mabasa L, van Vuuren D, Johnson R. Molecular 379 insights into the pathophysiology of doxorubicin-induced cardiotoxicity: a graphical 380 representation. Arch Toxicol. 2022 Jun;96(6):1541-1550. doi: 10.1007/s00204-022-03262-w. 381 [4] Swain SM, Whaley FS, Ewer MS. Congestive heart failure in patients treated with 382 doxorubicin: a retrospective analysis of three trials. Cancer. 2003 Jun 1;97(11):2869-79. doi: 383 10.1002/cncr.11407. 384 [5] Wallace KB, Sardão VA, Oliveira PJ. Mitochondrial Determinants of Doxorubicin-Induced 385 Cardiomyopathy. Circ Res. 2020 Mar 27;126(7):926-941. doi: 384 [5] Wallace KB, Sardão VA, Oliveira PJ. Mitochondrial Determinants of Doxorubicin-Induced 385 Cardiomyopathy. Circ Res. 2020 Mar 27;126(7):926-941. doi: 386 10.1161/CIRCRESAHA.119.314681. Epub 2020 Mar 26. 386 10.1161/CIRCRESAHA.119.314681. Epub 2020 Mar 26. 387 [6] Wenningmann N, Knapp M, Ande A, Vaidya TR, Ait-Oudhia S. Insights into Doxorubicin- 388 induced Cardiotoxicity: Molecular Mechanisms, Preventive Strategies, and Early 389 Monitoring Mol Pharmacol 2019 Aug;96(2):219-232 doi: 10 1124/mol 119 115725 387 [6] Wenningmann N, Knapp M, Ande A, Vaidya TR, Ait-Oudhia S. Insights into Doxorubicin- 388 induced Cardiotoxicity: Molecular Mechanisms, Preventive Strategies, and Early 389 Monitoring. Mol Pharmacol. 2019 Aug;96(2):219-232. doi: 10.1124/mol.119.115725. 390 [7] Jiang L, Gong Y, Hu Y, You Y, Wang J, Zhang Z, Wei Z, Tang C. Peroxiredoxin-1 391 Overexpression Attenuates Doxorubicin-Induced Cardiotoxicity by Inhibiting Oxidative 392 Stress and Cardiomyocyte Apoptosis. Oxid Med Cell Longev. 2020 Jul 29;2020:2405135. 393 doi: 10.1155/2020/2405135. 390 [7] Jiang L, Gong Y, Hu Y, You Y, Wang J, Zhang Z, Wei Z, Tang C. Peroxiredoxin-1 391 Overexpression Attenuates Doxorubicin-Induced Cardiotoxicity by Inhibiting Oxidative 392 Stress and Cardiomyocyte Apoptosis. Oxid Med Cell Longev. 2020 Jul 29;2020:2405135. 393 doi: 10.1155/2020/2405135. Manuscript to be reviewed Furthermore, in paclitaxol-treated MDA-MB-231 cells, hyperoside 350 inhibited cellr activity, facilitated apoptosis. And it also enhanced the sensitivity of MDA-MB- 351 231 cells to paclitaxel via blocking TLR4 signaling. Interestingly, meanwhile, hyperoside 352 inhibited paclitaxel-induced cytotoxicity in human normal mammary epithelial cells MCF-10A 353 (Sun T et al., 2020). Our research confirmed that hyperoside synergized with DOX to inhibit 354 survival rate and induce apoptosis of MDA-MB-231 cells, but the specific mechanism needs 355 further research. 356 p 340 Hyperoside is a kind of flavonol glycosides with antitumor property. On lung cancer cells in 341 vitro, hyperoside downregulated Bcl-2 level, upregulated Bax level, and increased the expression 342 of anti-tumor factors such as p53. In addition, in vivo experiments in mice have also 343 demonstrated its inhibitory effect on tumor angiogenesis (Liu YH et al., 2016). Fu et al. revealed 344 that hyperoside exerted anti-cancer function by inducing autophagy in lung cancer cells. And 345 further, this might be related to the inhibition of Akt/mTOR/p70S6K signaling pathway and the 346 activation of ERK1/2 pathway (Fu T et al., 2016). Also in breast cancer, hyperoside inhibited the 347 activity and migratory capacity of breast cancer cells, and activated mitochondrial apoptosis 348 pathway by inhibiting ROS/NF-κB signaling pathway, which in turn induced breast cancer cell 349 apoptosis (Qiu J et al., 2019). Furthermore, in paclitaxol-treated MDA-MB-231 cells, hyperoside 350 inhibited cellr activity, facilitated apoptosis. And it also enhanced the sensitivity of MDA-MB- 351 231 cells to paclitaxel via blocking TLR4 signaling. Interestingly, meanwhile, hyperoside 352 inhibited paclitaxel-induced cytotoxicity in human normal mammary epithelial cells MCF-10A 353 (Sun T et al., 2020). Our research confirmed that hyperoside synergized with DOX to inhibit 354 survival rate and induce apoptosis of MDA-MB-231 cells, but the specific mechanism needs 355 further research. 358 Our study suggests that hyperoside may reduce DOX-induced cardiomyocyte damage via 359 inhibition of the ASK1/p38 signaling pathway, while raising the sensitivity of cancer cells to PeerJ reviewing PDF \| (2022:10:78807:1:0:CHECK 26 Mar 2023) Manuscript to be reviewed Manuscript to be reviewed 360 DOX. These findings revealed the significant value for the attenuation of DOX toxicity in 361 normal cells and the synergistic effect of hyperoside in tumor cells. Due to these properties, 362 hyperoside may be a potential candidate for alleviating cardiotoxicity during DOX therapy. Our 363 study showed a protective role of hyperoside on myocardium and provided insight into how 364 hyperoside regulates cardiomyocyte apoptosis of DIC. However, hyperoside needs to be 365 comprehensively evaluated in DIC animal models. 366 367 Acknowledgments 368 We are grateful for the technical support from the Central Lab of Taizhou People’s Hospital, 369 China. We would also like to thank TopEdit (www.topeditsci.com) for its linguistic assistance 370 during the preparation of this manuscript. 371 372 References 373 [1] Herrmann J. Adverse cardiac effects of cancer therapies: cardiotoxicity and arrhythmia. Nat 374 Rev Cardiol. 2020 Aug;17(8):474-502. doi: 10.1038/s41569-020-0348-1. 375 [2] Sawicki KT, Sala V, Prever L, Hirsch E, Ardehali H, Ghigo A. Preventing and Treating 376 Anthracycline Cardiotoxicity: New Insights. Annu Rev Pharmacol Toxicol. 2021 Jan 377 6;61:309-332. doi: 10.1146/annurev-pharmtox-030620-104842. 378 [3] Sangweni NF, Gabuza K, Huisamen B, Mabasa L, van Vuuren D, Johnson R. Molecular 379 insights into the pathophysiology of doxorubicin-induced cardiotoxicity: a graphical 380 representation. Arch Toxicol. 2022 Jun;96(6):1541-1550. doi: 10.1007/s00204-022-03262-w. 381 [4] Swain SM, Whaley FS, Ewer MS. Congestive heart failure in patients treated with 382 doxorubicin: a retrospective analysis of three trials. Cancer. 2003 Jun 1;97(11):2869-79. doi: 383 10.1002/cncr.11407. 384 [5] Wallace KB, Sardão VA, Oliveira PJ. Mitochondrial Determinants of Doxorubicin-Induced 385 Cardiomyopathy. Circ Res. 2020 Mar 27;126(7):926-941. doi: 386 10.1161/CIRCRESAHA.119.314681. Epub 2020 Mar 26. 387 [6] Wenningmann N, Knapp M, Ande A, Vaidya TR, Ait-Oudhia S. Insights into Doxorubicin- 388 induced Cardiotoxicity: Molecular Mechanisms, Preventive Strategies, and Early 389 Monitoring. Mol Pharmacol. 2019 Aug;96(2):219-232. doi: 10.1124/mol.119.115725. 390 [7] Jiang L, Gong Y, Hu Y, You Y, Wang J, Zhang Z, Wei Z, Tang C. 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PeerJ reviewing PDF \| (2022:10:78807:1:0:CHECK 26 Mar 2023) Manuscript to be reviewed Manuscript to be reviewed Table 1(on next page) Table 1(on next page) Table 1: Primer sequences used in this research. Primer sequences used in this research. PeerJ reviewing PDF \| (2022:10:78807:1:0:CHECK 26 Mar 2023) Manuscript to be reviewed Manuscript to be reviewed Manuscript to be reviewed Manuscript to be reviewed 1 Table 1: 2 Primer sequences used in this research. Gene sequences (5’-3’) Tm (℃) Mus Bax F: AGACAGGGGCCTTTTTGCTAC 54.4 R: AATTCGCCGGAGACACTCG 53.6 Mus Bcl2 F: GCTACCGTCGTGACTTCGC 55.8 R: CCCCACCGAACTCAAAGAA 56.3 Mus cleaved caspase-3 F: CTCGCTCTGGTACGGATGTG 56.5 R: TCCCATAAATGACCCCTTCATCA 53.7 Mus GAPDH F: AGGTCGGTGTGAACGGATTTG 62.6 R: GGGGTCGTTGATGGCAACA 62.6 3 3 3 PeerJ reviewing PDF \| (2022:10:78807:1:0:CHECK 26 Mar 2023) Manuscript to be reviewed Figure 1 Figure 1: Optimal concentration of doxorubicin (DOX) and hyperoside for HL-1 cells. (A) Eﬀects of DOX on the survival rate of HL-1 cells. * P < 0.001 compared with the 0-μM group. (B) Eﬀects of hyperoside on the survival rate of HL-1 cells. * P < 0.001 compared with the 0-μM group. (C) Eﬀects of hyperoside on the survival rate of HL-1 cells induced by DOX. * P < 0.001 compared with the control group; ### P < 0.001 compared with the 0- μM group. (D) Quantitative analysis of cleaved caspase-3 expression represented by bar graph (n = 3). * P < 0.001, ** P < 0.01, * P < 0.05, compared with the 0-μM group. (E) Representative immunoﬂuorescence labeling images for cleaved caspase-3 (red) in HL-1 cells. Data are indicated as mean ± standard error of the mean (SEM) from three independent experiments. PeerJ reviewing PDF \| (2022:10:78807:1:0:CHECK 26 Mar 2023) PeerJ reviewing PDF \| (2022:10:78807:1:0:CHECK 26 Mar 2023) Manuscript to be reviewed Figure 3 Figure 3: Hyperoside attenuated doxorubicin (DOX)-induced apoptosis in HL-1 cells. Figure 2 Figure 2: Hyperoside attenuated doxorubicin (DOX)-induced oxidative stress injury in HL-1 cells. (A) Representative ﬂuorescence labeling images of DCFH-DA ﬂuorescent probe to detect (reactive oxygen species) ROS (green) in HL-1 cells. Scale bars, 50 μm. (B) Quantitative analysis of ROS depicted by bar graph (n = 3). (C-F) Biochemical methods to detect glutathione (GSH), catalase (CAT), superoxide dismutase (SOD), malondialdehyde (MDA) levels in HL-1 cells. (C) Relative activity of GSH depicted by bar graph (n = 3). (D) Relative activity of CAT depicted by bar graph (n = 3). (E) Relative activity of SOD depicted by bar graph (n = 3). (F) Relative expression level of MDA depicted by bar graph (n = 3). * P < 0.001, P < 0.01, * P < 0.05. Data are expressed as mean ± standard error of the mean (SEM) from three independent experiments. PeerJ reviewing PDF \| (2022:10:78807:1:0:CHECK 26 Mar 2023) PeerJ reviewing PDF \| (2022:10:78807:1:0:CHECK 26 Mar 2023) Manuscript to be reviewed PeerJ reviewing PDF \| (2022:10:78807:1:0:CHECK 26 Mar 2023) Figure 3: Hyperoside attenuated doxorubicin (DOX)-induced apoptosis in HL-1 cells. (A) Representative ﬂuorescence labeling images for terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling (TUNEL) (green) in HL-1 cells. Scale bars, 50 μm. (B) Quantitative analysis of TUNEL-positive cells depicted by bar graph (n = 3). (C) Representative immunoﬂuorescence labeling images of cleaved caspase-3 (red) in HL-1 cells. Scale bars, 50 μm. (D) Quantitative analysis of cleaved caspase-3 depicted by bar graph (n = 3). * P < 0.001. Data are expressed as mean ± standard error of the mean (SEM) from three independent experiments. PeerJ reviewing PDF \| (2022:10:78807:1:0:CHECK 26 Mar 2023) Manuscript to be reviewed Manuscript to be reviewed Manuscript to be reviewed PeerJ reviewing PDF \| (2022:10:78807:1:0:CHECK 26 Mar 2023) PeerJ reviewing PDF \| (2022:10:78807:1:0:CHECK 26 Mar 2023) Manuscript to be reviewed Figure 4 Figure 4: Hyperoside decreased the expression levels of apoptosis-related proteins in DOX-treated HL-1 cells. (A) Relative mRNA level of Bcl-2 associated X-protein (Bax)/Bcl-2 in HL-1 cells (n = 3). (B) Relative mRNA level of cleaved caspase-3 in HL-1 cells (n = 3). (C) The expression of cleaved caspase-3, Bcl-2, Bax and p53 in HL-1 cells assessed by western blot analysis. β-actin was used as the control. (D) Quantitative analysis of Bax/Bcl-2 depicted by bar graph (n = 3). (E) Quantitative analysis of cleaved caspase-3 depicted by bar graph (n = 3). (F) Quantitative analysis of p53 depicted by bar graph (n = 3). * P < 0.001, P < 0.01. Data are expressed as mean ± standard error of the mean (SEM) from three independent experiments. PeerJ reviewing PDF \| (2022:10:78807:1:0:CHECK 26 Mar 2023) Manuscript to be reviewed PeerJ reviewing PDF \| (2022:10:78807:1:0:CHECK 26 Mar 2023) Manuscript to be reviewed Figure 6 Figure 6: Hyperoside promotes the anticancer property of DOX Figure 5 Figure 5: Hyperoside inhibited apoptosis signal-regulating kinase 1 (ASK1)/p38 signaling pathway in doxorubicin (DOX)-treated HL-1 cells. β-actin was used as the control. (A) The expression of p-ASK1, t-ASK1, p-p38, and t-p38 in HL-1 cells examined by western blotting. (B) Quantitative analysis of p-ASK1/t-ASK1 depicted by bar graph (n = 3). (C) Quantitative analysis of p-p38/t-p38 depicted by bar graph (n = 3). * P < 0.001, ** P < 0.01, * P < 0.05. Data are expressed as mean ± standard error of the mean (SEM) from three independent experiments. PeerJ reviewing PDF \| (2022:10:78807:1:0:CHECK 26 Mar 2023) PeerJ reviewing PDF \| (2022:10:78807:1:0:CHECK 26 Mar 2023) Figure 6: Hyperoside promotes the anticancer property of DOX (A) CCK-8 assayed the eﬀect of DOX on the survival rate of MDA-MB-231 cells. (B) CCK-8 assayed the eﬀects of hyperoside on the survival rate of MDA-MB-231 cells induced by DOX. (C) Representative ﬂuorescence labeling images for terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling (TUNEL) (green) in MDA-MB-231 cells. Scale bars, 50 μm. (D) Quantitative analysis of TUNEL-positive cells depicted by bar graph (n = 3). * P < 0.001, P < 0.01, * P < 0.05. Data are expressed as mean ± standard error of the mean (SEM) from three independent experiments. PeerJ reviewing PDF \| (2022:10:78807:1:0:CHECK 26 Mar 2023) Manuscript to be reviewed PeerJ reviewing PDF \| (2022:10:78807:1:0:CHECK 26 Mar 2023) PeerJ reviewing PDF \| (2022:10:78807:1:0:CHECK 26 Mar 2023)
W2314418043.txt	https://zenodo.org/records/2213508/files/article.pdf	de		Der Name des Spinnwirtels im Akkadischen	Orientalistische Literaturzeitung	1,920	public-domain	1,761	153 Ürientalisüsche Literaturzeitnng 1920 Nr. 7/8. Täkil-ana-ilisu in Bruderschaftsverhältnis ge- zweckt. Dagegen ist eine neue Vermessung langt seien. Eine Legitimierung der beiden des Grundstückes in diesem Fall am Platz. Söhne war demnach nicht zu erweisen; 2. wird I I I 18. In der Lücke zu Beginn der Zeile als Grund gegen die Ansprüche der beiden an- kann mit gutem Sinn ergänzt werden la ba-li-e, geführt, hätten sie das Feld nicht in Besitz damit der vom Käufer gezahlte Preis „nicht genommen. Man wird dies wohl am besten verloren sei". verstehen, wenn die Zeugen argumentieren lässt: V 3. ύ-sa-ad-di-ma wird von King über"Wäre die Legitimierung des Lüsä-[. . . .] und setzt: when he made known his decision. Dies des Nimurta-näsir-[. . . .] durch Täkil-ana-ilisu ist aber sowohl grammatikalisch wie sachlich erfolgt, so hätten sie oder einer von ihnen unmöglich. Richtig müsste es heissen: usedlma. schon nach dem Tode des Sämi das Lehensfeld Auch sachlich stimmt die Uebersetzung nicht, in Besitz nehmen müssen, da doch Täkil-ana- da eine Entscheidung zu treffen nicht Sache ilisu jünger war als sie. Die Ansprüche des des Ahu-däru, sondern des Königs war. Das Kidin-Nimurta werden von den Zeugen eben- W o r t kommt vielmehr von nadü I I I 1: er liess falls nicht anerkannt. Hier heisst es, ummasu die Sache fallen, auf sich beruhen. Eben deslä zukkurat. Der Sinn dieser juristischen Wen- wegen kommt er auch nicht zum Gottesurteil. dung ist uns nicht bekannt. Wenn wir jedoch Auffallend ist, dass das Gottesurteil hier im von der Sache auf ihren Namen schliessen Zivilrechtsstreit Anwendung findet, nicht im dürfen, dann kann lä zukkurat nur den Grund Strafprozess (Mitteilung von Koschaker). angeben, weshalb die Mutter des Kidin-Nimurta das Feld nicht erhalten konnte. Wir haben L 4 (vgl. King pi. X X I V — X X V I I I ; SS. 19 ff.). oben schon gesehen, dass Lehensgüter auf weibI 14. Zur Phrase eklam sadädu vgl. meine liche Erben nicht übergehen konnten. Dem- Ausführungen Z A X X V I I , 1912, 249ff. Freinach konnte die Mutter des Kidin-Nimurta lich könnte auch an das Ziehen der 'Grenze jene Wirtschaft nicht erhalten, weil sie als wie L 11, I 3 gedacht sein«. Weib zur F ü h r u n g eines Lehensgutes „nicht (Schluss folgt.) berufen war". I I 48. Die Redensart res ekli nasü, die Der Marne des Spinnwirteis im Rkkadischen. Von A r t h u r TJngnad. sich auf den Grenzsteinurkunden öfters findet, ist von Hinke, dem King folgt, in dem Sinn Bei den Ausgrabungen in Babylon sind verstanden worden, „den Grenzstein wegnehmen" zahlreiche Spinnwirtel aus Ton und Stein ge(A new Boundary-Stone 197). Allein die von funden worden 1 . Wie die Babylonier selbst ihm vorgebrachten Argumente beweisen nichts. aber diesen Gegenstand bezeichnet haben, ist Denn dass nasü nur von tragbaren Sachen ge- m. W . bisher noch nicht bekannt. Ich möchte braucht werden kann, „emporheben", ist zwar dafür ein W o r t in Anspruch nehmen, f ü r das richtig f ü r die ursprüngliche Bedeutung einer bisher noch keine Erklärung gegeben ist, nämRedensart, nicht aber für die sich daraus ent- lich gäsu (fem.), Plur. gäsü und gäsdtu. Die wickelnde IJebertragung auf bestimmte Ver- Länge des ä ist nicht direkt zu erweisen, aber ; hältnisse. So ist in der Wendung „ p ü t . . . . na ü doch wahrscheinlich. Gegen die vorgeschlagene für jem. bürgen" natürlich von nichts Tragbarem Bedeutung könnte allerdings die Tatsache andie Rede, dagegen heisst sie ursprünglich „die geführt werden, dass als Determinativ in der Hand erheben für jemanden". Die Gleich- Regel isu dient. Doch kann das entweder so stellung von res ekli kasädu mit zittam akälu erklärt werden, dass isu hier wie oft ganz allbeweist keinesfalls, dass mit res ekli der gemein „Gerät" bezeichnet, oder wir müssen Grenzstein gemeint sein müsse. Im Gegenteil annehmen, dass Spinnwirtel tatsächlich vielfach entspricht resu dem synouymen Ausdruck aus Holz hergestellt wurden 2 . F ü r die zuerst kakkadu, der genau sowie das hebr. Ι&'ΝΊ „Summe" gegebene Erklärung spricht der Umstand, dass heissen kann. Die Wendung resu nasü ist voll- A T K 3 14 IV 9 'fga-sit sa sinnipi ή, d. h. „(Holz) ständig parallel mit dem hebr. NIM (Ex 30, Wirtel aus Elfenbein" erwähnt werden. An 12; Nu 1, 2; 26, 2 u. ö.), welches soviel heisst andern Stellen, wo gäsu in ATK aufgeführt wie „die Zahl, Summe aufnehmen, zählen". werden, fehlt das Determinativ, so 25 1 1 6 2 : 1 Somit kann an unserer Stelle mit vollständiger ga-su sa abni . . . 4 „Wirtel aus Stein"; 22 I I 6 1 : Berechtigung übersetzt werden, „das Ausmass selbigen Feldes erhob er". Uebrigens verlangt 1 Vgl. R. K o l d e w e y , Bas wiedererstehende Babylon auch der Sinn dasselbe. Denn es wäre wider- S. 252. 2 sinnig, den Grenzstein vor der Uebergabe des Bei unseren Bauernfrauen dienten früher sogar als Spinnwirtel Feldes an seinen Besitzer zu entfernen, da der Kartoffeln 3 Bie ΈΙ-Amarna Tafeln, hsg. v. K n u d t z o n . Grenzstein doch den Schutz des Feldes be1 Hier stand wohl noch der Name des Steines. Brought to you by \| New York University Bobst Library Technical Services Authenticated Download Date \| 8/1/15 9:38 PM Orientaiistiache Literaturzeitung 192Ö Nr. 7/8. Die absolute Gleichmässigkeit der TextX ga-su™"' rabati™' sa abnati™" „lOgrosse Wirtel aus Steinen; 25 II 65 ff. ga-su kaspimeS silberne anlage und die stereotypen Formulierungen Wirtel"; 29,82 :1ga-su /twrasii „goldener Wirtel". beweisen, dass die Abfassung solcher Protokolle Sicher aus Holz sind die imRitualtext Um. 311 + — wir dürfen weiter schliessen: die Abwick325 + 3 7 5 l , Z. 5 begegnenden 'fga-sa-a-ti ''sil- lung des amtlichen Schreibwerks überhaupt — li-ba-ni „Wirtel aus Süssholz 2 ", die aber viel- nach bestimmten von der assyrischen Heeresleicht nicht zu praktischen Zwecken dienten; verwaltung angeordneten Regeln zu geschehen es werden damit wohl wirteiförmige Holz- hatte, deren Ziel war, mit möglichster Kürze stückchen gemeint sein. des Ausdrucks, Uebersichtlichkeit der Buchung Das Wort findet sich mehrfach in den und Vollständigkeit aller benötigten Angaben Labartutexten, wo es in der Nachbarschaft von zu verbinden. fSAL =pilakku „Spindel" begegnet; so IV An die Spitze der Protokolle wurde Name R 5 5 , 1, R s . 2 8 : ".ga-su u.pilakku; 5 6 , 5 0 b : und Dienstrang des Offiziers gesetzt, dessen Ab".ga-su ''. du-di-it-tu pilakku; 5 8 I I 4 0 : u. ga-su teilung an der Musterung beteiligt oder inter3 amil se-bir-tu ".pilakku seb-r[uj „zerbrochenerWirtel"; essiert war; so 31,1: mA-me-A-tar rab hanse ama 79-7-8, 81 II 11 (IV R, Add. 11) ist der Zu- oder 132, l . f : mA-me-A-tar rab Jci-sir. Falls sammenhang zerstört: '1 ga-su ".mu-[. . . ] i . nicht etwa die beiden Titel rab hanse (31,1. 32,1) Endlich findet sich das Wort in Verbindung u n d rab kisir (34, a 7. b 2. 3 6 , 1 1 f. 1 3 1 , b 3. mit sipätu „Wolle"; so in dem altbab. Brief 5 . 132, a If b If.) identisch sein sollten, was schwer UM VII94, 27: sim u.ga-sumn sipatim „Be- anzunehmen wäre, liegt nahe zu vermuten, zahlung für die Wollwirtel 6 (habe ich be- dass der rab Μsir dem rab hanse unterstellt kommen)". Auch in derMitgifturkuude R ( = BE war; denn es finden sich wohl die Protokolle VI 1) 84, 24 f. finden wir für die Abteilungen mehrerer rab kisir auf einer II 'f ga-su sa sipätim Tafel vereinigt (so auf Nr. 34. 131. 132), daIII gasu SAG.DU (= kakkadi). gegen erhält der rab hanse jeweils einen geWas mitletzteren gemeint ist, bleibt vorläufig sonderten Bericht (s. Nr. 31. 32). Danach noch dunkel. Indes spricht das Material, aus scheint die erste grössere Einheit von 50 Gedem eine gäsu gefertigt werden kann, wie auch spannen einem rab hanse, sagen wir „Rittdie Beziehung, in der dieser Gegenstand einer- meister", unterstellt gewesen zu sein, unter seits zur Spindel, andrerseits zur Wolle steht, dem mehrere kleinere Abteilungen (kisru, etwa für die vorgeschlagene Bedeutung „Wirtel". zu je 10 Gespannen?) von je einem rab kisir befehligt wurden. Jeder einzelne Musterungsakt wurde in Dokumente des assyrischen Militarismus. 1 3 Zeilen protokolliert; Ζ. 1: Name und DienstVon O t t o S c h r o e d e r . bezeichnung amel narkabti „Wagenführer", Ζ. 2: Dass ein so hoch entwickeltes Heerwesen Heimatsort (Formel: sä a,Y), Z. 3: Zahl und wie das assyrische einen bis ins Einzelne geGattung der gemusterten Tiere, sowie Musteregelten Verwaltungsapparat gehabt haben rungsbefund. Die Entscheidung lautet gewöhnmuss, durfte man a priori vermuten; gleichlich kurz damik „tauglich" oder la damik „unwohl war die Bestätigung, die eine Reihe Tafelbrocken aus Assur bot, eine Ueberraschung, tauglich"; seltener wird die Untauglichkeit bed. h. wegen insofern wir in ihnen einen Einblick in das gründet: in α ini-su la damik, Augenkrankheit untauglich. Zur Erzielung ganz modern anmutende Musterungsgeschäft in Sachen des Pferdeersatzes gewinnen. Es möglichster Kürze wurden sogar die Ideogramme handelt sich um die 1U Tafeln und Bruchstücke, noch abgekürzt: sisu „Pferd" wird durch die ich K A V 7 Nr. 31—38. 131. 132 unter der V angedeutet. — Einige Beispiele für viele: amel narkabti Bezeichnung „Protokolle über Pferdemuste- 1. 3 1 , 2 — 4 : mAm-mu-a, sä "'Ka-ma-ni rungen" veröffentlicht habe. 1 stsu-su damik, 1 la damik m Z i m m e r n , Eitualtafeln No. 67 iS. 184). 2. 3 1 , 1 4 — 1 6 : It-ta-a-da, amel narkabti 1 Ygl. Z i m m e r n bei G e l d e r e n BA 4, 521. sä alSe i-li-ti * So ist wohl zu lesen. 4 sisum"-su la damku In dem Labarturelief Α (Frank, Beschwörungsreliefs m narkabti Tafel I) könnte der rechts vom Kopfe der Labartu in 3. 3 1 , 2 3 — 2 5 : Iii ab, amel der obersten Reibe ganz am Baude dargestellte napfsä al Se i-li-ti artige Gegenstand sehr gut ein Wirtel sein. imeriie-da-ni-u ina ini-sula damik ' Vgl. A. U n g n a d , Altbabylonisehe Briefe aus dem 1 Wo ausnahmsweise die Angaben in 2 Zeilen zuMuseum zu Philadelphia, Stuttgart 1920, S. 62 * So ist gegen meine Fassung auf Grund von Ε 84, sammengedrängt (32, 29f.) oder in 4 Z e i l e n auseinT 24 zu übersetzen. = Keilschrifttexte aus Assur andergerissen (SB, a 2—5) erscheinen, ist der Platzmangel daran Schuld. verschiedenen Inhalts (35. Wies. Yeröff. der DOG.) 1 Brought to you by \| New York University Bobst Library Technical Services Authenticated Download Date \| 8/1/15 9:38 PM
https://openalex.org/W2012619336	https://estudiosamericanos.revistas.csic.es/index.php/estudiosamericanos/article/download/561/564/564	Spanish; Castilian	null	Un ejemplo de mundialización: El movimiento de biombos desde el Pacífico hasta el Atlántico (s. XVII-XVIII)	Anuario de estudios americanos	2,012	cc-by	14,145	Anuario de Estudios Americanos, 69, 1, enero-junio, 31-62, Sevilla (España), 2012 ISSN: 0210-5810. DOI: 10.3989/aeamer.2012.1.01 Anuario de Estudios Americanos, 69, 1, enero-junio, 31-62, Sevilla (España), 2012 ISSN: 0210-5810. DOI: 10.3989/aeamer.2012.1.01 1 Sobre el movimiento de personas, objetos e ideas en la Edad Moderna: Gruzinski, 2010; Russell Wood, 1998. 2 Subrahmanyam, 1993 y 1997, 735-762. 3 Castelló, 1970; Sanabrais, 2006, 69-106; Sebastián, 1992; Curiel, 1999, 9-32. A case of globalization: the circulation of folding screens from the Pacific to the Atlantic (17th-18th centuries) Alberto Baena Zapatero Centro de História de Além-Mar (Universidade Nova de Lisboa), Portugal El artículo analiza la circulación de objetos entre Asia, América y Europa a través de un estudio de caso: la producción y comercio de biombos. El análisis de las redes eco- nómicas asociadas al tráfico de obras de arte supera los límites nacionales o imperiales y nos obliga a romper con los planteamientos tradicionales de centro y periferia. Asimismo, las relaciones personales que implicó el proceso de encomienda de biombos o la difusión de estos objetos a través del movimiento de personas por cuatro continentes, muestran cómo el desarrollo de la economía mundial o la formación de imperios de dimensiones pla- netarias incentivó los intercambios en todos los niveles y dio como resultado el nacimiento de una nueva cultura material de carácter mundial. PALABRAS CLAVE: Biombos; Comercio; Mundialización; Globalización. This article analyzes the circulation of goods between Asia, America, and Europe employing a case-study perspective: the manufacturing and commerce of folding screens. Study of the economic networks related to art trade overcomes national or imperial bounda - ries and calls for a breakdown of conventional centre-periphery schemes. This article there- fore studies personal relationships in the commission of screens and in the diffusion of the finished goods through a survey of the movements of people in four continents. My main aim is to show that the development of a world-economy and the formation of world-scale empires fostered exchanges of all kinds and resulted in a new material culture of a truly global character. KEYWORDS: Folding screens; Commerce; Globalization. 31 ALBERTO BAENA ZAPATERO EL MOVIMIENTO DE BIOMBOS DESDE EL PACÍFICO HASTA EL ATLÁNTICO En cuanto al otro gran virreinato americano, es importante señalar que hay pocos trabajos que traten del mobiliario en Perú durante este periodo y, en aquellos con los que contamos, apenas se encuentra alguna referencia a la existencia de biombos en las casas principales.4 No obstante, a tenor de la abundancia de estas piezas detectada en la investigación realizada en los protocolos notariales de la ciudad de Lima durante el siglo XVIII, conside- ramos de la mayor importancia recuperar su presencia en los ajuares domésticos, ya que amplían considerablemente el marco geográfico que hasta ahora se ha manejado. Además, siendo el estudio artístico de estos objetos de una gran rele- vancia, nos proponemos un examen más detallado de esta fuente desde un enfoque tanto económico, que incluya esta mercancía en el conjunto del tráfico mundial de objetos de lujo, como social, poniendo de manifiesto los motivos del éxito de estos muebles en los salones de tres continentes. En este sentido, los biombos constituyen un buen ejemplo de la necesidad de observar los fenómenos históricos no sólo desde una perspectiva más amplia sino, además, interdisciplinar y transversal. Asimismo, las redes personales que implicó el proceso de encomienda de obras de arte o la difu- sión de los biombos por medio del movimiento mundial de personas, pue- den mostrar cómo la extensión del tráfico comercial o la formación de imperios de dimensiones planetarias incentivaron los intercambios en todos los niveles, dando como resultado el nacimiento de una nueva cultura mate- rial de la que los biombos constituyen una de sus mejores expresiones. q y j p Por último, resulta necesario subrayar las dificultades que implica el estudio del comercio internacional de biombos, ya que su tránsito abarca- ba un amplio espectro geográfico y se trataba de una mercancía minorita- ria en los cargamentos. Normalmente es difícil cuantificar los productos inmersos en el trato transpacífico porque en los registros de mercancías sólo se solían computar los cajones o fardos que se llevaban, sin especifi- car su contenido, y una gran parte de los intercambios se realizaron de manera clandestina y no dejaron ninguna pista. Por lo tanto, la información con la que contamos no nos permite establecer series anuales de exporta- ción de biombos o estudiar la evolución de sus precios. 4 Crespo, 2006 y Germaná, 2008, 189-206. Introducción La conquista de América y el desarrollo de nuevas rutas directas con Asia, abren un campo de estudio que abarca cuatro continentes y las prin- cipales civilizaciones de la Edad Moderna. Las transformaciones que sufrió el mundo durante este periodo nos invitan a preguntarnos hasta qué punto muchos de los fenómenos asociados a la globalización son recientes o si, por el contrario, cuentan con antecedentes que es necesario revisar.1 En este sentido, los biombos suponen la excusa perfecta para indagar en lo que Sanjay Subrahmanyam definió como connected histories, las relaciones históricas que se dieron entre sociedades contemporáneas, y tratar de eva- luar las implicaciones culturales y sociales que propiciaron los nuevos caminos iniciados por portugueses y españoles.2 La importancia de considerar los biombos como un producto más inserto en el comercio internacional resulta evidente si tenemos en cuenta el hecho de que hasta el momento existen pocas investigaciones que se hayan ocupado detenidamente de la producción, transporte y venta de estos muebles, dedicando la mayoría del esfuerzo a los textiles o la porcelana. Algunos trabajos de historiadores norteamericanos y portugueses han exa- minado los biombos dentro del conjunto del arte namban en Japón, sin embargo, se ha prestado poco interés a la producción china de estas piezas destinadas a la exportación. Además, abundan en los catálogos de exposi- ciones análisis particulares sobre los principales ejemplares conservados hasta la fecha, que se limitan a describirlos desde un punto de vista estéti- co (iconografía, estilo, símbolos, materiales…), poniendo poca atención en su significado histórico y desligando de manera artificial el nacimiento de una nueva economía mundial de sus manifestaciones artísticas. Para el caso de Nueva España, tan sólo el libro de Teresa Castelló y Marita Martínez publicado hace cuarenta años o, más recientemente, los estudios de Gustavo Curiel, Santiago Sebastián y Sofía Sanabrais, le con- ceden una consideración pormenorizada a este asunto.3 No obstante, estos autores se fijan fundamentalmente en los biombos de factura mexicana, dejando de lado el tráfico de este tipo de muebles desde su origen en Asia hasta América o Europa. 32 Anu. estud. am., 69, 1, enero-junio, 2012, 31-62. ISSN: 0210-5810. DOI: 10.3989/aeamer.2012.1.01 EL MOVIMIENTO DE BIOMBOS DESDE EL PACÍFICO HASTA EL ATLÁNTICO ALBERTO BAENA ZAPATERO bienes de los territorios implicados en el comercio, que conservan la evi- dencia de estas relaciones. Así, aportaremos datos de diferente naturaleza y cronología con el propósito de demostrar el éxito constante de esta mer- cancía en el tráfico transoceánico, su progresiva inclusión en la producción local americana y su posterior exportación a Europa. 5 Yuste, 1995, 231-264; Chaunu, 1976. EL MOVIMIENTO DE BIOMBOS DESDE EL PACÍFICO HASTA EL ATLÁNTICO No obstante, con- tamos con varias fuentes indirectas que transmiten valiosas informaciones con las que cubrir estas lagunas: crónicas, libros de sobordo, registros de aduanas, los propios biombos conservados y, sobre todo, los inventarios de 33 Anu. estud. am., 69, 1, enero-junio, 2012, 31-62. ISSN: 0210-5810. DOI: 10.3989/aeamer.2012.1.01 El comercio internacional en Manila Durante la Edad Moderna se consolidaron dos rutas que articularon la mayoría del comercio internacional en el Pacífico. La primera de estas vías fue la que iniciaron los portugueses una vez que lograron circunnavegar África y conectar con la India, China y Japón; la segunda fue la que esta- blecieron los castellanos a través del trato entre Manila, México y Sevilla, pero que en la práctica también se extendió desde Nueva España por el resto de América. Si bien existen numerosos testimonios que permiten ras- trear la presencia de muebles en la primera de esas rutas, en esta ocasión nos vamos a referir fundamentalmente al «camino español», menos cono- cido en lo que se refiere a su relación con el conjunto del comercio asiáti- co, en especial el de obras de arte. Dentro del importante volumen de mercancías que cada año llegaban hasta Manila desde los diferentes centros productores de Asia, los biombos sólo supusieron una pequeña parte de un comercio dominado por telas, especias y porcelanas.5 Su transporte en largas distancias fue realizado mediante un sistema relativamente sencillo que facilitaba su protección durante el viaje. Normalmente estas piezas se introducían plegadas dentro de cajones que, en muchas ocasiones, eran elaborados especialmente para este cometido, ya que debían tener un largo mayor del habitual. Podemos hacernos una idea de cómo pudo ser el porte de estos objetos en los barcos europeos gracias a las recomendaciones que hizo Alessandro Valignano a Nunes Rodrigues sobre la forma en que debía transportar los que conduciría como regalo la embajada de japoneses. Sobre este asunto afirmaba que tení- an que ser trasladados en una caja grande que él mismo había llevado para Japón y que adquirió en Cochim, debiendo ser almacenados en un local 34 Anu. estud. am., 69, 1, enero-junio, 2012, 31-62. ISSN: 0210-5810. DOI: 10.3989/aeamer.2012.1.01 EL MOVIMIENTO DE BIOMBOS DESDE EL PACÍFICO HASTA EL ATLÁNTICO donde «la lluvia no entrase y el agua no llegase».6 Así, no es extraño encon- trar a personajes que conservaban estos recipientes para mantener en buen estado sus adquisiciones o alusiones a cajones de biombos en los registros de mercancías de los barcos, algunos incluso decorados con pintura.7 g p Respecto al arribo de biombos a Manila a lo largo de los siglos XVII y XVIII, es posible afirmar que generalmente pudieron hacerlo a través de cuatro tipos de intermediarios: portugueses, españoles, chinos o japoneses. 6 Canepa, 2008, 24. 7 Archivo General de Indias de Sevilla (AGI), Filipinas, 170, 4, 32r. Entre los bienes embar- gados al maestre de campo don Esteban Eguiño el 3 de junio de 1720 aparece «Un cajón pintado con su biobo dentro de papel nuevo». 8 Sección Contaduría de la Real Hacienda de Filipinas, AGI. Datos obtenidos a partir de la investigación realizada en el proyecto «Prosopografía de las comunidades lusófonas residentes e de passagem nas Filipinas (1582-1654)» financiado por Fundaçao para a Ciência e a Tecnologia, Portugal. 9 Chaunu, 1976, 142-169. 6 Canepa, 2008, 24. El comercio internacional en Manila La actividad realizada por los mercaderes lusos como introductores de pro- ductos asiáticos en Filipinas queda demostrada por la llegada al puerto de Manila (Cavite) de navíos con esta bandera desde Macao, Nagasaki e inclu- so de Goa, principales centros portugueses de redistribución en la región. A pesar de que entre las condiciones aceptadas por Felipe II en las Cortes de Tomar, al poco tiempo de proclamarse rey de Portugal, se estableció una separación administrativa y comercial entre los dos imperios, los barcos fletados en los puertos lusos llegaron con asiduidad hasta Cavite para hacer negocio. Entre 1577 y 1643 han quedado registradas 63 embarcaciones cuyo origen era Macao, además de otras siete que arribaron en 1612 y que, aunque se conoce que eran tripuladas por naturales de este reino, se ignora si procedían de Goa o de Macao.8 Para este periodo, además, no fue extra- ño que los barcos portugueses cargados con productos chinos se dirigiesen a Manila desde Nagasaki, consiguiendo así un trato fiscal más favorable al que recibían cuando lo hacían directamente desde Macao. Después de la ruptura de la unión ibérica en 1640, las naves lusas con- tinuaron llegando a Filipinas. Si bien es cierto que la guerra o el clima de hostilidad entre estos dos reinos pudieron haber afectado al comercio des- de Macao, puesto que se registra un parón en la entrada de barcos desde este puerto entre 1643 y 1672, a partir de esta fecha se restablece la rela- ción nuevamente.9 No obstante, la ausencia de barcos comandados por capitanes portugueses durante estos años no debe indicar necesariamente que quedasen al margen del comercio con Manila, ya que pudieron seguir con su actividad de manera clandestina o a través de intermediarios como 35 Anu. estud. am., 69, 1, enero-junio, 2012, 31-62. ISSN: 0210-5810. DOI: 10.3989/aeamer.2012.1.01 ALBERTO BAENA ZAPATERO los chinos, los japoneses o los armenios. Desde finales del siglo XVII, Macao deberá enfrentar la fuerte competencia de Cantón como principal plaza europea en China. Entre los objetos que transportaron en sus bodegas los barcos de ban- dera portuguesa se encontraban los preciados biombos, como demuestran las crónicas que se refieren al envío de este tipo de muebles desde los puer- tos lusos del Pacífico hacia la India y Europa. 10 Fróis, 1984, 313. 11 Álvarez Taladriz, 1953, 36-37. 12 AGI, Filipinas 20, 11, 70, citado en Gil, 2011, 325-326. 12 AGI, Filipinas 20, 11, 70, citado en Gil, 2011, 325-326. Anu. estud. am., 69, 1, enero-junio, 2012, 31-62. ISSN: 0210-5810. DOI: 10.3989/aeamer.2012.1.01 El comercio internacional en Manila El padre Luis Frois hizo alu- sión a los biombos japoneses afirmando que «ya se han mandado algunos a Portugal y a Roma, y van cada año para la India muchos»,10 mientras que el padre Juan Rodríguez Tsuzu, en su Historia de la Iglesia del Japón, comenta sobre los biombos que son «cosa muy acomodada que de acá lle- van para Europa, de que hay muy varios y ricos en el Fuchu».11 Entre el conjunto de productos que pasaban de Macao a Cavite también se encon- tró este tipo de muebles. A inicios del siglo XVII, el gobernador de Filipinas Juan de Silva envió varias veces a Macao a su criado Pedro Angulo con la misión de comprar diferentes objetos de lujo orientales, entre los que se refieren los biombos.12 Muchos de ellos fueron realizados en los talleres locales, como aconteció con el biombo del Diluvio Universal, actualmente en la colección del Museo Soumaya de la ciudad de México, o en talleres situados en otras partes de China. En este sentido, pueden considerarse una prueba de su difusión los que encontramos regis- trados en los inventarios de bienes de distintas ciudades de América o Europa, y a los que nos referiremos más adelante. Estos muebles no serían una excepción, ya que la relación comercial y de encargo de obras de arte a Macao desde Filipinas y Nueva España fue algo habitual. Recordemos que la reja del coro de la catedral de México fue fundida en este centro por- tugués por un artesano chino. Al margen de los portugueses, los castellanos afincados en Manila siempre tuvieron un gran interés por establecer tratos comerciales directa- mente con los productores chinos que les permitiesen el acceso directo al lucrativo intercambio con el continente. Durante las últimas décadas del siglo XVI y primeras del XVII, la diplomacia castellana se esforzó infruc- tuosamente por conseguir un enclave comercial estable en la costa de Fujian, similar al que disfrutaban los portugueses en Macao. Ante el fraca- so de este proyecto, en parte por los esfuerzos diplomáticos lusos, los cas- 36 Anu. estud. am., 69, 1, enero-junio, 2012, 31-62. ISSN: 0210-5810. DOI: 10.3989/aeamer.2012.1.01 EL MOVIMIENTO DE BIOMBOS DESDE EL PACÍFICO HASTA EL ATLÁNTICO tellanos enfocaron su relación comercial en la región de Cantón. El comercio internacional en Manila A partir de finales del siglo XVII y conforme Macao y los portugueses perdían su papel tradicional como intermediarios, esta ciudad fue cobrando importan- cia como centro redistribuidor de productos chinos. Resulta difícil encon- trar fuentes que nos proporcionen un panorama general de las mercancías implicadas en el tráfico entre Cantón y Manila, no obstante, cuando tene- mos la suerte de conservar el testimonio de los productos que transporta- ban los barcos castellanos, vemos que aparecen los biombos entre los cajo- nes de sus bodegas. En el registro y descarga efectuados en 1769 a la chalupa Nuestra Señora del Carmen, que venía desde Cantón a Manila a cargo del capitán don Antonio Pacheco, encontramos «dos cajones largos sin número ni marca que contienen dentro dos beobos de maque blanco cada uno con doce hojas», valorados en 25 pesos cada uno.13 j p Por otra parte, las autoridades permitieron la constante llegada de población china al puerto de Cavite y su instalación permanente en un barrio de las afueras de la ciudad, el Parián, desde el que realizaban toda su actividad económica. El enorme número de sangleyes fue siempre un motivo de recelo por parte de la comunidad española y derivó en conflic- tos violentos. Una parte de esta población era de comerciantes pero otra estaba compuesta por trabajadores de distintos oficios. Por lo tanto, resul- ta difícil dictaminar si todos los biombos chinos que aparecen en los regis- tros de bienes, tanto de México como de España, fueron producidos por artesanos afincados en el continente o si una parte de ellos no serían fabri- cados en los talleres de sangleyes ubicados en Manila. Como ejemplo de este problema, debemos señalar que existen diferentes teorías sobre el ori- gen del antes citado Biombo del Diluvio. A pesar de que tradicionalmente se atribuye su producción a la escuela de Macao, otros autores como LeRoy han apuntado que pudo realizarse en Manila, elaborado por artistas asiáti- cos conversos al servicio de la Corona española.14 Asimismo, contamos con dos testimonios que pueden darnos una pista de la existencia de una manu- factura de biombos en Manila, encuadrada seguramente en la producción de todo tipo de muebles de maque, o por lo menos de la presencia de talle- res donde se remataban las piezas mandadas desde China. 13 AGI, Filipinas, 942, 5, 145v y 187v. 14 LeRoy, 1999, 119-134. Anu. estud. am., 69, 1, enero-junio, 2012, 31-62. ISSN: 0210-5810. DOI: 10.3989/aeamer.2012.1.0 15 AGI, Filipinas, 942, 5, 145v y 187v. 16 AGI, Filipinas, 170, 4, 14v. 17 Castelló y Martínez, 1970, 12. 18 En el caso de los biombos transportados por holandeses podían denominarse como «de Bantam», en referencia al puerto de Java desde el que se exportaban a Europa. p 17 Castelló y Martínez, 1970, 12. El comercio internacional en Manila El primer indi- cio lo encontramos en el cargamento del Nuestra Señora del Carmen, don- de junto a materiales para producir camapes o taburetes de maque, aparece 37 Anu. estud. am., 69, 1, enero-junio, 2012, 31-62. ISSN: 0210-5810. DOI: 10.3989/aeamer.2012.1.01 ALBERTO BAENA ZAPATERO 16 AGI, Filipinas, 170, 4, 14v. 15 AGI, Filipinas, 942, 5, 145v y 187v. ALBERTO BAENA ZAPATERO DOI: 10.3989/aeamer.2012.1.01 EL MOVIMIENTO DE BIOMBOS DESDE EL PACÍFICO HASTA EL ATLÁNTICO mucho más próximos. Este hecho explicaría que los biombos registrados en Manila nunca sean calificados como «de la India» o «de Bantam», sino que siempre se hace alusión a su factura china o japonesa. En cuanto a los biombos producidos en Japón, éstos podían alcanzar Manila a través de los japoneses que se trasladaban a Filipinas para nego- ciar directamente o por medio de los europeos que tenían tratos en Nagasaki. A inicios del siglo XVII, algunos españoles se dirigían hasta Japón para comprar muebles, como sucedió con los que se vendieron en 1613 al inglés John Saris.19 Sin embargo, los que más abundaron en este trá- fico hasta mediados del siglo XVII fueron los japoneses y portugueses. Así lo recogió Antonio de Morga, quien no pudo pasar por alto en su crónica sobre las Filipinas la importancia de estos comerciantes, señalando a los biombos entre las mercancías habituales que se enviaban a Nueva España: De Japón, vienen asimismo cada año del puerto de Nangasaque, con los Nortes de fin de octubre, y por el mes de marzo, algunos navios de mercaderes, japones y portugue- ses, que entran y surgen en Manila, por la misma orden; la gruesa que traen es harina de trigo, muy buena para el abasto de Manila, cecinas estimadas; algunas sedas tejidas de matices, curiosas, biouos al olio y dorados, finos y bien guarnecidos; [...]. En esto, se hacen también algunos empleos, sin que se cobren derechos reales destos navios, y lo mas se gasta en la tierra, y dello sirve para cargazones a la Nueva España.20 Entre 1590 y 1635 anclaron en Manila 58 embarcaciones japonesas cargadas de productos y se calcula que hubo una población permanente de 500 personas de esta nacionalidad, la mayoría comerciantes y cristianos.21 La relación no se vio interrumpida por los enfrentamientos religiosos o el aislamiento del país. Sin embargo, después de 1635 la población japonesa se reduce y se aprecia una entrada mucho menor de sus barcos, siendo que desde esta fecha hasta el año 1700 se registraron tan sólo 13, evidencia de las dificultades impuestas por la nueva coyuntura política.22 A partir de mediados del siglo XVII los holandeses sustituyeron a los portugueses como distribuidores de objetos japoneses. 19 Satow, 1967. 20 Morga, 2007, 289-290. 21 Chaunu, 1976, 142-169. Morga, 2007, 299. En los registros de la Hacienda de Filipinas de finales de la década de 1630 se pueden observar los pagos que hicieron numerosos japoneses para obte- ner una licencia con la que se les permitiese residir en Manila. AGI, Contaduría, 1217, 77v-78r. 22 Chaunu, 1976, 142-169. ALBERTO BAENA ZAPATERO «un cajón con cincuenta y dos hojas de loza de maque colorado para dos beobos», lo que podría indicar que se mandaban materias primas para la fabricación posterior de estos muebles.15 Asimismo, entre las propiedades del maestre de Campo de la ciudad de Manila, don Esteban de Eguiño, se incluyen «doce tablas de maque negro para biobo de estrado» que pudieron ser montadas por algún artesano especializado o reutilizadas en la elabora- ción de otro tipo de muebles como en ocasiones sucedía en Europa.16 Los talleres chinos, dada la creciente demanda occidental de muebles de lujo que se venía desarrollando desde la segunda mitad del siglo XVI, comenzaron a elaborar biombos expresamente para la exportación. Coetáneamente al inicio de la producción en Cantón de porcelana para los mercados europeos, se realizaron un tipo de «biombos de encargo» con temas occidentales sacados de pinturas o grabados europeos que eran inter- pretados a la manera oriental. Se trataba de asuntos y composiciones nue- vos para los artesanos asiáticos, pero que se adaptaban al gusto de sus clientes tanto europeos como americanos. Por lo tanto, estos biombos se integran dentro de un fenómeno más amplio de desarrollo de una produc- ción artesanal china destinada a cubrir las exigencias de la élite de estos dos continentes. Desde finales del XVII se produjo un incremento de la exportación de los biombos chinos llamados erróneamente de Coromandel. Esta denomi- nación deriva de la costa de la India del mismo nombre, sobre el golfo de Bengala, desde donde eran embarcados a Europa, pero la mayoría procedía de las provincias chinas de Fujian, Zhejiang, Jiangsu y Anhui.17 Esta con- fusión explica que la mayoría de los biombos que encontramos en Portugal y muchos de los que aparecen en España sean registrados en las fuentes como «de la India».18 Es de suponer que, al ser un importante centro redis- tribuidor de biombos del norte de China, alguno pudo ser embarcado con destino a Manila, desde donde su paso a Acapulco resultaría muy atracti- vo. No obstante, a tenor de las fuentes es más probable suponer que los biombos que salían de las costas del golfo de Bengala se destinasen funda- mentalmente a Europa, mientras que, para el caso de Filipinas y América, este tipo de muebles chinos llegaría directamente desde Cantón o Macao, 38 Anu. estud. am., 69, 1, enero-junio, 2012, 31-62. ISSN: 0210-5810. 23 Archivo General de la Nación de la ciudad de México (AGN), Civil, 1835, 6 y AGN, Civil, 1863, 19 y también Archivo Histórico de Notarías de la ciudad de México (AHNMéx.), Toribio Cobian, esc. 110, 726, 1655. 24 AGN, Civil, 1327, exp. 1, 18r. Además, Gustavo Curiel, en la lista que publicó sobre biom- bos consignados en documentos notariales de la ciudad de México, cita dos biombos japoneses más: en 1679 un «biobo grande del Japón, todo dorado» y en 1683 otro biombo japonés de ocho hojas con «flo- res y animales», Curiel, 1999, 9-32. 25 AGN, Civil, 114, exp. 1. ALBERTO BAENA ZAPATERO No obstante, la VOC, al margen del contrabando, necesitó de intermediarios autorizados para tratar en Cavite si deseaba introducir sus mercancías en el circuito español. 39 Anu. estud. am., 69, 1, enero-junio, 2012, 31-62. ISSN: 0210-5810. DOI: 10.3989/aeamer.2012.1.01 25 AGN, Civil, 114, exp. 1. ALBERTO BAENA ZAPATERO Si queremos rastrear la presencia de biombos japoneses entre las pro- piedades de los personajes más ricos de Filipinas o Nueva España, nos encontramos con el problema de la falta de interés de los productores de las fuentes conservadas por especificar el origen de los objetos o de hacerlo con rigor geográfico. En muchos de los inventarios de bienes analizados sólo aparece el tamaño y el precio de los biombos, sin más explicación, o se califica libremente a todos los llegados a través del galeón de Manila con el adjetivo genérico de «chinos», lo que impide distinguir los diferentes lugares de fabricación. No obstante, también se encuentran casos donde se especifica su procedencia japonesa, lo que probaría la larga relación comer- cial entre Nueva España y Japón. En los inventarios de bienes de la ciudad de México aparecen consignados varios de estos muebles. En 1626, el mer- cader Pedro de Burgos cuenta con un biombo de Japón «pintado y dorado» que se califica como viejo, lo que indicaría la existencia temprana en el virreinato de este tipo de muebles; a mediados del siglo XVII, Ana María Gomes de la Madriz o la viuda del capitán Tomas Aguirre Hirsnaba tenían cada una un biombo japonés de ocho hojas;23 mientras que en 1733, entre las propiedades de Josefa del Castillo, mujer del capitán José Fernández de Córdoba, se enlista un biombo japonés entre varios de factura novohispa- na.24 Pero quizás el testimonio más evidente de que el vínculo entre Manila y Japón se mantuvo después de que el país se cerrara para los católicos lo hallamos en el registro de propiedades del antiguo gobernador de Filipinas, don Fausto Crusat y Góngora, que en 1706 contaba con once biombos nipones entre sus propiedades.25 En el caso de Perú, merced a la exclusividad de la que gozaba Acapulco en el trato con Filipinas, la mayoría de las mercancías asiáticas que arribaron al Callao lo hicieron a través del comercio irregular con Nueva España. Después de algunos contactos directos a finales del siglo XVI, tendremos que esperar hasta el siglo XVIII para volver a encontrar cargamentos venidos directamente desde Asia. En las dos primeras déca- das de este siglo, los trastornos ocasionados por la guerra de sucesión espa- 40 Anu. estud. am., 69, 1, enero-junio, 2012, 31-62. ISSN: 0210-5810. ALBERTO BAENA ZAPATERO DOI: 10.3989/aeamer.2012.1.01 EL MOVIMIENTO DE BIOMBOS DESDE EL PACÍFICO HASTA EL ATLÁNTICO ñola provocaron que las flotas de galeones fueran sustituidas por barcos franceses sueltos, quienes aprovecharon la coyuntura para encaminarse directamente a Cantón e inundar el Callao con mercancías chinas. Además, la Compañía de Filipinas gozó de una oficina permanente en Lima y varios de sus barcos llegaron a sus costas cargados con productos de Asia. Es pro- bable que, como sucedía con los buques españoles y portugueses que nave- gaban en el Pacífico, en sus bodegas se incluyese algún mueble de lujo. Por lo tanto, alguno de los biombos chinos que hemos documentado en Lima para el siglo XVIII también pudo haber utilizado esta vía de acceso. 27 AGI, Contratación, 368, 7, 1, 64v y 65v. 26 De Vivero, 2003, 27. El comercio de biombos entre Filipinas y Nueva España Manila supuso la puerta por la que el conjunto de mercancías asiáti- cas entraban cada año en Nueva España a cambio de la tan codiciada plata americana. El éxito de los biombos como producto suntuoso inserto en el comercio transpacífico entre los puertos de Cavite y Acapulco resulta indiscutible. La referencia de Rodrigo de Vivero y Velasco a los biombos que ya habían llegado al virreinato antes de su retorno a principios del siglo XVII demuestra una vez más la pronta propagación de estos objetos y el interés por establecer una importación estable de los mismos: Hase trato diversamente qué efectos buenos al servicio de Dios y del rey nuestro señor podía surtir abrirse trato desde Japón a la Nueva España, porque pinturas, biombos, escritorios, y lo que otra vez se trajo, no es mercadería para ordinario, pero esta mis- ma razón me hace mayor fuerza para tener por buena la contratación, porque si la Nueva España cambia lo inútil y lo superfluo, como son paños, añil, granos, cueros, fieltros, sombreros, vino... y por eso se le retorne plata, oro, que tanto abunda y tan- to es menester acá, no hace fuerza la razón contraria que se funda en que los géneros del Japón no sean necesarios en la Nueva España.26 En los casos en los que contamos con la lista de mercancías transpor- tadas en la nao de China, comprobamos la existencia constante de biom- bos. Gaspar Álvarez, secretario de gobernación y guerra de las Islas Filipinas, envió a Acapulco en 1611 y en 1617 varios cajones de biombos.27 En 1680, el capitán don Luis Morales registró en el galeón Capitana Santelmo «dos biobos grandes divididos en seis cajoncillos, de a cuatro 41 41 Anu. estud. am., 69, 1, enero-junio, 2012, 31-62. ISSN: 0210-5810. 28 AGI, Escribanía, 411A, 286v. 29 AGI, Escribanía, 411A, 393r y 398r. 30 AGN, Marina, 1, 11, 114r. 31 AGI, Filipinas, 967, 47r, 47v, 55v, 66r, 72v, 81r. 32 Ibidem, 121v. 33 En los barcos que arribaron a Acapulco en 1612 se requisaron «dos pares de biovos», AGI, Contaduría, 902. 34 «Normas para el comercio entre Filipinas y Nueva España», AGI, Filipinas, 342, 9, 223r-228r. 35 Sobre la influencia de los comerciantes mexicanos en Filipinas ver: Yuste, 2007. Anu. estud. am., 69, 1, enero-junio, 2012, 31-62. ISSN: 0210-5810. DOI: 10.3989/aeamer.2012.1.01 El comercio de biombos entre Filipinas y Nueva España DOI: 10.3989/aeamer.2012.1.01 ALBERTO BAENA ZAPATERO tablas cada uno»,28 mientras que en 1684 el capitán Francisco Antonio de Velasco o Tomas Enríquez incluyeron varios cajones de biombos entre los bienes que remitieron al virreinato.29 Ya en el siglo XVIII, el sargento mayor, don Domingo Antonio de Otero Bermudes, apuntó en el libro de sobordo del galeón Santo Cristo de Burgos que en 1716 realizó el trayecto hasta Nueva España dos cajones de biombos «regulados por cuatro pie- zas».30 Asimismo, en 1771 el Consulado de Manila realizó un informe en el que se recogían las mercancías que se despacharon en los galeones que fueron a Nueva España entre 1736 y 1740, apareciendo consignados en las listas varios biombos y rodaestrados de doce tablas, con la evolución de sus precios.31 En este mismo documento se registraron para el año de 1770 rodaestrados de veinticuatro hojas maqueados y un «beobo de maque colo- rado con flores doradas de dos caras de 24 hojas».32 A estos datos oficiales debemos sumar todos aquellos biombos que llegaron de forma ilegal y que, por lo tanto, son también muy difíciles de consignar en las fuentes.33 Asimismo, los intentos por regular el tráfico de estas piezas son la mejor prueba de su presencia habitual. En la real cédula de 1726 dirigida al marqués de Torrecampo, gobernador de Filipinas, en la que se le infor- ma sobre la reglamentación del comercio de las islas con Nueva España, se hace alusión a los biombos, a cómo debían embarcarse y a la tasa que tenían que pagar. De donde se deduce que esta mercancía ocupó un papel lo suficientemente significativo dentro del conjunto de las exportaciones como para que se reparara en la misma.34 Una vez llegaban los cargamentos a Acapulco, estos se distribuían entre comerciantes mexicanos y de otras partes de América en la feria que se celebraba en esta misma ciudad. Dadas las conexiones que tenían los novohispanos en Manila, la mayoría de los productos continuaban el viaje hacia la ciudad de México.35 La importancia de este trato quedó evidencia- da por el hecho de que la ruta que unía el puerto del Pacífico y la capital se 42 Anu. estud. am., 69, 1, enero-junio, 2012, 31-62. ISSN: 0210-5810. DOI: 10.3989/aeamer.2012.1.01 Anu. estud. am., 69, 1, enero-junio, 2012, 31-62. ISSN: 0210-5810. DOI: 10.3989/aeamer.2012.1.01 EL MOVIMIENTO DE BIOMBOS DESDE EL PACÍFICO HASTA EL ATLÁNTICO denominó «camino de la China». 36 AGN, Indiferente Virreinal, caja-exp. 6450-090 y caja-exp. 6450-091 (Agradezco a la arqueóloga Andreia Martins Torres que me facilitara esta referencia). 37 Viera, 1952, 41. El comercio de biombos entre Filipinas y Nueva España Además, la Real Aduana también nos ha dejado algunas evidencias de la entrada de biombos asiáticos. Entre los géneros de China introducidos en la ciudad por el navío San José de Gracia en 1784 y 1787, se registraron varios «biombos maqueados en tabla y con figuras doradas» a 96 pesos la pieza.36 Desde inicios del siglo XVIII, en la Plaza Mayor de la ciudad de México se situó el Parián, conocido así por ser el lugar donde se vendían los productos que llegaban desde Filipinas. Entre estas mercancías se podían hallar muebles de lujo, muy demandados por los ricos criollos para adornar sus palacios. Juan de Viera nos narra como «en ese mercado hay camas, biombos y estrados… en fin se puede poner una casa dentro de una hora para recibir potentados».37 En varias pinturas del siglo XVIII en las que aparece representado el Parián podemos comprobar que los biombos estuvieron entre los objetos puestos a la venta, como sucede en el cuadro de la Plaza Mayor conservado en el Museo Nacional de Historia de la ciu- dad de México, fechado para 1766, o en el perteneciente a la Fundación Banamex. El análisis de los inventarios de bienes, testamentos y dotes de las familias más prósperas del virreinato durante los siglos XVII y XVIII reve- la la popularidad de los biombos asiáticos entre el mobiliario de los princi- pales palacios de la capital mexicana. A la fascinación occidental por todos aquellos objetos lejanos, rodeados de un halo de «exotismo», se unía en Nueva España la dificultad de acceder a los productos europeos y el lugar privilegiado que, por el contrario, ocupaba en el comercio internacional con Asia. Los tapices, que en los palacios del viejo continente rodeaban las paredes de los cuartos principales, eran escasos y muy caros en América, por lo que habitualmente fueron sustituidos por biombos que recubrían los muros del salón de estrado, mucho más accesibles. Los propietarios novohispanos de estos objetos formaban parte de un grupo de «nuevos ricos» surgidos de la conquista, poblamiento y explota- ción de América que, ante la falta de títulos nobiliarios o linajes que res- paldasen su ascenso social, buscaron símbolos de estatus que expresaran la posición recientemente adquirida. En este sentido, los biombos llegaron en el momento preciso al sitio adecuado y toda familia rica que se preciase 43 Anu. estud. am., 69, 1, enero-junio, 2012, 31-62. ISSN: 0210-5810. 38 Curiel, 2000, 65-101. 39 Romero de Terreros, 1957, 60. 38 Curiel, 2000, 65-101. 39 Romero de Terreros, 1957, 60. Anu. estud. am., 69, 1, enero-junio, 2012, 31-62. ISSN: 0210-5810. DOI: 10.3989/aeamer.2012.1.01 El comercio de biombos entre Filipinas y Nueva España DOI: 10.3989/aeamer.2012.1.01 ALBERTO BAENA ZAPATERO Biombo chino de exportación, Museo Amparo, ciudad de Puebla, México. Biombo chino de exportación, Museo Amparo, ciudad de Puebla, México. contaba con varios de estos muebles. La marquesa doña Teresa Francisca María de Guadalupe Retes, por ejemplo, tuvo ocho biombos repartidos por sus casas, uno seguro de origen chino,38 mientras que entre los objetos de valor del Conde de Xala se listan un biombo de maque de China de 240 pesos y «otro dicho de China con once hojas».39 Es interesante consignar que durante el siglo XVII, paralelamente al comercio con Filipinas, se desarrolla en Nueva España una manufactura de biombos propia que adquiere un volumen muy importante, hasta competir en el mercado de exportaciones con los de manufactura asiática. El naci- miento de esta producción en el virreinato, al margen de la influencia que 44 Anu. estud. am., 69, 1, enero-junio, 2012, 31-62. ISSN: 0210-5810. DOI: 10.3989/aeamer.2012.1.01 EL MOVIMIENTO DE BIOMBOS DESDE EL PACÍFICO HASTA EL ATLÁNTICO eventualmente pudiera tener la llegada de algún artista foráneo, se debió a una serie de factores internos y a las consecuencias derivadas del papel cen- tral que ocupaba Nueva España en el tráfico internacional. Dejando a un lado las consideraciones artísticas, hay que subrayar la oportunidad econó- mica que la demanda de este mueble abría para los artesanos locales. Al eli- minar el gasto derivado del transporte y los intermediarios, los biombos «achinados» ofrecían precios mucho más baratos que los chinos o japone- ses, normalmente entre los 5 y los 16 pesos, y sólo en algunos casos hechos «al remedo de maque» o de «maqué fingido» fueron valorados entre los 30 y los 50 pesos, muy lejos de los 100 pesos que costaba de media un biom- bo asiático nuevo. Lo mismo sucedía con los ejemplares mexicanos pinta- dos con temas occidentales, que contaban con un valor un poco más alto que los «achinados» pero aún muy lejos del precio de los que llegaban en el galeón de Manila. El éxito progresivo de esta producción hizo que duran- te el siglo XVIII se extendiera tanto su uso que resulta muy difícil no encontrar varios biombos entre las propiedades de cualquier español con cierta fortuna, si bien los chinos fueron más exclusivos y, por lo tanto, menos frecuentes. 40 En Perú encontramos biombos hechos en «pintura del reino» o en «pintura de Lima», ade- más de otros ejemplares en vaqueta dorada de Huamanga. AGL, Protocolos notariales, 76, esc. Orencio de Ascarrunz, 1750, f.553r; AGL, Protocolos notariales, 871, esc. Agustín Gerónimo de Portalanza, 1761, 338v. En Colombia se conservan dos biombos con temas costumbristas hechos en la tierra: López Pérez, 1998. El comercio de biombos entre Filipinas y Nueva España Durante esta centuria, además de la producción novohispana, vemos surgir manufacturas en otras partes de América como Perú, Guatemala o Colombia que se adaptaron perfectamente al gusto local y a los materiales utilizados comúnmente en los muebles de la tierra.40 Expresión artística de una nueva identidad criolla, no podemos detenernos en este asunto, pero conviene apuntar que en el futuro el estudio de los temas representados en sus hojas podrá arrojar luz sobre la posible influencia que las obras novo- hispanas tuvieron en el resto de América. y 44 Así, Simón de Gaviola, caballero de la orden de Santiago, y su mujer tuvieron cuatro biom- bos (AHPS, 14, 1, 1653, 1203r y 1214v.); don Francisco Tello y Portugal, marqués de Sauseda y caba- llero de la orden de Alcántara, tuvo «dos biombos de China» en su poder (AHPS, 13, 2, 1702, 1623- 1634); doña Ana María Fernández Altamirano, propietaria de una hacienda de olivares cerca de la ciudad, «un biombo de ocho hojas pintado» (AHPS, 1, 1, 1721, 325v.); o doña Ana Tello de Guzmán, viuda de don Lorenzo Dávila y Medina, conde de Valhesmoro, «un biombo de lienzo pintado» (AHPS, 1, 1, 1727, 445r). 42 Los biombos fueron habituales entre las propiedades de los miembros del Consejo de Indias, de la Casa de Contratación o del Consulado de Sevilla: Domingo de Urbizu, miembro del Consejo Real de Hacienda y alguacil mayor de la Casa de Contratación, presenta varios biombos en su ajuar doméstico; Ignacia Calzado, mujer de Esteban Torrado de Guzmán, contador y secretario del Consulado de Sevilla, «un biombo grande de liensso pintado» (ambos citados por Sanz y Dabrio, 1974, LVII, 89-150); además encontramos otro ejemplar propiedad de Juan de Torres y la Vega Ponce de León, conde de Miraflores de los Ángeles y presidente de la Casa de Contratación, que en la almone- da celebrada por su muerte sería vendido a Manuel de Torres, miembro de la Real Audiencia de la ciu- dad de Sevilla, por 120 reales de vellón. Archivo Histórico Provincial de la Ciudad de Sevilla (AHPS), 1, 1, 1721, 498r. 41 Juan Gil ha publicado un estudio en el que presenta una relación de los productos de Extremo Oriente que llegaron hasta Sevilla durante el siglo XVII en base a la documentación del Archivo de Indias de Sevilla. Gil, 2011. 43 Sanz y Dabrio, 1974, 89-150. El viaje por el Atlántico y por el Pacífico Sur Una parte de las mercancías que llegaban con el Galeón de Manila hasta la ciudad de México se reenviaban hacia Veracruz para ser embarca- das con dirección a España. Desde finales del siglo XVI y principios del 45 Anu. estud. am., 69, 1, enero-junio, 2012, 31-62. ISSN: 0210-5810. DOI: 10.3989/aeamer.2012.1.01 ALBERTO BAENA ZAPATERO ALBERTO BAENA ZAPATERO XVII, Europa se vio inundada por una avalancha de productos asiáticos de carácter suntuario y de uso cotidiano, destinados en la mayoría de los casos a regalos o a colecciones particulares. Los puertos de entrada de estas mer- cancías fueron Lisboa y Sevilla, donde pronto se extendió entre las princi- pales familias enriquecidas por el comercio el gusto por los objetos veni- dos de lugares lejanos.41 El estudio de los libros notariales del Archivo de Protocolos de Sevilla refleja el auge que tuvieron los biombos entre los enseres domésticos de los comerciantes y funcionarios de la ciudad, espe- cialmente entre aquellos relacionados con la carrera de Indias durante los siglos XVII y XVIII.42 Si bien coincidimos con María Jesús Sanz y María Teresa Dabrio en destacar cómo entre aquellos individuos implicados en el tráfico con América se encontraban riquísimos ajuares, superiores muchas veces a los de los nobles de la ciudad, discrepamos respecto a su opinión de atribuir a este grupo una preferencia por los objetos importados de Asia frente al supuesto gusto más tradicional de los aristócratas.43 Una lectura atenta de los inventarios de bienes de Sevilla demuestra que el interés por estas obras no fue exclusivo de los comerciantes y funcionarios sino que, por el con- trario, también se extendió entre las familias nobles.44 Asimismo, desde el inicio de las relaciones ibéricas con Asia, la rea- leza española y la portuguesa tuvieron un gran interés por acceder a las mejores piezas que ofrecían aquellas culturas lejanas. Muchas fueron 46 Anu. estud. am., 69, 1, enero-junio, 2012, 31-62. ISSN: 0210-5810. DOI: 10.3989/aeamer.2012.1.01 EL MOVIMIENTO DE BIOMBOS DESDE EL PACÍFICO HASTA EL ATLÁNTICO adquiridas como regalos llevados por las embajadas o enviadas por los fun- cionarios reales desplazados a aquellas regiones que buscaban ganar el favor en la Corte, mientras que otras fueron simplemente compradas por encargo. La unión de las Coronas ibéricas propició que, durante sesenta años, las dos rutas que ligaban Europa con Asia quedaran bajo un mismo monarca, lo que facilitó aún más el tráfico de objetos «exóticos» hacia la Corte de los Felipes. Después de la ruptura hispano-lusa de 1640, los reyes españoles siguieron demandando este tipo de muebles para la decoración de sus palacios, como puede apreciarse por su popularidad dentro de las colecciones reales de los siglos XVII y XVIII. 48 AHPS, 14, 2, 1683, 229-235. Además, la localización más antigua que se conoce del biom- bo del palo del volador del Museo de América de Madrid es el Hospital de la Caridad de Sevilla, en cuya hermandad ingresaban los personajes mas importantes de la ciudad, alguno de los cuales debió donar la obra. Sandoval, 2007. 46 Arte y Saber, 1999, Inv. 207. 45 Fernández Baytón, 1975, 118 y 124. 46 Arte y Saber, 1999, Inv. 207. 47 Entre las pinturas del Palacio Real de Madrid, Carlos III contaba con un «un biombo de cha- rol negro con varias pinturas de aves y árboles de doce hojas»; en el real Sitio de San Lorenzo había cinco biombos de tela, hechos en damasco carmesí, filipichín encarnado y bayeta encarnada; en San Ildefonso un biombo de damasco amarillo; en el real sitio del Pardo, en el cuarto del rey se encontra- ban «dos biombos de ocho hojas cubiertos de damasco carmesí con galón de oro y tachuela dorada», mientras que en el de las infantas niñas había «uno de ocho hojas de doblar de caoba, cubierto su bas- tidor de damasco carmesí, trenzas y borlas correspondientes, que sirve a SS. AA delante de las chime- neas» Fernández Miranda, 1988, 1, 220 y 448 y 2, 353, 371 y 421. 48 AHPS, 14, 2, 1683, 229-235. Además, la localización más antigua que se conoce del biom- bo del palo del volador del Museo de América de Madrid es el Hospital de la Caridad de Sevilla, en cuya hermandad ingresaban los personajes mas importantes de la ciudad, alguno de los cuales debió donar la obra. Sandoval, 2007. 45 Fernández Baytón, 1975, 118 y 124. 54 Fueron propietarios de biombos chinos en Lima durante el siglo XVIII los siguientes per- sonajes: el sargento mayor Gaspar Fernández de Montejo (Archivo General de la Nación en Lima, AGL, Protocolos notariales, 1, esc. J. Agüero, 1732, 629v.), Isabel Carrillo de la Presa, dueña de un molino de pólvora y del navío La Soledad (AGL, Protocolos notariales, 83, esc. O. Ascarrunz, 1765, f. 302r.), Juana Ignacia de Gomendio, poseedora de un cajón de textiles (AGL, Protocolos notaria- les, 2, esc. Joseph de Agüero, 1734/35, 921r.), el Maestre de Campo Francisco de Oyague (Archivo Riva Agüero, ARA, Sección colonial, C-58, 38r), o el miembro del Consejo de Indias y oidor decano de la Real Audiencia de Perú, Álvaro de Navia Bolaño, conde del valle de Oselle (AGL, Protocolos notariales, 509, González Mendoza, 1757/1760, 468r y 651r). g 50 Hay otros seis biombos en este inventario, dos de ellos también de las Indias, AHPM, 7684, 1653, 283-284. ALBERTO BAENA ZAPATERO Así, Carlos II, en el inven- tario de pinturas del Palacio Real de Madrid, tenía un biombo de nueve hojas de charol «de la india», seguramente denominado así por haber sido importado desde la costa de Coromandel.45 Entre los bienes de Isabel de Farnesio de 1745 se mencionan «dos biombos de charol y nácar con pintu- ra fina» heredados de su tía Mariana de Neoburgo, la última esposa de Carlos II.46 Carlos III, por su parte, contó con numerosos biombos reparti- dos entre sus diferentes palacios, algunos para su uso personal y otros para las infantas.47 Al margen de los biombos chinos y japoneses que alcanzaron la Península, muchos de los producidos en Nueva España también partieron rumbo a Europa durante los siglos XVII y XVIII. De esta forma, es posi- ble encontrar estos muebles mexicanos incluidos en el ajuar doméstico de las familias nobles tanto de Sevilla como de Madrid. En el inventario de 1683 del palacio sevillano de doña Inés María de Molina y Bermúdez se lista un impresionante biombo de estrado de las Indias de veintiséis pane- les de lienzo pintado.48 En Madrid, por su parte, en el inventario de 1646 de 47 Anu. estud. am., 69, 1, enero-junio, 2012, 31-62. ISSN: 0210-5810. DOI: 10.3989/aeamer.2012.1.01 49 AHPM, 5892, 841r, 11 de agosto de 1646. 50 Hay otros seis biombos en este inventario, dos de ellos también de las Indias, AHPM, 7684, 1653, 283-284. 51 Arte y Saber, 1999, Inv. 207. 52 Calderón, 2003, 586 53 Bonialian, 2011, 15-16 54 Fueron propietarios de biombos chinos en Lima durante el siglo XVIII los siguientes per- sonajes: el sargento mayor Gaspar Fernández de Montejo (Archivo General de la Nación en Lima, AGL, Protocolos notariales, 1, esc. J. Agüero, 1732, 629v.), Isabel Carrillo de la Presa, dueña de un molino de pólvora y del navío La Soledad (AGL, Protocolos notariales, 83, esc. O. Ascarrunz, 1765, f. 302r.), Juana Ignacia de Gomendio, poseedora de un cajón de textiles (AGL, Protocolos notaria- les, 2, esc. Joseph de Agüero, 1734/35, 921r.), el Maestre de Campo Francisco de Oyague (Archivo Riva Agüero, ARA, Sección colonial, C-58, 38r), o el miembro del Consejo de Indias y oidor decano de la Real Audiencia de Perú, Álvaro de Navia Bolaño, conde del valle de Oselle (AGL, Protocolos notariales, 509, González Mendoza, 1757/1760, 468r y 651r). 53 Bonialian, 2011, 15-16 49 AHPM, 5892, 841r, 11 de agosto de 1646. 52 Calderón, 2003, 586 51 Arte y Saber, 1999, Inv. 207. 55 Además, encontramos biombos mexicanos entre las propiedades de: Diego de Carbajal, corredor mayor del reino (AGL, Protocolos notariales, 64 esc. Arredondo 1731, 260v-266v y 291r-297r), Fernando Carrillo, sargento del batallón de milicias de la ciudad de Lima, (AGL, Protocolos notariales, 67, esc. Antonio José de Ascarrunz, 1748) o Josefa Jiménez Lobatón y Salazar, madre de don José de Rezabal y Ugarte, regente de la Real Audiencia de Santiago de Chile (AGL, Protocolos notariales, 144, esc. Lucas de Bonilla, 1794, 91r-120v). ALBERTO BAENA ZAPATERO Álvaro de Bazán, marqués de Santa Cruz, se incluían «dos biombos traídos de las Indias» tasados en 600 reales,49 mientras que entre los bienes inven- tariados en 1653 al conde de Monterrey se citan «cuatro biombos de las Indias».50 Por último, en la testamentaría de Isabel de Farnesio también aparece un biombo que representaba la conquista de México, de veinticua- tro paneles con pintura y madre perla, prueba de que estos objetos fueron valorados tanto como sus homólogos orientales.51 También desde la costa occidental de Nueva España, a pesar de las restricciones del siglo XVI y a la prohibición posterior del tráfico entre virreinatos, partieron rumbo al Callao muchos productos llegados en el Galeón de Manila. Los comerciantes peruanos idearon distintas formas de acceder a estas mercancías, como el trasbordo en puertos intermedios o el contrabando a bordo de navíos que transportaban azogue.52 Como señala Bonialian, un número importante de barcos peruanos navegaban cada año hasta los puertos de Acapulco, Zihuatanejo, Huatulco, Realejo y Sonsonate, transportando plata, azogue, cacao y vino, y retornaban tiempo después cargados con mercancías europeas, mexicanas y asiáticas.53 Entre estos objetos se encontraron los biombos, tanto los de origen chino como los de factura mexicana. Como sucede con el resto de territorios analiza- dos, nuevamente encontramos a ricos comerciantes y funcionarios entre los dueños de estos objetos. La existencia de varios ejemplares asiáticos en Lima durante el siglo XVIII demuestra cómo el virreinato peruano estuvo conectado con el comercio de Extremo Oriente, bien fuese directamente o, más probablemente, a través de su relación con Nueva España.54 Además, la presencia de biombos mexicanos en Sudamérica nos indica que la pro- ducción en este territorio tuvo un volumen notable, que fueron valorados 48 Anu. estud. am., 69, 1, enero-junio, 2012, 31-62. ISSN: 0210-5810. DOI: 10.3989/aeamer.2012.1.01 Anu. estud. am., 69, 1, enero-junio, 2012, 31-62. ISSN: 0210-5810. DOI: 10.3989/aeamer.2012.1.01 EL MOVIMIENTO DE BIOMBOS DESDE EL PACÍFICO HASTA EL ATLÁNTICO más allá de sus fronteras y que alcanzaron no sólo Europa, como se pensa- ba hasta ahora, sino también el resto del continente americano.55 ) 56 «Memoria de mercaderías que trae el embajador de Japón», AGI, Filipinas, 7, 7, 88. Embajadas La llegada de los primeros biombos a Nueva España y Europa fue paralela a los contactos diplomáticos entre los reinos ibéricos y Japón, y a la consolidación del comercio en el Pacífico, tanto en su vertiente occiden- tal como a través del Galeón de Manila. Los primeros ejemplares de los que tenemos noticia no arribaron a Europa de la mano de los comerciantes sino de la iniciativa de los religiosos. Fueron los japoneses que componían la embajada que partió de Nagasaki junto al jesuita Alejandro Valignano rum- bo a Lisboa en 1582, quienes dieron a conocer estos muebles. Entre los pre- sentes embarcados por la embajada se encontraban dos pares de biombos, los dos primeros ofrecidos a Felipe II y los otros dos al papa Gregorio XIII. A principios del siglo XVII, tan sólo algunos años después de que esta misión visitase la Corte de Felipe II y el Vaticano, entre la relación de obje- tos que el embajador de Japón traía con el fin de venderlos para su despa- cho en Madrid, se hace mención de «Treynta Caxones de biobos de oro fino, cada Caxon dos biobos».56 Prueba de que, nuevamente, los propios japoneses colaboraron en la difusión por Europa de un mueble de lujo que se consideraba típicamente oriental, pero que poco a poco iba ganando un espacio en los salones de los principales palacios de occidente. En América, la primera noticia que tenemos sobre una presencia ofi- cial de japoneses data de 1610. Rodrigo de Vivero, después de su mandato como gobernador de Filipinas, naufragó en Japón durante su regreso a México y el shogun Ieyasu le ofreció un buque construido en sus astilleros para que continuase su viaje de retorno. Junto a Rodrigo de Vivero embar- caron una embajada de 23 japoneses encabezados por Tanaka Shosuke con 49 Anu. estud. am., 69, 1, enero-junio, 2012, 31-62. ISSN: 0210-5810. DOI: 10.3989/aeamer.2012.1.01 ALBERTO BAENA ZAPATERO el cometido de aprender la técnica del beneficiado de la plata y de consoli- dar una relación comercial directa entre ambos reinos. 57 «Carta de D. Juan de Silva al rey», AGI, Filipinas, 20, 4, 36, 15 y 16. 58 Castelló y Martínez, 1970, 27. 59 Gutiérrez, 1947, 74. 60 AGN, Tierras, 3283, 2, 54-61. Los biombos también fueron denominados arrimadores de estrado en Nueva España. 59 Gutiérrez, 1947, 74. EL MOVIMIENTO DE BIOMBOS DESDE EL PACÍFICO HASTA EL ATLÁNTICO EL MOVIMIENTO DE BIOMBOS DESDE EL PACÍFICO HASTA EL ATLÁNTICO 61 Gruzinski, 2010. 62 Baena, 2007, 441-450. Anu. estud. am., 69, 1, enero-junio, 2012, 31-62. ISSN: 0210-5810. DOI: 10.3989/aeamer.2012.1.01 Embajadas Además, ese mismo año, el gobernador de Filipinas, Juan de Silva, informó al rey que había recibido del citado shogun varios biombos de poca calidad como gesto de amistad.57 En 1611, Tanaka emprendió el camino de vuelta a Japón uniéndose a la expedición de Sebastián Vizcaíno, primer embajador enviado al país nipón por Felipe III con el propósito de impulsar los lazos económicos y políticos. En estas misiones diplomáticas eran obligatorios los regalos y en este apartado tuvieron un protagonismo especial los biombos, lo que vino a establecer desde el primer momento su papel como objetos distinguidos. Iyesasu respondió a los presentes de la misión española enviando al virrey Velasco el Joven diez biombos tejidos de oro, tres armaduras de samurai y una carta geográfica del Japón, obsequios que recibió en 1614 su sucesor en el cargo, el marqués de Guadalcázar, quien finalmente los haría llegar a don Luis de Velasco.58 Adquiridos por los virreyes, los biombos pronto ocuparon un lugar principal entre los muebles de las casas reales de Chapultepec. En 1640, por ejemplo, se dispusieron dos ejemplares chinos «que cubrían la música e instrumentos» mientras se servía el banquete de recepción al virrey Diego López Pacheco.59 La vida en la corte virreinal constituía en México el modelo aristocrático que trataban de imitar las principales familias novo- hispanas, por lo tanto, el hecho de que llegaran biombos entre las ofrendas de las comitivas japonesas o su uso en los palacios oficiales, hizo que se extendiera la imagen de este mueble como elemento de prestigio y que muchas de las familias más ricas de la ciudad quisieran hacerse con uno para lucir en sus salones. Asimismo, en la cultura política de la época se incorporó la imagen del biombo como mueble especialmente apropiado para regalar si se quería obtener algún favor o simplemente agradar al des- tinatario. Ejemplo de ello son los biombos que enviaron los virreyes novo- hispanos a la península o el «arrimador de estrado» que en 1663 ofreció el gobernador de Nuevo México, Diego de Peñalosa, al contador de la ciudad de México, Carlos de Sigüenza.60 50 Anu. estud. am., 69, 1, enero-junio, 2012, 31-62. ISSN: 0210-5810. DOI: 10.3989/aeamer.2012.1.01 Funcionarios reales, comerciantes y mercaderes La conformación de la Monarquía Católica durante el siglo XVI pro- dujo un conglomerado de relaciones planetarias que conllevaría la consti- tución de burocracias móviles y un sistema económico de dimensiones mundiales. Serge Gruzinski ha llamado la atención sobre una «elite mun- dializada» que se movió a través de las fronteras de los dominios españo- les y portugueses, ambos íntimamente relacionados.61 Este grupo de indivi- duos propició de manera inconsciente una nueva cultura que superó los límites imperiales y que se vio influida por todas aquellas civilizaciones con las que entró en contacto. A lo largo de la Edad Moderna, el transpor- te de biombos acompañó al movimiento de personas, en especial al de mer- caderes y cargos reales desplegados por cuatro continentes, sirviendo como testimonio de los cambios producidos. p Virreyes y gobernadores fueron unos grandes consumidores de obras de arte, bien fuese para incorporar a su propio ajuar o para enviar a la Península. Así, después de su mandato en Nueva España, muchos transpor- taron hasta su nuevo destino los biombos que habían adquirido. Los temas representados en las hojas de estos muebles podían ser varios: los lugares más emblemáticos de la capital, como la Alameda, el paseo de Iztacalco o la Plaza Mayor; hechos históricos relevantes como la conquista de México, que solían acompañarse por una vista aérea de la urbe por su otra cara; representaciones de algunas de las prácticas más pintorescas de la tierra, como los desposorios indígenas y los ritos prehispánicos asociados al fes- tejo de los mismos; o historias mitológicas apropiadas para espacios priva- dos.62 Estos biombos pudieron servir a sus dueños de recuerdo de su paso por aquella ciudad, de los méritos adquiridos en el embellecimiento de la urbe, o simplemente como símbolo de cosmopolitismo ante sus invitados peninsulares. Muchos de los temas elegidos encajaban perfectamente con el gusto aristocrático de la época, que buscaba saciar la curiosidad por conocer «otros mundos» a través de la representación en sus salones de escenas costumbristas. De cualquier manera, los biombos no constituyeron un caso especial y deben ser relacionados con los enconchados, trabajo típicamente novohispano del que se han documentado envíos a la Península 51 Anu. estud. am., 69, 1, enero-junio, 2012, 31-62. ISSN: 0210-5810. 63 La profesora García Saiz ha señalado a los virreyes conde de Galve y conde de Moctezuma como posibles comitentes de estas obras (García Saiz, 1999). Asimismo, Sonia Ocaña defiende la idea de la vinculación entre esta técnica y la identidad criolla (Ocaña, 2008). 64 El cuadro que representa la plaza mayor de México de Cristóbal de Villalpando fue propie- dad del conde de Galve y los descendientes del duque de Alburquerque conservan otra pintura sobre México (Martínez del Río, 1994). 65 AHPM, 2320, 21 de septiembre de 1617, 92r-93v. 66 Martínez del Rio, 1994, 133-150. Funcionarios reales, comerciantes y mercaderes DOI: 10.3989/aeamer.2012.1.01 ALBERTO BAENA ZAPATERO con el tema de la conquista,63 o con los cuadros pintados con vistas de la plaza de la ciudad de México que fueron encargados por funcionarios u ofrecidos por la ciudad al final de su estancia.64 El primer ejemplo que encontramos de funcionarios que se traslada- ron desde México cargando con un biombo producido en la tierra es el que aparece consignado en Madrid en el inventario de bienes de 1617 del virrey Luis de Velasco el Joven o hijo. Puesto que se indica que el biombo era «de las Indias», lo lógico es pensar que lo adquirió en México antes de su par- tida en 1612, fecha que nos sugiere cómo para inicios del siglo XVII ya estaba activa la producción de biombos en Nueva España.65 El biombo, que representa la «Vista de la Plaza Mayor de la ciudad de México y del paseo de Iztacalco», descubierto en España y actualmente en la colección Rivero Lake, muestra en su parte superior el escudo del marqués de Cadereita, virrey de la Nueva España entre 1635 y 1640. Puede que el interés de este gobernante fuera mostrar la ciudad en todo su esplen- dor, dado que emprendió numerosas obras para mejorar su drenaje y para reparar los daños producidos por un terremoto. Incluso, parece que en la Plaza Mayor pudo representarse a sí mismo saliendo con su comitiva de Palacio en un ambiente festivo. El biombo, en el que aparecen por un lado «Las artes liberales» y por el otro «Los cuatro elementos», fue pintado por Juan Correa siguiendo las indicaciones del virrey fray Payo Enríquez de Ribera, arzobispo y virrey entre 1667 y 1679. Una vez se hubo cumplido su periodo de gobierno en las Indias, fray Payo trasladaría esta pieza entre su ajuar hasta la Península, donde permanecería hasta su incorporación a la colección del Museo Franz Mayer.66 Otro virrey que aprovechó su breve estancia en la ciudad de México para hacerse con alguno de estos muebles fue José Sarmiento y Valladares, conde de Moctezuma y de Tula, el cual contó entre sus propie- dades con dos biombos de dos haces cada uno, con temas sobre «La con- quista de México» y una «Vista de la ciudad» el primero, mientras que en el otro se representaría «La defensa de Viena frente a los turcos» por una 52 Anu. estud. 63 La profesora García Saiz ha señalado a los virreyes conde de Galve y conde de Moctezuma como posibles comitentes de estas obras (García Saiz, 1999). Asimismo, Sonia Ocaña defiende la idea de la vinculación entre esta técnica y la identidad criolla (Ocaña, 2008). 63 La profesora García Saiz ha señalado a los virreyes conde de Galve y conde de Moctezuma como posibles comitentes de estas obras (García Saiz, 1999). Asimismo, Sonia Ocaña defiende la idea de la vinculación entre esta técnica y la identidad criolla (Ocaña, 2008). 64 El cuadro que representa la plaza mayor de México de Cristóbal de Villalpando fue propie- dad del conde de Galve y los descendientes del duque de Alburquerque conservan otra pintura sobre México (Martínez del Río, 1994). 64 El cuadro que representa la plaza mayor de México de Cristóbal de Villalpando fue propie- dad del conde de Galve y los descendientes del duque de Alburquerque conservan otra pintura sobre México (Martínez del Río, 1994). 67 «Inventario de bienes del virrey depuesto Iturrigaray, 22 de septiembre de 1808-17 de mayo de 1809 confiscados por las Reales cajas» en García, 1985, 2, 471. 68 AGN, Civil, 114, 1. 69 Gemelli Careri, 2002, 6 y 7. 70 Calderón, 2003, 587. 71 Arquivo Histórico Ultramarino, Conselho Ultramarino, c.16, 3. ALBERTO BAENA ZAPATERO Con los gobernadores de Filipinas sucedió algo parecido. Don Fausto de Crusat y Góngora debió de ser un gran aficionado al arte oriental y entre las propiedades que en 1701 le acompañaron a su vuelta a México se encontraban numerosos muebles japoneses, 21 escritorios, 10 de ellos de maque fino; abundante porcelana, abanicos y quimonos chinos; y hasta tres camas de ébano de estilo salomónico hechas en la India. Entre estos obje- tos llama la atención la presencia de 11 biombos japoneses de diferentes tamaños y materiales (maqué, madre perla, oro y piedra ágata), destacando un conjunto de cuatro biombos de 6 hojas cada uno valorado en 625 pesos.68 Por su número y valor puede que muchas de estas piezas fueran transportadas con la idea de venderse al llegar a su nuevo destino, sirvien- do como promotor inconsciente de influencias culturales. En el caso de Perú, también se pudieron utilizar las embarcaciones que debían trasladar a las dignidades eclesiásticas o civiles destinadas al virreinato para transportar mercancías ilegales venidas desde Asia. El via- jero italiano Gemelli Careri pudo comprobar cómo en 1697 coincidieron en el puerto de Acapulco la nao de China con el navío que debía conducir al nuevo virrey del Perú, Conde de Cañete, hasta Lima.69 Esta situación fue aprovechada por los comerciantes peruanos para transbordar una gran par- te de los productos asiáticos de la nao hasta el barco del representante real, seguramente con su complicidad.70 Además, entre los inventarios de bienes de los capitanes portugueses y holandeses implicados en el comercio del Pacífico encontramos escrito- rios y cajas japonesas, lo cual nos pone en relación con el gusto por acu- mular obras de arte que pudieron desarrollar en sus viajes. No olvidemos que por su situación privilegiada dentro del comercio mundial tuvieron acceso a las mejores piezas y a precios más baratos que los compradores habituales. En Portugal fue común encontrar biombos entre el ajuar domés- tico de aquellas personalidades que tuvieron relación con el Estado da India. El alférez mayor Joam de Meneses, por ejemplo, murió cuando vol- vía a Lisboa desde Goa transportando entre sus pertenencias dos biombos,71 el hidalgo García de Mello Torres, que tuvo derecho a un viaje a China, dejó en testamento dos biombos japoneses de cuatro hojas cada uno, mien- tras que Diego Valente, que fue obispo del Japón, cuando falleció tenía tres 54 Anu. estud. Funcionarios reales, comerciantes y mercaderes am., 69, 1, enero-junio, 2012, 31-62. ISSN: 0210-5810. DOI: 10.3989/aeamer.2012.1.01 EL MOVIMIENTO DE BIOMBOS DESDE EL PACÍFICO HASTA EL ATLÁNTICO Biombo con «Vista de la Plaza Mayor de la ciudad de México y del paseo de Iztacalco», colección Rivero Lake Biombo con «Vista de la Plaza Mayor de la ciudad de México y del paseo de Iztacalco», colección Rivero Lake cara y «Una escena de caza» en su reverso. Este ejemplar resulta muy interesante si tenemos en cuenta que es uno de los pocos que fue hecho con la técnica del enconchado, típica del virreinato, y que se elaboró en una coyuntura política especialmente sensible como fue la sucesión de Carlos II. Por último, entre los objetos que se incautaron al virrey Iturrigaray en 1808 se hallaban «siete biombos de diversas calidades y pinturas», mien- tras que entre los muebles de su propiedad que se registraron en el palacio de Chapultepec aparecía «un biombo de moda con tallas doradas».67 Estos bienes, de no haber sido retenidos, también hubieran acabado junto a Iturrigaray en España como prueba de su paso por la capital novohispana. 53 Anu. estud. am., 69, 1, enero-junio, 2012, 31-62. ISSN: 0210-5810. DOI: 10.3989/aeamer.2012.1.01 72 Citado por Mendes Pinto, 1993, 20-21. 73 Okamoto, 1993. 74 AGI, Filipinas, 170, 4, 32r y 39r. 75 En el siglo XVII, el capitán Felipe de Navarijo, o la hija del capitán Cristóbal Gomes de la Madriz, Ana María Gomes de la Madriz, disfrutaron de biombos asiáticos en sus casas de la capital (AGN, Civil, 1802, 1 y AGN, Civil, 1863, 19). Mientras que los mercaderes Pedro de Burgos, propie- tario de una tienda de ropa y telas, o Álvaro de Lorenzana, miembro del Consulado de México, tuvie- ron varios ejemplares chinos (AGN, Civil, 1835, 6; AGN, Bienes Nacionales, 1294, 1, 1653). Ya en el siglo XVIII, el comerciante Nicolás de Arteaga y el dueño de un cajón en la plaza mayor, Miguel de Ibarburo, también tuvieron varios «rodaestrados de china» (AGN, Bienes Nacionales, 404, 5; AGN, Civil, 323, 1). Anu. estud. am., 69, 1, enero-junio, 2012, 31-62. ISSN: 0210-5810. DOI: 10.3989/aeamer.2012.1.01 74 AGI, Filipinas, 170, 4, 32r y 39r. 73 Okamoto, 1993. 72 Citado por Mendes Pinto, 1993, 20-21. 76 AGN, Civil, 1998, 3. 77 Berenice Ballesteros ha probado el éxito que tuvo durante el siglo XVII el menaje asiático entre los miembros del Consulado de México; los inventarios consultados para el XVIII muestran características similares. Ballesteros, 2008. 78 AGI, México, 263, 131. 79 AGN, Bienes Nacionales, 445, 4. 80 AHPS, oficio 9, libro 1, 187-191, 23 de febrero de 1691. 81 Boxer, 1968, 22. 82 Sato, 2002, 18, 31-34. 77 Berenice Ballesteros ha probado el éxito que tuvo durante el siglo XVII el menaje asiático entre los miembros del Consulado de México; los inventarios consultados para el XVIII muestran características similares. Ballesteros, 2008. ALBERTO BAENA ZAPATERO am., 69, 1, enero-junio, 2012, 31-62. ISSN: 0210-5810. DOI: 10.3989/aeamer.2012.1.01 EL MOVIMIENTO DE BIOMBOS DESDE EL PACÍFICO HASTA EL ATLÁNTICO pares de biombos.72Así, Yoshitomo Okamoto mantiene que los mercaderes que frecuentaban Nagasaki «were of course the pioneers in popularizing namban tastes».73 Su objetivo al juntar estos objetos pudo no estar sólo en la mera ostentación sino que es posible que les apeteciese también recordar los lugares por los que habían pasado. Asimismo, en el caso de los ejem- plares producidos para la exportación en los que aparecen europeos llegan- do hasta las costas de Asia, puede que sus dueños quisieran conservar un testimonio de su actividad comercial, origen de su riqueza y de su forma- ción cosmopolita. Entre los capitanes españoles de Manila podemos observar una cos- tumbre parecida a sus homólogos holandeses y portugueses por acumular objetos orientales. Por ejemplo, entre los bienes embargados en 1720 al maestre de Campo don Esteban Eguiño, quien formase parte de una com- pañía de comercio que se dedicaba a la compra de objetos asiáticos para reenviarlos en la nao de China hasta Acapulco, encontramos cuatro biom- bos de papel pintado.74 En Nueva España, si bien el gusto por los produc- tos orientales se extendió rápidamente entre el conjunto de la población, este interés fue si cabe más acentuado entre aquellos que tenían o habían tenido algún tipo de relación con el comercio del Pacífico, sirviendo en un principio como introductores y difusores.75 Resulta especialmente ilustrativo de la relación directa que se estable- ció entre aquellos personajes que tenían algún tipo de vinculación con Filipinas y el origen oriental de la mayoría de piezas que componían su ajuar doméstico, el caso del capitán Andrés de Acosta, quien en 1622 man- tenía relaciones con las islas e importaba cajones de mercaderías de China. Este comerciante, aparte de tener dos biombos entre sus bienes personales, contaba con un escritorio, un baúl, un escritorillo, un bufete y varias cajas de espejos de Japón, además de tres sobrecamas de la India bordadas de oro 55 Anu. estud. am., 69, 1, enero-junio, 2012, 31-62. ISSN: 0210-5810. DOI: 10.3989/aeamer.2012.1.01 ALBERTO BAENA ZAPATERO 76 AGN, Civil, 1998, 3. 82 Sato, 2002, 18, 31-34. ALBERTO BAENA ZAPATERO y seda, un pabellón de tafetán, un cajoncillo, quimonos y abanicos de China, e incluso una esclava china de catorce años.76 Los objetos incluidos en este inventario de bienes prueban la facilidad y el interés con el que un mercader afincado en Nueva España accedía a todo tipo de mercancías asiáticas.77 También entre los funcionarios enriquecidos hubo quien quiso acceder a estas piezas que el comercio internacional ponía a su disposición, como el tesorero de la Casa de la Moneda de la ciudad de México, Luis Moreno de Monroy, que en 1622 declaraba tener entre sus propiedades «un biobo grande y dos pequeños» valorados en 70 pesos;78 o la mujer del con- tador Francisco de Castro y Prado, que en 1694 poseía un «biobo de China con diez tablas».79 Ya en la Península, como vimos, los comerciantes sevi- llanos presentaban una situación muy similar. Entre las pertenencias con- signadas al capitán don Martín de la Mata, quien participase del tráfico con Nueva España durante gran parte de la segunda mitad del siglo XVII, encontramos un biombo de estrado de «18 hojas pintadas con la historia de Cortés y Moctezuma».80 Por último, en el lado japonés, los mercaderes enriquecidos con el comercio abierto con occidente fueron uno de los mayores grupos consu- midores de biombos namban.81 En este sentido, algunos autores han seña- lado cómo las pinturas más antiguas donde aparecen representados los por- tugueses llegando a Japón evidencian la temática comercial a través del énfasis puesto en la representación de las actividades mercantiles, resaltan- do la nao de trato portuguesa y los productos que se descargaban. De esta forma, el éxito de estas composiciones en los biombos de la escuela Kano reflejaría el gusto y los intereses de una clase mercantil emergente.82 Aparte de lo señalado, otro aspecto importante a tener en cuenta a la hora de estudiar el movimiento de personas y biombos a nivel mundial son las redes tejidas por las órdenes religiosas a través de cuatro continentes. Muchas obras de arte circularon entre Asia, América y Europa de la mano de los frailes mendicantes. Los jesuitas, responsables de la misión en 56 Anu. estud. am., 69, 1, enero-junio, 2012, 31-62. ISSN: 0210-5810. ALBERTO BAENA ZAPATERO DOI: 10.3989/aeamer.2012.1.01 EL MOVIMIENTO DE BIOMBOS DESDE EL PACÍFICO HASTA EL ATLÁNTICO Japón, adquirieron biombos durante su residencia en aquellas islas y los colocaron en sus celdas, como se indica en las «obedencias» de 1612 en las que se prohibía que los misioneros decoraran sus cuartos con este tipo de muebles.83 Es de suponer que, en sus cambios de residencia, muchos sirvie- ron de intermediarios en su divulgación por el mundo. Además, para el siglo XVIII hemos documentado biombos en varias haciendas jesuitas de Nueva España. El estudio detenido de las propiedades y encomiendas aso- ciadas a la Iglesia pueden revelar nuevos datos asociados al movimiento de objetos. 83 Boxer, 1974, 215. Anu. estud. am., 69, 1, enero-junio, 2012, 31-62. ISSN: 0210-5810. DOI: 10.3989/aeamer.2012.1.01 84 Balbuena, 2006, 91. Consideraciones finales La apertura y consolidación de dos rutas entre Europa y Asia vino incentivada por el desarrollo de una enorme demanda de productos orien- tales por parte de las sociedades occidentales. El análisis pormenorizado de las fuentes demuestra la importancia que tuvo el tráfico de objetos de lujo dentro del comercio internacional y cómo, entre los muebles más valorados y cotizados, se encontraron los biombos. Su transporte desde China o Japón hasta Portugal y Castilla a través de la India o de América, prueba la exis- tencia de una «primera mundialización» de las relaciones económicas con base en las coronas ibéricas. El largo camino recorrido por los biombos nos revela un conglomerado de conexiones que los imperios, si no las inventa- ron, sí las propiciaron o estimularon, creando las condiciones para que pudieran llevarse a cabo. Así, el tráfico de esta mercancía permite identifi- car los intermediarios más destacados del comercio con Manila, destacan- do portugueses, chinos y japoneses, además de los principales puertos des- de donde se importaban: Macao, Cantón y Nagasaki. Además, la ruta mundial seguida por los biombos nos permite deste- rrar el eurocentrismo propio de los análisis históricos más tradicionales. La importancia que llegó a adquirir el comercio a través del Galeón de Manila demuestra que Europa no siempre fue la explicación última de todos los fenómenos sino que en ocasiones fueron otras las regiones que llevaron la iniciativa o marcaron el volumen y características de los productos inclui- dos en los intercambios. Por lo tanto, el tráfico de biombos nos permite redefinir los términos de centro y periferia, ya que, en lo que se refiere a la 57 Anu. estud. am., 69, 1, enero-junio, 2012, 31-62. ISSN: 0210-5810. DOI: 10.3989/aeamer.2012.1.01 EL MOVIMIENTO DE BIOMBOS DESDE EL PACÍFICO HASTA EL ATLÁNTICO Por otra parte, la posible producción de biombos por artesanos de ori- gen chino o japonés en Manila nos pone en relación no sólo con el movi- miento de mercancías a través de estas rutas sino también con el significa- tivo flujo de personas que se dio entre estos territorios y su influencia en las producciones regionales. Del lado de los europeos, descubrimos un gru- po de funcionarios reales y comerciantes que se movieron por los dominios mundiales de la Monarquía Católica divulgando inconscientemente una cultura material nueva que unas veces resultaba simplemente ecléctica y otras sincrética. El estudio de los inventarios de bienes de estos personajes nos acerca la imagen de unos individuos que no sólo buscaron la riqueza inmediata sino que, además, muchos supieron valorar y apreciar las expre- siones artísticas de aquellos lugares por los que pasaron. Por lo tanto, los biombos fueron los testigos mudos del movimiento mundial de personas que la gestión política, económica o religiosa de los territorios que compo- nían el Imperio español puso en marcha. Presentado el 26 de junio de 2011 Aceptado el 1 de febrero de 2012 Presentado el 26 de junio de 2011 Aceptado el 1 de febrero de 2012 ALBERTO BAENA ZAPATERO DOI: 10.3989/aeamer.2012.1.01 EL MOVIMIENTO DE BIOMBOS DESDE EL PACÍFICO HASTA EL ATLÁNTICO ALBERTO BAENA ZAPATERO «ruta española» del Pacífico, el centro no fue Europa sino Nueva España, y fue precisamente el Consulado de México quien controló la Nao de China. Siendo esto así, cobran sentido los versos laudatorios de Bernardo de Balbuena quien, en su Grandeza Mexicana, reivindicaba el lugar de este territorio como ombligo del mundo: En ti se juntan España y China Italia con Japón, y finalmente un mundo entero en trato y disciplina.84 En ti se juntan España y China Italia con Japón, y finalmente un mundo entero en trato y disciplina.84 Ya sea como regalo diplomático o como mercancía de encargo, no es posible entender el éxito de los biombos en el tráfico mundial y su fácil integración a culturas diferentes si no se rompe con la «retórica de la alte- ridad». De esta forma, resulta necesario hacer hincapié en los puntos en común, las circulaciones de todo tipo y las conexiones entre los distintos lugares, por muy lejanos que parezcan. El comercio entre europeos y asiá- ticos se hizo en unas condiciones muy diferentes a las que se establecieron con las civilizaciones precolombinas conquistadas, China y Japón fueron valoradas desde occidente como culturas refinadas con las que era posible establecer una relación más o menos de igualdad. El resultado de este equi- librio fue la difusión de los biombos entre los ajuares domésticos de las familias principales de Asia, América y Europa, ejemplo de una cultura material de carácter mundial que compartía determinados valores asocia- dos a la búsqueda de prestigio u ostentación. Este fenómeno integrador propició que, independientemente de que variaran los temas representados en sus hojas, los biombos ocupasen un lugar preeminente en los espacios principales de los palacios de tres continentes. El nacimiento en el siglo XVII de una producción de biombos en Nueva España y su exportación al resto de América y Europa explican la vitalidad de los procesos propiciados por los intercambios económicos. De igual manera, el desarrollo posterior de otras manufacturas locales en América como las de Perú, Guatemala o Colombia, reflejan la madurez artística que para el siglo XVIII se había alcanzado en el continente. La asi- milación y reinterpretación de los biombos dentro de la cultura material americana dio como resultado unas dinámicas artísticas originales que se pueden asociar al gusto e identidad criollos. 58 Anu. estud. am., 69, 1, enero-junio, 2012, 31-62. ISSN: 0210-5810. Bibliografía Aguiló, Marí Paz: El mueble en España. Siglos XVI y XVII, Madrid, CSIC, 1993. Alva Rodríguez, Inmaculada: «La centuria desconocida: el siglo XVII» en Aguiló, Marí Paz: El mueble en España. 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Bonialian, Mariano: «México, epicentro semiinformal del comercio hispanoame- ricano (1680-1740)», América latina en la historia económica, 35, México, enero-junio de 2011, 7-28. 59 Anu. estud. am., 69, 1, enero-junio, 2012, 31-62. ISSN: 0210-5810. DOI: 10.3989/aeamer.2012.1.01 ALBERTO BAENA ZAPATERO Boxer, Charles: Fidalgos in the Far East (1550-1570), Hong Kong, Oxford University Press, 1968. Boxer, Charles: The Christian Century in Japan, 1549-1650, Berkeley, University of California Press, 1974. Calderón, Francisco R.: Historia económica de la Nueva España en tiempo de los Austrias, México, Fondo de Cultura Económica, 2003. Canepa, Teresa: «Obras de arte namban para os mercados japonês, português e holandês» en Welsh, Jorge e Vinhais, Luisa (eds.): Depois dos bárbaros II. Arte Namban para os mercados japonês, português e holandês, Londres, 2008, 14-29. Castelló, Teresa y Martínez, María Josefa: Biombos mexicanos, México, Instituto Nacional de Antropología e Historia, 1970. 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https://openalex.org/W1879648027	https://www.regepe.org.br/regepe/article/download/38/26	Portuguese	null	ORIENTAÇÃO EMPREENDEDORA COMO INDICADOR DO GRAU DE EMPREENDEDORISMO CORPORATIVO: FATORES QUE CARACTERIZAM OS INTRAEMPREENDEDORES E INFLUENCIAM SUA PERCEPÇÃO.	Revista de Empreendedorismo e Gestão de Pequenas Empresas	2,013	cc-by	11,064	ORIENTAÇÃO EMPREENDEDORA COMO INDICADOR DO GRAU DE EMPREENDEDORISMO CORPORATIVO: FATORES QUE CARACTERIZAM OS INTRAEMPREENDEDORES E INFLUENCIAM SUA PERCEPÇÃO ORIENTAÇÃO EMPREENDEDORA COMO INDICADOR DO GRAU DE EMPREENDEDORISMO CORPORATIVO: FATORES QUE CARACTERIZAM OS INTRAEMPREENDEDORES E INFLUENCIAM SUA PERCEPÇÃO Aurimar Barcelos França - FACCAMP1 Jeferson Saraiva - FACCAMP2 Marcos Hashimoto - FACCAMP3 Resumo: O presente artigo analisa a percepção dos colaboradores em relação ao nível de orientação empreendedora (OE) como um indicador apropriado para o diagnóstico e mensuração do grau de empreendedorismo corporativo. Trata-se de uma pesquisa exploratória, quantitativa, realizada com funcionários de uma empresa de médio porte. Os achados indicam que a percepção dos colaboradores é significativamente influenciada pela idade e tempo de serviço. Estes resultados ajudam a minimizar vieses de pesquisa e corroboram para que as empresas identifiquem com mais facilidade seus intraempreendedores e apoiem suas iniciativas inovadoras. Palavras-chave: Orientação Empreendedora (OE), Intraempreendedorismo, Empreendedorismo Corporativo. FRANÇA, A. B.; SARAIVA, J.; HASHIMOTO, M. Orientação empreendedora como indicador do grau de empreendedorismo corporativo: fatores que caracterizam os intraempreendedores e influenciam sua percepção. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n. 3, 2012. 78 1 E.mail: aurimar.franca@yahoo.com.br. Endereço: R. Mário Leandro Luiz de Faria, 429, CEP: 13214-593 - Jundiaí SP 2 E.mail: jefuza@gmail.com 3 E.mail: prof.hashimoto@uol.com.br ENTREPRENEURIAL ORIENTATION AS AN INDICATOR OF THE DEGREE OF CORPORATE ENTREPRENEURSHIP: FACTORS THAT FEATURING THE INTRAPRENEURS AND INFLUENCING THEIR PERCEPTION Abstract: This article examines the perceptions of employees relative to the level of entrepreneurial orientation (EO) as an appropriate indicator for the diagnosis and measurement of the degree of corporate entrepreneurship. This is an exploratory and quantitative research, performed with employees of a medium-sized company. The findings indicate that the perception of employees is significantly influenced by the age and time of service. These results help to minimize research biases and corroborate for companies more easily to identify their intrapreneurs and support their innovative initiatives. Keywords: Entrepreneurial Orientation (EO), Intrapreneurship, Corporate Entrepreneurship. 78 Introdução Algumas organizações que buscam melhorar o desempenho e gerar novos negócios encontram na sua capacidade de inovar, as oportunidades que propiciarão maior competitividade nos mercados em que atuam. O caminho da inovação não é fácil para muitas organizações cujos negócios não são baseados em tecnologia. Contudo, o escopo da inovação permite que qualquer empresa possa inovar por meio de mudanças nos seus processos e serviços nos diversos níveis da organização. Neste sentido, a Orientação Empreendedora (OE) representa um caminho viável para muitas destas organizações trilharem o caminho da inovação como fator de diferenciação e competitividade. A OE implica na implantação de ações que promovam um clima favorável para iniciativas de natureza empreendedora pelos seus colaboradores. Entretanto, não se pode generalizar um programa ou uma estratégia única e ideal de OE, também conhecida como Empreendedorismo Corporativo, pois as organizações são diferentes em seus modelos de negócios, histórias, cultura, processos e pessoas (HASHIMOTO, 2010). Nessa esteira, o objetivo do presente artigo é analisar a percepção dos colaboradores em relação ao nível de OE e as variáveis que influenciam essa percepção, a fim de tornar esse indicador apropriado para o diagnóstico e mensuração periódica dos avanços empreendedores de uma organização. FRANÇA, A. B.; SARAIVA, J.; HASHIMOTO, M. Orientação empreendedora como indicador do grau de empreendedorismo corporativo: fatores que caracterizam os intraempreendedores e influenciam sua percepção. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n. 3, 2012. 79 O sucesso de um programa que promova a OE depende de diversos fatores internos e externos, dentre eles destaca-se a atuação das pessoas envolvidas no processo, principalmente dos funcionários que estão no nível intermediário das organizações (HASHIMOTO, 2010), a necessidade de entender as variáveis que podem interferir na percepção desses colaboradores em relação à OE, ajudam na determinação de ações mais eficazes no processo de implantação destes programas e aumentam as chances de atingir os resultados esperados. Diversos autores concluíram que existe uma forte correlação positiva entre a OE e o desempenho das organizações (CHILD, 1972; COVIN; COVIN, 1989; COVIN; SLEVIN, 1990; ZAHRA; COVIN, 1995; DESS et al., 1997; WIKLUND, 1999; LUMPKIN; DESS, 2001; FRANÇA, A. B.; SARAIVA, J.; HASHIMOTO, M. Orientação empreendedora como indicador do grau de empreendedorismo corporativo: fatores que caracterizam os intraempreendedores e influenciam sua percepção. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n. 3, 2012. Introdução 79 79 SADLER-SMITH et al., 2003; RICHARD, 2004; TARABISHY et al., 2005; WIKLUND; SHEPHERD, 2005; FRISHMMAR; HÖRTE, 2007; HASHIMOTO, 2009). Portanto, faz sentido direcionar esforços para a medição periódica e confiável da OE. Convém ponderar que pouca ênfase tem sido dada na literatura sobre os fatores moderadores (RAUCH, A. et al., 2009) que influenciam a percepção dos empregados em relação à OE da organização.Determinar os fatores que afetam na medição do grau de empreendedorismo é fundamental para se obter resultados confiáveis e aceitos por toda a organização. As empresas que querem manter viva essa mentalidade empreendedora são aquelas em que a “auditoria da saúde empreendedora” é adotada e valorizada (IRELAND; KURATKO; MORRIS, 2006). Esse é um instrumento por meio do qual uma organização pode avaliar o grau em que seus funcionários estão preparados para se envolver em comportamento empreendedor através da inovação, a assunção de riscos, ações autônomas e proativas. Segundo Hashimoto (2009), a criação de condições para o surgimento de iniciativas empreendedoras por parte dos funcionários é fundamental na implantação de uma estratégia de OE. Por isso, as pessoas são fundamentais nesse processo e representam a base das observações empíricas do presente trabalho. Nessa linha, a pesquisa aqui apresentada foi estruturada para medir qual o nível de significância das variáveis independentes: idade, sexo, nível hierárquico, tempo de serviço e área de atuação dos colaboradores em relação à percepção da OE da organização. A figura 1 demonstra as relações que geraram as hipóteses desta pesquisa: FRANÇA, A. B.; SARAIVA, J.; HASHIMOTO, M. Orientação empreendedora como indicador do grau de empreendedorismo corporativo: fatores que caracterizam os intraempreendedores e influenciam sua percepção. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n. 3, 2012. 80 FRANÇA, A. B.; SARAIVA, J.; HASHIMOTO, M. Orientação empreendedora como indicador do grau de empreendedorismo corporativo: fatores que caracterizam os intraempreendedores e influenciam sua percepção. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n. 3, 2012. 80 80 FRANÇA, A. B.; SARAIVA, J.; HASHIMOTO, M. Orientação empreendedora como indicador do grau de empreendedorismo corporativo: fatores que caracterizam os intraempreendedores e influenciam sua percepção. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n. 3, 2012. 81 Figura 1. Mapa teórico dos fatores que influenciam a OE. Fonte: Elaborado pelos autores. Nível Hierárqui- co (H2) Tempo de serviço (H3) Área de atuação (H4) Gênero (H1) Idade (Ho) OE Desempenho da organização Impacta Figura 1. Mapa teórico dos fatores que influenciam a OE. Fonte: Elaborado pelos autores. Nível Hierárqui- co (H2) Tempo de serviço (H3) Área de atuação (H4) Gênero (H1) Idade (Ho) OE Desempenho da organização Impacta Idade (Ho) Impacta Desempenho da organização Fonte: Elaborado pelos autores. Fonte: Elaborado pelos autores. Para avaliar os fatores / variáveis estruturadas na figura 1, definiu-se as seguintes hipóteses: H0a: A percepção da OE não é significativamente influenciada pela idade dos colaboradores. H0a: A percepção da OE não é significativamente influenciada pela idade dos colaboradores. H0b: A idade dos colaboradores exerce influência sobre sua percepção da OE. H1a: A percepção da OE não é significativamente influenciada pelo gênero dos colaboradores. H1b: O gênero dos colaboradores exerce influência sobre sua percepção da OE. H1b: O gênero dos colaboradores exerce influência sobre sua percepção da OE. H3a: A percepção da OE não é significativamente influenciada pelo tempo de serviço dos colaboradores. H3b: O tempo de serviço dos colaboradores exerce influência sobre sua percepção da OE. H4a: A percepção da OE não é significativamente influenciada pela área de atuação dos colaboradores. H4b: A área de atuação dos colaboradores exerce influência sobre sua percepção da OE. H4b: A área de atuação dos colaboradores exerce influência sobre sua percepção da OE. As hipóteses foram testadas em uma amostra de funcionários de uma empresa familiar de capital fechado, fabricante de alimentos, sediada no estado de São Paulo. OE. H2a: A percepção da OE não é significativamente influenciada pelo nível hierárquico dos colaboradores. H2a: A percepção da OE não é significativamente influenciada pelo nível hierárquico dos colaboradores. H2b: O nível hierárquico dos colaboradores exerce influência sobre sua percepção da OE. 81 FRANÇA, A. B.; SARAIVA, J.; HASHIMOTO, M. Orientação empreendedora como indicador do grau de empreendedorismo corporativo: fatores que caracterizam os intraempreendedores e influenciam sua percepção. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n. 3, 2012. 82 Fundamentação Teórica De acordo com Hashimoto (2010, p. 1), “o primeiro uso do termo empreendedorismo foi registrado por Richard Cantilon, em 1755, para explicar a receptividade ao risco de comprar algo por um determinado preço e vendê-lo em um regime de incerteza”. Jean Baptiste Say em 1803 ampliou essa definição, dizendo que empreendedorismo está relacionado àquele que “transfere recursos econômicos de um setor de produtividade mais baixa para um setor de produtividade mais elevada e de maior rendimento”. Diversos autores ao longo do tempo têm estudado e definido o que é empreendedorismo sob diferentes perspectivas. Algumas correntes associavam empreendedores com inovação. Os economistas (tem-se como exemplo Joseph Schumpeter), definem o empreendedor como aquele que destrói a ordem econômica existente pela introdução de novos produtos e serviços, pela criação de novas formas de organização ou pela exploração de novos recursos e materiais, sob a ótica desse estudioso do tema dentro do enfoque econômico. Para ele, o empreendedor é a essência da inovação no mundo (SCHUMPETER, 1982).No contexto comportamentalista os estudiosos têm se concentrado nas características FRANÇA, A. B.; SARAIVA, J.; HASHIMOTO, M. Orientação empreendedora como indicador do grau de empreendedorismo corporativo: fatores que caracterizam os intraempreendedores e influenciam sua percepção. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n. 3, 2012. 82 Diversos autores ao longo do tempo têm estudado e definido o que é empreendedorismo sob diferentes perspectivas. Algumas correntes associavam empreendedores com inovação. Os economistas (tem-se como exemplo Joseph Schumpeter), definem o empreendedor como aquele que destrói a ordem econômica existente pela introdução de novos produtos e serviços, pela criação de novas formas de organização ou pela exploração de novos recursos e materiais, sob a ótica desse estudioso do tema dentro do enfoque econômico. Para ele, o empreendedor é a essência da inovação no mundo (SCHUMPETER, 1982).No contexto comportamentalista os estudiosos têm se concentrado nas características FRANÇA, A. B.; SARAIVA, J.; HASHIMOTO, M. Orientação empreendedora como indicador do grau de empreendedorismo corporativo: fatores que caracterizam os intraempreendedores e influenciam sua percepção. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n. 3, 2012. 82 82 de comportamento dos empreendedores como criação e intuição, por exemplo. Pode-se destacar McClelland (1972) dentro do grupo de autores que aponta como um dos traços mais importantes do empreendedor, a motivação de realização ou impulso para melhorar. Fundamentação Teórica Em seus estudos ele discutia o motivo que levava algumas sociedades a serem mais dinâmicas que outras.A Academia tem direcionado seus estudos no campo do empreendedorismo tanto para os indivíduos como para grupos e organizações empresariais. O empreendedorismo é concebido sob o escopo dos indivíduos, por estar geralmente associado à criação de algo novo, ou seja, uma invenção que tenha um valor reconhecido. Já no contexto das organizações, o empreendedorismo é visto como um meio de crescimento e renovação para organizações já existentes, na forma do empreendedorismo corporativo (GUTH; GINSBERG, 1990), mais recentemente denominado genericamente como Orientação Empreendedora, que se refere ao processo empreendedor no nível da organização. A OE emerge da perspectiva da escolha estratégica de Child (1972), o qual afirma que as oportunidades de desenvolvimento de novos negócios podem ser alcançadas de maneira bem sucedida e de forma intencional (VAN DE VEM; POOLE, 1995). Portanto, o conceito envolve as intenções e ações de atores-chave que atuam num processo generativo dinâmico voltado para a (re)criação de um negócio no nível da organização, onde se destacam os estudos de Miller & Friesen (1982) e Covin e Slevin (1990, 1991).A OE foi originalmente concebida por Miller (1983) por meio de três dimensões: receptividade a riscos, inovação e pró-atividade. Elas variam de forma independente uma da outra (COVIN, 2006) em força e efeito sobre o desempenho organizacional, e são comumente utilizadas para medir o nível de empreendedorismo das organizações (KREISER; MARINO; WEAVER, 2002). Pró-atividade é o processo de agir em antecipação de necessidades futuras, mudanças e desafios que podem levar a novas oportunidades (LUMPKIN; DESS, 1996). A receptividade a riscos é definida como se aventurar em território desconhecido ou circunstâncias sem saber quais serão os resultados (COVIN; SLEVIN, 1991). Finalmente, inovação reflete a unidade para lidar com atividades que resultem em novas ideias e com experimentos que podem levar a novos processos, produtos e serviços (LYON; LUMPKIN; DESS, 2000).O desenvolvimento FRANÇA, A. B.; SARAIVA, J.; HASHIMOTO, M. Orientação empreendedora como indicador do grau de empreendedorismo corporativo: fatores que caracterizam os de comportamento dos empreendedores como criação e intuição, por exemplo. Pode-se destacar McClelland (1972) dentro do grupo de autores que aponta como um dos traços mais importantes do empreendedor, a motivação de realização ou impulso para melhorar. Em seus estudos ele discutia o motivo que levava algumas sociedades a serem mais dinâmicas que outras.A Academia tem direcionado seus estudos no campo do empreendedorismo tanto para os indivíduos como para grupos e organizações empresariais. O empreendedorismo é concebido sob o escopo dos indivíduos, por estar geralmente associado à criação de algo novo, ou seja, uma invenção que tenha um valor reconhecido. Já no contexto das organizações, o empreendedorismo é visto como um meio de crescimento e renovação para organizações já existentes, na forma do empreendedorismo corporativo (GUTH; GINSBERG, 1990), mais recentemente denominado genericamente como Orientação Empreendedora, que se refere ao processo empreendedor no nível da organização. A OE emerge da perspectiva da escolha estratégica de Child (1972), o qual afirma que as oportunidades de desenvolvimento de novos negócios podem ser alcançadas de maneira bem sucedida e de forma intencional (VAN DE VEM; POOLE, 1995). Portanto, o conceito envolve as intenções e ações de atores-chave que atuam num processo generativo dinâmico voltado para a (re)criação de um negócio no nível da organização, onde se destacam os estudos de Miller & Friesen (1982) e Covin e Slevin (1990, 1991).A OE foi originalmente concebida por Miller (1983) por meio de três dimensões: receptividade a riscos, inovação e pró-atividade. Elas variam de forma independente uma da outra (COVIN, 2006) em força e efeito sobre o desempenho organizacional, e são comumente utilizadas para medir o nível de empreendedorismo das organizações (KREISER; MARINO; WEAVER, 2002). Pró-atividade é o processo de agir em antecipação de necessidades futuras, mudanças e desafios que podem levar a novas oportunidades (LUMPKIN; DESS, 1996). A receptividade a riscos é definida como se aventurar em território desconhecido ou circunstâncias sem saber quais serão os resultados (COVIN; SLEVIN, 1991). Finalmente, inovação reflete a unidade para lidar com atividades que resultem em novas ideias e com experimentos que podem levar a novos processos, produtos e serviços (LYON; LUMPKIN; DESS, 2000).O desenvolvimento FRANÇA, A. B.; SARAIVA, J.; HASHIMOTO, M. Orientação empreendedora como indicador do grau de empreendedorismo corporativo: fatores que caracterizam os intraempreendedores e influenciam sua percepção. FRANÇA, A. B.; SARAIVA, J.; HASHIMOTO, M. Orientação empreendedora como indicador do grau de empreendedorismo corporativo: fatores que caracterizam os intraempreendedores e influenciam sua percepção. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n. 3, 2012. 83 Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n. 3, 2012. 83 83 de numerosas tipologias do comportamento empreendedor sugere que a OE pode ser mais bem caracterizada por várias dimensões em diversas combinações presentes quando uma empresa se engaja na criação e expansão de um negócio. Lumpkin e Dess (1996) destacam que as três dimensões são fundamentais na compreensão do processo empreendedor para as quais, as combinações dependem do tipo de oportunidade empreendedora que a empresa procura e acrescentam mais duas dimensões, a autonomia e a agressividade competitiva. A autonomia se refere à liberdade e poder para implementar o que for necessário para antecipar e agir sobre uma oportunidade com perseverança, adaptabilidade e tolerância a erros, (MORRIS; ALLEN; SCHINDEHUTTE; AVILA, 2006) seja para a introdução de novos produtos e serviços no mercado ou a adoção de novas técnicas, processos e tecnologia (COVIN; MILES, 1999). Nesse contexto, o intraempreendedor tem independência para agir, define os objetivos e metas, toma decisões, busca e gerencia recursos, escolhe as estratégias de ação, traça planos de ação para alcançar resultados relevantes para o negócio (HASHIMOTO, 2009). Já a agressividade competitiva (COVIN; COVIN, 1989) reflete um intenso esforço de uma organização para superar seus rivais e está relacionada com a reação às ameaças do mercado, principalmente em relação à concorrência. Embora Lumpkin e Dess (1996) afirmem que as cinco dimensões da OE sejam centrais para entender o processo empreendedor, este poderá ocorrer em diferentes combinações em uma organização. Isto porque, deve-se ter em mente o tipo de oportunidade empreendedora que a organização persegue, ou mesmo apenas algumas dimensões podem estar presentes. Os fatores externos como, por exemplo, o ambiente de negócios, ou os fatores internos como a estrutura organizacional ou ainda, as características dos fundadores ou líderes da organização, poderão ser contingentes o quanto cada uma das dimensões será útil para predizer a natureza e o sucesso de um empreendimento. Assim, eles propõem a multidimensionalidade do constructo OE, no qual cada dimensão deve ser analisada de forma independente e nenhuma das dimensões seja obrigatória na caracterização da OE, nem sequer determinante. Morris (1998) corrobora com essa abordagem, apontando que a variação de cada uma das dimensões depende do contexto e da situação.A maioria FRANÇA, A. B.; SARAIVA, J.; HASHIMOTO, M. Orientação empreendedora como indicador do grau de empreendedorismo corporativo: fatores que caracterizam os intraempreendedores e influenciam sua percepção. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n. 3, 2012. 84 dos estudos sobre a OE tem adotado as três dimensões propostas por Miller (1983), entre eles: Covin e Slevin (1989, 1991); Covin e Covin (1990); Guth e Ginsbert (1990); Morris, Lewis e Sexton (1994); Zahra e Covin (1995); Wiklund (1999); Richard et al. (2004); Mello et al. (2004); Wiklund e Shepherd (2005); Covin, Green e Slevin (2006). A OE e o gerenciamento empreendedor são conceitos análogos utilizados para caracterizar uma organização empreendedora, ou seja, uma organização que possui uma postura empreendedora. Covin e Slevin (1989) afirmam que organizações com postura empreendedora promovem frequentemente, e de forma extensiva, a inovação em seus produtos e tecnologias. Elas são agressivamente competitivas e seus gerentes possuem propensão a assumir riscos calculados para implantar as mudanças.Lumpkin e Dess (1996), ao tratar da OE relacionada à performance, também destacam os fatores moderadores dessa relação. Eles apontam que fatores organizacionais e fatores ambientais influenciam a OE, que por sua vez, reflete na performance da organização. Em sentido semelhante, Miller (1983) aponta que o empreendedorismo é integralmente relacionado às variáveis de ambiente, estrutura, estratégia e personalidade do líder, e que este relacionamento varia sistemática e logicamente de um tipo de organização para outro. E ainda, na amplitude de estudos empíricos sugerem que a orientação empreendedora da empresa provém da liderança e da orientação de seus principais executivos (SADLER-SMITH et al., 2003; TARABISHY et al., 2005). A teoria de gestão estratégica também atesta que os gerentes de alto escalão têm impacto efetivo na formulação estratégica (GRONHAUG, KAUFMANN, 1988; WIKLUND, 1999). É bem verdade que estratégias empresariais devem construir uma cultura voltada para o desenvolvimento do intraempreendedorismo ou empreendedorismo corporativo, que é visto como um processo de estimular ideias inovadoras e processos (SCOTT; ROSA; KLANDT, 1998), muitas vezes com foco na criação de riqueza. Empreendedorismo corporativo é um tema de atração para muitos gestores em empresas e tornou-se um assunto de interesse devido ao seu efeito positivo sobre o desempenho das organizações. Pesquisas anteriores (MILLER, 1983; KURATKO et al., 1990; GUTH; GINSBERG, 1990) identificaram um conjunto de pré-requisitos para o dos estudos sobre a OE tem adotado as três dimensões propostas por Miller (1983), entre eles: Covin e Slevin (1989, 1991); Covin e Covin (1990); Guth e Ginsbert (1990); Morris, Lewis e Sexton (1994); Zahra e Covin (1995); Wiklund (1999); Richard et al. (2004); Mello et al. (2004); Wiklund e Shepherd (2005); Covin, Green e Slevin (2006). FRANÇA, A. B.; SARAIVA, J.; HASHIMOTO, M. Orientação empreendedora como indicador do gra de empreendedorismo corporati o fatores q e caracteri am os de numerosas tipologias do comportamento empreendedor sugere que a OE pode ser mais bem caracterizada por várias dimensões em diversas combinações presentes quando uma empresa se engaja na criação e expansão de um negócio. Lumpkin e Dess (1996) destacam que as três dimensões são fundamentais na compreensão do processo empreendedor para as quais, as combinações dependem do tipo de oportunidade empreendedora que a empresa procura e acrescentam mais duas dimensões, a autonomia e a agressividade competitiva. A autonomia se refere à liberdade e poder para implementar o que for necessário para antecipar e agir sobre uma oportunidade com perseverança, adaptabilidade e tolerância a erros, (MORRIS; ALLEN; SCHINDEHUTTE; AVILA, 2006) seja para a introdução de novos produtos e serviços no mercado ou a adoção de novas técnicas, processos e tecnologia (COVIN; MILES, 1999). Nesse contexto, o intraempreendedor tem independência para agir, define os objetivos e metas, toma decisões, busca e gerencia recursos, escolhe as estratégias de ação, traça planos de ação para alcançar resultados relevantes para o negócio (HASHIMOTO, 2009). Já a agressividade competitiva (COVIN; COVIN, 1989) reflete um intenso esforço de uma organização para superar seus rivais e está relacionada com a reação às ameaças do mercado, principalmente em relação à concorrência. Embora Lumpkin e Dess (1996) afirmem que as cinco dimensões da OE sejam centrais para entender o processo empreendedor, este poderá ocorrer em diferentes combinações em uma organização. Isto porque, deve-se ter em mente o tipo de oportunidade empreendedora que a organização persegue, ou mesmo apenas algumas dimensões podem estar presentes. Os fatores externos como, por exemplo, o ambiente de negócios, ou os fatores internos como a estrutura organizacional ou ainda, as características dos fundadores ou líderes da organização, poderão ser contingentes o quanto cada uma das dimensões será útil para predizer a natureza e o sucesso de um empreendimento. Assim, eles propõem a multidimensionalidade do constructo OE, no qual cada dimensão deve ser analisada de forma independente e nenhuma das dimensões seja obrigatória na caracterização da OE, nem sequer determinante. Morris (1998) corrobora com essa abordagem, apontando que a variação de cada uma das dimensões depende do contexto e da situação.A maioria 84 A OE e o gerenciamento empreendedor são conceitos análogos utilizados para caracterizar uma organização empreendedora, ou seja, uma organização que possui uma postura empreendedora. Covin e Slevin (1989) afirmam que organizações com postura empreendedora promovem frequentemente, e de forma extensiva, a inovação em seus produtos e tecnologias. Elas são agressivamente competitivas e seus gerentes possuem propensão a assumir riscos calculados para implantar as mudanças.Lumpkin e Dess (1996), ao tratar da OE relacionada à performance, também destacam os fatores moderadores dessa relação. Eles apontam que fatores organizacionais e fatores ambientais influenciam a OE, que por sua vez, reflete na performance da organização. Em sentido semelhante, Miller (1983) aponta que o empreendedorismo é integralmente relacionado às variáveis de ambiente, estrutura, estratégia e personalidade do líder, e que este relacionamento varia sistemática e logicamente de um tipo de organização para outro. E ainda, na amplitude de estudos empíricos sugerem que a orientação empreendedora da empresa provém da liderança e da orientação de seus principais executivos (SADLER-SMITH et al., 2003; TARABISHY et al., 2005). A teoria de gestão estratégica também atesta que os gerentes de alto escalão têm impacto efetivo na formulação estratégica (GRONHAUG, KAUFMANN, 1988; WIKLUND, 1999). É bem verdade que estratégias empresariais devem construir uma cultura voltada para o desenvolvimento do intraempreendedorismo ou empreendedorismo corporativo, que é visto como um processo de estimular ideias inovadoras e processos (SCOTT; ROSA; KLANDT, 1998), muitas vezes com foco na criação de riqueza. Empreendedorismo corporativo é um tema de atração para muitos gestores em empresas e tornou-se um assunto de interesse devido ao seu efeito positivo sobre o desempenho das organizações. Pesquisas anteriores (MILLER, 1983; KURATKO et al., 1990; GUTH; GINSBERG, 1990) identificaram um conjunto de pré-requisitos para o Pesquisas anteriores (MILLER, 1983; KURATKO et al., 1990; GUTH; GINSBERG, 1990) identificaram um conjunto de pré-requisitos para o FRANÇA, A. B.; SARAIVA, J.; HASHIMOTO, M. Orientação empreendedora como indicador do grau de empreendedorismo corporativo: fatores que caracterizam os intraempreendedores e influenciam sua percepção. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n. 3, 2012. 85 85 86 86 percepções do ambiente competitivo e da OE se correlacionam de forma diferenciada em empresas familiares, dependendo da geração no comando, e é geralmente mais forte em empresas de segunda geração da família. Estudos anteriores sugerem que fatores demográficos como gênero (HISRICH; O'BRIEN, 1981; SEXTON; BOWMAN-UPTON, 1990; CHAGANTI; PARASUMAN, 1996), idade (COOPER, 1973; HOWELL, 1972; SHAPERO, 1971), etnia (WALDINGER et al., 1990;. CHAGANTI; GREENE, 2002), nacionalidade (HOFSTEDE, 1980), experiência de trabalho (TIMMONS; SPINELLI, 2007) e experiência empresarial (KRUEGER, 1993; BROCKHAUS, 1982), podem ser importantes para entender as ações e intenções empreendedoras dos indivíduos dentro ou fora das organizações.Para Rauch, A. et al. (2009), a variância dos resultados em estudos de OE devem levar em consideração a melhoria das escalas de medição, ou examinar fatores moderadores relevantes que podem afetar a relação entre OE e o desempenho da organização. Potenciais variáveis moderadoras incluem a idade da empresa, o dinamismo ambiental, cultura nacional, a estratégia perseguida e estrutura organizacional. O estudo feito por estes autores sugere que é hora de abrir as pesquisas sobre OE para novas ideias e continuar analisando o papel dessas variáveis moderadoras (ver LUMPKIN; DESS, 1996), aumentando assim, a compreensão do espírito empresarial e reforçando a base científica. percepções do ambiente competitivo e da OE se correlacionam de forma diferenciada em empresas familiares, dependendo da geração no comando, e é geralmente mais forte em empresas de segunda geração da família. Estudos anteriores sugerem que fatores demográficos como gênero (HISRICH; O'BRIEN, 1981; SEXTON; BOWMAN-UPTON, 1990; CHAGANTI; PARASUMAN, 1996), idade (COOPER, 1973; HOWELL, 1972; SHAPERO, 1971), etnia (WALDINGER et al., 1990;. CHAGANTI; GREENE, 2002), nacionalidade (HOFSTEDE, 1980), experiência de trabalho (TIMMONS; SPINELLI, 2007) e experiência empresarial (KRUEGER, 1993; BROCKHAUS, 1982), podem ser importantes para entender as ações e intenções empreendedoras dos indivíduos dentro ou fora das organizações.Para Rauch, A. et al. (2009), a variância dos resultados em estudos de OE devem levar em consideração a melhoria das escalas de medição, ou examinar fatores moderadores relevantes que podem afetar a relação entre OE e o desempenho da organização. Potenciais variáveis moderadoras incluem a idade da empresa, o dinamismo ambiental, cultura nacional, a estratégia perseguida e estrutura organizacional. O estudo feito por estes autores sugere que é hora de abrir as pesquisas sobre OE para novas ideias e continuar analisando o papel dessas variáveis moderadoras (ver LUMPKIN; DESS, 1996), aumentando assim, a compreensão do espírito empresarial e reforçando a base científica. FRANÇA, A. B.; SARAIVA, J.; HASHIMOTO, M. Orientação empreendedora como indicador do grau de empreendedorismo corporativo: fatores que caracterizam os empreendedorismo corporativo, relacionados com o ambiente (ameaças e oportunidades), o comportamento de alta e média gestão (visão, apoio, empenho e estilo), a organização (cultura, estratégia, estrutura e modos de trabalho) e desempenho (HEINONEN, 1999). Esses fatores são amplamente discutidos na literatura e reconhecidos como importantes na promoção do empreendedorismo corporativo (GUTH E GINSBERG, 1990; COVIN; SLEVIN, 1991; ZAHRA, 1991; HORNSBY et al., 1993; THOMPSON, 1999; ANTONCIC; HISRICH, 2001; KURATKO et al., 2005), em especial, o papel vital dos gerentes de nível médio em criar um ambiente que encoraje a inovação e empreendedorismo interno (HORNSBY et al., 2002; KURATKO et al., 2005). Veenker et al. (2008) estudaram a relação entre características organizacionais e as condições presentes no empreendedorismo corporativo em organizações holandesas e concluíram os fatores: a centralização, a cooperação entre colegas, a cooperação entre os departamentos e a formalização, interferem na percepção do empreendedorismo corporativo.Existem ainda outros fatores, como por exemplo: as experiências empreendedoras anteriores que afetam diretamente a viabilidade percebida e conveniência das ações empreendedoras (KRUEGER, 1993). Estudos também consideram que vieses cognitivos, tais como excesso de confiança e ilusão de controle, influenciam diretamente as percepções relacionadas ao empreendedorismo e a percepção de risco (KEH, et al., 2002; SIMON et al., 2000). Outras pesquisas sobre cultura e empreendedorismo sugerem que os valores culturais como: a distância de alta direção, o individualismo, a baixa incerteza, podem aumentar a viabilidade percebida e a conveniência de potenciais oportunidades empreendedoras (BUSENITZ; LAU, 1996; MCGRATH; MACMILLAN, 1992; MCGRATH et al., 1992, MITCHELL et al., 2002).Traços da personalidade também podem influenciar a percepção do indivíduo em relação às oportunidades empreendedoras e contribuem para uma melhor compreensão do processo de tomada de decisão (YAN, 2010). Existe também uma interação entre conhecimento, aprendizagem e cognição do indivíduo nas atividades e ações para o processo empreendedor (HAYNIE et al., 2012).Um estudo recente sobre orientação empreendedora desenvolvido por Cruz e Nordqvist (2012), mostra que as FRANÇA, A. B.; SARAIVA, J.; HASHIMOTO, M. Orientação empreendedora como indicador do grau de empreendedorismo corporativo: fatores que caracterizam os intraempreendedores e influenciam sua percepção. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n. 3, 2012. número total de colaboradores da organização pesquisada. Segundo Hashimoto (2010), para que a amostra que identifica a OE seja válida, a população estudada deve ter representatividade. Para essa estratificação, os respondentes foram classificados e codificados de acordo com o perfil de cada um respectivamente, dentro das categorias descritas na tabela 1. número total de colaboradores da organização pesquisada. Segundo Hashimoto (2010), para que a amostra que identifica a OE seja válida, a população estudada deve ter representatividade. Para essa estratificação, os respondentes foram classificados e codificados de acordo com o perfil de cada um respectivamente, dentro das categorias descritas na tabela 1. Tabela 1 - Perfil dos respondentes Perfil dos respondentes Idade Códigos % Entre 20 e 29 anos 1 28 Entre 30 e 39 anos 2 67 Entre 40 e 49 anos 3 3 Superior a 50 anos 4 3 Gênero Códigos % Feminino 0 31 Masculino 1 69 Nível hierárquico Códigos % Gerência/Coordenação 1 8 Supervisão 2 14 Operacional 3 78 Tempo de serviço na organização Códigos % Menor ou igual a 1 ano 1 17 Entre 1 e 5 anos 2 42 Entre 6 e 10 anos 3 16 Entre 11 e 20 anos 4 6 Superior a 20 anos 5 0 Áreas de atuação Códigos % Administrativa 1 8 Industrial 2 56 Qualidade 3 28 Engenharia 4 8 Fonte: Os Autores. FRANÇA, A. B.; SARAIVA, J.; HASHIMOTO, M. Orientação empreendedora como indicador do grau de empreendedorismo corporativo: fatores que caracterizam os intraempreendedores e influenciam sua percepção. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n. 3, 2012. 88 A empresa escolhida é familiar, de primeira geração, capital fechado, fabricante de alimentos e está no mercado a mais de 40 anos. Atualmente, conta com duas unidades fabris, dois centros de distribuição e um faturamento anual de 80 a 120 milhões de reais. O estudo foi realizado entre os meses de Dezembro de 2011 a Janeiro de 2012. A unidade pesquisada está sediada no interior do estado de São Paulo e possui em torno 350 colaboradores diretos.As proposições utilizadas no questionário foram adaptadas, testadas e aplicadas por Hashimoto (2009). Estão embasadas na literatura científica considerando o questionário de OE para mensurar a postura estratégica das organizações, com base em medidas desenvolvidas por A empresa escolhida é familiar, de primeira geração, capital fechado, fabricante de alimentos e está no mercado a mais de 40 anos. Metodologia FRANÇA, A. B.; SARAIVA, J.; HASHIMOTO, M. Orientação empreendedora como indicador do grau de empreendedorismo corporativo: fatores que caracterizam os intraempreendedores e influenciam sua percepção. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n. 3, 2012. 87 O presente estudo refere-se a uma investigação de natureza exploratória que examina a relação entre o perfil do colaborador e sua percepção da OE de uma organização. Foi adotado o método de pesquisa quantitativo, no qual os dados empíricos foram coletados através de um questionário com questões de múltipla escolha em escala Likert de 5 pontos. Cada respondente assinalou a opção mais adequada com relação às proposições descritas, dando nota de 1 a 5, sendo 1 igual a discordo totalmente e 5 concordo totalmente. A amostra foi composta por 36 respondentes selecionados aleatoriamente e convidados individualmente a emitir sua percepção com relação às proposições. Essa amostra representa 10,29% do O presente estudo refere-se a uma investigação de natureza exploratória que examina a relação entre o perfil do colaborador e sua percepção da OE de uma organização. Foi adotado o método de pesquisa quantitativo, no qual os dados empíricos foram coletados através de um questionário com questões de múltipla escolha em escala Likert de 5 pontos. Cada respondente assinalou a opção mais adequada com relação às proposições descritas, dando nota de 1 a 5, sendo 1 igual a discordo totalmente e 5 concordo totalmente. A amostra foi composta por 36 respondentes selecionados aleatoriamente e convidados individualmente a emitir sua percepção com relação às proposições. Essa amostra representa 10,29% do FRANÇA, A. B.; SARAIVA, J.; HASHIMOTO, M. Orientação empreendedora como indicador do grau de empreendedorismo corporativo: fatores que caracterizam os intraempreendedores e influenciam sua percepção. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n. 3, 2012. 87 87 Atualmente, conta com duas unidades fabris, dois centros de distribuição e um faturamento anual de 80 a 120 milhões de reais. O estudo foi realizado entre os meses de Dezembro de 2011 a Janeiro de 2012. A unidade pesquisada está sediada no interior do estado de São Paulo e possui em torno 350 colaboradores diretos.As proposições utilizadas no questionário foram adaptadas, testadas e aplicadas por Hashimoto (2009). Estão embasadas na literatura científica considerando o questionário de OE para mensurar a postura estratégica das organizações, com base em medidas desenvolvidas por FRANÇA, A. B.; SARAIVA, J.; HASHIMOTO, M. Orientação empreendedora como indicador do grau de empreendedorismo corporativo: fatores que caracterizam os intraempreendedores e influenciam sua percepção. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n. 3, 2012. 88 88 A escala utilizada nesta pesquisa se concentra em quatro dimensões da OE: receptividade a riscos, inovação, pró-atividade e autonomia. As variáveis independentes: idade, gênero, nível hierárquico, tempo de serviço e área de atuação dos colaboradores, foram analisadas em relação às variáveis dependentes que fizeram parte deste constructo, sendo formatadas em 14 proposições (P01 a P14) para mensurar o nível de OE da organização, conforme apresentado na Tabela 2. Tabela 2- Proposições utilizadas no questionário Tabela 2 Proposições utilizadas no questionário Fonte: Adaptado de Hashimoto (2009). Código Proposições P01 Sou estimulado (a) a contribuir para melhorar a forma como é feito o meu trabalho. P02 Sei o que devo fazer para crescer profissionalmente nesta empresa. P03 Esta empresa ouve e coloca em prática as sugestões de seus funcionários. P04 Tenho pouca liberdade para contribuir com críticas e sugestões ao (à) meu (minha) chefe. P05 Sinto-me apoiado (a) pelo meu (minha) chefe quando decido assumir novos desafios. P06 Participo pouco das decisões que afetam a mim e ao meu trabalho. P07 Os (As) chefes sabem demonstrar como podemos contribuir com os objetivos da empresa. P08 As pessoas que trabalham nesta empresa tem dificuldade em saber o que devem fazer para torná-la cada vez melhor. P09 Meu (Minha) chefe ouve e respeita a opinião da sua equipe. P10 As pessoas que trabalham na empresa estão sempre procurando inovar aquilo que fazem. P11 Sinto que nesta empresa as pessoas podem assumir riscos quando tentam promover melhorias no seu trabalho. P12 Nesta empresa as pessoas têm autonomia para tomar decisões sem precisar consultar o (a) chefe. avaliaram se as proposições pertenciam ou não ao tema e se não existiam ambiguidades. Todas as proposições foram validadas e aprovadas. Utilizou-se a análise do alfa de Cronbach para verificar a integridade interna e a consistência do questionário. Para analisar os dados obtidos através das respostas e testar as hipóteses formuladas, utilizou-se regressão linear simples e teste de correlação linear de Pearson. P13 Sou reconhecido (a) e recompensado (a) quando tenho iniciativas inovadoras. P14 Nesta empresa há pouca tolerância aos erros cometidos pelos empregados (as). FRANÇA A B ; SARAIVA J ; HASHIMOTO M Orientação empreendedora como indicador As proposições P04, P06, P08 e P14, foram invertidas negativamente para evitar vieses e aumentar a confiabilidade das respostas.Foram usadas técnicas estatísticas para testar as hipóteses definidas à luz das teorias atuais existentes sobre OE. Os dados foram trabalhados em planilhas Excel e nos softwares SPSS e Bioestat. A validação do questionário foi realizada por meio da análise de quatro Juízes especialistas e estudiosos do tema empreendedorismo e administração, que FRANÇA, A. B.; SARAIVA, J.; HASHIMOTO, M. Orientação empreendedora como indicador do grau de empreendedorismo corporativo: fatores que caracterizam os intraempreendedores e influenciam sua percepção. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n. 3, 2012. 89 As proposições P04, P06, P08 e P14, foram invertidas negativamente para evitar vieses e aumentar a confiabilidade das respostas.Foram usadas técnicas estatísticas para testar as hipóteses definidas à luz das teorias atuais existentes sobre OE. Os dados foram trabalhados em planilhas Excel e nos softwares SPSS e Bioestat. A validação do questionário foi realizada por meio da análise de quatro Juízes especialistas e estudiosos do tema empreendedorismo e administração, que FRANÇA, A. B.; SARAIVA, J.; HASHIMOTO, M. Orientação empreendedora como indicador do grau de empreendedorismo corporativo: fatores que caracterizam os intraempreendedores e influenciam sua percepção. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n. 3, 2012. 89 do grau de empreendedorismo corporativo: fatores que caracterizam os intraempreendedores e influenciam sua percepção. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n. 3, 2012. 89 89 FRANÇA, A. B.; SARAIVA, J.; HASHIMOTO, M. Orientação empreendedora como indicador do grau de empreendedorismo corporativo: fatores que caracterizam os intraempreendedores e influenciam sua percepção. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n. 3, 2012. 90 explicada pela variável preditiva, devendo outros fatores atuar como preditores da OE. Quadro 1 - Resultados do teste de regressão linear simples – variáveis: idade e gênero. Nível de significância p < 0,05. Variáveis independentes Idade Gênero F (regressão) 4,8952 / p = 0,0318 0,04161 / p = 0,8257 R2 (ajustado) 0,1001 -0,0280 Coeficient e de regressão -3,6741 / t = -2,2125 / p = 0,0337 -0,5091 / t = -0,2147 / p = 0,8313 Fonte: Os Autores. Quadro 1 - Resultados do teste de regressão linear simples – variáveis: idade e gênero. Nível de significância p < 0,05. Fonte: Os Autores. Analisando o gênero dos colaboradores, o valor de F (regressão) não é significativo (Tabela 2, p = 0.8257). Aceitando-se a hipótese de nulidade (H1a) e rejeitando a hipótese H1b, percebe-se que a OE não é significativamente influenciada pelo gênero dos colaboradores.Para os níveis hierárquicos pesquisados (Gerência e Coordenação, Supervisão e Operacional), o valor de F (regressão) não é significativo (Tabela 3, p = 0.2095), aceitando-se a hipótese de nulidade (H2a) e rejeitando a hipótese H2b, constatando-se que a percepção da OE não é significativamente influenciada pelo nível hierárquico dos colaboradores. Quadro 2 - Resultados do teste de regressão linear simples – variáveis: nível hierárquico e tempo de serviço. Nível de significância p < 0,05. Variáveis independentes Nível hierárquico Tempo de serviço F (regressão) 1,6186 / p = 0,2095 5,4487 / p = 0,0242* R2 (ajustado) 0,0174 0,1128 Coeficien te de regressão 2,2077 / t = 1,2722 / p = 0,2118 -0,29236 / t = -2,3342 / p = 0,0256 Fonte: Os Autores. FRANÇA, A. B.; SARAIVA, J.; HASHIMOTO, M. Orientação empreendedora como indicador do grau de empreendedorismo corporativo: fatores que caracterizam os intraempreendedores e influenciam sua percepção. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n. 3, 2012. 91 Avaliando o tempo de serviço em relação ao nível de OE, o valor de F (regressão) é muito significativo (Tabela 3, p = 0.0242), rejeitando-se a hipótese de nulidade (H3a) e confirmando-se a hipótese H3b, ou seja, a percepção da OE é influenciada pelo nível hierárquico dos colaboradores. Constata-se que o nível de OE diminui na medida em que o tempo de serviço aumenta. Análise dos Resultados A verificação da integridade interna do instrumento de coleta de dados utilizado apresentou um alfa de Cronbach de 0,71, validando o questionário utilizado, pois um valor de α de pelo menos 0,7 reflete uma fidedignidade aceitável (CRONBACH, 1951; NUNNALLY, 1978). Os respondentes atribuíram notas de 1 a 5 para cada uma das 14 proposições descritas no questionário. Portanto, o nível de OE da organização pode ser expresso e atingir as pontuações:  Nível baixo de OE: notas de 14 a 28 pontos.  Nível médio de OE: notas de 29 a 42 pontos.  Nível alto de OE: notas de 43 a 70 pontos. Analisando as notas atribuídas pelos respondentes para cada uma das 14 proposições, chega-se ao nível de OE da organização que, na média geral, ficou em 48,56 pontos, demonstrando que há um nível alto de OE. Dos 36 respondentes, 19,4% acredita que a organização está com um nível médio de OE e 80,6% acredita que a organização tem um nível alto de OE. Constata-se desta forma, que a maioria dos respondentes percebe a empresa como sendo intraempreendedora.Com relação à variável independente idade dos colaboradores, o valor de F (regressão) é significativo (Tabela 2, p = 0.0318), rejeitando-se a hipótese de nulidade (H0a) e confirmando-se a hipótese H0b. Nessa linha, nota-se que a percepção da OE é influenciada pela idade dos colaboradores. Verificou-se que o nível de OE diminui na medida em que a idade do colaborador aumenta e pelo coeficiente de determinação ajustado (R2 = 0.1001), verifica-se que somente 10,01% da variável dependente é FRANÇA, A. B.; SARAIVA, J.; HASHIMOTO, M. Orientação empreendedora como indicador do grau de empreendedorismo corporativo: fatores que caracterizam os intraempreendedores e influenciam sua percepção. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n. 3, 2012. 90 90 determinação ajustado (R2 = 0.1128), verifica-se que somente 11,28% da variável dependente é explicada pela variável preditiva, devendo outros fatores atuar como preditores da OE.Quando se correlaciona o grau de percepção da OE com a área de atuação dos colaboradores (Tabela 4), verifica-se que os resultados mostram fraca correlação positiva entre o nível de OE e as áreas contempladas na pesquisa (r = 0.1322 e p = 0.4422), aceitando-se assim, a hipótese de nulidade H4a: A percepção da OE não varia significativamente entre as diferentes áreas de atuação dos colaboradores respondentes. determinação ajustado (R2 = 0.1128), verifica-se que somente 11,28% da variável dependente é explicada pela variável preditiva, devendo outros fatores atuar como preditores da OE.Quando se correlaciona o grau de percepção da OE com a área de atuação dos colaboradores (Tabela 4), verifica-se que os resultados mostram fraca correlação positiva entre o nível de OE e as áreas contempladas na pesquisa (r = 0.1322 e p = 0.4422), aceitando-se assim, a hipótese de nulidade H4a: A percepção da OE não varia significativamente entre as diferentes áreas de atuação dos colaboradores respondentes. Quadro 3 - Teste correlação linear de Pearson. Teste de correlação linear de Pearson entre OE e área de atuação n (pares) 36 R (Pearson) 0,1322 IC 95% -0,21 a 0,44 IC 99% -0,31 a 0,52 R2 0,0175 T 0,7774 GL 34 (p) 0,4422 Poder 0,05 0,1885 Poder 0,01 0,0498 Fonte: Os Autores. Conclusões Pelo coeficiente de Avaliando o tempo de serviço em relação ao nível de OE, o valor de F (regressão) é muito significativo (Tabela 3, p = 0.0242), rejeitando-se a hipótese de nulidade (H3a) e confirmando-se a hipótese H3b, ou seja, a percepção da OE é influenciada pelo nível hierárquico dos colaboradores. Constata-se que o nível de OE diminui na medida em que o tempo de serviço aumenta. Pelo coeficiente de FRANÇA, A. B.; SARAIVA, J.; HASHIMOTO, M. Orientação empreendedora como indicador do grau de empreendedorismo corporativo: fatores que caracterizam os intraempreendedores e influenciam sua percepção. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n. 3, 2012. 91 FRANÇA, A. B.; SARAIVA, J.; HASHIMOTO, M. Orientação empreendedora como indicador do grau de empreendedorismo corporativo: fatores que caracterizam os intraempreendedores e influenciam sua percepção. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n. 3, 2012. 91 91 Conclusões FRANÇA, A. B.; SARAIVA, J.; HASHIMOTO, M. Orientação empreendedora como indicador do grau de empreendedorismo corporativo: fatores que caracterizam os intraempreendedores e influenciam sua percepção. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n. 3, 2012. 92 A proposta descrita nesse artigo está baseada em uma pesquisa de campo, onde se analisou as correlações e o nível de significância entre grau de percepção da OE dos diferentes atores dentro de uma organização familiar de médio porte.De acordo com os resultados desta pesquisa, a percepção da OE na organização pode variar também de acordo com a idade e o tempo de serviço dos colaboradores, sendo que, quanto maior a idade e/ou o tempo de serviço, menor o grau de percepção da OE da organização. Isso significa que os colaboradores mais velhos e os mais antigos de casa tendem a ser mais orientados à atitude empreendedora (HASHIMOTO, 2009). Contudo, a variável moderadora experiência de trabalho pode A proposta descrita nesse artigo está baseada em uma pesquisa de campo, onde se analisou as correlações e o nível de significância entre grau de percepção da OE dos diferentes atores dentro de uma organização familiar de médio porte.De acordo com os resultados desta pesquisa, a percepção da OE na organização pode variar também de acordo com a idade e o tempo de serviço dos colaboradores, sendo que, quanto maior a idade e/ou o tempo de serviço, menor o grau de percepção da OE da organização. Isso significa que os colaboradores mais velhos e os mais antigos de casa tendem a ser mais orientados à atitude empreendedora (HASHIMOTO, 2009). Contudo, a variável moderadora experiência de trabalho pode 92 influenciar negativamente na percepção de oportunidade de um novo empreendimento (YAN, 2010). Esse fato leva a uma suposição de que a percepção da OE também tende a ser mais criteriosa e exigente na medida em que os funcionários adquirem mais experiência, levando-os a atribuir notas mais baixas quando questionados sobre o grau de empreendedorismo da organização.Esses resultados corroboram com a teoria de que o grau de OE pode ser influenciado por diversos fatores, entre eles: a idade, tempo de casa, gênero (sexo), nível hierárquico, salário e nível de instrução dos colaboradores. Isto sem considerar a influência do ambiente externo, visto que tais variáveis dificilmente podem ser isoladas para uma avaliação mais isenta e livre de vieses (HASHIMOTO, 2009; ZAHRA; COVIN, 1995; ANTONCIC; HISRICH 2001).As hipóteses determinadas foram testadas a um nível de significância de 5% e o resumo da análise das hipóteses contra os resultados obtidos estão na Tabela 3. Tabela 3 - Resumo da análise das hipóteses. Hipótese Descrição Conclusão H0a A percepção da OE não é significativamente influenciada pela idade dos colaboradores. Rejeitada H0b A idade dos colaboradores exerce influência sobre sua percepção da OE. Não rejeitada H1a A percepção da OE não é significativamente influenciada pelo gênero dos colaboradores. Não rejeitada H1b O gênero dos colaboradores exerce influência sobre sua percepção da OE. Rejeitada H2a A percepção da OE não é significativamente influenciada pelo nível hierárquico dos colaboradores. Não rejeitada H2b O nível hierárquico dos colaboradores exerce influência sobre sua percepção da OE. Rejeitada H3a A percepção da OE não é significativamente influenciada pelo tempo de serviço dos colaboradores. Rejeitada H3b O tempo de serviço dos colaboradores exerce influência sobre sua percepção da OE. Não rejeitada H4a A percepção da OE não é significativamente influenciada pela área de atuação dos colaboradores. Não rejeitada H4b A área de atuação dos colaboradores exerce influência sobre sua percepção da OE. Rejeitada Fonte: Os Autores. FRANÇA, A. B.; SARAIVA, J.; HASHIMOTO, M. Orientação empreendedora como indicador do grau de empreendedorismo corporativo: fatores que caracterizam os intraempreendedores e influenciam sua percepção. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n. 3, 2012. 93 Essas comprovações podem corroborar para a tomada de decisões com relação às estratégias de implantação da OE, podendo ser avaliadas através de uma Essas comprovações podem corroborar para a tomada de decisões com relação às estratégias de implantação da OE, podendo ser avaliadas através de uma 93 FRANÇA, A. B.; SARAIVA, J.; HASHIMOTO, M. Orientação empreendedora como indicador do grau de empreendedorismo corporativo: fatores que caracterizam os intraempreendedores e influenciam sua percepção. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n. 3, 2012. 94 tomada de decisões pouco assertivas rumo ao empreendedorismo corporativo e a melhoria no desempenho dos negócios. sistemática de medição do indicador OE que, segundo os autores citados nesse estudo (CHILD,1972; COVIN; COVIN, 1989; COVIN; SLEVIN, 1990; ZAHRA; COVIN, 1995; DESS et al., 1997; WIKLUND, 1999; LUMPKIN; DESS, 2001; SADLER-SMITH et al., 2003; RICHARD, 2004; TARABISHY et al., 2005; WIKLUND; SHEPHERD, 2005; FRISHMMAR; HÖRTE, 2007; HASHIMOTO, 2009), tem correlação positiva com o desempenho organizacional e influencia o desempenho financeiro das organizações. Para Moreno e Casillas (2008), existe ainda uma correlação entre inovatividade de uma organização e sua OE, consequentemente influenciando a estratégia que a organização irá utilizar, levando a concluir que a mensuração correta e confiável da OE torna-se um importante indicador para a organização empreendedora. A partir dos resultados apresentados neste artigo, abre-se um campo para novas pesquisas teóricas e empíricas a respeito da percepção dos colaboradores acerca da OE da empresa, levando-se em consideração outras variáveis independentes inter-relacionadas que não foram aqui analisadas. Desta maneira, ressalta-se a limitação da presente pesquisa que utilizou uma amostra pequena, levando em consideração a opinião de colaboradores de uma única organização e um conjunto de variáveis abrangendo somente cinco independentes (idade, gênero, nível hierárquico, tempo de serviço e área de atuação).Levando-se em consideração estas limitações, abre-se a possibilidade de replicação desta pesquisa em outras organizações de diferentes tipos, negócios e tamanhos, possibilitando testes mais abrangentes das hipóteses aqui descritas, levantando outras a serem testadas para examinar a relação entre o perfil do colaborador e a percepção dele em relação à OE. Desta maneira, haverá espaço para a geração e generalização de teorias sobre OE nas organizações. Por derradeiro, recomenda-se que os gestores de organizações que desejam ou estão em processo de implantação de uma cultura empreendedora, adotem o indicador OE como uma métrica para a mensuração do nível de empreendedorismo da organização. Todavia, é preciso levar em consideração e tomar o cuidado de entrevistar pessoas com diferentes perfis de idade e tempo de serviço para não criar um viés indesejável nos resultados dessas pesquisas internas, o que poderia levar à FRANÇA, A. B.; SARAIVA, J.; HASHIMOTO, M. Orientação empreendedora como indicador do grau de empreendedorismo corporativo: fatores que caracterizam os intraempreendedores e influenciam sua percepção. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n. 3, 2012. 94 94 tomada de decisões pouco assertivas rumo ao empreendedorismo corporativo e a melhoria no desempenho dos negócios. 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W4386272662.txt	https://zenodo.org/records/4019116/files/EU%20Krise.pdf	de		Die Krise der Europäischen Union (EU) mit Beginn 2008 und möglichen Lösungsversuchen. Hegemonieprojekte in der Krise.	Zenodo (CERN European Organization for Nuclear Research)	2,014	cc-by	10,873	FRANZ ZEILNER DIE KRISE DER EUROPÄISCHEN UNION (EU) MIT BEGINN 2008 UND MÖGLICHEN LÖSUNGSVERSUCHEN. HEGEMONIEPROJEKTE IN DER KRISE. Paper, Jänner 2014 Seite 1 „DAS EUROPÄISCHE PARLAMENT IST ALS EINZIGE DIREKT GEWÄHLTE INSTITUTION IN DER EU DIE VERTRETUNG DER BÜRGER/INNEN. EINE STARKE DEMOKRATISCHE BESONDERS LEGITIMIERUNG IST DAHER – EINE HOHE WICHTIG D.H. WAHLBETEILIGUNG. Seite 2 INHALTSVERZEICHNIS I. EINLEITUNG II. HISTORISCHES ZUR EUROPÄISCHEN UNION (EU) 1. Abschnitte bis zur „Osterweiterung“ 1.1. Der erste Ansatz 1922 1.2. Die Gründung des Europarates (Council of Europe) 1949 1.3. Die Europäische Gemeinschaft für Kohle und Stahl (EGKS) und die Europäische Wirtschaftsgemeinschaft (EWG): Das Entstehen der Europäischen Gemeinschaften (EG) 1.4. Die Erweiterung der Europäischen Union 2. Das Inkrafttreten der Währungsunion 1999 3. Grundlegendes zu den EU-Konvergenzkriterien 4. Der Euro und die Eurozone 4.1. Der Euro 4.2. Die Eurozone III. DIE EUROKRISE 1. Ursachen 2. Eine multiple Krise 3. Die Einleitung von Defizitverfahren Seite 3 IV. HEGEMONIEPROJEKTE IN DER KRISE 1. Der Begriff Hegemonie 2. Das europäische Institutionengefüge in einer Hegemoniekrise 3. Die fünf Hegemonieprojekte in der Krise der Europäischen Union 3.1. Das neoliberale Hegemonieprojekt 3.2. Das national-soziale Hegemonieprojekt 3.3. Das national-konservative Hegemonieprojekt 3.4. Das Proeuropäisch-soziale Hegemonieprojekt 3.5. Das linksliberal-alternative Hegemonieprojekt 4 Das Vertrauen in die Europäische Union ist durch die Krise betroffen V. DIE STRATEGISCHE NEUORIENTIERUNG IN DE KRISE DER EUROPÄISCHEN UNION 1. Eine Krise des neoliberalen Projektes 2. Die vier strategischen Suchprozesse innerhalb des bisherigen neoliberalen Hegemonieprojektes VI. DIE DEMOKRATISCHE FRAGE 1. Die Demokratie wurde zur Disposition gestellt 2. Das Fehlen einer europäischen Zivilgesellschaft 3. Die Demokratie in Zeiten der Eurokrise: Politische Reaktionen von Vertretern der Politik 3.1. Diskussionsveranstaltung im österreichischen Parlament: 3.2. Reden bzw. Aussagen von Abgeordneten zum Thema (Auswahl): Seite 4 VII. MASSNAHMEN GEGEN DIE EUROKRISE 1. Die Institutionen „European Finacial Stability Facility“ (EFSF) und „European Financial Stabilisation Mechanism“ (EFSM). 2. Maßnahmen der Europäischen Union und ihrer Mitgliedstaaten VIII. EIN AUSBLICK 1. Die Ansichten von Viviane Reding und Olli Rehn zur Krise 2. Die unterschiedlichen Wirtschaftsmodelle Europas werden durch eine Einheitswährung nicht harmonisiert 2.1. Studie: Vergleich mit den USA 2.2. Förderprogramme zur Forcierung der Wirtschaft: 3. Die Vergabe von Firmenkrediten (Darlehen) durch Banken 4. Die Wahl zum Europäischen Parlament am 25. Mai 2014 IX. ZUSAMMENFASSUNG, BEMERKUNGEN X. VERWENDETE LITERATUR Seite 5 I. EINLEITUNG Die Inhalte der gegenständlichen Arbeit mit dem Titel „Die Krise der Europäischen Union (EU) mit Beginn 2008 und möglichen Lösungsversuchen. Hegemonieprojekte in der Krise“ sind für das Thema Europäische Union, die auch krisenanfällig ist, wesentlich. Das behandelte Thema ist sehr komplex und umfasst auch aktuelle Hegemonieprojekte, die strategische Neuorientierung in der Krise und die demokratische Frage im Kontext der Europäischen Integration. Im historischen Rückblick betrachtet können die ersten Ansätze für ein gemeinsames Europa bereits 1922 festgestellt werden. Aus der Idee von Coudenhove-Kalergi entwickelte sich sehr viel in Richtung europäische Gemeinschaften bis zur Gründung der Europäischen Union (EU) und deren Weiterentwicklungen. Das betrifft vor allem auch Erweiterungen der EU, die seit deren Gründung erfolgt und weiterhin Thema sind. Bereits im Jahre 1999 trat die Währungsunion in Kraft und damit auch der Euro ins Leben. Parallel zur Währungsunion – weil die makroökonomischen Daten der einzelnen Teilnahmeländer sehr unterschiedlich waren – wurde ein Stabilitäts- und Wachstumspakt geschlossen. Hier verpflichteten sich die Vertragsstaaten ihre Schulden zu reduzieren und ihre Wettbewerbsfähigkeit zu erhöhen. Das ist in der Realität bei manchen Staaten aber nicht geschehen, es ergab allerdings keine Probleme solange der Euro stabil war. Auf dem internationalen Finanzmarkt wurden Darlehen aufgenommen, die dann von manchen Staaten nicht zurückgezahlt wurden, womit deren Schulden zunahmen. Als wesentliche Folge der dann eingetretenen Krise verstießen EU-Mitgliedstaaten auch gegen die MaastrichtKriterien. Das tangiert das jährliche Haushaltsdefizit und die Gesamtverschuldung. Die EU-Konvergenz-kriterien hatten und haben jedoch das Ziel ausschließlich auf Stabilität zu setzen, sie sollen vor allem den wirtschaftlichen Zusammenhalt der Euroländer sichern. Die Geschichte der Europäischen Integration kann grundsätzlich als Erfolgsgeschichte gesehen werden, sie ist aber auch eine Geschichte unterschiedlicher Krisen. Das bedeutet, dass die Europäische Union auch krisenanfällig ist. Die aktuelle Krise in der Eurozone und das bisherige Krisenmanagement haben innerhalb der Gesellschaften, wie auch zwischen den Staaten der Europäischen Union, aber integrationsgeschichtlich beispiellose Spannungen und Konflikte hervorgebracht. Die Europäische Union steckt seit 2008 nicht nur in einer Wirtschafts- und Eurokrise bzw. in einer Schuldenkrise, sondern Seite 6 auch in einer Vertrauenskrise. Autoren der Forschungsgruppe Europa bezeichnen sie auch als massive Vielfachkrise. Diese Krisen verstärken sich auch gegenseitig und betreffen die Euro-Krise, die Staatskrisen wie beispielsweise in Griechenland, den Legitimationsverlust der EU, die Krise des politischen Integrationsprozesses, das Nichtfunktionieren von Projekten und das Fehlen eines neuen attraktiven europäischen Projektes und wirken sich auf den europäischen Integrationsprozess und auf grundlegende Werte der Demokratie negativ aus. Die national eingefärbten Kontroversen um nachhaltige Gegenstrategien und die sozialen Verwerfungen in Krisenländern drohen nicht nur die Eurozone zu spalten sondern gefährden auch das Projekt eines vereinigten Europa. Der Protest gegen „Brüssel“ schlägt in manchen Ländern auch bereits in manifeste Tendenzen zur Rationalisierung um. Diese Entwicklung berührt zudem auch eine weitere wesentliche Krise, nämlich jene der Demokratie. Auch das Fehlen einer ausgeprägten europäischen Zivilgesellschaft macht die EU besonders anfällig für wirtschaftliche Krisen. Das inkludiert auch das Wir-Gefühl bzw. das Gefühl „Wir sind Europäer“, das vorhanden sein sollte. Dem stehen in der Gegenwart aber auch nationale Tendenzen entgegen. Die Eurokrise veranlasste die Vertreter der Mitgliedstaaten der EU bzw. die Regierenden bereits auch dazu, eine Phase der autoritären Koordinierung der nationalen Wirtschafts- und Fiskalpolitiken einzuleiten. Bisherige Integrationsschritte wie etwa der Fiskalpakt, das Europäische Semester oder auch die geplanten bilateralen Arrangements zwischen EU-Mitgliedsländern und der Europäischen Kommission brachten der Kommission einen Machtzuwachs, dagegen verloren die nationalen Parlamente und das Europäische Parlament an Einfluss. Gerade jetzt ist eine breite öffentliche Debatte über alternative Vorschläge zur Zukunft Europas gefragt. Es ist eine Situation entstanden, die deutlich macht, dass die Europäische Einigung kein irreversibles Projekt ist und auch ein Auseinanderbrechen der Eurozonen nicht ausgeschlossen werden kann. Die gegenständliche Arbeit behandelt die aktuelle Situation der sogenannten „Eurokrise“ und in diesem Kontext Grundlegendes zur Europäischen Union wie etwa die EU-Konvergenzkriterien usw. Schwerpunkte liegen in den Bereichen Hegemonieprojekte in der Krise, strategische Neuorientierung in der Krise der Europäischen Union, die demokratische Frage und Maßnahmen gegen die Eurokrise. Ein kurzer Ausblick rundet die gegenständliche Publikation ab. Seite 7 II. HISTORISCHES ZUR EUROPÄISCHEN UNION (EU) 1. Abschnitte bis zur „Osterweiterung“ 1.1. Der erste Ansatz 1922 1922 begannen die ersten Ansätze bzw. Überlegungen ein gemeinsames Europa zu schaffen. Der österreichisch-ungarische Schriftsteller Richard Coudenhoive-Kalergi sah damals in der Gründung eines europäischen Staatenbundes die einzige Möglichkeit einen weiteren (Welt-) Krieg zu verhindern. 1922 wurde auch bereits die Paneuropa-Union gegründet* (Richter 1993; S 788 ff; vgl. auch „Die Paneuropäische Union“ in: Der Standard vom 16.August 2009). Die 1922 gegründete Paneuropa-Union ist die älteste noch existente europäische Einigungsbewegung. Diese Panbewegung hatte ihren historischen Sitz in der Wiener Hofburg, wo das Zentralbüro war. Im Sinne des europäischen Föderalismus tritt sie für ein politisch und wirtschaftlich geeintes, demokratisches und friedliches Europa auf Grundlage des christlich-abendländischen Wertefundaments ein (Richter 1993, S 788 ff; vgl. auch „Die Paneuropäische Union“ in: Der Standard vom 16.August 2009). Aus dieser Idee von Coudenhove-Kalergi entstand dann 1923 die Pan-EuropaBewegung. Massive politische Gegensätze nach dem Ersten Weltkrieg und der Beginn des Zweiten Weltkrieges standen dann aber diesen Überlegungen entgegen. Erst nach Ende des Zweiten Weltkrieges wurde die Idee eines gemeinsamen Europas wieder aufgegriffen. Im Jahre 1946 thematisierte beispielsweise der englische Premierminister Winston Churchill die „Vereinigten Staaten von Europa“. 1950 schlug dann der französische Außenminister Robert Schumann in einer seiner Reden eine wirtschaftliche Zusammenarbeit europäischer Staaten vor. Die Idee eines gemeinsamen Europas war auch geprägt von Überlegungen, mögliche zukünftige Kriege zwischen den europäischen Staaten zu verhindern. Das betraf damals nur „westliche“ Staaten, weil der Zusammenbruch des Kommunismus erst 1989 erfolgte Seite 8 bzw. dann das Thema Osterweiterung relevant wurde (Gutschner/Rohr 2008, S 74). Die Rede des französischen Außenministers Robert Schumann vom 9.Mai 1950 wird als „Geburtstag“ der Europäischen Union gesehen. Der 5.Mai wird auch jährlich als Europatag gefeiert (Gutschner/Rohr 2006, S 74) 1.2. Die Gründung des Europarates (Council of Europe) 1949: Der Europarat ist eine 1949 durch den Vertrag von London gegründete europäische internationale Organisation europäischer Staaten. Der Rat, ein Forum für europäische und aussereuropäische Probleme, besteht aus dem Ministerkomitee, welches sich aus den Aussenministern der Mitgliedstaaten zusammensetzt. Belgien, Dänemark, Frankreich, Großbritannien, Irland, Italien, Luxemburg, Niederlande, Norwegen und Schweden waren damals die Staaten des Europarates mit Sitz in Strassburg (Keller 1999, S 1 ff). 1.3. Die Europäische Gemeinschaft für Kohle und Stahl (EGKS) und die Europäische Wirtschaftsgemeinschaft (EWG): Das Entstehen der Europäischen Gemeinschaften (EG) 1951 wurde auf Initiative Frankreichs die Europäische Gemeinschaft für Kohle und Stahl gegründet. Damit erfolgte der erste Schritt zu einer wirtschaftlichen Vereinigung einiger europäischer Staaten. 1957 erfolgte dann der nächste Schritt mit dem Vertrag von Rom, Deutschland, Frankreich, Italien und die Benelux-Staaten (Mitglieder der EGKS) gründeten die Europäische Wirtschaftsgemeinschaft. Diese europäischen Staaten beschlossen damals auch die gemeinsame Nutzung der Atomenergie (EURATOM): Im Jahre 1967 wurden dann die Europäische Gemeinschaft für Kohle und Stahl, die zusammengelegt, Europäische damit Wirtschaftsgemeinschaft entstanden die und Europäischen die EURATOM Gemeinschaften. Wesentliches Ziel der Europäischen Gemeinschaften war es, einen freien Binnenmarkt der Mitgliedstaaten zu schaffen. Absicht war es auch, dass die EG vor allem weltweit mit den USA und Japan konkurrieren können. Es wurde auch vereinbart, dass nicht nur eine wirtschaftliche, sondern auch eine politische Gemeinschaft bzw. Union geschaffen werden sollte. Das erfolgte mit dem Vertrag Seite 9 von Maastricht der am 1.November 1995 in Kraft trat (Europäische Union, EU). (Gutschner/Rohr 2008, S 74). Im Wesentlichen wurde in diesem Vertrag geregelt, dass sich EU-Bürger/innen im EU-Gebiet frei bewegen können, es sollte eine Wirtschafts- und Währungsunion geschaffen werden (Einführung einer gemeinsamen Währung: Euro), eine Europäische Zentralbank sollte errichtet werden, das Europäische Parlament sollte gestärkt werden, die gemeinsame Außen- und Sicherheitspolitik sollte intensiviert werden. Ein Zeichen fortschreitender Einigung war dann die Einführung des Euro als alleiniges Zahlungsmittel im Jahr 2002, vorerst in zwölf Mitgliedstaaten. Nur Staaten die bestimmte Bedingungen erfüllen, können den Euro als Zahlungsmittel einführen (Gutschner/Rohr 2008, S 74 f). 2. Das Inkrafttreten der Währungsunion 1999 Die Währungsunion trat 1999 in Kraft und damit auch der Euro ins Leben. Die elf Staaten, die damals dieses gemeinsame Projekt starteten konnten aber nicht als optimaler Währungsraum gesehen werden. Der Zusammenschluss der nationalen Währungen zum Euro erfolgte nicht nur aus wirtschaftlichen, sondern besonders auch aus politischen Gründen. Beispielsweise sollte der Euro auch dazu dienen, Deutschland verstärkt in die europäischen Strukturen einzubinden, das galt auch für andere Länder (http://www.bpb. de/ internationales/ europa/europa-kontrovers/ 47840/einleitung, S 1). Europa galt viele Jahrzehnte lang als Erfolgsgeschichte. Die Europäische Union wurde sogar als Wegbereiterin des Friedens betrachtet, die Unionsbürgerschaft, die Währungsunion und der Europäische Gerichtshof galten als Beginn einer postnationalen Konstellation. Die Europäische Union sah man nicht nur als Voraussetzung für ein soziales Europa im Zeitalter der Globalisierung, sondern besonders auch als Möglichkeit zur Problemlösung (Buckel et al 2012, S 7; vgl. auch Habermas 2011, S 117 ff). Die Währungsunion bzw. die europäische Wirtschafts- und Währungsunion (WWU) ist das Ergebnis einer fortschreitenden wirtschaftlichen Integration in der Europäischen Union die sich in drei Stufen entwickelte. Sie war und ist wesentlich, weil sie die Koordinierung der Wirtschafts- und Fiskalpüolitik sowie die gemeinsame Währungspolitik regelt. Es wurde vorgesehen, dass letztendlich alle Mitgliedstaaten, Seite 10 welche die Voraussetzungen erfüllen, den Euro als einheitliche Währung der Union einführen. Es wurde nur für das Vereinigte Königreich und Dänemark eine Ausnahmeregelung getroffen (Artikel 3 des Vertrages über die Europäische Union (EUV); Artikel 3,5,119,144,219 und 282 bis 284 des Vertrages über die Arbeitsweise der Europäischen Union (AEUV). Parallel zur Währungsunion wurde – weil die makroökonomischen Daten der einzelnen Teilnehmerländer sehr unterschiedlich waren - ein Stabilitäts- und Wachstumspakt geschlossen, in dem die Vertragsstaaten sich verpflichteten ihre Schulden zu reduzieren und ihre Wettbewerbsfähigkeit zu erhöhen. Das ist in der Realität dann aber nicht geschehen und ergab auch keinerlei Probleme solange der Euro stabil war. Dieser hatte phasenweise sogar eine starke Stellung gegenüber dem US-Dollar erreicht. Die Inflationsrate war niedrig sowie auch die Zinsen für Staatsanleihen, weshalb auf dem internationalen Finanzmarkt Darlehen aufgenommen wurden. Die meisten Staaten zahlten dann ihre Schulden aber nicht zurück, sondern vergrößerten diese noch (http://www.bpb.de/internationales/ europa/europa-kontrovers/ 47840/ einleitung, S 1). Als wesentliche Folge der dann eingetretenen Finanzkrise verstoßen derzeit mehrere EU-Mitgliedstaaten gegen die Maastricht-Kriterien. Das betrifft das jährliche Haushaltsdefizit aber auch die Gesamtverschuldung. Die EU-Konvergenzkriterien hatten und haben aber das Ziel ausschließlich auf Stabilität zu setzen. 1992 verpflichteten sich die Mitgliedstaaten der Europäischen Union im Vertrag von Maastricht auch zur Einhaltung dieser Kriterien die folgend nun auch dargestellt werden (Weindl/ Woyke 1999, S 17). 3. Grundlegendes zu den EU-Konvergenzkriterien Für die konkreten Teilnehmerstaaten erfolgte der Beitritt zur Eurozone unter gleichzeitigem Verzicht auf den autonomen Einsatz von Geld- und Wechselkurspolitik. Für die Eurokrise hat diese Tatsache jedenfalls zwei relevante Handlungseinschränkungen zur Folge: 1) Eine gemeinsame Währung entzieht den Mitgliedstaaten die Möglichkeit, durch Geldpolitik asymmetrische makroökonomische Schocks zu vermindern. Wenn nur einige Mitgliedstaaten der Währungsunion von makroökonomischen Schocks Seite 11 betroffen sind, ist eine nur diese Staaten betreffende expansive Geldpolitik nicht möglich. 2) Mit dem Beitritt haben die Teilnahmeländer darauf verzichtet, durch kompetitive Währungsabwertung ihre Wettbewerbsfähigkeit zu verbessern (Obstfeld/Rogoff 1996,S 633f) Vor dem Beitritt zum Euroraum erfolgte auch eine „Konvergenzprüfung“, wodurch besonders gewährleistet werden sollte, dass die Bedeutung der beiden Problembereiche minimiert werden. Bereits vor Einführung der gemeinsamen Währung wurde ein Konvergenzprozess* verpflichtend, der eventuelle Notwendigkeiten zu asymmetrischer Reaktion beschränken sollte. Die Eurozone sollte damals dadurch forciert einem optimalen Währungsraum angenähert werden (Frankfurter Allgemeine Zeitung (FAZ) vom 21.10.2004; vgl. auch Mundell 1961, S 657 ff). Bei Errichtung der EU-Konvergenzkriterien, die nach Meinung mancher Experten unzureichend sind, wurde auch übersehen, dass nicht nur der staatliche Sektor, sondern auch der private Sektor durch exzessive Verschuldung und Wettbewerbsverschlechterungen zum Problemfall werden kann (Buckel et al 2012, S 14 ff; vgl. auch Eichengreen 2012, S 2) 1992 haben sich, wie bereits erwähnt, die EU-Mitgliedstaaten durch den Vertrag von Maastricht gegenseitig erstmals zu den EU-Konvergenzkriterien bzw. den MaastrichtKriterien, die aus fiskalischen und monetären Vorgabewerten bestehen, verpflichtet (Weindl/Woyke 1999, S 12 ff; vgl. auch http://de.wikipedia.org/wiki/EU-Konvergenzkriterien, S 1). Diese Kriterien haben unter anderem das Ziel, in der EU und insbesondere in der Eurozone eine Angleichung der Leistungsfähigkeit der einzelnen nationalen Wirtschaftsräume in der EU zu befördern und damit auch eine grundsätzliche wirtschaftliche Stabilität und Solidarität der EU zu gewährleisten. Die Mehrzahl der sogenannten Konvergenzkriterien sind in Art. 126 und Art. 140 AEU-Vertrag geregelt (Art. 126 und Art. 140 AEU-Vertrag). Seite 12 Das sind konkret folgende Kriterien: - Die Preisniveaustabilität: Die Inflationsrate darf nicht mehr als 1,5 Prozentpunkte über derjenigen der drei preisstabilsten Mitgliedstaaten liegen - Die Finanzlage der öffentlichen Hand: Der staatliche Schuldenstand darf nicht mehr als 60 % des Bruttoinlandsprodukts betragen. Das jährliche Haushaltsdefizit darf nicht mehr als 3 % des Bruttoinlandsprodukts betragen. - Die Wechselkursstabilität: Der Staat muss mindestens zwei Jahre lang ohne Abwertung am Wechselkursmechanismus II teilgenommen haben. Dabei darf die Währung des Landes nur in einer bestimmten Wechselkursbandbreite (meist 15 %) vom Eurokurs abweichen. Bei größeren Abweichungen muss die Zentralbank des Landes intervenieren. - Langfristige Zinssätze: Der Zinssatz langfristiger Staatsanleihen darf nicht mehr als 2 Prozentpunkte über dem Durchschnitt der drei preisstabilen Mitgliedstaaten liegen (Art. 126 AEU-Vertrag). Im Bereich der Wissenschaft bzw. der Nationalökonomie ist es allerdings umstritten, ob diese Konvergenzkriterien auch tatsächlich geeignet sind, um den wirtschaftlichen Zusammenhalt der Euroländer zu gewährleisten. Die Theorie optimaler Währungsräume nimmt zusätzlich noch weitere Kriterien in den Fokus, wie etwa die Intensität des Handels zwischen den verschiedenen Staaten. Diese Konvergenzkriterien sehen keinerlei gemeinschaftliche Konjunkturpolitik vor, sie zielen ausschließlich auf Stabilität ab (Art. 126 AEU-Vertrag; vgl. auch http://de.wikipedia.org/wiki/EUKonvergenz- kriterien, S 1 ff). Diese Thematik bzw. Problematik untersuchten auch die Ökonomen Kevin O’Rourke und Alan Tayler und publizierten das Ergebnis auch in einem Aufsatz im „Journal of Economic Perspektives“. Sie sehen in der Euro-Zone keinen einheitlichen Währungsraum, besonders weil die Konjunkturzyklen der einzelnen Wirtschaftsräume zu unterschiedlich sind. Für das Funktionieren einer gemeinsamen Währung über einen so großen Raum, müssten die Wirtschaftszyklen wie beispielsweise in den USA „Synchron“ verlaufen (http://deutsche-wirtschaftsnachrichten.de2013/08/24/studie-der-zerfall-der-euro-zon..., S 1 ff; vgl. auch O’Rourke/Tayler, Journal of Economic Perspektives). Seite 13 4. Der Euro und die Eurozone 4.1. Der Euro: Gemäß Art. 3 Abs. 4 EUV ist der Euro die Währung der Europäischen Wirtschaftsund Währungsunion, eines in Art. 127-144 AEUV geregelten Politikbereichs der Europäischen Union (Art. 3 Abs. 4 EUV; Art. 127-144 AEUV). Der Euro wird von der Europäischen Zentralbank emittiert und ist die offizielle Währung in jenen Mitgliedstaaten die zusammen die Eurozone bilden, sowie in weiteren europäischen Staaten. Am 1.Jänner 1999 wurde der Euro als Buchgeld, am 1.Jänner 2002 dann als Bargeld eingeführt. Er ist nach dem US-Dollar die bedeutendste Reservewährung der Welt. Die gemeinsame Währung hat in ihrer Gestaltung, ihrer Verwendung und den politischen Strategien auch eine eigene Geschichte. Die Europäische Zentralbank und die Europäische Kommission wachen gemeinsam darüber, dass der Euro seinen Wert und seine Stabilität behält (Art. 3 Abs. 4 EUV; Art. 127-144 AEUV). 4.2. Die Eurozone: Der Begriff Eurozone ist in den Gründungsverträgen der Europäischen Union nicht vorhanden. Es ist aber für die Mitglieder der EU die Formulierung enthalten „Mitgliedstaaten deren Währung der Euro ist“ (Art. 136 ff. AEU-Vertrag). Das Amt für Veröffentlichungen der Europäischen Union verwendet die Begriffe Euro-Währungsgebiet oder Euroraum. Damit werden die am Euro teilnehmenden Länder als Ganzes bezeichnet. Im allgemeinen Sprachgebrauch hat sich vielfach aber die Bezeichnung Eurozone etabliert. Im weitesten Sinne wird Eurozone auch für alle Staaten verwendet, die den Euro nutzen. Diese Staaten müssen nicht Mitglied der Europäischen Union sein. Solche Staaten haben aber ein Währungsabkommen mit einem Mitgliedstaat der Europäischen Union, sie können auch den Euro einseitig eingeführt haben oder den Kurs ihrer Währung an die Leitwährung Euro gekoppelt haben (Länder, Sprachen, Währungen, in: Internationale Regeln für Veröffentlichungen. Amt für Veröffentlichungen der Europäischen Union, S 1 ff). , Im engeren Sinn zählen zur Eurozone aber nur jene Mitgliedstaaten der EU, welche den Euro als Währung eingeführt haben. Für diese Staaten war es notwendig, dass Seite 14 sie die EU-Konvergenzkriterien erfüllen. Am 1.Jänner 1999 – Einführung des Euro als Buchgeld - erfüllten diese Kriterien elf der damals fünfzehn EU-Mitgliedstaaten. (Länder, Sprachen, Währungen, in: Internationale Regeln für Veröffentlichungen. Amt für Veröffentlichungen der Europäischen Union, S 1 ff). , III. DIE EUROKRISE 1. Ursachen Vereinfacht dargestellt ist die Euro – Krise eine durch die Welt-Finanzkrise 2007/ 2008 verstärkte Haushalts- und Verschuldungskrise mehrerer Mitgliedstaaten der Eurozone. Grundlegende Ursachen liegen sicher auch in Konstruktionsfehlern der Währungsunion selbst. Das wird aber kontrovers diskutiert (https://lobbypedia. de/ wiki/eurokrise, S 1). Eine grundlegende Ursache ist auch, dass ein Währungsraum errichtet wurde und dadurch eine gemeinsame Geldpolitik erfolgt. Im Bereich der Wirtschafts- und Fiskalpolitik agiert aber jeder Staat selbstständig. Jeder Staat hat deshalb seine eigene Lohn- und Steuerpolitik wodurch es auch zu Unterschieden in der Lohnentwicklung kommt die bereits zu starken Leistungsbilanz Ungleichgewichten im Euroraum geführt haben (Grunert 2011, S 1 ff). Leistungsbilanzdefizite führten auch zum Verlust der Kreditwürdigkeit der Defizitländer. Ein Leistungsbilanzdefizit wird grundsätzlich durch massive private Verschuldung von Unternehmen und Privathaushalten finanziert. Die private Verschuldung mündet wiederum in eine Verschuldung des Staates weil dieser für die Darlehen haftet. Besonders verschuldet sich der Staat durch Rettungsprogramme von Banken. In dieser Situation sind Spanien, Irland und Italien, die zwar vor der Finanzkrise 2007/2008 keine besonders hohe Staatsverschuldung hatten, jedoch aber eine kreditfinanzierte Immobilienblase (Grunert 2011, S 1 ff). Akut wurde die Euro-Krise im Jahre 2010 mit der griechischen Finanzkrise. Damals legte Griechenland die tatsächliche Höhe seiner defizitären Haushaltslage und die Höhe der Verschuldung offen, konnte sich aber nicht mehr selbst bzw. aus eigener Kraft im notwendigen Ausmaß refinanzieren. Schulden bzw. Zinsen waren nicht mehr zu bewältigen. Es stiegen damals auch die Zinsen auf griechische Staatsanleihen im Zuge der Offenlegung rapide an. Neben Griechenland hatten auch Irland, Portugal, Seite 15 Spanien und Italien hohe Haushaltsdefizite und Verschuldungsgrade und konnten sich kaum aus eigener Kraft am Kapitalmarkt finanzieren (http://lobbypedia. de/wiki/Eurokrise, S 1). Diese Situation betreffend wurde die aktuelle Diskussion in den ersten Jahren der Eurokrise, vor allem deren Bewältigung, grundsätzlich kontrovers geführt. Überwiegend war und ist der Diskurs aber von einer einseitigen, allein auf Griechenland bezogenen Problemansicht geprägt. Nach dieser Ansicht waren (frühere) griechische Regierungen Schuld an dieser Situation, insbesondere weil sie Jahr für Jahr gegen die Defizitgrenzen des Stabilitätspaktes verstießen (Scharpf 2011, S 324 ff).. Der drohende Staatsbankrott Griechenlands hätte den Euro gefährdet und möglicherweise negative Auswirkungen auf die Europäische Union gehabt, weshalb Griechenland (später auch noch Irland und Portugal) durch Garantien und verbilligte Kredite des europäischen Rettungsschirmes „gerettet“ werden mussten. Die Krisenländer mussten drakonische Sanierungspläne akzeptieren die von einer „Troika“ aus Europäischer Kommission, Europäischer Zentralbank und Internationalem Währungsfonds definiert und im Vollzug überwacht werden (Scharpf 2011, S 324 ff). 2. Eine multiple Krise Die Eurokrise ist eine multiple Krise. Sie ist nun eine bereits seit dem Jahre 2008 andauernde Krise innerhalb der Eurozone. Sie weist gleichzeitig Aspekte einer Staatsschuldenkrise, einer Bankenkrise bzw. Finanzkrise und einer Wirtschaftskrise auf (Buckel et al 2012, S 7 ff; vgl. auch http://de.wikipedia.org/wiki/Eurokrise, S 1). Mit der im Jahre 2008 beginnenden Eurokrise sind besonders auch drei große Ungleichgewichte im Euroraum, die sich in der Europäischen Wirtschafts- und Währungsunion aufgebaut haben, akut geworden. Das divergierende Wettbewerbsfähigkeit und eine sind erstens eine Tendenz zu Zahlungsbilanzungleichgewichten, zweitens ein zu gering kapitalisierter und wenig krisenrobuster Bankensektor und drittens Überschuldung im öffentlichen und in einigen Ländern auch im privaten Sektor. Die drei Probleme verstärken sich durch die wechselseitigen Abhängigkeiten und haben bereits auch zu einer Vertrauenskrise im Bereich der Europäischen Union, vor allem aber auch zu vielen öffentlichen Diskussionen geführt (Keuschnigg et al 2012, S 1 f). Seite 16 Im Bereich der wissenschaftlichen Diskussion ist die Gewichtung der unterschiedlichen Krisenfaktoren wesentliches Thema, das wird aber kontrovers diskutiert. Die im Bereich der Nationalökonomie konventionelle Erklärung für die Ursachen der Eurokrise ist hier zumindest zweiseitig. Die Eurokrise wird auf wirtschaftlicher Seite als Währungskrise der ersten Generation interpretiert. Die wesentliche Begründung dafür ist, dass in einigen Ländern der Europäischen Union eine vermehrte staatliche bzw. private Kreditaufnahme zu relativ höherer Inflation als in anderen Ländern der Europäischen Union geführt hat (http://de.wikipedia.org/wiki/Eurokrise, S 2). Aufgrund der Euro-Währungsunion war ein Ausgleich der unterschiedlichen Preisentwicklungen durch die natürliche Wechselkurskorrektur nicht möglich. Dadurch kam es in einigen Euro-Ländern zu anhaltend hohen Leistungsbilanzdefiziten, Leistungsbilanzüberschüssen. D.h., es bei traten anderen dagegen makroökonomische zu anhaltend hohen Ungleichgewichte auf (http://de.wikipedia.org/wiki/ Eurokrise, S 2). Auf politischer Seite führte die Euro-Währungsunion dazu, dass einzelstaatliche Geldpolitik nicht möglich ist. Grundsätzlich sei deshalb als schnelle Reaktion auf Wirtschaftskrisen nur das Instrument der Fiskalpolitik vorhanden. Fiskalpolitik belastet aber jedenfalls den Staatshaushalt stärker als Geldpolitik (http://de.wikipedia.org/ wiki/Eurokrise,, S 2 f). Zudem sahen Experten als weitere Ursache für die Euro-Krise, dass durch den Wegfall der Wechselkursunsicherheit aufgrund der Euro-Währungsunion die Zinsen in Euro-Ländern mit traditionell höherer Inflation stark sanken (Boyer 2013, S 533 ff vgl. auch http://de.wikipedia.org/wiki/Eurokrise, S 3). Dies verursachte eine gesteigertes Kreditaufnahme- und Investitionsverhalten. Begünstigt wurde diese Situation auch durch mangelhafte Banken- und Kapitalmarktregulierung, wodurch sogenannte „Wirtschaftsblasen“ entstanden deren Platzen die Konjunkturprogramme und Rettungsmaßnahmen auslösten (Boyer 2013, S 533 ff). 3. Die Einleitung von Defizitverfahren Die ab dem Jahre 2007/2008 beginnende Finanzkrise mit ihren direkten Kosten sowie die dadurch bewirkten Verwerfungen führte in der Europäischen Union dann zu sogenannten „Defizitverfahren.“ Die EU-Kommission kündigte bereits im Februar Seite 17 2009 die Einleitung von Defizitverfahren gegen die fünf Euro-Länder Frankreich, Spanien, Irland, Griechenland und Malta und gegen das Nicht-Euro Land Lettland an. Die Vorgaben des Stabilitäts- und Wachstumspaktes erfüllte aber auch das NichtEuro-Land Großbritannien nicht. 2009 folgten Defizitverfahren gegen Polen, Rumänien und Litauen und die Verlängerung für Ungarn. Ebenfalls 2009 wurden wie tere Defizitverfahren eröffnet wovon auch Österreich betroffen war. Neben Österreich auch noch gegen Deutschland, Belgien, Italien, die Niederlande, Portugal, die Slowakei, Slowenien und Tschechien. Infolge der Finanzkrise hatten damals 20 der insgesamt 27 Mitgliedstaaten Wachstumspaktes nicht der EU erfüllt die Kriterien des stabilitäts- und (http://de.wikipedia.org/wiki/Stabilitäts- _und_Wachstumspakt, S 1 ff). IV. HEGEMONIEPROJEKTE IN DER KRISE 1. Der Begriff Hegemonie Im Bereich der Wissenschaften, hier vor allem in der politikwissenschaftlichen Forschung, wird der Begriff „Hegemonie“ unterschiedlich definiert und verwendet. Vielfach wird darunter aber die Vormachtstellung eines Staates verstanden, aktuell auch im Kontext der globalen Weltordnung. Es ist aber sicher vorteilhaft sich auf einen differenzierten Hegemoniebegriff unter Einbeziehung ökonomischer, politischer, rechtlicher und kultureller Aspekte zu beziehen, insbesondere im Kontext von Analysen multipler Krisenparameter bzw. Krisenphänomene in den Mitgliedsländern der Europäischen Union (Gramsci 1991, S 120; GH 1 120). Die Hegemoniekrise innerhalb der Europäischjen Union kann an mehreren Parametern festgemacht werden, beispielsweise auch wenn zwei bedeutende iuristische Institutionen, sich gegenseitig ihr Macht streitig machen, über einen konkreten Rechtsfall zu entscheiden (Gramsci 1991, S 120; GH 1 120). 2. Das europäische Institutionengefüge in einer Hegemoniekrise Dass sich das europäische Institutionengefüge in einer Hegemoniekrise befindet ist vielfach Expert/innenmeinung, wird aber kontrovers diskutiert. Bereits Antonio Gramsci stellte fest, dass Hegemoniekrisen durch eine Neuzusammensetzung des Seite 18 „dialektischen Verhältnisses von Zwang und Konsens“ entstehen. Er sah die „normale“ Ausübung der Hegemonie auf dem klassisch gewordenen Feld des parlamentarischen Regimes ausgezeichnet durch eine Kombination von Zwang und Konsens, ohne dass der Zwang den Konsens zu sehr überwiegt, sondern im Gegenteil vom Konsens der Mehrheit, wie er in den sogenannten Organen der öffentlichen Meinung zum Ausdruck kommt, getragen werde (Gramsci 1991, S 120; GH 1 120). Gegenwärtig fehlt es vielfach aber an diesem Konsens. Verfolgt man den Verlauf der aktuellen Krise in Europa wird erkennbar, dass die hegemoniale Phase des Neoliberalismus bereits auch im imperialen Zentrum an ihr Ende zu kommen scheint. Um die herrschenden Machtverhältnisse aufrechtzuerhalten soll Zwang auch ein Mittel sein um notfalls die fehlende Zustimmung zu ersetzen. Bisher erfolgte die Durchsetzung von Projekten des Neoliberalismus (der Binnenmarkt und die Währungsunion) im Rahmen der Europäischen Union auf Konsens, sie haben nun an Zustimmung verloren. Die vorhandene große Krise lässt auch die imaginären Bilder des Neoliberalismus verblassen (http://arranca.org/ausgabe/46/die- neuzusammensetzung-von-zwang-und-konsens-in-d.., S 1). 3. Die fünf Hegemonieprojekte in der Krise der Europäischen Union Grundsätzlich können fünf Hegemonieprojekte in der Krise der Europäischen Union festgelegt werden. Das sind das neoliberale Hegemonieprojekt, das national-soziale Hegemonieprojekt, das national-konservative Hegemonieprojekt, das proeuropäischsoziale Hegemonieprojekt und das linksliberal-alternative Hegemonieprojekt. Alle diese Hegemonieprojekte stehen im wissenschaftlichen Diskurs bzw. im Konflikt um die Europäische Integration (Buckel et al 2012, S 24 ff). 3.1. Das neoliberale Hegemonieprojekt: Die Autoren gingen davon aus, dass es grundsätzlich möglich ist, ein über längere Zeit relativ stabiles neoliberales Projekt zu identifizieren. In diesem Hegemonieprojekt war es grundlegende Strategie, die auch bis zum Ausbruch der Krise 2008 zentral blieb, den wettbewerbsstaatlichen Umbau vieler gesellschaftlicher Bereiche und aller staatlichen Aufgaben zu betreiben (Buckel et al 2012, S 25 f). Seite 19 Zu diesen gesellschaftlichen Bereichen zählt auch der Agrarbereich. Es wurde hier aber verabsäumt, so Expert/innen, produktionssteuernde Instrumente einzuführen. Ideologische Differenzen verschärften sich hier im Laufe der Jahre, so dass bereits in den 1980er Jahren drei unterschiedliche ideologische Lager erkennbar waren. Das waren die überzeugten Neoliberalen, die verdeckten Neoliberalen und die Marktregulierer (Choplin et al 2011, S 41). Die überzeugten Neoliberalen vertreten die Ansicht, dass dort produziert werden soll, wo es am billigsten ist. Der Marktmechanismus und die freie Konkurrenz stellen sicher, dass es zu einem maximalen Wohlstand der Bevölkerung kommen wird. Liberalisierung bedeutet hier, dass der Staat l so wenig als möglich eingreifen soll. Das entsprach besonders den Interessen von Import-ExportUnternehmen. Die verdeckten Neoliberalen vertreten ähnliche Ideologien, sie befürworten aber eine starke Einmischung des Staates. Besonders soll die Modernisierung von Strukturen und Techniken staatlich gefördert werden (Choplin et al 2011, S 41). Der Hauptkontext war und ist hier das politische Projekt des Binnenmarktes, dessen vier Grundfreiheiten die Grundlage ist. Adressaten der Grundfreiheiten sin hauptsächlich die Mitgliedstaaten (Buckel et al 2012, S 25 f). Der Europäische Binnenmarkt wird aber nicht alleine durch die genannten vier Grundfreiheiten vollendet. Diesbezüglich wurden auf europäischer Ebene auch bereits verschiedene Maßnahmen gesetzt (Europäischer Binnenmarkt-Wikipedia). Die Flexibilisierung von Produktions- und Arbeitsverhältnissen, die Finanzialisierung der Ökonomie und der Abbau staatlicher Regelungen waren die politischen Leitlinien des Binnenmarktes. Es ging hier im Wesentlichen um die Internationalisierung von Kapital, Handel, Produktions- und Versorgungsketten und auch um die Mobilität des Produktionsfaktors Arbeit im Kontext einer neokolonialen internationalen Arbeitsteilung, die von der Ausnutzung von Niedriglohnsektoren profitierte (Buckel et al 2012, S 25). Soziale Basis dieses Projekts sind die Schlüsselsektoren des globalisierten, postfordistischen Akkumulationsregimes (Buckel et al 2012, S 25). Dieses Hegemonieprojekt wird getragen von den Schlüsselbranchen des gegenwärtigen Akkumulationsregimes, den transnationalen Industriekonzernen und der Finanzindustrie, die gleichzeitig auch auf ideologisch-diskursiver Ebene eine Seite 20 Dominanz aufweisen. Think Tanks sind hier wesentlich sowie auch die neoklassisch dominierten Wirtschaftswissenschaften (Buckel et al 2012, S 24). Der Umsatz transnationaler Konzerne entspricht etwa dem Bruttosozialprodukt mittlerer Volkswirtschaften. Grundsätzlich werden auch Milliardengewinne in extensive Lobbyisten Netzwerke investiert wodurch staatliche politische Entscheidungen beeinflusst werden können (Buckel et al 2012, S 25 f). Die vorhandenen Ressourcen des neoliberalen Hegemonieprojektes zur Zielerreichung sind sehr hoch. Aufgrund dessen und der politischen Stellung sind die Akteur/innen auch in der Lage eine „systemrelevante Leistungsverweigerung wirkungsvoll anzudrohen“. Das bedeutet beispielsweise auch Entscheidungen und Investitionen von der Akzeptanz ihrer Forderungen abhängig zu machen. Zudem verfügen sie auch über große materielle Ressourcen (Buckel et al 2012, S 25). 3.2. Das national-soziale Hegemonieprojekt: Bei diesem Hegemonieprojekt steht sozialer Ausgleich und soziale Umverteilung im Mittelpunkt der politischen Strategie. Das europäische Sozialmodell, das hier keine gesamteuropäische Perspektive verfolgt, steht im Mittelpunkt. Bestehende nationale Systeme sollen aufrecht bleiben und sozialpolitische Errungenschaften geschützt werden. Hauptargument dafür ist der existente neoliberale Globalisierungsdruck. Man vertritt die Ansicht, dass eine Strategie der Europäisierung kaum Aussicht auf Erfolg hat (Buckel et al 2012, S 26). Schwerpunkte dieses Projekts sind der nationale Wohlfahrtsstaat, die nationale Arbeitsgesetzgebung, die staatliche Unterstützung einheimischer Unternehmen, korporatistische Strukturen usw. (Buckel et al “2012, S 26). Die mit dieser Thematik befasste gegenständliche Forschungsgruppe vertritt zudem auch die Ansicht, dass die sozialstrukturelle Basis dieses Projekts in den Resten des hegemonialen Blocks der fordistischen Produktions- und Lebensweise liegt (Buckel et al 2012, S 26). Diese fordistische Produktions- und Lebensweise war für Jahrzehnte dominierend. Im Fordismus hatten die Arbeiter/innen, die mit vielen ihrer Kolleg/innen in großen Gruppen arbeiteten, nur geringe Qualifikationen benötigt. Dies führte aber auch Seite 21 dazu, dass untereinander die Solidarität bzw. der Zusammenhalt gestärkt wurde (http://de.wikipedia.org/wiki/Postfordismus, S 1). Der Postfordismus löste dann in den 1970er Jahren den Fordismus ab, der sich als bestimmende Wirtschaftsform im Kapitalismus durchgesetzt hatte. Im Bereich der Politikwissenschaft geht man davon aus, dass der Fordismus nicht in der Lage war, seine Produktionsreserven auszuschöpfen. Begründet wird dies damit, dass der Fordismus nach seiner Durchsetzung in der Triade (Nordamerika , Westeuropa, Japan) nicht mehr fähig war, die Hegemonie der USA, als treibende Kraft des westlichen Kapitalismus, auf Dauer zu sichern (http://de.wikipedia.org/wiki/Postfordismus, S 1 f). Im Fordismus nach tayloristischer Prägung war das Produktionswissen in der Maschine die von Arbeiter/innen nur bedient wurde, eingebaut. Die Maschine war sozusagen nur das Investitionsobjekt, welchem mechanisch das Wissen um die Produktion eingeschrieben war (http://de.wikipedia.org/wiki/Postfordismus, S 2). In modernen Produktionsprozessen und unter dem grundsätzlich schnellen Wechsel der Produktionszyklen ist der Wert um das Wissen – in unserer Wissensgesellschaft - zur Herstellung der Produktionsmittel nun von größerem Wert als die Produktionsmittel selbst (http://de.wikipedia.org/wiki/ Postfordismus, S 2). 3.3. Das national-konservative Hegemonieprojekt: Dieses Hegemonieprojekt befürwortet ein Europa der souveränen Nationen. Grundlegender Bezugspunkt ist die Nation selbst und wird als Selbstzweck und als Eigenwert gesehen. Im Unterschied dazu sieht das national-soziale Projekt eine Solidargemeinschaft. Kleine und mittelständische Sektoren und Unternehmen bilden hier die Basis, die Mitarbeiter/innen bilden auch die politische Landschaft (Buckel et al 2012, S 27). Die Ressourcen dieses Projektes liegen besonders in der Verankerung in einem nationalen, konservativen Milieu und der Fähigkeit, dieses über rechtspopulistische Kampagnen, Wahlkämpfe, Debatten usw. zu mobilisieren. Zudem sind auch ökonomische Ressourcen vorhanden bzw. wesentlich wie etwa die direkte Unterstützung von regional und national ausgerichteten Kapitalfraktionen. Dieses Projekt geht aber auch in vielen Ländern im Rahmen konservativer Parteien und Stiftungen Bündnisse Seite 22 mit Akteuren des neoliberalen Hegemonie-projektes ein und profitiert auch von deren Ressourcen (Buckel et al 2012, S 28). Beispielsweise haben die drei konservativen Parteien der EU, Deutschland (CDU/CSU), Großbritannien (Conservative Party) und Spanien (Partido Popular) jeweils neoliberal ausgerichtete Wirtschaftsflügel und stärker national-konservative Strömungen, sie haben auch wichtige Ressourcen diesbezüglich zur Verfügung (CDU/CSU; Conservative Party; Partido Popular). 3.4. Das Proeuropäisch-soziale Hegemonieprojekt: Akteur/innen dieses Projektes verfolgen eine Strategie die auf Rettung des europäischen Sozialmodells durch eine gesamteuropäische Sozialpolitik gerichtet ist. Dieses Projekt, das weniger Gewicht auf den Nationalstaat legt, ist in der Suche nach sozialem Ausgleich dem national-sozialen Hegemonieprojekt ähnlich. Die Grundannahme lautet: Die Krise des Wohlfahrtsstaates im Kontext neoliberaler Globalisierungsprozesse kann nur durch eine Europäisierung der Wirtschafts- und Sozialpolitik und einer europäischen Tarifpolitik begegnet werden (Buckel et al 2012, S 28). Das zentrale Argument lautet: Wenn sich Kapitalakteure internationalisieren und politische und ökonomische Prozesse bereits auf Scales oberhalb des Nationalstaates stattfinden, dann könne eine sozial ausgleichende Politik sich nicht auf die nationale Ebene beschränken. Zu den Argumenten gehört auch die Stärkung der europäischen Wirtschaft und ihrer Institutionen zu unterstützen. Dadurch soll in der geopolitischen Konkurrenz zwischen Europa, USA und Asien eine bessere Position erreicht werden und so die Voraussetzungen für Erhaltung und Ausbau des europäischen Sozialmodells gesichert werden (Buckel et al 2012, S 28). Seit der Krise des Fordismus ist die soziostrukturelle Positionierung dieses Projekts aber durch die Schwächung der organisierten Arbeiter/innen gekennzeichnet. Faktum ist, dass sich die EU-Arbeitgeberverbände weigern Koordinierungsregeln für Lohnpolitik einzuführen und die Prekarisierung der Arbeitsverhältnisse in den nationalen Gesellschaften negative Auswirkungen haben. Viele Kompetenzen und Einflussmöglichkeiten verbleiben auf der nationalen Ebene. Wesentlich ist auch, dass die Ressourcen des proeuropäisch-sozialen Hegemonieprojekts dadurch begrenzt sind, weil Akteure und Fraktionen noch zu unverbunden sind. Eine europäische Zivilgesellschaft mit europäischer Öffentlichkeit ist kaum vorhanden (Buckel et al 2012, S 29). Seite 23 3.5. Das linksliberal-alternative Hegemonieprojekt: Bei diesem Projekt ist ein liberaler normativer und politischer Citizenship Ansatz, der Träger eines politischen Liberalismus ist, wesentlich. Dieser Liberalismus umfasst Toleranz mit Menschen- und Bürger/innenrechten, mit Minderheitenrechten und sozialem Ausgleich. Die Autoren vertreten hier die Ansicht, dass sich in diesem Hegemonieprojekt die Ziele jener zivilgesellschaftlichen Kräfte verdichten, die nicht primär an ökonomischen Fragen im engeren Sinn ansetzen. Akteur/innen kommen aus der Richtung des politischen Liberalismus und der neuen Linken (Buckel et al 2012, S 29f). Viele davon kommen aus der dritten Welle der Frauenbewegung und setzen sich beispielsweise für Antirassismus, für Umweltschutz und die Rechte von Kindern, alten Menschen usw. ein. Die wesentliche soziale Basis dieses Projektes kommt von den Grünen. Vertreten wird eine ausgesprochen proeuropäische Strategie, die EU wird als Konstellation postnationaler Bürger/innenschaft gesehen. Über europäische Richtlinien wird beispielsweise versucht progressive Veränderungen durchzusetzen, die auf nationaler Ebene nicht möglich wären (Buckel et al 2012, S 30). Die ökonomischen Ressourcen sind im Vergleich mit anderen Projekten aber schwach. Besondere Ressourcen liegen im hohen sozialen und kulturellen Kapital der Akteur/innen und auch in der Unterstützung aus diesem Bereich wie etwa der Wissenschaft usw. (Buckel et al 2012, S 30). 4 Das Vertrauen in die Europäische Union ist durch die Krise betroffen Auch das Vertrauen in die Europäische Union ist durch die Krise betroffen. Ursachen eines allgemeinen Vertrauensverlustes in die Politik sind insbesondere im Krisenmanagement der vergangenen Jahre begründet. Vor allem hat die Eurokrise und Reaktionen der Politik auf diese das Vertrauen vieler Menschen in die gemeinsame Währung negativ beeinflusst. Dazu haben auch die Bankenrettungspakete und die sinkenden Einnahmen durch die Rezession beigetragen. Sie haben Schuldenstände explodieren lassen und damit auch die Spielräume für materielle Zugeständnisse erheblich reduziert. Seite 24 Die Subalternen der EU-Mitgliedstaaten sind nun auch im Fokus von Austeritätsbzw. Sparprogrammen, welche durch das europäische Institutionengefüge verordnet wurden (http://arranca.org/ ausgabe/46/ die-neuzusammensetzung-von-zwang-undkonsens-in-d..., S 2). Diese Maßnahmen und Projekte haben Auswirkungen auf das Vertrauen der Bevölkerung in die Europäische Union. Das steht auch in Zusammenhang damit, dass die Eurokrise viel umfassender ist als nur eine Staatsschuldenkrise. Eine Staatsschuldenkrise kann grundsätzlich mittels kurzfristiger Rettungsmechanismen, mittelfristiger Sparmassnahmen und langfristiger Strukturreformen bewältigt werden. Die gegenwärtige Krise ist auch eine Vertrauenskrise in die Politik, vor allem in den Euro als gemeinsame Währung (Heinen 2012, S 1 ff). Dass die Eurokrise auch eine Vertrauenskrise ist kann durch Ergebnisse von Umfragen, wie beispielsweise durch jene des Bankenverbandes, wo 91 % der Befragten angaben nicht daran zu glauben, dass die Staatsverschuldung in den kommenden zehn bis fünfzehn Jahren sinken werde (Bankenverband 2012). Vor allem kann eine Vertrauenskrise auch durch das Eurobarometer belegt werden. Im Jahre 2012 zeigt Eurobarometer, dass 59 % der deutschen Bevölkerung europäischen Institutionen misstrauen (EU 57 %). Ein Vergleich mit dem Jahre 2007 zeigt den Trend auf, hier waren es 36 % (EU 32 %) (Eurobarometer 2007/2012). Im letzten erschienenen ARD-Deutschland-Trend gaben 75 % der befragten Personen an, dass sie glauben, der schlimmste Teil der Eurokrise stehe noch bevor. Besondere Sorgen zeigten sie bezüglich ihrer Ersparnisse, das gaben damals 75 % der Befragten an (Infratest –Dimap 2013). V. DIE STRATEGISCHE NEUORIENTIERUNG IN DER KRISE DER EUROPÄISCHEN UNION 1. Eine Krise des neoliberalen Projektes Die aktuelle Krise der Europäischen Union stellt sich hegemonietheoretisch gesehen als Krise des neoliberalen Projekts dar. Wesentlich ist hier die einseitig ökonomische Integrationsweise der EU. Diese fokussiert auf die ökonomische Vergemeinschaftung Seite 25 über Binnenmarkt, Währungsunion und Austeritätspolitiken gemäß den MaastrichtKriterien und auch auf die periphere Integration Osteuropas (Forschungsprojekt „Staatsprojekt Europa“ 2012, S 30). Die Maastricht-Kriterien bzw. die Konvergenzkriterien wurden im Vertrag von Maastricht festgelegt. Demnach kann ein Staat nur an der Europäischen Wirtschaftsund Währungsunion teilnehmen, wenn er die fiskalischen und monetären Maastricht Kriterien erfüllt (Statistik Austria – Maastricht-Indikatoren). Folgende fiskalische Maastricht Kriterien sind zu erreichen: Das öffentliche Defizit darf 3% des BruttoInlandsproduktes (BIP) nicht übersteigen. Der öffentliche Schuldenstand darf maximal 60 % des BIP erreichen. Folgende monetären Maastricht – Kriterien sind zu erreichen: Die nationale Inflationsrate darf maximal eineinhalb (1,5) Prozentpunkte über derjenigen der drei preisstabilsten EU-Staaten liegen. Der langfristige Zinssatz darf höchstens zwei (2) Prozentpunkte höher als in den preisstabilsten EU-Staaten liegen (Statistik Austria-Maastricht-Indikatoren). Gleichzeitig mit diesen Zielen wurde aber die Wirtschafts- und Sozialpolitik nicht in derselben Weise auf die Europäische Ebene verlagert. Daher ist die EU gegenwärtig auch institutionell nicht in der Lage durch makroökonomische Steuerungs- und Ausgleichsmechanismen auf die Staatsschuldenkrise der Mitgliedstaaten zielführend zu reagieren. Die neoliberale Politik ist auch gegenwärtig im Bereich der EU aktuell. Es ist aber hier zu beachten, dass gesellschaftliche Konflikte zunehmen und auch die Bereitschaft vorhanden ist Elemente neoliberaler Krisenlösungsstrategien auch autoritär gegen die Bevölkerungen durchzusetzen (Buckel et al 2012, S 30f). 2. Die vier strategischen Suchprozesse innerhalb des bisherigen neoliberalen Hegemonieprojektes 1) Besonders verfolgen die starken neoliberalen Strömungen der europäischen sozialdemokratischen Parteien nach wie vor eine proeuropäisch-neoliberale Strategie. Ein Beispiel dafür ist bzw. war Peer Steinbrück (Buckel et al 2012, S 31). Diese proeuropäisch-neoliberale Strategie steht momentan aber vor der Situation, dass die gegenwärtige Krise die europäische Integration auch in der Richtung beeinträchtigt hat, dass der Nationalismus in Europa grundsätzlich zunimmt (http:// www. bpb..de/apuz/154378/halbe-hegemonie-das-deutsche-dilemma?p=all, S 3). Seite 26 Konkrete Entwicklungen sind hier aber noch nicht vorhersehbar. Die Strategie der Regulierung ist bei diesen Strömungen wesentlich. Sie verbindet sich mit Strategien der Versicherungsbranchen und der Vermögensbesitzer, die besonders den Zerfall des Euro verhindern wollen. Um die Gemeinschaftswährung zu erhalten verfolgen die Akteur/innen die Strategie, neue europäische Institutionen zu etablieren. Das wären beispielsweise eine Wirtschaftsregierung, ein europäisches Finanzministerium oder ein ausgebauter europäischer Rettungsschirm oder auch ein europäischer Währungsfonds. Eurobonds stehen hier nicht zur Diskussion (Buckel et al 2012, S 32). Eurobonds stehen in keinem Zusammenhang mit der europäischen Gemeinschaftswährung Euro. Es sind Anleihen mittlerer und längerer Laufzeit, die für den internationalen Kapitalmarkt (Euromarkt) errichtet wurden. Sie werden aber von einem anderen Land emittiert als jenem Land, aus dem die konkrete Währung stammt. In der diese aufgelegt werden. Die Emission von Eurobonds erfolgt grundsätzlich in Form von Inhaberschuld-verschreibungen durch internationale Bankkonsortien. Die gängigsten Anleihewährungen sind der US-Dollar, der Yen, Schweizer Franken, Pfund Sterling, sowie auch der Euro. Schuldner sind private Unternehmen von hoher Bonität, Staaten usw. (http://de. wikipedia.org/ wiki/ Eurobond_(Euromarkt-Anleihe), S 1). 2) Die autoritär-neoliberale Strategie wird besonders von der deutschen Exportindustrie und von transnationalen Konzernen verfolgt. Sie sind zudem auch im European Round Table of Industrialists und Business Europe organisiert. Der Binnenmarkt ist das entscheidende politische Projekt. Eurobonds werden hier befürwortet. Diese Strategie versucht das alte neoliberale Projekt, den „disziplinierenden Neoliberalismus“ zu retten (Gill 1998, S 1 ff). Der „disziplinierende Neoliberalismus“ unterwirft aber zunehmend viele Bereiche des Lebens der Marktdisziplin. Auf politisch – institutioneller Ebene wird dieser disziplinierende Neoliberalismus durch einen neuen Konstitutionalismus, der versucht politische Entscheidungen zu entdemokratisieren und eine neue neoliberale Politik durch internationale Abkommen zu zementieren, durch einen neuen Konstitutionalismus verankert. Ein wesentliches Beispiel dafür ist das bei den EUKonvergenzkriterien bereits dargestellte Maastricht Kriterium. Damit werden nationale Regierungen zu fiskalischer Disziplin gezwungen und auch eine alternative Wirtschaftspolitik beinahe unmöglich gemacht (Neogramscianismus-Wikipedia; vgl. auch http://de.wikipedia. org/wiki/Neogramscianismus, S 3). Seite 27 3) Die national-neoliberale Strategie wird grundsätzlich verfolgt von Österreich, Deutschland, Finnland und den Niederlanden, sie sind Gegner bzw. radikale Ablehner proeuropäischer Krisenlösungen. Sie sind gegen jede Form von Eurobonds und gegen eine expansive Geldpolitik der EZB (Buckel et al 2012, S 30 ff). 4) Die Strategie der peripheren Statthalter ist die Position der Regierungen in Staaten im Bereich der europäischen Peripherie. Dazu zählen Spanien, Portugal, Italien, Irland und Griechenland. Sie implementieren unabhängig von ihrer Parteizugehörigkeit seit langem neoliberale Politiken. Sie sind mittlerweile aber durch ihre Notlage in eine politische Abhängigkeit von den beiden großen Mitgliedstaaten Frankreich und Deutschland geraten (Buckel et al 2012, S 30 ff). VI. DIE DEMOKRATISCHE FRAGE 1. Die Demokratie wurde zur Disposition gestellt Die Finanzinstitutionen Europäische Zentralbank (EZB) und der Internationale Währungsfonds (IWF) stehen ausserhalb jedes Einflussbereiches demokratiacher Wahlen. Sie werden im Kontext der Rettungspolitik jedoch zu konkreten Akteuren innenpolitischer Maßnahmen. Die beiden anderen beteiligten Institutionen sind nur indirekt demokratisch legitimiert, sie werden von gewählten Regierungen entsandt. Die Regierungen der Euroländer beschlossen die verschuldeten Staaten durch Gewährung von Krediten (Darlehen) vor ‚Zahlungsunfähigkeit oder auch vor einem eventuellen Ausstieg zu bewahren. Dazu musste die „Nichtbeistandsklausel“ – welche die Haftung für Schulden einzelner Mitgliedstaaten ausschließt - außer Kraft gesetzt werden (Art. 125 AEU-Vertrag). Im Meorandum of understanding wurde festgehalten, daß die Kreditnehmer bzw. die Schuldenländer zu Sparmaßnahmen verpflichtet werden (Memorandum of underständing). Zusammengefasst haben die Euroländer konkrete innenpolitische Bereiche in die Sphäre des europäischen Projekts gegeben. Als eine Folge muss im Schuldenfall jeder Mitgliedstaat Sparmaßnahmen ergreifen, für einen Ausstieg aus dem Euro gibt es keine Regelung. Dafür ist aber keine demokratische Basis vorhanden. Mehr Seite 28 Demokratie bzw. mehr Freiheit für die Mitgliedstaaten der EU zur Zeit der Krise sind Ziele die nicht zu verwirklichen sind. Weil Interessen der Herrschenden in der Krise gefährdet sind, wird die bürgerliche Demokratie sogar zur Disposition gestellt. Beispielsweise wurden in den Defizitstaaten die üblichen demokratischen Spielregeln zur Durchsetzung der massiven Kürzungspolitik weitgehend außer Kraft gesetzt. Regierungen von Defizitstaaten unterschrieben für die Zusage von Hilfszahlungen der anderen Euroländer sogar Memoranden womit der sogenannten „Troika“, die aus der Europäischen Kommission, der Europäischen Zentralbank und dem Internationalen Währungsfonds besteht, weitreichende Kontrollrechte eingeräumt wurden. Wie weit hier Werte der Demokratie beeinträchtigt werden steht zur Diskussion (http:// www.andreas-wehr.eu/von-der-euro-krise-zur-krise-der- demokratie-202.html, S 2). Die deutsche Bundeskanzlerin Angela Merkel proklamierte in Anspielung auf die Worte von Willi Brandt „Mehr Demokratie wagen“ in ihrer ersten Regierungserklärung vor etwa nun bereits mehreren Jahren den Satz: „Lassen sie uns mehr Freiheit wagen“. Beide Aussagen richtig verstanden bedeuten aber eigentlich dasselbe. Merkel erklärte damals auch, dass für sie ein Volk mehr als eine Ansammlung von Individuen sei, nämlich eine „Schicksalsgemeinschaft“ (Willy Brandt-Wikipedia; Regierungserklärung: mehr Freiheit wagen/ZEIT ONLINE-Die Zeit). Unter diesem Motto setzte damals Willy Brandt innenpolitische Reformen durch, die Staat und Gesellschaft weiter liberalisierten und viele Bürger/innen auch ermutigten sollte selbst politisch aktiv zu werden. Das ist auch für eine funktionierende Zivilgesellschaft in der Europäöischen Union wesentlich (Willy Brandt-Wikipedia; Bundeskanzler Willy Brandt Stiftung: Willy Brandt). 2. Das Fehlen einer europäischen Zivilgesellschaft Solche Kontrollrechte bedeuten aber auch eine Einschränkung der Freiheit. Bezogen auf die Hegemonieprojekte ist es zum aktuellen Zeitpunkt auch nicht vorhersehbar, welche Konstellation sich durchsetzen wird, welches Staatsprojekt sich dabei herausbildet und vor allem auch, wie das institutionelle Gefüge des europäischen Staatsapparate-Ensembles verändern wird. Nach Ansicht der Forschungsgruppe lässt sich nicht einmal sagen, ob das europäische Staatsprojekt die Krise überleben wird. In diesem Staatsprojekt ist eine europäische Zivilgesellschaft kaum noch vorhanden (Buckel et al 2012, S 42). Besonders das Fehlen einer europäischen Zivilgesellschaft macht die EU aber anfällig für wirtschaftliche Krisen. Ein Vergleich der Wachstumsraten mit der Seite 29 Akzeptanz der EU in der Bevölkerung weist eine deutliche Parallele auf. Sinken die Wachstumsraten in der EU, dann nimmt in der Regel auch ihre Akzeptanz bei der Bevölkerung ab. Das Staatsprojekt Europa ist in seiner inneren Struktur, die zu einer Distanzierung der Bevölkerung von den Entscheidungsträgern führt, grundlegend undemokratisch verfasst (Buckel et al 2012, S 42). 3. Die Demokratie in Zeiten der Eurokrise: Politische Reaktionen von Vertretern der Politik 3.1. Diskussionsveranstaltung im österreichischen Parlament: Die geänderten Entscheidungsprozesse in der EU vor dem Hintergrund der Finanzund Schuldenkrise und damit auch die demokratische Frage standen bereits im Jahre 2011 im Rahmen einer Diskussionsveranstaltung im Österreichischen Parlament auf der Tagesordnung. Anlass war besonders der Umstand, dass die aktuelle Krise eine politische Reaktion notwendig macht, die sich nicht an bewährten Mustern orientieren kann, weil die von den Märkten erzeugte Dynamik zunehmend raschere, bisher auch nicht erprobte Interventionen erfordert. EU-Kommissar a.D. Franz Fischler, Universitätsprofessorin Sonja Puntscher-Riekmann, der Abgeordnete Ewald Stadler und der Abgeordnete zum Europäischen Parlament, Hannes Swoboda, suchten im Rahmen dieser Veranstaltung nach Antworten auf die Fragen: Wer entscheidet tatsächlich? Wie ist es um die Mitwirkung des Parlaments bestellt? Kommt die Demokratie unter die Räder? Sind wir am Weg zur Eurokratur? (Parlamentskorrespondenz Nr. 1192 vom 05.12. 2011). 3.2. Reden bzw. Aussagen von Abgeordneten zum Thema (Auswahl): Die Hauptaussage von Franz Fischler lautete: „Die Europäische Kommission muss demokratischer werden. Er gab grundsätzlich zu bedenken, dass Demokratie nicht von einem Tag auf den anderen funktionieren könne, sie lebe von einem Dialog mit der Bevölkerung“. Eine Änderung des EU-Vertrages, die sich ausschließlich auf einen Sanktionsmechanismus für das Schuldenmachen reduziert lehnte er jedenfalls ab (Parlamentskorrespondenz Nr. 1192 vom 05.12. 2013). Seite 30 Sonja Puntscher-Rieckmann betonte besonders, dass die EU Solidarität brauche. Der Vertrag von Lissabon habe zwar mehr Demokratie geschaffen, er liefere ihrer Ansicht nach aber zu wenig Handhabe für eine Krise dieses Ausmaßes. Die EU sei eine große Währungsunion und überlasse die Wirtschaftspolitik aber den Mitgliedstaaten, die von gegenseitigem Misstrauen geprägt sind und bei Lösungsmodellen nach intergouvernementalen Mechanismen außerhalb der EUVerträge greifen. Sie umriss so das Wesentliche der Ausgangslage für die aktuelle Krise und deren möglichen Lösungsversuchen (Parlamentskorrespondenz Nr. 1192 vom 05.12. 2011. Der Nationalratsabgeordnete Ewald Stadler sah in der EU in erster Linie ein Demokratieproblem und stellte fest, die Union fürchte derzeit nichts stärker als die Bevölkerungen. Er sah im Euro von Anfang an einen Geburtsfehler und trat dafür ein, die Weiterentwicklung der EU zu „entschleunigen“. Man solle sich mehr Zeit nehmen und die Menschen einbinden, zumal man an einer demokratischen Diskussion nicht vorbeikommen werde (Parlamentskorrespondenz Nr. 1192 vom 05.12. 2011). Hannes Swoboda, der Abgeordnete zum Europäischen Parlament, betonte besonders, dass eine Euro-Kernunion die EU zerstören würde. Er führte den Übergang der Macht auf die Märkte auch auf die reale Politik zurück, die sich seiner Meinung nach durch ständige Deregulierungen selbst entmachtet habe. Er trat für die Schaffung einer echten Währungsunion ein, erteilte aber jedenfalls dem Konzept einer Euro-Kernzone eine klare Absage (Parlamentskorrespondenz Nr. 1192 vom 05.12. 2011). Zur demokratischen Frage sei nun abschließend auch festgestellt: In der demokratischen Frage laufen die unterschiedlichen Stränge alternativer Projekte zusammen. Sie muss der Ausgangspunkt für eine Auseinandersetzung mit der Krise und zugleich der Krisenlösung sein. Der Kampf um Demokratie muss der Ausgangspunkt für eine Transformation sein. Für einen radikalen Transformismus, der darauf abzielt, die bestehenden Kräfteverhältnisse so zu verschieben, dass die Kämpfe um Emanzipation auch weiter vorangetrieben werden können (Buckel et al 2012, S 42). Seite 31 VII. MASSNAHMEN GEGEN DIE EUROKRISE 1. Die Institutionen „European Finacial Stability Facility“ (EFSF) und „European Financial Stabilisation Mechanism“ (EFSM). Um bestmöglich sicherzustellen, dass den Euro-Mitgliedstaaten nicht das Geld ausgeht, wurde die Institution „European Finacial Stability Facility“ (EFSF) gegründet. Im Bereich der öffentlichen Diskussion bzw. in den Medien wurde diese Institution auch als „Rettungsschirm“ bekannt. In Not geratenen Euroländern wird durch den EFSF dadurch geholfen, indem auf dem internationalen Finanzmarkt Kredite bzw. Darlehen aufgenommen werden, die das konkrete Land nicht mehr erhalten würde. Diese werden dann an dieses Land weiterleitet (http://www.bpb.de./ internationales/europa/ europa-kontrovers/47840/einleitung, S 2). Alle Eurostaaten stehen hinter dem EFSF* und bürgen mit einer Summe von 440 Milliarden Euro für die Anleihen. Zusätzlich haften noch weitere 250 Milliarden Euro vom Internationalen Währungsfonds. Dazu kommen noch weitere 60 Milliarden Euro, die von der Europäischen Kommission auf dem Kreditmarkt aufgenommen und dem Rettungsschirm bei Bedarf zur Verfügung gestellt werden können. Das ist der sogenannte „European Financial Stabilisation Mechanism“ (EFSM). Beide Institutionen, der EFSF und der EFSM waren nur vorübergehend etabliert und wurden 2013 durch den „European Stability Mechanism“ (ESM), einer Art Europäischer Währungsfonds, abgelöst (http://www. bpb.de./internationales/ europa/europa-kontrovers/47840/ einleitung, S 2). Noch vor Gründung des EFSF zur Kreditgewährung von Staaten erhielt Griechenland im Jahre 2010 Kredite vom IWF und den Euro-Mitgliedstaaten im Rahmen von zwei Hilfspaketen. Ab 2013 sollte dann auch der dauerhafte Stabilitätsmechanismus ESM funktionieren (http://lobbypedia.de/wiki/ Eurokrise, S 2 vgl. auch http://www.taz.de/Glossar-zur-Eurorettung/I79059/). Der Europäische Stabilitätsmechanismus ESM, als Teil des „Euro-Rettungsschirms“, ist eine internationale Finanzinstitution die ihren Sitz in Luxemburg hat. Der ESM soll überschuldete Mitgliedstaaten der Eurozone durch Notkredite und durch Bürgschaften unterstützen. Dadurch soll besonders die Zahlungsunfähigkeit dieser Länder verhindert werden (Europäischer Stabilitätsmechanismus-Wikipedia). Seite 32 Neben EFSF und ESM hat die EU-Kommission auch fünf Verordnungen und eine Richtlinie erlassen, das ist die sogenannte „Six Pack Maßnahme“ zur Erneuerung des Stabilitäts- und Wachstumspaktes. Es sind hier schärfere Strafen für Budgetsünder vorgesehen, die auch schneller verhängt werden können (EUInfothek vom 29.09.2011). Diese Maßnahmen der EU basieren aber nicht auf einem Interessenausgleich zwischen den Mitgliedstaaten, sondern auf einer marktkonformen Durchsetzung eines radikalen Sparpakets. Zu diesem Zweck wird auch über Rechtsakte verfügt, wie beispielsweise dem Fiskalpakt oder die Six Pack Maßnahme. Auf Ebene der EU wurde ein Exekutivföderalismus errichtet, der strenge Durchgriffsmöglichkeiten auf die nationalen Haushalte schafft (http://www.freitag. de/autoren/ henrihaiti/europaeische-integration-in-der-hegemoniekr..., S 1). Zu den hier dargestellten Maßnahmen gegen die Euro-Krise gibt es auch noch sogenannte „Alternativvorschläge“. Beispielsweise ist ein solcher Alternativvorschlag die Einführung eines Süd-Euros. Experten gehen davon aus, dass bei einer bereits bei Einführung des Süd-Euros erfolgten Abwertung (etwa zwischen 30 und 40 %) gegenüber dem verbleibenden Nord – Euro die Wettbewerbsfähigkeit schon ausgeglichen wäre (Flassbeck/Spieker 2010, S 3 ff). Dass die Euro-Krise nicht nur eine Staatsschuldenkrise ist, sondern eine viel tiefere Krise, die besonders durch Leistungsbilanzungleichgewichte entstanden ist, ist mittlerweile überwiegende Meinung. Außenhandelsdefizite von Staaten stehen mit Außenhandelsüberschüssen von anderen Staaten im Zusammenhang. Südliche Krisenländer Europas haben daher nur dann eine reale Möglichkeit ihr staatliches Budgetdefizit zu senken, wenn sie ihre Leistungsbilanz verbessern. Bei einem unveränderten Leistungsbilanzdefizit bedeutet eine Senkung des staatlichen Budgetdefizits automatisch ein steigendes Finanzierungsdefizit des privaten Sektors. Das sind Unternehmen und Haushalte. In diesem Fall werden öffentliche durch private Defizite ersetzt die langfristig gesehen wieder zu staatlichen Defiziten werden können bzw. werden (http://lobbypedia.de/wiki/Eurokrise, S 2). Seite 33 2. Maßnahmen der Europäischen Union und ihrer Mitgliedstaaten Die Europäische Union reagiert auf die globale Wirtschaftskrise mit Maßnahmen auf verschiedenen Ebenen. Das sind: - eine Haushaltskonsolidierung und eine verstärkte wirtschaftspolitische Steuerung - eine Reform und Korrektur des Finanzsektors - der Förderung von Wachstum und der Schaffung von Arbeitsplätzen - die wirtschaftliche Lage in den einzelnen Ländern (http://europa. eu/ newsroom/highlights/finacial-crisis/index_de.htm, S 1). Die 2011 beschlossene „Economic Governance“ und der „Fiskalpakt“ lassen auch den Schluss zu, dass der Neoliberalismus trotz Einbußen seiner führenden Position noch immer vorhanden ist. Diese Einbußen erfolgten besonders durch Zwangsmaßnahmen auf nationalstaatlicher Ebene, aber auch auf Ebene der EU. Dadurch soll die europäische Wirtschaftspolitik mit einer europäischen Schuldenbremse, einem Verfahren zur wettbewerblichen Restrukturierung und einem automatischen Austeritätsmechanismus positiv beeinflusst werden. Es ist auch beabsichtigt, die derzeitigen Austeritäts- und Restrukturierungsprogramme der EUProblemstaaten auf alle Mitgliedstaaten auszudehnen und durch Rechtsgrundlagen dauernd zu sichern. Diese Maßnahmen sollen auch gegen den Widerstand der Bevölkerung durchgesetzt werden können (http://arranca.org/ausgabe/46/die- neuzusammensetzung-von-zwang-und-konsens-in-d..., S 3). VIII. EIN AUSBLICK 1. Die Ansichten von Viviane Reding und Olli Rehn zur Krise Nach Ansicht von Viviane Reding, Vizepräsidentin der Europäischen Kommission, und Olli Rehn, Mitglied der Europäischen Kommission, kann seit Ende des Jahres 2013 festgestellt werden, dass die EU begonnen hat die aktuelle Krise zu überwinden. Für viele EU-Bürger seien die schwierigen Zeiten aber noch nicht vorbei. Viele hielten das Auseinanderbrechen der Eurozone für eine reale Seite 34 Möglichkeit. Seit 1. Jänner 2014 verwenden aber sogar mehr Länder den Euro. Lettland ist nun als achtzehntes Land der gemeinsamen Währung beigetreten (Die Presse, Donnerstag 2.Jänner 2014, S 22). Lettland kämpfte sich noch vor zwei Jahren aus einer tiefen Rezession heraus. Es wurde unterstützt durch ein EU-IWF-Programm und hat gegenwärtig die am schnellsten wachsende Wirtschaft in der EU (Die Presse, Donnerstag 2.Jänner 2014). Nach Ansicht der beiden Autoren ist auch Irland nach Jahren der Krise – verursacht durch den katastrophalen Zusammenbruch seiner Banken und seines Immobilienmarkts im Jahre 2008 - durch ein Reformprogramm gestärkt worden. Irland konnte am 15. Dezember 2013 den Euro-Rettungsschirm verlassen. Lettland und Irland würden zeigen, dass Europas Strategie zur Bekämpfung der Krise wirksam ist (Die Presse, Donnerstag 2.Jänner 2014). Das Kernelement dieser Strategie ist, dass es Solidarität nur im Gegenzug für Solidarität gibt. Mitgliedstaaten mit finanziellen Schwierigkeiten wird von anderen Staaten geholfen. Der politische Wille ist die kumulierten Ungleichgewichte mittels Reformen zu beseitigen und die Finanzlage auf eine tragfähige Grundlage zu stellen (Die Presse, Donnerstag 2.Jänner 2014). Neben diesen beiden Ländern gäbe es auch in anderen Ländern positive Entwicklungen. Beispielsweise werde Spanien ab Jänner 2014 keine finanzielle Unterstützung mehr benötigen. Nach Ansicht der Autoren sind drei Punkte entscheidend um aus der Krise gestärkt hervorzugehen. Erstens müsse Europa seine Strukturreformen fortsetzen, zweitens braucht Europa ein gut funktionierendes Bankensystem um Investitionen zu fördern und den notwendigen Strukturwandel zu finanzieren und drittens seien solide Rahmenregeln für eine europäische Wirtschaftsund Finanzpolitik notwendig 2. Die unterschiedlichen Wirtschaftsmodelle Europas werden durch eine Einheitswährung nicht harmonisiert 2.1. Studie: Vergleich mit den USA Neben dieser beispielsweise positiven Perspektive der beiden EU-Kommissionsmitglieder gibt es allerdings auch sehr gegenteilige, die Krise betreffende Studien. Seite 35 Beispielsweise eine von Ökonomen durchgeführte und am 24.08. 2013 in den Deutschen Wirtschafts-Nachrichten veröffentlichte Studie. Ein Vergleich mit den USA zeigte hier, dass die unterschiedlichen Wirtschaftsmodelle Europas durch eine Einheitswährung nicht harmonisiert werden. Die Geldpolitik der EZB hätte die immense Verschuldung der Euro-Staaten verstärkt. Die Löhne in den einzelnen Ländern könnten gar nicht genug sinken, um die Krisenländer wieder wettbewerbsfähig zu machen. Es sei nun für Reformen zu spät (Deutsche Wirtschafts Nachrichten, 24.08. 2013). 2.2. Förderprogramme zur Forcierung der Wirtschaft: Im Jahr der Veröffentlichung dieser Studie hat die EU jedenfalls auch Förderprogramme, besonders zur Forcierung der Wirtschaft, beschlossen. Für das Jahr 2014 bzw. grundsätzlich für die Zukunft ist auch „Mehr Erfolg weniger Bürokratie“ ein Schlagwort der Europäischen Union. Im November 2013 hat das Europäische Parlament die Programme „Horizon 2020“ und „COSME“ beschlossen. Diese Förderprogramme sollen es besonders Klein- und Mittelbetrieben bzw. Unternehmen (KMU) ermöglichen mehr in Forschung und Innovation zu investieren (Horizon 2020; COSME). Das Programm „Horizon 2020“ soll mit etwa 70 Milliarden Euro Forschung und Innovation innerhalb der EU fördern. Künftig bekommen Europas Wissenschaftler zwischen 70 und 100 Prozent der direkten Kosten rückerstattet, zudem sollen Förderanträge schneller abgewickelt werden. Neu ist das KMU-Instrument innerhalb der Forschungsförderung das speziell auf die Anforderungen von kleinen und mittleren Unternehmen im Forschungsbereich zugeschnitten ist (Rübig 2013). Das Programm „COSME“ soll mit etwa zwei Milliarden Euro die Wettbewerbsfähigkeit von KMU stärken. Neben finanzieller Unterstützung sollen auch politische Maßnahmen wie Bürokratieabbau und leichterer Zugang zu Finanzierung vorangetrieben werden. Es soll auch das Programm „Erasmus für Jungunternehmer“ im Rahmen von COSME ausgebaut werden (Rübig 2013). 3. Die Vergabe von Firmenkrediten (Darlehen) durch Banken Zu diesen dargestellten Förderprogrammen für die Wirtschaft bzw. für Unternehmen ist die ergänzende Feststellung notwendig, dass Banken derzeit so wenig Firmenkredite vergeben wie dies bisher noch nie der Fall war. Was die Konjunktur betrifft sehen manche Experten die Zinssenkung der Europäischen Zentralbank Seite 36 (EZB) sogar als wirkungslos. Die Europäische Zentralbank hat den Leitzins* auf ein Rekordtief von 0,25 Prozent gesenkt um dadurch auch die Wirtschaft anzukurbeln (Die Presse Samstag, 4.Jänner 2014). Durch diese Maßnahme sollen Wachstum, ein Konjunkturaufschwung und mehr Arbeitsplätze geschaffen werden. Beispielsweise sind Darlehen im November 2013 im Vergleich zum Vorjahrsmonat um 3,9 Prozent gesunken. In den südeuropäischen Krisenstaaten ist die Situation besonders dramatisch. Firmenkredite gingen in Spanien um 13, 5 Prozent zurück, in Italien um 5,9 Prozent (Die Presse Samstag, 4. Jänner 2014). Die nationalen Banken in den diversen Ländern geben aber das günstige EZB-Geld nicht an die Unternehmen weiter, sondern kaufen vielfach Staatsanleihen. Ein Grund dafür ist, dass Banken im Gegensatz zu Firmenkrediten Eurostaatsanleihen in ihrer Bilanz mit einem Risikogewicht von null Prozent darstellen dürfen. Die Forderung des Internationalen Währungsfonds, Staatshaushalte und Banken zu entflechten, wird dadurch nicht erfüllt, sondern genau der gegenteilige Effekt bewirkt (Die Presse Samstag, 4.Jänner 2014). Die EZB hat bereits mehrfach erklärt, dass sie die Kreditvergabe ankurbeln könnte, indem sie von Banken mit solcher Praxis eine Art Strafzins „für das Parken von Geld“ einführen wolle. Die Banken lehnen solche Absichten jedoch grundsätzlich ab (Die Presse Samstag, 4.Jänner 2014). 4. Die Wahl zum Europäischen Parlament am 25. Mai 2014 Im Jahre 2014 wird besonders der 25.Mai ein entscheidender Tag für Europa werden. An diesem Tag findet auch in Österreich die Wahl zum Europäischen Parlament statt. In der gesamten Europäischen Union sind etwa 360 Millionen Bürger/innen aufgerufen, ihre Volksvertretung zu wählen. Das Europäische Parlament, gestärkt durch den Vertrag von Lissabon 2009, ist die demokratisch legitimierte Kontrollinstanz Gesetzgebungsbereichen. und zudem Besonders beim mitentscheidend EU-Budget – in mit fast allen mehrjährigem Finanzrahmen – spielt das Parlament eine entscheidende Rolle, es handelt hier auf Augenhöhe mit dem Rat (Vertrag von Lissabon 2009). *EU-Wahlumfrage Hajek vom 12.12.2013: Als Ergebnis erster Umfragen zur Wahl zum Europäischen Parlament am 25. Mai 2014 ist ein Trend in Richtung konservative Parteien feststellbar. Seite 37 Konservative und auch Rechte könnten demnach gegenüber Links-Parteien einen Vorteil haben. Das soll zumindest in Österreich so sein. Die ÖVP habe hier die meisten Wählerstimmen zu erwarten, die FPÖ läge etwa gleich mit der SPÖ. Die Grünen stagnieren eher. Für eine Überraschung könnten hier aber die NEOS sorgen. Das Team Stronach, Hans Peter Martin u.a. werden sehr wahrscheinlich keinerlei Rolle spielen. Zu sehr ähnlichen Ergebnissen kam auch Karmasin mit der Umfrage vom 7.12.2013 (Quelle: Hajek/ATV/FORMAT) IX. ZUSAMMENFASSUNG, BEMERKUNGEN Bereits 1922 begannen die ersten Ansätze ein gemeinsames Europa zu schaffen. Aus der Idee von Coudenhove-Kalergi entstand dann sehr viel für dieses gemeinsame Europa bzw. die Europäischen Gemeinschaften bis zur Gründung der Europäischen Union (EU) und deren Erweiterungen. Als Staatenbund bzw. als (teilweise) supranationale Organisation wollte die Europäische Union sich nicht durch Gewalt ausdehnen sondern durch Überzeugung. Die sogenannte Euro-Krise, für deren Ursachen besonders ökonomische Ungleichgewichte im EU-Binnenmarkt gesehen werden, hat die Europäische Union aber vom Vorbild – lange Zeit waren besonders auch die Erweiterungs- und Nachbarschaftspläne Europas ein Vorzeigeprojekt - zum Unterstützungsfall des Weltwährungsfonds gemacht. Vereinfacht dargestellt ist die Euro – Krise eine durch die Welt – Finanzkrise 2007/2008 verstärkte Haushalts- und Verschuldungskrise mehrerer Mitgliedstaaten der Eurozone. Grundlegende Ursachen liegen auch in „Konstruktionsfehlern“ der Währungsunion selbst. Durch die griechische Finanzkrise im Jahre 2010 wurde sie dann besonders akut. Sie kann als multiple Krise bezeichnet werden, weil zu dem bereits dargestellten Institutionengefüges auch eine vorhanden ist. Hegemoniekrise Die fünf des grundsätzlich europäischen möglichen Hegemonieprojekte in der Krise mit unterschiedlichen politischen Strategien lassen zum gegenwärtigen Zeitpunkt nicht erkennen, welches Projekt sich letztendlich durchsetzen wird. Die aktuelle Krise der Europäischen Union stellt sich hegemonietheoretisch gesehen aber jedenfalls als Krise des neoliberalen Projektes dar. Wesentlich ist hier die einseitig ökonomische Integrationsweise der EU. Diese fokussiert auf die ökonomische Vergemeinschaftung über Binnenmarkt, Währungsunion und Austeritätspolitiken gemäß den Maastricht Kriterien und auch die periphere Integration Osteuropas. Seite 38 Die Krise der EU als Hegemoniekrise in Europa und die Bemühungen zur Problemlösung erfüllen jedenfalls auch den Begriff „autoritärer Wettbewerbsetatismus“. Alle Lösungsversuche müssen auch auf eine Stärkung bzw. Wiedererlangung der ursprünglichen neoliberalen Gesellschaftsformation gerichtet sein bzw. diese jedenfalls mitberücksichtigen. Bei Betrachtung der fünf dargestellten Hegemonieprojekte ist es auch Erkennbar, dass sich der Kampf um Hegemonie im Unterschied zum klassischen Gramscianismus nicht mehr alleine auf nationaler Ebene abspielt, sondern auf mehreren Ebenen. Das sind vor allem die Europäische Gemeinschaft und die Weltordnung. Von besonderer Bedeutung sind hier die Produktionsmittel bzw. die materiellen Kapazitäten, Ideologien und Theorien bzw. Ideen und Institutionen und Organisationen. Die „Marktdisziplin“ ist hier besonders stark ausgeprägt und führt auch zu massiven Nachteilen für die Natur bzw. unseren Lebensraum. Die Hegemonie des Neoliberalismus beruht in zunehmender Weise nicht mehr nur auf Konsens, sondern auch auf der Ausübung von Zwang. Die Forderung nach Demokratie ist aber im Bereich der Nationalstaaten und auch auf Ebene der Europäischen Union von erheblicher Bedeutung. Für den Euroraum wird es von besonderer Bedeutung werden die Zahlungsfähigkeit der „Eurostaaten“ zu gewährleisten und das verlorene Vertrauen wieder zurückzugewinnen. Mit welchen Mitteln diese Ziele zu erreichen sind ist aber relativ unklar. . Beispielsweise sind hier Eurobonds, Europäische Währungsfonds usw. in Diskussion. Für alle Lösungsversuche gibt es Befürworter, aber auch Gegner, sie werden jedenfalls kontrovers diskutiert. Nach Ansicht von Viviane Reding und Olli Rehn kann seit Ende des Jahres 2013 festgestellt werden dass die EU begonnen hat die aktuelle Krise zu überwinden. Das ist aber zumindest sehr differenziert zu betrachten. Die Auswirkungen der gegenständlichen Eurokrise müssen politikfeldspezifisch fokussiert und beurteilt werden. Die Eurokrise hat bereits politikfeldspezifische Diskurse bewirkt und beispielsweise auch zu Prognosen über eine (mögliche) Abschwächung der Bedeutung der Europäischen Union im weltweiten Mächtespiel geführt. Ein wesentlicher Bedeutungsverlust der Europäischen Union, als globaler Akteur mit unterschiedlichen Bereichen und Intensitäten der Macht, ist mit dieser Krise aber nicht erfolgt. Für die politischen Akteure bzw. die Entscheidungsträger ist es sehr wesentlich die Ursachen, alle möglichen Zusammenghänge, vor allem auch die kausalen Folgen sehr genau zu untersuchen. Es sollte alles überlegt und getan Seite 39 werden um in Zukunft solche Krisen zu verhindern oder zumindest abzuschwächen. Europa galt viele Jahre als Erfolgsgeschichte, in diese Richtung sollte gemeinsam gearbeitet werden. Das erfordert neben den Akteuren der Europäischen Union auch eine starke, (politisch) gebildete und interessierte Zivilgesellschaft mit der Einstellung „Wir sind Europäer“. Mag.Franz Zeilner (Jänner 2014) Seite 40 X. VERWENDETE LITERATUR - Artikel 3 Abs. 4 EUV. - Artikel 125 AEU-Vertrag. - Artikel 126 und 140 AEU-Vertrag. - Artikel 136 ff. AEU-Vertrag - Artikel 3 des Vertrages über die Europäische Union (EUV); Artikel 3,5,119,144,219 und 282 bis 284 des Vertrages über die Arbeitsweise der Europäischen Union (AEUV). - Art. 127-144 AEUV. - Bankenverband (2012). Euro, Banken und Politik im Spiegel der Öffentlichkeit. Ber lin, Oktober 2012. - Buckel Sonja/Cuevas Romero Manuel Jose/Eberhardt Pia/Georgi Fabian/Huke Nikolai/Kannankulam John/Kaspakrek Bern/Krämer Anna/Lüddemann Da na/Oberndorfer Lukas/Pichl Maximilian/Ressel Saida/Tsianos S. Vassilis/Wissel Jens/Wolff Sebastian, Forschungsgruppe „Staatsprojekt Europa“ (Hrsg.), Die EU in der Krise. Zwischen autoritärem Etatismus und europäischem Frühling, Müns ter 2012. - Bundeskanzler Willy Brandt Stiftung: Willy Brandt. - Der Standard vom 16.August 2009. „Die Paneuropäische Union“ - Deutsche Wirtschafts Nachrichten, 24.08. 2013. - Die Presse Donnerstag, 2.Jänner 2014. - Die Presse Samstag, 4.Jänner 2014. - CDU/CSU, Conservative Party, Partido Popular. - Choplin Gerard/Strickner Alexandra/Trouve Aurelie (Hg.), Ernährungssouveränität. Für eine andere Agrar- und Lebensmittelpolitik in Europa, Wien 2011. - Eichengreen B. J. et al, Right-wing political extremism in the Great Depression, El. R. 2012. - EU-Infothek vom 29.09.2011. - Eurobarometer 2007/2012. - Europäischer Binnenmarkt - Wikipedia. - Flassbeck/Spiecker, Der Staat als Schuldner – Quadratur des Bösen? Wirtschafts dienst 91. Jahrgang Heft 7, Wien New York 2011. - Frankfurter Allgemeine Zeitung (FAZ) vom 21.10.2004. Seite 41 - Gill Bernhard, paradoxe Natur. Zur Vieldeutigkeit der Unterscheidung von Natur und Gesellschaft, Opladen 1998. - Gramsci Antonio, Gefängnishefte – Kritische Gesamtausgabe, Hrg. Von Klaus Bochmann & Wolfgang Fritz Haug, Hamburg (GH), 1991. - Grunert Günter, Politik Unterrichten, 1/2011. - Gutschner Peter/Rohr Christian (2008). Geschichte.aktuell2 für die BHS, 2.Auflage, Veritas Verlag, Linz. - Habermas Jürgen, Zur Verfassung Europas, Berlin 2011. - Hajek/ATV/FORMAT. - Heinen N. (2012). Mission Vertrauen. Wege aus der Eurokrise. Halle. - Heinrich Böll Stiftung. - Horizon 2020/COSME. - Illing Falk, Die Euro-Krise, Analyse der europäischen Strukturkrise, Springer VS, Wiesbaden 2013 (e-book). - Infratest-Dimap (2013). ARD-DeutschlandTrend April 2013. Berlin. - Keller Horst (1999). Werte statt Grenzen. Der Europarat – Wegbereiter und Vor denker. NDV Neue Darmstädter Verlagsanstalt, Rheinbreitbach. - Keuschnigg Christian/Weyerstraß Klaus/Hofer Helmut/Schwarzbauer Wolf gang//Skriner Edith, Szenarien für die Krise des Euroraums und die Folgen für Ös terreich, Institut für Höhere Studien/Institut for Advanced Studies, Studie im Auf trag der Wirtschaftskammer Österreich,Vienna 2012. - Länder, Sprachen, Währungen, in: Internationale Regeln für Veröffentlichungen. Amt für Veröffentlichungen der Europäischen Union. - Memorandum of understanding (MoU) bzw. Letter of Intent („LoI“) (Grundsatzver einbarung). - Mundell R. (1961). 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https://openalex.org/W2619338960	https://figshare.com/ndownloader/files/8504801	unk	null	Structural features of PhoX, one of the phosphate-binding proteins from Pho regulon of Xanthomonas citri	PloS one	2,017	cc-by	8	S1 Fig S1 Fig S1 Fig S1 Fig
https://openalex.org/W1994060820	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0066605&type=printable	English	null	Biochemical Characterization of Hypothetical Proteins from Helicobacter pylori	PloS one	2,013	cc-by	10,232	Abstract Competing Interests: Author Brian P Anton is an employee of New England Biolabs. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. Competing Interests: Author Brian P Anton is an employee of New England Biolabs. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials * E-mail: steffen@bu.edu (MS); mferrer@icp.csic.es (MF) Abstract The functional characterization of Open Reading Frames (ORFs) from sequenced genomes remains a bottleneck in our effort to understand microbial biology. In particular, the functional characterization of proteins with only remote sequence homology to known proteins can be challenging, as there may be few clues to guide initial experiments. Affinity enrichment of proteins from cell lysates, and a global perspective of protein function as provided by COMBREX, affords an approach to this problem. We present here the biochemical analysis of six proteins from Helicobacter pylori ATCC 26695, a focus organism in COMBREX. Initial hypotheses were based upon affinity capture of proteins from total cellular lysate using derivatized nano-particles, and subsequent identification by mass spectrometry. Candidate genes encoding these proteins were cloned and expressed in Escherichia coli, and the recombinant proteins were purified and characterized biochemically and their biochemical parameters compared with the native ones. These proteins include a guanosine triphosphate (GTP) cyclohydrolase (HP0959), an ATPase (HP1079), an adenosine deaminase (HP0267), a phosphodiesterase (HP1042), an aminopeptidase (HP1037), and new substrates were characterized for a peptidoglycan deacetylase (HP0310). Generally, characterized enzymes were active at acidic to neutral pH (4.0–7.5) with temperature optima ranging from 35 to 55uC, although some exhibited outstanding characteristics. Citation: Choi H-P, Juarez S, Ciordia S, Fernandez M, Bargiela R, et al. (2013) Biochemical Characterization of Hypothetical Proteins from Helicobacter pylori. PLoS ONE 8(6): e66605. doi:10.1371/journal.pone.0066605 Editor: Ivo G. Boneca, Institut Pasteur Paris, France Received January 17, 2013; Accepted May 8, 2013; Published June 18, 2013 Copyright: 2013 Choi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, use, distribution, and reproduction in any medium, provided the original author and source are credited. et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted roduction in any medium, provided the original author and source are credited. Funding: The cloning and biochemical testing of recombinant proteins was funded by RC2-GM092602 from the National Institutes of Health (NIGMS), awarded to COMBREX (SK, MS). Affinity enrichment and testing of native proteins was funded by the Spanish Ministry of Economy and Competitiveness (former MICINN), within the ERA NET PathoGenoMics2 call, grant number 0315441A and BFU2008-04501-E/BMC (MF). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Biochemical Characterization of Hypothetical Proteins from Helicobacter pylori Han-Pil Choi1, Silvia Juarez2, Sergio Ciordia2, Marisol Fernandez2, Rafael Bargiela3, Juan P. Albar2, Varun Mazumdar4, Brian P. Anton5, Simon Kasif1,4, Manuel Ferrer3, Martin Steffen1,6 1 Dept of Biomedical Engineering, Boston University, Boston, Massachusetts, United States of America, 2 Proteomic Facility, CNB-National Centre for Biotechnology, CSIC, Darwin 3, Madrid, Spain, 3 Spanish National Research Council (CSIC), Institute of Catalysis, Madrid, Spain, 4 Bioinformatics Program, Boston University, Boston, Massachusetts, United States of America, 5 New England Biolabs, Ipswich, Massachusetts, United States of America, 6 Dept of Pathology and Laboratory Medicine, Boston University School of Medicine, Boston, Massachusetts, United States of America Biochemistry of Six Proteins from H. pylori The annotations of 1082 proteins are based on sequence homology of unknown stringency, and without identification of the original experimental source. Though the number and percentage of experimentally supported gene annotations will increase as the GSDB becomes more comprehensive, it is apparent that H. pylori is an appropriate organism for further biochemical characterization. The annotations of 1082 proteins are based on sequence homology of unknown stringency, and without identification of the original experimental source. Though the number and percentage of experimentally supported gene annotations will increase as the GSDB becomes more comprehensive, it is apparent that H. pylori is an appropriate organism for further biochemical characterization. then concentrated using centrifugal evaporation under vacuum. Coated nano-particles were stored at –20uC until use. Protein lysate and coated nano-particles were incubated for 5 min, allowing proteins to bind the potential substrates. Nano- particles with captured protein(s) were separated by ultrafiltration through low-adsorption hydrophilic 500000 nominal molecular weight limit (NMWL) cutoff membranes (regenerated cellulose, Amicon), followed by a wash step with PBS (1:20 diluted). Proteins which bind the attached substrates with high affinity are expected to be isolated. We have observed that those proteins that are most efficiently captured or those with high affinity (Km below 2 mM) and modest reaction rates (lower than 50 min21), while proteins with higher reaction rates are captured somewhat less efficiently. The nano-particles with their bound protein were dried under vacuum, and stored at –20uC. They were then analyzed in two ways. (1) For protein identification, nano-particles were incubated with trypsin (Roche, sequencing grade) overnight at 37uC. Tryptic peptides were collected by ultrafiltration through 10 kDa cutoff membranes, and evaporated to dryness. Peptides were analyzed using RP-LC-MALDI TOF/TOF mass spectrometry (see details below). (2) For biochemical testing of isolated protein material, proteins are recovered from the nano-particles after incubation in PBS for 24 h at 4uC. No degradation in enzymatic activity was observed, when compared to shorter incubations for test enzymes. Released proteins were separated from nano-particles by ultra- filtration through low-adsorption hydrophilic 500000 NMWL cutoff membranes. Proteins were purified by high-performance gel filtration chromatography (FPLC) using a 25 ml Superose-12 pre- packed HR 10/30 gel-filtration column and PBS buffer (0.4 ml min21). Protein solutions, at concentrations of at least 0.1 mg ml21, were stored at –20uC. A full description of the affinity capture methodology will be presented elsewhere. Biochemistry of Six Proteins from H. pylori The high specificity of the protein capture method can be observed in Figure S1. Several efforts to increase the throughput of biochemical validation have already been quite successful. Yakunin and coworkers [4,5] developed a set of "entry-level" assays designed to identify the general activity of a protein (phosphatase, dehydrogenase, protease etc.), which is then followed by the use of more specific substrates. A complementary approach uses a mixture of different substrates simultaneously (substrate cocktails) to speed up the characterization of new enzymes [6]. Impressively, Cravatt and coworkers [7,8] have pioneered the use of ‘‘activity- based protein profiling,’’ enriching enzymes of a particular class using affinity labels and identifying them by mass spectrometry. They and others have applied this technique to 7 classes of enzymes. In this work, we utilized new methodology (to be described in detail in a forthcoming manuscript) to generate hypotheses. The method utilizes nano-particles coated with substrate analogs to enrich proteins from cell lysates of H. pylori. The focus on this paper is the biochemical characterization of the enriched proteins. The isolated proteins were examined in two ways. First, an aliquot of the isolated material was digested with trypsin, and analyzed by mass spectrometry. A second aliquot of isolated protein was utilized in biochemical assays to confirm suspected activities. The combined results at this stage provide putative functions and probable protein identities, and were treated as hypotheses, which we then explored using standard recombinant DNA technology and traditional in vitro biochemistry: the proteins identified by mass spectrometry were cloned, expressed in Escherichia coli, purified using an epitope tag and subjected to in vitro biochemical analysis. Materials and Methods Proteins were analyzed essentially as in [11]. Briefly, trypsin, proteomics grade (Sigma) in 25% acetonitrile (ACN)/50 mM ammonium bicarbonate solution was added to the protein solution, and reactions were stopped after 4 hours by adding 0.5% trifluoroacetic acid (TFA). Tryptic eluted peptides were dried by speed-vacuum centrifugation and resuspended in 4 ml of MALDI solution (30% ACN/15% isopropanol/0.1% TFA). A 0.8 ml aliquot of each peptide mixture was deposited onto a 386- well OptiTOFTM Plate (AB SCIEX, Foster City, CA, USA) and allowed to dry at room temperature. A 0.8 ml aliquot of matrix solution (3 mg/mL a-cyano-4-hydro-cinnamic acid in MALDI solution) was then deposited onto dried digest and allowed to dry at room temperature. For MALDI-TOF/TOF analysis, data was automatically acquired using an ABI 4800 MALDI TOF/TOF mass spectrometer (AB SCIEX, Foster City, CA, USA) in positive ion reflector mode. Spectra were smoothed and corrected to zero baseline using ABI 4000 Series Explorer Software v3.6. Spectra were internally calibrated with the mass signals of trypsin autolysis to achieve a typical mass measurement accuracy of ,25 ppm. Spectra were analyzed using MASCOT software v.2.2.04 (Matrix Science, London, UK) using a custom protein database containing all possible H. pylori protein sequences. The mass tolerance for precursors was set to 6 50 ppm, and to 6 0.3 Da for MS/MS fragment ions. Peptide identifications were accepted when scored at greater than 95.0% probability by the Mascot algorithm [12]. Protein identifications were treated as hypotheses for further biochemical characterization. Chemicals and strains Chemicals, biochemicals and solvents used for enzymatic tests were of the purest grade available and were purchased from Sigma-Aldrich Co. (St. Louis, MO). H. pylori ATCC 26695 was grown on Trypticase Soy Agar (TSA) plates at 37uC, under 5% CO2 for 48 hours. Protein lysates were generously provided by Dr. Xuesong Zhang and Dr. Martin Blaser using the BugBuster Protein extraction reagent (Novagen; Darmstadt, Germany), and stored at a protein concentration above 0.4 mg ml21 at –80uC until use. Introduction [3]; (2) traced the annotated functions of 15% of microbial proteins in COMBREX to their experimental sources; (3) developed a novel gene recommendation system to encourage new experiments for those proteins that would have implications for the largest number of additional proteins; and (4) directly funded the experimental testing of numerous microbial proteins using a novel small-grant model. Our ability to accurately assign gene function lags far behind the tremendous progress made in DNA sequencing. The value of completed sequence would be significantly enhanced if we were able to more completely interpret the complement of genes within genomes. Today, we are typically unable to assign functions to approximately 30% of genes in a new microbial genome, about the same fraction that was not annotated in the first sequenced genome, Haemophilus influenzae Rd in 1995 [1]. Given the rapid increases in the production of DNA sequence data associated with microbiomes and meta-genomes, this issue becomes ever more acute. Based on a community consensus, COMBREX emphasizes the results from rigorous, direct biochemical experiments, and is therefore willing to entertain hypotheses for specific gene function from the broadest spectrum of sources - experimental, computa- tional, and even ‘‘intuition-based’’ hypotheses based on detailed experience. This mindset was adopted in recognition of the difficulties associated with experimentally approaching the ‘‘hy- pothetical protein.’’ To address this general issue, we initiated COMBREX (COMputational BRidges to EXperiments, http://combrex.bu. edu) in order to coordinate the efforts of computational and experimental biologists, and to serve as a standard repository for protein function data (predictions, hypotheses and experimental validations) [2]. As part of this effort, COMBREX has: (1) created the Gold Standard Database of experimentally characterized proteins (GSDB) in conjunction with UniProt, NCBI and JCVI Helicobacter pylori is a major human pathogen that has been associated with the development of gastritis, gastric ulcers and stomach cancer. Despite its clinical importance, and the large amount of literature devoted to various aspects of its biology, COMBREX documented experimental support for the functions of only 399 of the nearly 1600 proteins-coding genes in H. pylori. PLOS ONE \| www.plosone.org 1 June 2013 \| Volume 8 \| Issue 6 \| e66605 Biochemistry of Six Proteins from H. pylori Cloning, expression and purification of recombinant proteins Cloning, expression and purification of recombinant proteins the range of pH 4.0–9.5, and the temperature dependence in the range of 30–70uC. The buffers used were: citrate (pH 4.0–5.0), acetate (pH 5.0–6.0), 2-(N-morpholino) ethanesulfonic acid (MES) (pH 6.0–7.0), 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) (pH 7.0–8.0), Tris-HCl (pH 8.0–9.0) and glycine (pH 9.0–9.5), all at 100 mM. The pH was adjusted at 25uC. In all cases, one unit of the enzyme was defined as the amount of enzyme that catalyzed the formation of 1 mmol of reaction product per minute at 30uC. Three independent experiments were performed for each parameter and graphs were plotted using mean values and standard deviations wherever appropriate. In all cases, for the characterization of isolated protein material, the absorbance was measured using a BioTek Synergy HT spectro- photometer. The following assays were performed. p Genomic DNA was extracted from H. pylori ATCC 26695 by DNA Maxi Kit (Qiagen) according to manufacturer’s protocol. The recombinant plasmids for the (His)6-tagged proteins were constructed by PCR amplification of the corresponding gene from H. pylori ATCC 26695 genomic DNA. The oligonucleotide primers used for PCR were designed to complement the TOPO-Cloning system from Invitrogen or In-Fusion Cloning system from Clontech and were as follows: HP0267 (Fwd 59-CAC CAT GCA AGA AAT CAT AGG AGC GTC-39 and Rev 59- TTA GAT CAC CCT TTT CCC CCC TAA AAA C-39), HP0310 (Fwd 59-CAC CAT GGC AAA AGA AAT TTT AGT GG-39 and Rev 59-CTA TTT TTT TCT AGG GTT TCG-39), HP0959 (Fwd 59-ACC ACG GTG GTC ATA TGG CGT TAG TTA AGG AAG TGT TGG TAG-39 and Rev 59-GTT AAC CTT ACT CGA GTT AAA TGA TTT GCA AGG GGT TTT TAA AAT TCT C-39), HP1037 (Fwd 59-CAC CAT GAA AGG ATT AGA AAG AGA ATC G-39 and Rev 59-TCA CAA AAG CTC AGA CCT AGA-39), HP1042 (Fwd 59-CAC CAT GAT GCA AGT TTA CCA CCT TTC-39 and Rev 59-TTA AGC GTT GTT GAA GAT TTC-39), and HP1079 (Fwd 59-CAC CAT GAT TCA GTC TGT TCG CAT C-39 and Rev 59-TTA ACC AAA AAG ATT CTC TTC-39). The amplified gene inserts were cloned into the pET200/D-TOPO vector using the Champion pET Directional TOPO Expression Kit from Invitro- gen, or into the pCOATexp mH6 vector (a gift from Shaorong Chong, New England Biolabs) after digestion with NdeI and XhoI using the In-Fusion HD cloning. Recombinant plasmids were transformed into One Shot TOP10 Chemically Competent E. Cloning, expression and purification of recombinant proteins coli (Invitrogen) or Stellar Competent E. coli (Clontech). From the transformed E. coli, recombinant plasmids were purified by QuickClean II Plasmid Miniprep Kit (GenScript), and sequenced by GENEWIZ to confirm the identity of the construct. The purified recombinant plasmids for the (His)6-tagged proteins were transformed into BL21(DE3) Competent E. coli (New England Biolabs) or T7 Express lysY/Iq Competent E. coli (New England Biolabs). Single colonies were grown overnight in LB broth containing ampicillin or kanamycin at 37uC. Larger volume cultures were inoculated from the overnight growth. When these cultures achieved an OD600 value of ,0.6, recombinant protein expression was induced by 0.5 mM isopropyl-b-D-thiogalactopyr- anoside (IPTG). After additional incubation at ,20uC, cells were harvested and stored at –80uC. The frozen cell pellets were resuspended in lysis buffer (50 mM Tris-HCl, pH 8.0, 300 mM NaCl, 10 mM imidazole, 10% glycerol, 5 mM b-mercaptoetha- nol) containing EDTA-free Protease Inhibitor Cocktail (Roche), lysed via sonication, and then 0.1% Triton X-100 was added. Recombinant proteins were purified from the cell-free supernatant by using Ni-NTA agarose (Qiagen). After washing, recombinant proteins were eluted with imidazole (50 mM Tris-HCl, pH 8.0, 300 mM NaCl, 250 mM imidazole, 10% glycerol, 5 mM b- mercaptoethanol, 0.1% Triton X-100). The purified proteins were analyzed by SDS-polyacrylamide gel electrophoresis. Protein concentration was determined using the Bio-Rad Bradford Protein Assay. Biochemical characterization 1. Acetyl esterase (HP0310) – Esterase kinetic parameters were initially assayed using p-nitrophenyl (pNP) esters [13] with minor modifications. Briefly, kinetic parameters were calculated by adding 0–12 nM enzyme solution and 0–2 mM pNP ester to 190 ml buffer. The reaction was followed spectrophotometrically measuring the release of p-nitrophenol at 405 nm. Substrates tested included pNPacetate, pNPpropionate and pNPbutyrate. The standard acetyl esterase assay was performed in 100 mM sodium acetate buffer, pH 6.0 and 30uC. Under our experimental conditions, the absorption coefficient for pNP was measured as 3021 M21 cm21. For other pHs, the absorption coefficient was calculated. The release of acetate from acetylated polymeric substrates chitin, chitosan and xylan was measured using phenol red as pH indicator [14]. Each well in a 96-well microtiter plate contained 0–12 nM enzyme solution, 0–100 mg ml21 substrate, and 0.911 mM phenol red in 200 ml of 5 mM N-(2-hydroxyethyl)- piperazine-N’-(3-propanesulfonic acid (EPPS) buffer (pH 8.0). The acetate release was colorimetrically monitored at 550 nm. Cloning, expression and purification of recombinant proteins Stock solutions (500 mg ml21 in dimethylsulfoxide, DMSO) of substrates were prepared immediately prior to use. Under our experimental conditions, the absorption coefficient for phenol red was measured as 8450 M21 cm21. Acetylated xylan and chitosan were prepared by incubating the substrates with pyridine:acetic anhydride (1:1) at 25uC for 48 hrs followed by the recovery of acetylated products by evaporation with ethanol [10]. The standard acetyl esterase assay contained [E]o = 12 nM, [acetylated xylan] = 10 mg ml21, 5 mM EPPS buffer, pH 8.0, 0.45 mM phenol red in a total volume of 200 ml, at 30uC. 2. Adenosine deaminase (HP0267) – The deamination of adenine or adenosine was determined in a 96-well microtiter plate using a coupled assay with glutamate dehydrogenase (GDH; Roche Applied Science, Mannheim, Germany) [15], in which the formation of ammonia was followed at 340nm in the presence of 0–12 nM enzyme solution, 0–2 mM adenine or adenosine, 0.15 mM NADH, 25 mM R-ketoglutarate and 5 mg ml21 GDH in a final volume of 200 ml. The standard adenosine aminohy- drolase assay contained [E]o = 12 nM, [adenosine] 1 mM, [NADH] 0.15 mM, [R-ketoglutarate] 25 mM, [GDH] 5 mg ml21, 100 mM HEPES (pH 7.5), at 30uC. For purified recombinant protein, biochemical activity was assayed using two methods, the first being the coupled GDH assay, and second by following the decrease of absorbance of the substrate adenosine at 265 nm using Infinite M 200 PRO (TECAN) in the presence of 50 mM potassium phosphate, pH 7.6, and 0.045 mM adenosine or adenine. Kinetic parameters were determined at 25uC using 90 nM of enzyme and various concentrations of adenosine (ranging from 0 to 1 mM) [16,17]. Protein enrichment via affinity purifications As the focus of this paper is the in vitro biochemical characterization of recombinant proteins, a full description of these methods will be described in detail in a forthcoming publication. Briefly, total cell lysates containing 10 mg total protein in 100 ml phosphate buffered saline (PBS) were incubated for 5 mins with 10 mg gold nano-particles coated with various substrate analogs. Nano-particles were produced as indicated [9]. Nano- particles (,10 mg in 100 ml of PBS) were mixed with 100 ml of a solution containing potential substrates (0.2 mg ml21 in PBS) in closed vials and incubated overnight at 4uC in an orbital shaker. The substrates utilized were adenine, acetylated xylan [10], guanosine-5’-triphosphate (GTP)/dihydroneopterin triphosphate (both at equal amount), succinyl-Ala-Ala-Ala-p-nitroanilide, cyclic adenosine monophosphate (cAMP) and GTP. The suspension was June 2013 \| Volume 8 \| Issue 6 \| e66605 PLOS ONE \| www.plosone.org 2 Biochemistry of Six Proteins from H. pylori Results After 15 min incubation at room temperature, the mixture was centrifuged and the excess of iodine was eliminated from the supernatant by the addition of 100 ml of 2.0% ascorbic acid (in the above buffer), followed by the addition of 120 ml of 1 N NaOH and alkaline phosphatase (12.5 units; Roche Applied Science, Mannheim, Germany) at 37uC for 100 min. Reaction product was detected spectrofluorometrically: lex of 350 nm and lem of 450 nm [22]. The standard GTP cyclohydrolase assay contained [E]o = 12 nM, [GTP] 0.2 mM, 100 mM sodium citrate (pH 4.0), at 30uC. For purified recombinant protein, cyclohydrolase activity was measured by monitoring absorbance at 330 nm, with the following reaction conditions: 0.2 mM GTP in 100 mM Tris-HCl, pH 8.0, 100 mM KCl, and 2.5 mM EDTA [23]. y y ( ) HP1037 - HP1037 is annotated as a ‘‘hypothetical protein’’ with a predicted mass of 40.8 kDa, and with two identified domains, Creatinase_N and Peptidase_M24 (Pfam). HP 1037 has previously been predicted as an aminopeptidase based on sequence homology to characterized proteins [24]. This protein was isolated using nano-particles coated with succinyl-Ala-Ala-Ala-p-nitroanilide. As shown in Table 1, the protein was able to cleave five different aminopeptides tested, with N-succinyl-Ala-Ala-Ala-Pro-Phe-p-ni- troanilide being preferred. This result can be explained by the up to 9-fold greater kcat value and the up to 5.5-fold lower Km value for this substrate. Both, p-nitroanilide (PNA) and methylcoumarine amide derivatives were accepted as substrates, with substrates containing multiple alanines (i.e. N-succinyl-Ala-Ala-Ala-Pro-Phe- p-nitroanilide and succinyl-Ala-Ala-Ala-p-nitroanilide) being pre- ferred (Table 1): kcat values from one to two orders of magnitude higher. In addition, further tests of enzyme activity with different peptide substrates suggest cleavage specificity for peptides containing the following amino acids, Ala, Pro, Phe, Arg Phe, Leu, Val and/or Tyr. With N-Succinyl-Ala-Ala-Ala-Pro-Phe-p- nitroanilide, HP1037 demonstrated maximum activity at pH 4.0– 5.0 and about 40uC. Therefore, HP1037 should be considered an ‘‘aminopeptidase’’ (EC 3.4.11.-). 6. Phosphodiesterase (HP1042 assay) – Phosphodiesterase activity was measured in a 96-well microtiter plate by following the hydrolysis of the chromogenic substrate bis-p-nitrophenyl phos- phate (bis-pNPP) in a reaction mixture containing enzyme solution (0-20 nM) and a substrate solution (1–5 mM), and 1.0 mM MnCl2 in a final volume of 200 ml, at 410 nm. Results Total protein lysate was prepared from H. pylori ATCC 26695 grown on TSA plates, and the clarified lysate was incubated with six substrate-coated nano-particles as described in Materials and Methods. Captured proteins were digested with trypsin and analyzed by mass spectrometry, or, alternatively isolated from nano-particles and tested for biochemical activities suggested by the attached substrates. For those cases in which appropriate biochemical activity was observed, the genes encoding the proteins identified by mass spectrometry were cloned into a vector containing sequence for an N-terminal His-tag epitope using standard recombinant DNA methodology. Proteins were ex- pressed in E. coli BL21(DE3), and purified using immobilized metal affinity chromatography. Purified recombinant protein was tested for the biochemical activity initially observed. The detailed functional roles of six previously unannotated proteins are described below. 4. ATPase/GTPase activity (HP1079) 2 The hydrolysis of nucleotide triphosphates (NTP) was tested in 200 ml mixtures containing enzyme solution (0–20 nM), NTP (0–5.0mM), 10 mM MgCl2, and 10% glycerol (v v21) [19]. Reactions were incubated at the indicated temperature and pH for 30 min, after which reactions were stopped by the addition of 80 ml of a color reagent (3:1 ratio of 0.045% malachite green and 4.2% ammonium molybdate in 4 N HCl) and 100 ml of 34% citric acid solution. After incubation at room temperature for 30 min, the absorbance was measured at 660 nm. The standard NTPase assay contained [E]o = 12 nM, [ATP] 3 mM, 100 mM sodium acetate (pH 6.0), at 30uC. For purified recombinant protein, optimal ATP hydrolysis was observed 20 mM Tris-HCl (pH 8.0), 0.57 mM EDTA, 5 mM MgCl2, 133 mM NaCl, 3 mM KCl, 3 mM ATP. The mixture containing enzyme was incubated at 37uC for 1 hr. Liberated Pi was assayed with Malachite Green Phosphate Assay Kit (BioAssay Systems) according to manufacturer’s protocol [20,21]. HP0959 - HP0959 is annotated as a ‘‘hypothetical protein’’ with a predicted mass of 26.8 kDa. This protein was isolated using nano-particles coated with both GTP and dihydroneopterin triphosphate, and it was subsequently tested for its ability to hydrolyze GTP to form dihydroneopterin triphosphate. The protein did show a high affinity (Km of 0.04060.005 mM) and activity (kcat of 2593629.0 min21) for the substrate (Table 1); no product was detected when GTP was replaced by GDP, GMP, ATP, CTP and UTP. Biochemical characterization Biochemical characterization was performed twice, once on isolated protein material and once on recombinant purified protein. Km, Vmax, and kcat values were determined using nonlinear regression to fit the values for initial velocity and substrate concentration to the Michaelis-Menten equation. Unless otherwise indicated, the pH dependence of a reaction was tested in 3. Aminopeptidase (HP1037) – Enzyme activities toward amino acidic derivatives (Xaa) of p-nitroanilide (Xaa-pNA) derivatives were determined in a 96-well microtiter plate by adding an 3. Aminopeptidase (HP1037) – Enzyme activities toward amino acidic derivatives (Xaa) of p-nitroanilide (Xaa-pNA) derivatives were determined in a 96-well microtiter plate by adding an June 2013 \| Volume 8 \| Issue 6 \| e66605 June 2013 \| Volume 8 \| Issue 6 \| e66605 PLOS ONE \| www.plosone.org 3 Biochemistry of Six Proteins from H. pylori enzyme solution (0–20 nM) and a substrate solution in DMSO (1– 20 mM) in a final volume of 200 ml and continuously monitored the increase in absorption at 405 nm caused by the release of p- nitroaniline [18]. The initial activity rate was determined from the linear part of the optical density profile (e405nm of 10600 M21 cm21). The hydrolysis of Phe-Arg-methylcoumarine amide and succinyl-Leu-Leu-Val-Tyr-methylcoumarine amide was recorded under the same conditions, but monitoring the free amino methyl coumarine fluorimetrically (lex = 380 nm and lem = 460 nm). Buffer was supplemented with 5 mM calcium chloride and 0.1% w v21 Brij. The standard aminopeptidase assay contained [E]o = 12 nM, [N-succinyl-Ala-Ala-Ala-Pro-Phe-p-nitroanilide] 3 mM, 100 mM sodium citrate (pH 4.5), at 30uC. For purified recombi- nant protein, the substrate tested was N-Succinyl-Ala-Ala-Pro- Phe-p-nitroanilide and the buffer used was 100 mM citrate buffer, pH 4.5, the observed optimum for the isolated material. All other aspects of the assay were identical. containing 0–5 mM of substrate, and 0.1 mM MnCl2. After 20 min incubation at the indicated temperature and pH with enzyme solution (0–20 nM), 1 unit of alkaline phosphatase (Roche Applied Science, Mannheim, Germany) was added and the reaction was incubated for an additional 10 min at 30uC. Liberated Pi was assayed with malachite green reagent, as described above. The standard phosphodiesterase assay contained [E]o = 12 nM, [bis- pNPP] 2.0 mM, 100 mM sodium citrate (pH 4.5), at 30uC. Results HP0959 was observed to have peak activity at about 35uC, and approximately at pH 4.0, with a strong decrease above those values (Figure 1). Biochemical activity was also confirmed using the method of [23] by monitoring absorbance at 330 nm. Therefore, HP0959 should be considered as a ‘‘GTP cyclohydrolase l’’ (EC 3.5.4.16). 5. GTP cyclohydrolase (HP0959) – The conversion of GTP to dihydroneopterin triphosphate, cyclohydrolase enzymatic activity, was tested in a 96-well microtiter plate with 500 mM GTP, 2.5 mM ethylenediaminetetraacetic acid (EDTA), 0.05 M KCl, and 10% glycerin (v v21) in 100 ml. Reactions were carried out for 2 hrs in darkness. The reaction was terminated by adding 1.1 ml of the above buffer (lacking GTP) and 100 ml ml of an acidic iodine solution (1% I2 and 2% potassium iodide in 1 N HCl) [22]. After 15 min incubation at room temperature, the mixture was centrifuged and the excess of iodine was eliminated from the supernatant by the addition of 100 ml of 2.0% ascorbic acid (in the above buffer), followed by the addition of 120 ml of 1 N NaOH and alkaline phosphatase (12.5 units; Roche Applied Science, Mannheim, Germany) at 37uC for 100 min. Reaction product was detected spectrofluorometrically: lex of 350 nm and lem of 450 nm [22]. The standard GTP cyclohydrolase assay contained [E]o = 12 nM, [GTP] 0.2 mM, 100 mM sodium citrate (pH 4.0), at 30uC. For purified recombinant protein, cyclohydrolase activity was measured by monitoring absorbance at 330 nm, with the following reaction conditions: 0.2 mM GTP in 100 mM Tris-HCl, pH 8.0, 100 mM KCl, and 2.5 mM EDTA [23]. 5. GTP cyclohydrolase (HP0959) – The conversion of GTP to dihydroneopterin triphosphate, cyclohydrolase enzymatic activity, was tested in a 96-well microtiter plate with 500 mM GTP, 2.5 mM ethylenediaminetetraacetic acid (EDTA), 0.05 M KCl, and 10% glycerin (v v21) in 100 ml. Reactions were carried out for 2 hrs in darkness. The reaction was terminated by adding 1.1 ml of the above buffer (lacking GTP) and 100 ml ml of an acidic iodine solution (1% I2 and 2% potassium iodide in 1 N HCl) [22]. Results The measurement of phosphodiesterase activity against several 29,39- and 39,59-cyclic nucleoside monophosphates was based on the alkaline phospha- tase-sensitive nucleotide product as follows: 200 ml mixtures June 2013 \| Volume 8 \| Issue 6 \| e66605 PLOS ONE \| www.plosone.org 4 Biochemistry of Six Proteins from H. pylori Table 1. Kinetic parameters of native pure proteins directly isolated from Helicobacter pylori. Table 1. Kinetic parameters of native pure proteins directly isolated from Helicobacter pylori. Enzyme Substratea Km (mM)bc Vmax (mmol ml21 min21) kcat (min21) kcat/Km (s21 mM21) HP0267 Adenosine (Native protein) 0.3260.19 (0.032–2) 33.7563.99 11016151 (0.3–10800) 57.3 (30–150) Adenine (Native protein) 2.0460.16 (0.0147–6.6) 0.00360.001 0.4760.13 (330–804) 0.004 6-Chloroadenine n.d. n.d. n.d. Adenosine (Recombinant protein) 1.2960.24 (0.0147–6.6) 0.0360.001 331610 (0.3–10800) 4.28 (30–150) HP0310 Chitin 0.8560.14 6.9060.78 775658 15.2 Chitosan 0.8560.11 5.0160.21 968613 18.9 Acetylated xylan 0.4160.03 73.764.5 2549689 103 pNPacetate 0.1860.05 (0.00186–0.9) 37.461.7 1209655 (2856) 112 HP0959 GTP 0.04060.005 (0.004–980) 1.4260.06 2593629 (0.066–0.21) 1080 HP1037 NSAAAPPpNA 1.3860.09 18.261.5 1145639 13.8 PGPApNA 7.5660.27 1.4260.06 23.260.6 0.05 SAAApNA 1.1260.30 1.5460.13 12265 1.8 PAMCA 7.4360.36 0.7560.09 10.260.4 0.02 SLLVTMCA 4.0660.13 0.7860.07 4.660.2 0.02 HP1042 bis-pNPP 0.5360.04 1.1360.10 1085612 34.1 cAMP 0.8860.12 (0.000079–7) 2.0160.07 2008673 (0.0558–34020) 38 cGMP 0.8260.15 (0.00002–1) 5.1360.20 957682 (252–40020) 19.5 HP1079 ATP 1.0260.05 (0.001–0.98) 319621 9.361.2 (1.14–7980) 0.15 GTP 0.5860.05 (0.0008–0.85) 122611 0.5760.12 (0.108–1260) 0.02 aAbbreviations: bis-pNPP, bis-p-nitrophenyl phosphate; NSAAAPPpNA, N-succinyl-Ala-Ala-Ala-Pro-Phe-p-nitroanilide; PGPApNA, pyroglutamyl-Pro-Arg-p-nitroanilide; SAAApNA, succinyl-Ala-Ala-Ala-p-nitroanilide; PAMCA, Phe-Arg-methylcoumarine amide; SLLVTMCA, succinyl-Leu-Leu-Val-Tyr-methylcoumarine amide. bKinetic parameters determined at 30uC as described in Figure 1 legend and Materials and Methods. cPreviously reported numbers for the kinetic parameters are obtained from Brenda [26], and listed in parentheses. doi:10.1371/journal.pone.0066605.t001 Km (mM)bc Vmax (mmol ml21 min21) kcat (min21) kcat/Km (s21 mM21) aAbbreviations: bis-pNPP, bis-p-nitrophenyl phosphate; NSAAAPPpNA, N-succinyl-Ala-Ala-Ala-Pro-Phe-p-nitroanilide; PGPApNA, pyroglutamyl-Pro-Arg-p-nitroanilide; SAAApNA, succinyl-Ala-Ala-Ala-p-nitroanilide; PAMCA, Phe-Arg-methylcoumarine amide; SLLVTMCA, succinyl-Leu-Leu-Val-Tyr-methylcoumarine amide. bKinetic parameters determined at 30uC as described in Figure 1 legend and Materials and Methods. cPreviously reported numbers for the kinetic parameters are obtained from Brenda [26], and listed in parentheses. doi:10.1371/journal.pone.0066605.t001 (EC HP1042 - HP1042 is annotated as a ‘‘hypothetical protein’’ with a predicted mass of 40.2 kDa, and one domain DHH phosphatase family (Pfam). The protein was isolated using nano-particles coated with cAMP. The protein showed significant activity (kcat of 1085611.7 min21) towards the synthetic substrate bis-p-nitrophe- nyl phosphate (bis-pNPP), a general substrate for phosphodiester- ases and nucleases. The biochemical activity of HP1042 against naturally occurring phosphodiesters such as cyclic nucleotides and phospholipids was tested further. Results No phosphohydrolase activity was found toward various phosphatidylcholine and phosphorylat- ed sugars, but it was observed for the cyclic nucleotides cAMP and cyclic guanosine monophosphate (cGMP) (Table 1), with a (kcat/ Km)cAMP/(kcat/Km)cGMP factor of ,2. cAMP was found as the best substrate tested, mainly due to the about 2-fold greater kcat value as compared to bis-pNPP and cGMP. In all cases, the protein had an absolute requirement for Mn2+ (maximum activity was achieved when [MnCl2] . 5 mM). The apparent dissociation (Kd) constant for Mn2+, calculated from the dependence of phosphodiesterase activity of HP1042 on Mn2+ concentration, was determined to be Kd of 2.2660.15 mM. With bis-pNPP and cAMP as substrates, HP1042 demonstrated maximum activity at pH from 4.0 to 4.5, and at temperatures ranging from 30 to 40uC (Figure 1). According to activity features, the protein HP1042 should be considered as a 3’,5’-cyclic-nucleotide phosphodiesterase (EC 3.1.4.17). as a 3’,5’-cyclic-nucleotide phosphodiesterase (EC HP1079 - HP1079 is annotated as a ‘‘hypothetical protein’’ with a predicted mass of 42.9 kDa, possessing an ATPase domain and observed to interact with urease [25]. In this assay, it was found to be able to bind to nano-particles coated with both ATP and GTP with most efficient capture by GTP-coated nano-particles. However, kcat/Km was found to greater for ATP than GTP by a factor of ,7.5 (Table 1). Hydrolysis of other nucleotides, CTP, GTP, TTP and ADP, were also studied, but the activity levels were too low to be determined adequately. We further analyzed different divalent metal ions (Mg2+, Cu2+, Co2+, Zn2+, Ca2+, Mn2+ and Ni2+ - 8 mM each) in an ATPase assay to define the cofactor requirement of HP1079. Only Ca2+ was found to affect enzyme activity, but contradictory results were observed with the native protein (slight increase in ATPase activity) and the recombinant protein (inhibition by Ca2+), which require further clarification. With both substrates, HP1079 demonstrated maximum activity at pH from 5.5 to 6.5 (being optimal at pH 6.0), and 45uC (Figure 1). Therefore, the protein HP1079 should be considered as an ATPase/GTPase. HP0267 - HP0267 is annotated as a ‘‘chlorohydrolase’’ with a predicted mass of 45.5 kDa, and a protein domain Amidohydro_1 from the amidohydrolase family. HP0627 protein was isolated HP0267 - HP0267 is annotated as a ‘‘chlorohydrolase’’ with a predicted mass of 45.5 kDa, and a protein domain Amidohydro_1 from the amidohydrolase family. Results HP0627 protein was isolated June 2013 \| Volume 8 \| Issue 6 \| e66605 June 2013 \| Volume 8 \| Issue 6 \| e66605 PLOS ONE \| www.plosone.org 5 Biochemistry of Six Proteins from H. pylori Figure 1. Optimal temperature (upper) and pH (lower) for hypothetical proteins from H. pylori. The heat map colors represent the relative percentages of activity (in terms of kcat) as compared to the maximum (100%) within each enzyme. kcat values were determined using nonlinear regression to fit the values for initial velocity and substrate concentration to the Michaelis-Menten equation as described in Materials and Methods. The pH dependence of a reaction was tested in the range of pH 4.0–9.5 at 30uC, and the temperature dependence in the range of 30–70uC at the optimal pH (4.0 for HP0142 and HP0959; 6.0 for HP0179 and HP0310; 7.5 for HP0267; and 4.5 for HP1037). The buffers used were: citrate (pH 4.0–5.0), acetate (pH 5.0–6.0), MES (pH 6.0–7.0), HEPES (pH 7.0–8.0), Tris-HCl (pH 8.0–9.0) and glycine (pH 9.0–9.5), all at 100 mM. Reaction conditions: [E]o = 0–12 nM, [substrate: HP0310 (pNPacetate and acetylated xylan), HP0267 (adenosine), HP1037 (N-succinyl-Ala-Ala-Ala-Pro-Phe-p-nitroanilide), HP1079 (ATP), HP0959 (GTP), HP1042 (bis-pNPP)] ranging from 0 to 20 mM. Three independent experiments were performed for each parameter and graphs were plotted using mean values. doi:10.1371/journal.pone.0066605.g001 Figure 1. Optimal temperature (upper) and pH (lower) for hypothetical proteins from H. pylori. The heat map colors represent the relative percentages of activity (in terms of kcat) as compared to the maximum (100%) within each enzyme. kcat values were determined using nonlinear regression to fit the values for initial velocity and substrate concentration to the Michaelis-Menten equation as described in Materials and Methods. The pH dependence of a reaction was tested in the range of pH 4.0–9.5 at 30uC, and the temperature dependence in the range of 30–70uC at the optimal pH (4.0 for HP0142 and HP0959; 6.0 for HP0179 and HP0310; 7.5 for HP0267; and 4.5 for HP1037). The buffers used were: citrate (pH 4.0–5.0), acetate (pH 5.0–6.0), MES (pH 6.0–7.0), HEPES (pH 7.0–8.0), Tris-HCl (pH 8.0–9.0) and glycine (pH 9.0–9.5), all at 100 mM. Reaction conditions: [E]o = 0–12 nM, [substrate: HP0310 (pNPacetate and acetylated xylan), HP0267 (adenosine), HP1037 (N-succinyl-Ala-Ala-Ala-Pro-Phe-p-nitroanilide), HP1079 (ATP), HP0959 (GTP), HP1042 (bis-pNPP)] ranging from 0 to 20 mM. Three independent experiments were performed for each parameter and graphs were plotted using mean values. Results doi:10.1371/journal.pone.0066605.g001 (Table 1) [26]. However, the ratio Kcat/Km is lower for the recombinant protein than previous observations. While the precise reasons for the differences in activity are unclear, there does appear to be slight differences in the catalytic ability of the native and recombinant proteins, even after confirmation of the cloned sequence, and they are currently being investigated. We tentatively suggest that HP0267 be considered as an ‘‘adenosine deaminase’’ (EC 3.5.4.4), with slight ability to hydrolyze adenine (EC 3.5.4.2 - and see Discussion). using nano-particles coated with adenine. The native protein was tested for its ability to hydrolyze adenine and a chloro- derivative, 6-chloroadenine, as well as adenosine (Table 1). The protein did show a high affinity (Km of 0.3260.19 mM) and activity (kcat of 11016151 min21) for adenosine. A very weak activity with adenine was detected, and a reduction in the catalytic efficiency with this substrate was mainly due to a 2330-fold reduction in the kcat as compared to adenosine. No activity toward chloroadenine was observed. Optimal activity with adenosine was observed around 45uC and pH 7.5, making it the only protein of the group tested to show greatest activity at neutral or alkaline conditions (retaining . 70% maximum activity at pH 9.0, but with only 6% activity at pH 4.5). However, for the purified recombinant protein, biochemical activity was only observed using adenosine as a substrate, but not for adenine. This was observed using two different assay methods, both of which produced reasonable Km and Kcat values when compared to other characterized enzymes using nano-particles coated with adenine. The native protein was tested for its ability to hydrolyze adenine and a chloro- derivative, 6-chloroadenine, as well as adenosine (Table 1). The protein did show a high affinity (Km of 0.3260.19 mM) and activity (kcat of 11016151 min21) for adenosine. A very weak activity with adenine was detected, and a reduction in the catalytic efficiency with this substrate was mainly due to a 2330-fold reduction in the kcat as compared to adenosine. No activity toward chloroadenine was observed. Optimal activity with adenosine was observed around 45uC and pH 7.5, making it the only protein of the group tested to show greatest activity at neutral or alkaline conditions (retaining . 70% maximum activity at pH 9.0, but with only 6% activity at pH 4.5). June 2013 \| Volume 8 \| Issue 6 \| e66605 Discussion The task of experimentally determining the function of genes for which very little is known is a formidable task, but at the same time, one of significant scientific need. The magnitude of the problem increases daily as ever more DNA sequence is deposited in public databases, and current progress appears slow. For Mycobacterium tuberculosis H37Rv, when its genome was sequenced in 1998, there were 912 genes designated as ‘‘conserved hypothetical’’ genes [32]. Today, there are still 906 genes with this annotation. Many of these genes may have well-supported hypotheses or preliminary data regarding their function in the hands of experts engaged in their study, but this information is not readily available to most scientists who might wish to understand the biology of a particular organism. This highlights the need for improved technologies and approaches to experimentally validate protein molecular function. We summarize in Table 2 information related to the function of the six genes and the corresponding gene products that were studied in this paper. Prior to these studies, four of the six genes were annotated as ‘‘hypothetical protein,’’ with one protein labeled as ‘‘chlorohydrolase’’ based upon very distant sequence similarity (21% sequence identity) to an experimentally validated gene, atrazine chlorohydrolase from Pseudomonas sp. strain ADP. The sixth protein, HP0310, had been previously characterized as a peptidoglycan deacetylase [27–30]. While the individual proteins were not named informatively, four of the proteins belonged to protein clusters with descriptive names (HP0267 - PRK08418, HP0310 - CLSK865125, HP1037 - CLSK872355, and HP1042 - CLSK872354), three of which were suggestive to the biochemical activity was eventually observed. Interestingly, these four proteins also possessed annotated domains (Pfam) [35] that in all four cases could have been utilized to predict biochemical activity. COMBREX has focused on testing protein function in vitro. Within the framework of the Gene Ontology (GO) hierarchy of functional descriptors [33], these functions would be classified under the category of ‘‘molecular function.’’ Predictions of biochemical function are frequently straightforward to test, and results can usually be obtained that support or refute the prediction unambiguously. Furthermore, once a biochemical function is ascertained, it can directly lead to very specific hypotheses of gene function in-vivo, allowing an experimentalist to query those functions classified by the GO hierarchy as falling under the heading ‘‘biological process.’’ The full computational methodology underlying the COMBREX framework is described in a subsequent publication (in review). Biochemistry of Six Proteins from H. pylori Synthetic substrate pNPace- tate was also efficiently hydrolyzed, at slightly higher efficiency, mainly due to a higher substrate affinity (about 2-fold lower Km value) as compared to other acetylated substrates. Using acetylated xylan and pNPacetate as substrates, the protein showed maximum activity at 50uC, although it retains about 80% of the activity at 30–55uC, and was active at acidic pH ranging from 5.0 to 6.0, being optimal at pH 6.0 (Figure 1). Taken together with previous work, the protein HP0310 should be considered as a peptidogly- can deacetylase (EC 3.5.1.104), while possessing general ‘‘acet- ylesterase’’ (EC 3.1.1.6), and specific ‘‘acetylxylan esterase’’ activity (EC 3.1.1.72). was observed for the release of reduced sugars when monitored using 3,5-dinitrosalicylic acid (DNS) according to Miller [31], nor by measuring the release of xylose by the D-xylose Rapid Assay kits (Megazyme, Bray, Ireland). By contrast, the enzyme was able to efficiently release acetate groups from acetylated xylan, acetylated chitin and acetylated chitosan (Table 1). Based on the kcat/Km values, the protein functioned more efficiently with xylan than chitin-like products by an order of magnitude. This result can be explained by up to 3.2-fold greater kcat values and about 2-fold lower Km value for acetylated xylan. Synthetic substrate pNPace- tate was also efficiently hydrolyzed, at slightly higher efficiency, mainly due to a higher substrate affinity (about 2-fold lower Km value) as compared to other acetylated substrates. Using acetylated xylan and pNPacetate as substrates, the protein showed maximum activity at 50uC, although it retains about 80% of the activity at 30–55uC, and was active at acidic pH ranging from 5.0 to 6.0, being optimal at pH 6.0 (Figure 1). Taken together with previous work, the protein HP0310 should be considered as a peptidogly- can deacetylase (EC 3.5.1.104), while possessing general ‘‘acet- ylesterase’’ (EC 3.1.1.6), and specific ‘‘acetylxylan esterase’’ activity (EC 3.1.1.72). Results However, for the purified recombinant protein, biochemical activity was only observed using adenosine as a substrate, but not for adenine. This was observed using two different assay methods, both of which produced reasonable Km and Kcat values when compared to other characterized enzymes HP0310 - The protein product associated with HP0310 has previously been identified as a peptidoglycan deacetylase PgdA [27], and shown to contribute to bacterial survival [28,29]. A crystal structure of the protein [30] reveals a structure compatible with this activity, though notes a smaller than expected substrate binding groove as crystallized. In our assay, this protein was isolated using nano-particles coated with acetylated xylan. It was first tested for its ability to hydrolyze acetylated xylan. No activity June 2013 \| Volume 8 \| Issue 6 \| e66605 PLOS ONE \| www.plosone.org 6 Biochemistry of Six Proteins from H. pylori Biochemistry of Six Proteins from H. pylori When assigning biochemical activity to a protein isolated from crude lysates, there is always the risk that the observed biochemical activity is the result of a minor constituent in the isolated sample. For this reason, we cloned each of the identified target genes, expressed them in E. coli, purified the overexpressed protein using an epitope tag, and confirmed their biochemical activity in vitro. For testing biochemical activity on recombinant proteins, a common pitfall is that cofactors or other protein subunits required for activity will not be present, leading to false negatives. In this paper, we attempted to minimize the combined risks by testing biochemical activity on protein isolated from crude total cell lysates, and on purified recombinant protein. In all cases reported here, we observed essential agreement on the biochemistry of each gene from the two protein sources, with one primary exception. When HP0267 is isolated directly from H. pylori lysate, it appears as a single clean band on a polyacrylamide gel (Figure S1), and is observed to hydrolyze adenosine very efficiently and to much lower extent adenine. However with the cloned protein, we were only able to observe activity towards adenosine, but that was with a rather low specificity constant. A recent study presents a metabolic model for purine metabolism in H. pylori [34] which accounts for, and observes, both activities, but does not identify the protein(s) responsible. According to the data shown by the isolated protein, we tentatively favor its assignment as an adenosine deaminase, capable of activity towards adenine, allowing for the possibility that both activities may be relevant in vivo, and note that genetics will likely soon provide clarification. was observed for the release of reduced sugars when monitored using 3,5-dinitrosalicylic acid (DNS) according to Miller [31], nor by measuring the release of xylose by the D-xylose Rapid Assay kits (Megazyme, Bray, Ireland). By contrast, the enzyme was able to efficiently release acetate groups from acetylated xylan, acetylated chitin and acetylated chitosan (Table 1). Based on the kcat/Km values, the protein functioned more efficiently with xylan than chitin-like products by an order of magnitude. This result can be explained by up to 3.2-fold greater kcat values and about 2-fold lower Km value for acetylated xylan. Discussion Locus COMBREX gene annotation COMBREX cluster annotation Protein Domains Function based on isolated native protein and recombinant protein Proposed EC Number HP0267 Chlorohydrolase Chlorohydrolase Amidohydro_1 Adenosine deaminase 3.5.4.4 HP0310 Hypothetical protein Polysaccharide deacetylase polysaccharide deacetylase Acetyl esterase 3.1.1.6 Acetylxylan esterase 3.1.1.72 HP0959 Hypothetical protein Hypothetical protein - GTP cyclohydrolase 3.5.4.16 HP1037 Hypothetical protein X-Pro aminopeptidase Creatinase_N Aminopeptidase 3.4.11.- Peptidase_M24 HP1042 Hypothetical protein Putative phospho-esterase RecJ-like DHH phosphatase Phosphodiesterase 3.1.4.17 HP1079 Hypothetical protein Hypothetical protein - ATPase/GTPase 3.6.-.- doi:10.1371/journal.pone.0066605.t002 We also explored the potential consequences of our biochemical assignments on whole-organism H. pylori metabolic models - the manually curated ilT341 flux balance model [39], and the biochemical assignments made from automated sequence analysis from Kyoto Encyclopedia of Genes and Genomes (KEGG) [40]. Three activities are not currently represented, even if their activities are expected: adenosine deaminase, EC 3.5.4.4; acetylxylan esterase, EC 3.1.1.72; phosphodiesterase, EC 3.1.4.17. Two of the activities have insufficient specificity, but peptidase and ATPase activity are well represented. Lastly, GTP cyclohydrolase I activity, EC 3.5.4.16, is present, assigned to HP0928, and we have now also assigned that activity to HP0959. Neither protein shares significant sequence similarity, nor do they have common conserved domains, so it is likely that their in vivo roles are quite different. The peptidoglycan deacetylase activity of HP0310 has a documented role in conferring resistance to host immune responses via reduced degradation by lysozyme [28]. The acetylxylan esterase activity of this enzyme has not been One of the additional benefits of experimentally validating the function of a protein annotated as ‘‘hypothetical protein’’ is that it then can serve as a source of experimental information that impacts other genes. This is one of the fundamental driving concepts behind COMBREX’s efforts towards establishing a Gold Standard Database of protein function (GSDB), identifying all proteins for which the precise amino acid sequence is known and its biochemical function has been published in the scientific literature. These proteins can then serve as the foundation for the annotation of newly sequenced genes. In Table 3, we highlight the potential reach of the experimental validations reported here. In column 4 we identify 725 proteins in COMBREX that have BLAST E values lower (better) than 1025 over 80% of the query and target full length sequence. These proteins are members of clusters (NCBI ProtClustDB [38]) that have a total of over 1200 proteins. Discussion One of the remarkable adaptations of H. pylori is the ability to thrive in the highly acidic milieu of the gastric environment, with typical pH values , 3.5. H. pylori maintains an internal pH ,8.0 [36,37] by maintaining high levels of activity of the enzyme urease, which produces ammonia and carbon dioxide, enabling it to buffer large amounts of acid. The pH of the periplasmic space is ,6.2 over a range of external pHs, providing a proton motive force of greater than 100 mV to enable the generation of ATP [37]. We observe four different profiles for enzyme activity with varying pH. We observe that HP0267 shows maximal adenosine deaminase activity at approximately pH 7.5. HP0310 and HP1079 show maximal acetyl esterase and ATPase activity respectively in a narrow pH range , 6.0. HP1037 exhibits aminopeptidase activity over a broad range of pH, with significant measurable activity at pH , 8.0, and maximal activity at pH , 5.0. Lastly, HP0959 and HP1042 show significant GTP cyclohydrolase and phosphodies- terase activity only at the very acidic pHs of # 4.0. This may indicate protein localization in the cellular interior for HP0267, in the periplasmic space for HP0310 and HP1079, with the enzymes HP0959 and HP1042 either secreted or at the cellular surface. Our main contribution reported in this paper is deciphering the function of six proteins that previously lacked functional annota- tion in the clinically important microorganism, H. pylori ATCC 26695. Initial hypotheses were generated by a high throughput screen using an affinity-based enrichment methodology to isolate proteins that might bind/act upon the attached substrates. In our case, gold-nano-particles are coated with select substrates, and proteins with affinity toward those substrates can be isolated. Proteins that were isolated in this manner were identified by mass spectrometry, and also used to test directly the biochemical activity of interest. June 2013 \| Volume 8 \| Issue 6 \| e66605 PLOS ONE \| www.plosone.org 7 Biochemistry of Six Proteins from H. pylori Table 2. Genes tested in this paper. References enriched from casts of two earthworm species. Appl Environ Microbiol 76: 5934–5946. 1. Fleischmann RD, Adams MD, White O, Clayton RA, Kirkness EF, et al. (1995) Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science 269: 496–512. enriched from casts of two earthworm species. Appl Environ Microbiol 76: 5934–5946. 14. Janes LE, Lo¨wendahl C, Kazlauskas RJ (1998) Quantitative Screening of Hydrolase Libraries Using pH Indicators: Identifying Active and Enantioselec- tive Hydrolases. Chemistry - A European Journal 4: 2324–2331. 2. Roberts RJ, Chang YC, Hu Z, Rachlin JN, Anton BP, et al. (2011) COMBREX: a project to accelerate the functional annotation of prokaryotic genomes. Nucleic Acids Res 39: D11–14. 15. Kamat SS, Bagaria A, Kumaran D, Holmes-Hampton GP, Fan H, et al. (2011) Catalytic mechanism and three-dimensional structure of adenine deaminase. Biochemistry 50: 1917–1927. 3. Madupu R, Richter A, Dodson RJ, Brinkac L, Harkins D, et al. (2012) CharProtDB: a database of experimentally characterized protein annotations. Nucleic Acids Res 40: D237–241. 16. Murphy J, Baker DC, Behling C, Turner RA (1982) A critical reexamination of the continuous spectrophotometric assay for adenosine deaminase. Anal Biochem 122: 328–337. 4. Kuznetsova E, Proudfoot M, Sanders SA, Reinking J, Savchenko A, et al. (2005) Enzyme genomics: Application of general enzymatic screens to discover new enzymes. FEMS Microbiol Rev 29: 263–279. 17. Bergmeyer HU (1983). Methods of Enzymatic Analysis, 3rd ed. Deerfield Beach, FL: Verlag Chemie. pp. 135–136. y 5. Proudfoot M, Kuznetsova E, Sanders SA, Gonzalez CF, Brown G, et al. (2008) High throughput screening of purified proteins for enzymatic activity. Methods Mol Biol 426: 331–341. 18. Hatanaka T, Yamasato A, Arima J, Usuki H, Yamamoto Y, et al. (2011) Extracellular production and characterization of Streptomyces X-prolyl dipeptidyl aminopeptidase. Appl Biochem Biotechnol 164: 475–486. 6. Goddard JP, Reymond JL (2004) Enzyme activity fingerprinting with substrate cocktails. J Am Chem Soc 126: 11116–11117. 19. Rangrez AY, Abajy MY, Keller W, Shouche Y, Grohmann E (2010) Biochemical characterization of three putative ATPases from a new type IV secretion system of Aeromonas veronii plasmid pAC3249A. BMC Biochem 11: 10. J 7. Simon GM, Cravatt BF (2010) Activity-based proteomics of enzyme superfam- ilies: serine hydrolases as a case study. J Biol Chem 285: 11051–11055. ilies: serine hydrolases as a case study. J Biol Chem 285: 11051–11 8. Cravatt BF, Wright AT, Kozarich JW (2008) Activity-based protein profiling: from enzyme chemistry to proteomic chemistry. Supporting Information Figure S1 SDS-PAGE analysis of Coomassie-stained proteins isolated using derivatized gold nano-particles. In four of the five instances only a single intense band is observed. In lane 5, two bands were observed, which were identified using mass spectrometry as HP0803 and HP0959. GTP cyclohydrolase activity was observed for HP0959, however HP0803 resisted multiple attempts at cloning and expression in E. coli, it was not pursued further. HP0803 only has one know protein domain, DUF3943 (PF13084), and so its reason for binding to the derivatized nano particle coated with guanosine-5’-triphosphate (GTP) and dihydroneopterin triphosphate is unexplained. (Data for HP0310 not shown.). (DOC) Acknowledgments We thank Xuesong Zhang and Martin Blaser for help in preparing protein lysate from H. pylori, and Richard Roberts and Alexander Yakunin for helpful discussion. Ultimately, biological understanding of a protein’s function incorporates information related to the specific role of a protein (its molecular function) and the biological process, or pathway, which its molecular function impacts. That is, most biologists desire information related to both its in vivo and in vitro role. We point out that once a biochemical or in vitro function of a protein is determined, genetic methods, such as phenotype observation on knockout strains, can quickly lead to very specific hypotheses about in vivo roles proteins may serve. These types of experiments are frequently amenable to existing high-throughput approaches, placing the onus on the scientific community of developing higher- Discussion Lastly, in the NR database, there are close to 11,000 proteins that have BLAST E values lower than 1025, many of which might have their annotations affected by these observations. Table 3. Extended impact of genes identified. Table 3. Extended impact of genes identified. Locus UniProt Proposed Function Similar Proteins in COMBREXa # Proteins in clusters with similar proteinsb Blast vs NRc HP0267 O25046 Adenosine deaminase 148 519 646 HP0310 O25080 Acetylesterase 97 108 1374 HP0959 O25613 GTP cyclohydrolase 121 160 1536 HP1037 O25681 Aminopeptidase 290 353 6867 HP1042 O25683 Phosphodiesterase 29 33 172 HP1079 O25711 ATPase/GTPase 40 56 254 Totals: 725 1229 10849 aThe number of proteins in COMBREX which have BLAST E values , 161025, and alignment over 80% of both the query and target sequence. bThe number of proteins contained in the protein families (from NCBI ProtClustDB [38]) that have at least one BLAST hit, as defined above. cThe number of proteins in the NCBI Non-Redundant database (NR) which have BLAST E values ,161025. doi:10.1371/journal.pone.0066605.t003 Table 3. Extended impact of genes identified. aThe number of proteins in COMBREX which have BLAST E values , 161025, and alignment over 80% of both the query and target sequence. bThe number of proteins contained in the protein families (from NCBI ProtClustDB [38]) that have at least one BLAST hit, as defined above. cThe number of proteins in the NCBI Non-Redundant database (NR) which have BLAST E values ,161025. doi:10.1371/journal.pone.0066605.t003 June 2013 \| Volume 8 \| Issue 6 \| e66605 PLOS ONE \| www.plosone.org 8 Biochemistry of Six Proteins from H. pylori documented to date for H. pylori, but it has been reported in eukaryotic and prokaryotic organisms. Acetylxylan esterases are important enzymes influencing the digestibility of plant cell-wall material by cleaving the ester bonds and thus removing the acetyl moieties from complex carbohydrates. It is possible that this enzyme might play a role during infection, considering that xylan- containing carbohydrate structures are among those commonly consumed by humans [41]. HP1042 was identified as a phosphodiesterase with EC number 3.1.4.17, an activity found widely in the annotations of multi-cellular, eukaryotic, and prokaryotic organisms, and plays an important role in purine metabolism. Purine nucleotide salvage pathways are an integral part of genome scale metabolism, required for utilizing purine compounds from the environment. This pathway has been demonstrated in H. Discussion pylori [34], but the proteins responsible for adenosine deaminase and adenine deaminase activity were not known. We find that HP0267 has strong adenosine deaminase activity, and much weaker adenine deaminase activity, and could potentially be responsible for one or both of these activities in vivo. A protein that possesses adenine deaminase activity exclusively could be a potential drug target, since interference with the purine nucleotide salvage pathway reduces growth, and the activity is not observed in humans. throughput biochemical approaches in hopes of enabling our knowledge of gene function to keep pace with our ability to identify new genes through DNA sequencing. Author Contributions Conceived and designed the experiments: MS M. Ferrer. Performed the experiments: HPC SJ SC MF RB JPA. Analyzed the data: HPC SJ SC MF RB JPA M. Ferrer MS VM. Wrote the paper: MS M. Ferrer. Performed the experiments on recombinant protein: HPC MS. Performed the experiments on native protein: SJ SC M. Fernandez RB JPA. Provided oversight of the informatics database used to derive the protein clusters and sequence homology for the studied proteins: SK BA. Contributed the conceptual implementation of COMBREX: SK MS. Biochemistry of Six Proteins from H. pylori 25. Stingl K, Schauer K, Ecobichon C, Labigne A, Lenormand P, et al. (2008) In vivo interactome of Helicobacter pylori urease revealed by tandem affinity purification. Mol Cell Proteomics 7: 2429–2441. 34. Miller EF, Vaish S, Maier RJ (2012) Efficiency of purine utilization by Helicobacter pylori: roles for adenosine deaminase and a NupC homolog. PLoS One 7: e38727. p 26. Scheer M, Grote A, Chang A, Schomburg I, Munaretto C, et al. (2011) BRENDA, the enzyme information system in 2011. Nucleic Acids Res 39: D670–676. 35. Punta M, Coggill PC, Eberhardt RY, Mistry J, Tate J, et al. (2012) The Pfam protein families database. Nucleic Acids Res 40: D290–301. p 36. Sachs G, Meyer-Rosberg K, Scott DR, Melchers K (1996) Acid, protons and Helicobacter pylori. Yale J Biol Med 69: 301–316. 36. Sachs G, Meyer-Rosberg K, Scott DR, Melchers K Helicobacter pylori. Yale J Biol Med 69: 301–316. 27. Wang G, Olczak A, Forsberg LS, Maier RJ (2009) Oxidative stress-induced peptidoglycan deacetylase in Helicobacter pylori. J Biol Chem 284: 6790–6800. 37. Sachs G, Scott DR, Weeks DL, Rektorscheck M, Melchers K (2001) Regulation of Urease for Acid Habitation. In: Mobley HLT, Mendz GL, Hazell SL, editors. Helicobacter pylori: Physiology and Genetics. Washington (DC): ASM Press. 28. Wang G, Maier SE, Lo LF, Maier G, Dosi S, et al. (2010) Peptidoglycan deacetylation in Helicobacter pylori contributes to bacterial survival by mitigating host immune responses. Infect Immun 78: 4660–4666. 38. Klimke W, Agarwala R, Badretdin A, Chetvernin S, Ciufo S, et al. (2009) The National Center for Biotechnology Information’s Protein Clusters Database. Nucleic Acids Res 37: D216–223. 29. Wang G, Lo LF, Forsberg LS, Maier RJ (2012) Helicobacter pylori peptidoglycan modifications confer lysozyme resistance and contribute to survival in the host. MBio 3: e00409–00412. 39. Thiele I, Vo TD, Price ND, Palsson BO (2005) Expanded metabolic reconstruction of Helicobacter pylori (iIT341 GSM/GPR): an in silico genome-scale characterization of single- and double-deletion mutants. J Bacteriol 187: 5818–5830. 30. Shaik MM, Cendron L, Percudani R, Zanotti G (2011) The structure of Helicobacter pylori HP0310 reveals an atypical peptidoglycan deacetylase. PLoS One 6: e19207. 31. Miller GL (1959) Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar. Analytical Chemistry 31: 426–428. 40. Kanehisa M, Goto S, Sato Y, Furumichi M, Tanabe M (2012) KEGG for integration and interpretation of large-scale molecular data sets. Nucleic Acids Res 40: D109–114. g g y y 32. References Annu Rev Biochem 77: 383– 414. 20. Taussky HH, Shorr E (1953) A microcolorimetric method for the determination of inorganic phosphorus. J Biol Chem 202: 675–685. 9. Abad JM, Mertens SF, Pita M, Fernandez VM, Schiffrin DJ (2005) Functionalization of thioctic acid-capped gold nanoparticles for specific immobilization of histidine-tagged proteins. J Am Chem Soc 127: 5689–5694. 21. Bonting SL, Simon KA, Hawkins NM (1961) Studies on sodium-potassium- activated adenosine triphosphatase. I. Quantitative distribution in several tissues of the cat. Arch Biochem Biophys 95: 416–423. 10. Fonseca Silva TC, Habibi Y, Colodette JL, Lucia LA (2011) The influence of the chemical and structural features of xylan on the physical properties of its derived hydrogels. Soft Matter 7: 1090–1099. p y 22. He A, Rosazza JP (2003) GTP cyclohydrolase I: purification, characterization, and effects of inhibition on nitric oxide synthase in nocardia species. Appl Environ Microbiol 69: 7507–7513. 11. Varela C, Mauriaca C, Paradela A, Albar JP, Jerez CA, et al. (2010) New structural and functional defects in polyphosphate deficient bacteria: a cellular and proteomic study. BMC Microbiol 10: 7. 23. Bracher A, Eisenreich W, Schramek N, Ritz H, Gotze E, et al. (1998) Biosynthesis of pteridines. NMR studies on the reaction mechanisms of GTP cyclohydrolase I, pyruvoyltetrahydropterin synthase, and sepiapterin reductase. J Biol Chem 273: 28132–28141. 12. Perkins DN, Pappin DJ, Creasy DM, Cottrell JS (1999) Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20: 3551–3567. 24. Zheng Y, Roberts RJ, Kasif S (2004) Segmentally variable genes: a new perspective on adaptation. PLoS Biol 2: E81. 13. Beloqui A, Nechitaylo TY, Lopez-Cortes N, Ghazi A, Guazzaroni ME, et al. (2010) Diversity of glycosyl hydrolases from cellulose-depleting communities June 2013 \| Volume 8 \| Issue 6 \| e66605 June 2013 \| Volume 8 \| Issue 6 \| e66605 June 2013 \| Volume 8 \| Issue 6 \| e66605 PLOS ONE \| www.plosone.org 9 Biochemistry of Six Proteins from H. pylori Cole ST, Brosch R, Parkhill J, Garnier T, Churcher C, et al. (1998) Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393: 537–544. 41. Gill SR, Pop M, Deboy RT, Eckburg PB, Turnbaugh PJ, et al. (2006) Metagenomic analysis of the human distal gut microbiome. Science 312: 1355– 1359. 33. (2012) The Gene Ontology: enhancements for 2011. Nucleic Acids Res 40: D559–D564. PLOS ONE \| www.plosone.org June 2013 \| Volume 8 \| Issue 6 \| e66605 PLOS ONE \| www.plosone.org 10
https://openalex.org/W2040125628	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0007743&type=printable	English	null	An Integrated Analysis of Molecular Acclimation to High Light in the Marine Diatom Phaeodactylum tricornutum	PloS one	2,009	cc-by	16,137	Abstract doi:10.1371/journal.pone.0007743 Editor: Markus Grebe, Umea˚ Plant Science Centre, Sweden Received July 5 2009; Accepted October 6 2009; Published November 3 2009 Editor: Markus Grebe, Umea˚ Plant Science Centre, Sweden Editor: Markus Grebe, Umea˚ Plant Science Centre, Sweden Received July 5, 2009; Accepted October 6, 2009; Published November 3, 2009 Received July 5, 2009; Accepted October 6, 2009; Published November 3, 2009 Received July 5, 2009; Accepted October 6, 2009; Published November 3, 2009 Nymark et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which pe ribution, and reproduction in any medium, provided the original author and source are credited. Copyright: 2009 Nymark et al. This is an open-access article distributed under the terms of the Creative Commons Attribut unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Functional Genomics (FUGE) program of the Norwegian Research Council (grant # 184146/S10) and a PhD grant from the Norwegian University of Science and Technology. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: atle.bones@bio.ntnu.no Marianne Nymark, Kristin C. Valle, Tore Brembu, Kasper Hancke, Per Winge, Kjersti Andresen, Geir Johnsen, Atle M. Bones* Department of Biology, Norwegian University of Science and Technology, Trondheim, Norway Department of Biology, Norwegian University of Science and Technology, Trondheim, Norway Department of Biology, Norwegian University of Science and Technology, Trondheim, Norway PLoS ONE \| www.plosone.org An Integrated Analysis of Molecular Acclimation to High Light in the Marine Diatom Phaeodactylum tricornutum Marianne Nymark, Kristin C. Valle, Tore Brembu, Kasper Hancke, Per Winge, Kjersti Andresen, Geir Johnsen, Atle M. Bones* Abstract Photosynthetic diatoms are exposed to rapid and unpredictable changes in irradiance and spectral quality, and must be able to acclimate their light harvesting systems to varying light conditions. Molecular mechanisms behind light acclimation in diatoms are largely unknown. We set out to investigate the mechanisms of high light acclimation in Phaeodactylum tricornutum using an integrated approach involving global transcriptional profiling, metabolite profiling and variable fluorescence technique. Algae cultures were acclimated to low light (LL), after which the cultures were transferred to high light (HL). Molecular, metabolic and physiological responses were studied at time points 0.5 h, 3 h, 6 h, 12 h, 24 h and 48 h after transfer to HL conditions. The integrated results indicate that the acclimation mechanisms in diatoms can be divided into an initial response phase (0–0.5 h), an intermediate acclimation phase (3–12 h) and a late acclimation phase (12–48 h). The initial phase is recognized by strong and rapid regulation of genes encoding proteins involved in photosynthesis, pigment metabolism and reactive oxygen species (ROS) scavenging systems. A significant increase in light protecting metabolites occur together with the induction of transcriptional processes involved in protection of cellular structures at this early phase. During the following phases, the metabolite profiling display a pronounced decrease in light harvesting pigments, whereas the variable fluorescence measurements show that the photosynthetic capacity increases strongly during the late acclimation phase. We show that P. tricornutum is capable of swift and efficient execution of photoprotective mechanisms, followed by changes in the composition of the photosynthetic machinery that enable the diatoms to utilize the excess energy available in HL. Central molecular players in light protection and acclimation to high irradiance have been identified. Citation: Nymark M, Valle KC, Brembu T, Hancke K, Winge P, et al. (2009) An Integrated Analysis of Molecular Acclimation to High Light in the Marine Diatom Phaeodactylum tricornutum. PLoS ONE 4(11): e7743. doi:10.1371/journal.pone.0007743 Editor: Markus Grebe, Umea˚ Plant Science Centre, Sweden Received July 5, 2009; Accepted October 6, 2009; Published November 3, 2009 Citation: Nymark M, Valle KC, Brembu T, Hancke K, Winge P, et al. (2009) An Integrated Analysis of Molecular Acclimation to High Light in the Marine Diatom Phaeodactylum tricornutum. PLoS ONE 4(11): e7743. Introduction The high photosynthetic flexibility of diatoms is strongly related to their high capacity of NPQ, which can reach a 5-fold higher level than in plants [14]. In addition, the intensity dependence of a rise in variable fluorescence in P. tricornutum suggests that light absorbed by the light-harvesting Chl a/c-Fucoxanthin complex is not preferentially delivered to PSII, but is more equally distributed between the photosystems. These results described by Owens [15] imply that, under both low and high irradiances, adjustments are made in the transfer of excitation energy to the PSII reaction centre which prevents prolonged loss of photosynthetic capacity. These differences in the photosynthetic mechanism of diatoms compared to higher plants may be central to the ecological success of diatoms in a variable light environment. Despite the different photoprotective mechanisms evolved by photosynthetic organisms, light above the saturation point for photosynthesis (the light- saturation index, Ek) can cause fatal oxidative damage to the PSII reaction centre and result in a decrease in photosynthetic efficiency or photoinhibition [16–18]. Photoinhibition occurs when the rate of damage exceeds the capacity of the PSII repair mechanisms [19]. The reaction centre-binding D1 protein is the PSII component most prone to photooxidative damage [20]. The complex mechanisms behind the degradation and repair of PSII and its components have been a subject of investigation for several decades [19, 21). Damaged D1 proteins must be removed and replaced by newly synthesized molecules for the PSII to recover, and an increased rate of D1 synthesis has been reported for several photosynthetic organisms in conditions of HL [19,22,23]. significant acclimation to HL conditions were apparent some 12 h after start of the HL treatment. The acclimation processes continued during the next 36 h of exposure to HL conditions. We have identified and categorised transcripts involved in the various phases of light acclimation at a genomic scale. Introduction a molecular one. The whole-genome sequencing of Thalassiosira pseudonana [4] and Phaeodactylum tricornutum [5] has made possible detailed studies of the genetic basis of the unique properties underlying the ecological and evolutionary success of diatoms. Functional genomics have currently made it possible to investigate the molecular processes behind acclimation to changing environ- mental conditions in marine organisms. Genomic approaches to this field of investigation are expected to provide new and essential information for studying and monitoring biodiversity, acclimation and adaptations to life in the ocean. The planktonic diatoms (Bacillariophyceae) account for ap- proximately 40% of the primary production in the world oceans [1]. They are the dominant group of phytoplankton in cold waters [2] and have to cope with highly unpredictable and rapid changes in irradiances (PAR) and spectral quality (El). In low light it is necessary to collect photons as efficiently as possible, and when the light intensity becomes supersaturating for photosynthesis, it becomes necessary to protect the organism from potential photo- oxidative damage to the photosynthetic machinery. In order to optimize growth and reproduction and to minimize photodamage, phytoplankton has developed a number of mechanisms to modulate the rate of photosynthesis in situ. The photoacclimational mechanisms describe the short-term adjustments in response to changing light climate (physiological acclimation), while the photoadaptational mechanisms indicate a long-term evolutionary outcome based on the genes of the given species (genetic adaptation). Both processes work together to maximize evolution- ary fitness under a given set of environmental conditions [3]. Over the last four decades, progress in understanding photosynthesis is gradually moving from a descriptive and physiological approach to Important and well-known short-term acclimational mecha- nisms include photochemical quenching (PQ) related to fraction of open reaction centres in PSII [6] and non-photochemical quenching (NPQ) of chlorophyll fluorescence related to pH and photoprotective carotenoids (PPCs), changes in the distribution of excitation energy between photosystems I (PSI) and II (PSII), and damage and repair of PSII [7]. NPQ is the most important short- term ‘‘safety valve’’ that is activated by a sudden increase in irradiance, and can be measured by a decrease in chlorophyll a (Chl a) fluorescence intensity under HL [8]. In this process, harmful excess energy is dissipated as heat radiation. Introduction It is established that NPQ occurs in the light harvesting system of PLoS ONE \| www.plosone.org PLoS ONE \| www.plosone.org November 2009 \| Volume 4 \| Issue 11 \| e7743 1 Genomics of Light Acclimation PSII, is triggered by DpH and modulated by de-epoxidation of xanthophylls [9–11]. In diatoms, the main xanthophyll cycle is the diadinoxanthin (DD) cycle, which involves a forward reaction that converts DD, a carotenoid with low light energy transfer efficiency [12,13] into diatoxanthin (DT) under conditions of HL. The high photosynthetic flexibility of diatoms is strongly related to their high capacity of NPQ, which can reach a 5-fold higher level than in plants [14]. In addition, the intensity dependence of a rise in variable fluorescence in P. tricornutum suggests that light absorbed by the light-harvesting Chl a/c-Fucoxanthin complex is not preferentially delivered to PSII, but is more equally distributed between the photosystems. These results described by Owens [15] imply that, under both low and high irradiances, adjustments are made in the transfer of excitation energy to the PSII reaction centre which prevents prolonged loss of photosynthetic capacity. These differences in the photosynthetic mechanism of diatoms compared to higher plants may be central to the ecological success of diatoms in a variable light environment. Despite the different photoprotective mechanisms evolved by photosynthetic organisms, light above the saturation point for photosynthesis (the light- saturation index, Ek) can cause fatal oxidative damage to the PSII reaction centre and result in a decrease in photosynthetic efficiency or photoinhibition [16–18]. Photoinhibition occurs when the rate of damage exceeds the capacity of the PSII repair mechanisms [19]. The reaction centre-binding D1 protein is the PSII component most prone to photooxidative damage [20]. The complex mechanisms behind the degradation and repair of PSII and its components have been a subject of investigation for several decades [19, 21). Damaged D1 proteins must be removed and replaced by newly synthesized molecules for the PSII to recover, and an increased rate of D1 synthesis has been reported for several photosynthetic organisms in conditions of HL [19,22,23]. PSII, is triggered by DpH and modulated by de-epoxidation of xanthophylls [9–11]. In diatoms, the main xanthophyll cycle is the diadinoxanthin (DD) cycle, which involves a forward reaction that converts DD, a carotenoid with low light energy transfer efficiency [12,13] into diatoxanthin (DT) under conditions of HL. Results To study the mechanisms of protection and acclimation to high irradiances in diatoms, LL acclimated cells were subjected to HL for 0.5 h, 3 h, 6 h, 12 h, 24 h and 48 h. Global transcriptional regulation, change in pigment metabolites and efficiency and capacity of photosynthesis were analyzed in the material harvested from the six time-points. Based on the resulting measurements, the cells seemed to respond to the treatment in three different phases designated the initial response phase (0–0.5 h), the intermediate acclimation phase (3–12 h) and the late acclimation phase (12–48 h). PLoS ONE \| www.plosone.org Transcriptional profiling of nuclear and plastid transcripts Transcriptional profiling of nuclear and plastid transcripts In addition to the signals from probes representing nuclear- encoded genes, signals from all probes representing chloroplast genes were also detected on the microarrays. This observation indicates that the oligo dT-promoter primer used during the cDNA synthesis step included in the cRNA amplification procedure has been able to hybridize to the poly(A)-rich tail added to endonucleolytically cleaved mature transcripts from chloroplast genes [29]. The poly(A) tail stabilizes nuclear-encoded mRNAs in eukaryotic cells, whereas the poly(A)-rich tail serves as a degradation signal in the chloroplast [29]. The ability to hybridize not only to the poly(A)-tails of the nuclear-encoded mRNAs, but also to the poly(A)-rich tails of the chloroplast-encoded mRNAs has thereby facilitated the generation of cDNA from both types of transcripts. Several chloroplast genes were found to be differen- tially regulated based on the microarray analyses. If the degradation rates of the chloroplast-encoded mRNAs are the same in cells grown in LL and HL, the expression ratios calculated from probes representing chloroplast genes will be indicative of the regulation of these genes. Established long-term acclimation responses to shifts in light conditions include adjustments of the amount and ratios of light harvesting pigments (LHPs) and alterations of the size of the photosynthetic unit (PSU), which are reflected in changes of the maximum photosynthetic capacity of the organism. HL-acclimat- ed cells generally have a low LHP content and a high amount of photoprotective carotenoids; the relationship is inversed for LL- acclimated cells [7,12,24,25]. Some species of phytoplankton acclimate to low irradiances by increasing the size of the PSU [26], defined as the ratio of light-harvesting pigments to P700 reaction centre Chl a [27]. Diatoms tend to have large PSU sizes when grown at high, growth-rate-saturating irradiances; in contrast to the smaller units reported for green algae [28] grown at optimal irradiances. When rates of photosynthesis are not limited by light [26], the larger PSU sizes observed for diatoms could represent an evolutionary adaptation to large, daily fluctuating light environ- ments in the ocean. Species with large PSU size growing in HL should respond rapidly when mixed vertically down to low light in deeper water. To determine the reliability of the microarray data from the chloroplast genes, a two-step qRT-PCR was performed on the RNA material used in the microarray analyses for time points 0.5 h, 3 h, 12 h and 24 h. Transcriptional profiling of nuclear and plastid transcripts The gene encoding Mg-protoporphyrin IX monomethyl e protoporphyrin IX monomethyl ester to divinyl protochlorophyllide a in higher plants [30] is absent in question mark in the figure. The abbreviations used are: GLURS: glutamyl-tRNA synthetase; HEMA: g semialdehyde 2,1-aminomutase, HEMB: porphobilinogen synthase; HEMC: hydroxymethylbilane syn HEME: uroporphyrinogen decarboxylase; HEMF: coproporphyrinogen III oxidase; PPO: protoporphyrin CHLM: Mg-protoporphyrin IX methyl transferase; POR: protochlorophyllide oxidoreductase; DVR: div CHLG: chlorophyll synthase; DXS: deoxyxylulose-5-phosphate synthase; DXR: 1-deoxy-D-xylulose 5-pho erythritol 4-phosphate cytidylyltransferase; CMK: 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase; sphate synthase; HDS: 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase; HDR: 4-hydroxy-3-m farnesyl diphosphate synthase; GGPS: geranylgeranyl pyrophosphate synthase; IDI: isopentenyl isomerise; CHLP: geranylgeranyl reductase. Figure 1. Hypothesized chlorophyll a and steroid biosynthetic pathways in P. tricornutum. Colored squares indicate the regulation pattern of genes encoding putative enzymes functioning in the two pathways after exposure to HL for 0.5 h, 3 h, 6 h, 12 h, 24 h and 48 h. Squares with a diagonal line inside indicate genes with an expression ratio greater than +/20.5 that are not significantly regulated. The asterisk marking the expression pattern of subunit I of Mg-chelatase (MgCh) indicates that the gene is chloroplast encoded. The scale on the right represents gene expression ratio values, log2 transformed. The gene encoding Mg-protoporphyrin IX monomethyl ester cyclase responsible for converting Mg- protoporphyrin IX monomethyl ester to divinyl protochlorophyllide a in higher plants [30] is absent in P. tricornutum, and this step is marked with a question mark in the figure. The abbreviations used are: GLURS: glutamyl-tRNA synthetase; HEMA: glutamyl-tRNA reductase; HEML: glutamate-1- semialdehyde 2,1-aminomutase, HEMB: porphobilinogen synthase; HEMC: hydroxymethylbilane synthase; HEMD: uroporphyrinogen-III synthase; HEME: uroporphyrinogen decarboxylase; HEMF: coproporphyrinogen III oxidase; PPO: protoporphyrinogen oxidase; MgCh: magnesium chelatase; CHLM: Mg-protoporphyrin IX methyl transferase; POR: protochlorophyllide oxidoreductase; DVR: divinyl protochlorophyllide a 8-vinyl reductase; CHLG: chlorophyll synthase; DXS: deoxyxylulose-5-phosphate synthase; DXR: 1-deoxy-D-xylulose 5-phosphate reductoisomerase; ISPD: 2-C-methyl-D- erythritol 4-phosphate cytidylyltransferase; CMK: 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase; ISPF: 2-C-methyl-D-erythritol 2,4-cyclodipho- sphate synthase; HDS: 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase; HDR: 4-hydroxy-3-methylbut-2-enyl diphosphate reductase; FDPS: farnesyl diphosphate synthase; GGPS: geranylgeranyl pyrophosphate synthase; IDI: isopentenyl pyrophosphate:dimethylallyl pyrophosphate isomerise; CHLP: geranylgeranyl reductase. doi:10.1371/journal.pone.0007743.g001 Figure 1. Hypothesized chlorophyll a and steroid biosynthetic pathways in P. tricornutum. Colored squares indicate the regulation pattern of genes encoding putative enzymes functioning in the two pathways after exposure to HL for 0.5 h, 3 h, 6 h, 12 h, 24 h and 48 h. Transcriptional profiling of nuclear and plastid transcripts The relative expression levels of eight chloroplast-encoded genes considered to be of great importance during the photoacclimation process were investigated by qRT- PCR using random primers during the cDNA synthesis. The results showed that relative expression levels obtained from the qRT-PCR analysis correlated well with those produced by the microarray analysis (Supplementary Figure S1). These results imply that the expression ratios obtained from probes representing chloroplast genes actually reflect the relative amounts of the chloroplast gene products. Axenically cultured P. tricornutum was used to investigate the processes of light acclimation in diatoms. We hypothesised that algae should have a dynamic and fast responding regulatory system that make acclimation to changing light conditions swift and consistent. To study the molecular mechanisms of light acclimation, we performed an integrated analysis combining time series studies of pigment metabolites, fluorometry-based analyses of activity and efficiency of photosystems, and studies of global transcriptional regulation through genome wide transcriptional profiling. The photoprotective carotenoids DD and DT were detected and transcriptional profiles changed dramatically after exposure to HL for only 0.5 h. Pulse Amplitude Modulated (PAM) fluorometry analyses of the photosynthetic capacity showed that Synthesis of chlorophyll a and steroids. An immediate response after transfer to HL conditions was a dramatic reduction in expression of transcripts encoding enzymes in the Chl a biosynthesis. In higher plants, Chl a is synthesized from glutamate in a 15 step biosynthetic pathway [30] through the cooperative action of a range of different enzymes. Transcripts for all genes encoding enzymes involved in the Chl a synthesis of higher plants, except for the gene encoding Mg-protoporphyrin IX monomethyl ester cyclase (MgCy), were identified in P. tricornutum (Figure 1). MgCy is also absent in Thalassiosira pseudonana [31]. In higher plants, MgCy is responsible for converting Mg-protoporphyrin IX monomethyl ester to divinyl protochlorophyllide. The majority of the enzymes involved in the Chl a biosynthetic pathway are PLoS ONE \| www.plosone.org November 2009 \| Volume 4 \| Issue 11 \| e7743 2 Genomics of Light Acclimation Figure 1. Hypothesized chlorophyll a and steroid biosynthetic pathways in P. tricornutum. C of genes encoding putative enzymes functioning in the two pathways after exposure to HL for 0.5 h, diagonal line inside indicate genes with an expression ratio greater than +/20.5 that are not sign expression pattern of subunit I of Mg-chelatase (MgCh) indicates that the gene is chloroplast enco expression ratio values, log2 transformed. Transcriptional profiling of nuclear and plastid transcripts During the intermediate acclimation phase, the transcript levels of the above mentioned nuclear encoded genes gradually recovered, and after 12 h most genes were back to LL levels. The majority of these genes were found to be moderately up-regulated in the late acclimation phase. Photosystems and electron transport chain. Oxidative photosynthesis is catalyzed by the four multi-subunit complexes PSI, PSII, the cytochrome b6f complex and F-ATPase [34]. In P. tricornutum the vast majority of the genes encoding subunits of the mentioned membrane-protein complexes are localized to the chloroplast genome [35], while some genes have been transferred to the nucleus. The transcript levels of eight genes localized to the chloroplast genome were analyzed by qRT-PCR, including two PSII genes (psbA and psbV), two PSI genes (psaA and psaE), and an ATP-dependent metalloprotease (ftsH_2). Both chloroplast- and nuclear-encoded genes representing proteins involved in photosynthesis and repair of photo-damaged PSII were differentially expressed during the acclimation period, as indicated on Figure 3. The nuclear-encoded genes PSBO, OEE3 (PSBQ’) and PSBU and the chloroplast encoded psbV, encoding putative subunits of the oxygen evolving complex of PSII [36], were all found to be constitutively repressed under HL conditions (Figure 3). This was also evident for the nuclear-encoded PSBM gene, encoding one of the small transmembrane proteins of the PSII reaction center [36]. The microarray data based on signals from polyadenylated chloroplast transcripts also indicated that PSII genes like psbE, psbH, psbY and psbX were down-regulated as a response to the HL treatment during some or all of the acclimation phases. These genes are all predicted to encode small transmembrane proteins located in the reaction center of PSII [36]. In addition, a conserved open-reading frame named ycf66 [35], positioned in a gene cluster between the chloroplast-encoded psbV and psbX genes, displayed the same regulation pattern as the two psb genes, being down-regulated at all times (data not shown). Two genes assumed to encode the PSB27 and the HCF136 proteins that function in assembly and repair of PSII in both chloroplasts [37,38] and cyanobacteria [39,40) showed an increase in expression level at the initial response phase. During the intermediate phase, the PSB27 and HCF136 genes both displayed a slight down-regulation, whereas the expression levels at the late acclimation phase was back to LL levels. Transcriptional profiling of nuclear and plastid transcripts Squares with a diagonal line inside indicate genes with an expression ratio greater than +/20.5 that are not significantly regulated. The asterisk marking the expression pattern of subunit I of Mg-chelatase (MgCh) indicates that the gene is chloroplast encoded. The scale on the right represents gene expression ratio values, log2 transformed. The gene encoding Mg-protoporphyrin IX monomethyl ester cyclase responsible for converting Mg- protoporphyrin IX monomethyl ester to divinyl protochlorophyllide a in higher plants [30] is absent in P. tricornutum, and this step is marked with a question mark in the figure. The abbreviations used are: GLURS: glutamyl-tRNA synthetase; HEMA: glutamyl-tRNA reductase; HEML: glutamate-1- semialdehyde 2,1-aminomutase, HEMB: porphobilinogen synthase; HEMC: hydroxymethylbilane synthase; HEMD: uroporphyrinogen-III synthase; HEME: uroporphyrinogen decarboxylase; HEMF: coproporphyrinogen III oxidase; PPO: protoporphyrinogen oxidase; MgCh: magnesium chelatase; CHLM: Mg-protoporphyrin IX methyl transferase; POR: protochlorophyllide oxidoreductase; DVR: divinyl protochlorophyllide a 8-vinyl reductase; CHLG: chlorophyll synthase; DXS: deoxyxylulose-5-phosphate synthase; DXR: 1-deoxy-D-xylulose 5-phosphate reductoisomerase; ISPD: 2-C-methyl-D- erythritol 4-phosphate cytidylyltransferase; CMK: 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase; ISPF: 2-C-methyl-D-erythritol 2,4-cyclodipho- sphate synthase; HDS: 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase; HDR: 4-hydroxy-3-methylbut-2-enyl diphosphate reductase; FDPS: farnesyl diphosphate synthase; GGPS: geranylgeranyl pyrophosphate synthase; IDI: isopentenyl pyrophosphate:dimethylallyl pyrophosphate isomerise; CHLP: geranylgeranyl reductase. doi:10.1371/journal.pone.0007743.g001 y y doi:10.1371/journal.pone.0007743.g001 PLoS November 2009 \| Volume 4 \| Issue 11 \| e7743 PLoS ONE \| www.plosone.org 3 Genomics of Light Acclimation represented in the P. tricornutum genome by a single gene. The enzymes encoded by multi-gene families are glutamyl-tRNA synthetase (two genes, GLURS_1-2), uroporphyrinogen decar- boxylase(HEME_1-2), coproporphyrinogen III oxidase (HEMF_1- 3), protochlorophyllide oxidoreductase (POR_1-4), and the H subunit (CHLH_1-2) of Magnesium-chelatase (MgCh). All genes encoding enzymes in the Chl a pathway are found in the nucleus, with the exception of the CHLI gene encoding the MgCh I subunit, which is chloroplast-encoded. The expression of all nuclear single copy genes and at least one of each type of the nuclear-encoded multi-copy genes involved in Chl a biosynthesis dropped dramatically after 0.5 h (Figure 1), indicating a strong down-regulation of every step in the synthesis of Chl a at this initial response phase. The gene encoding CHLI was not significantly affected by the HL treatment. Phytyl diphosphate produced by the steroid biosynthesis pathway functions together with monovinyl chlorophyllide a from the Chl a biosynthesis pathway as a substrate for chlorophyll synthetase in the last step in the formation of Chl a. Transcriptional profiling of nuclear and plastid transcripts Two genes encoding ATP-dependent metallo- proteases (FTSH1 and FTSH2) found to be involved in the degradation of photodamaged D1 protein in plants and cyano- bacteria [21], were also significantly induced immediately after transfer to HL. At the two latest phases of the acclimation period the FTSH genes were unaffected or slightly up-regulated. The psbA and psbD genes, encoding the D1 and D2 proteins that together form the core of the PSII reaction center [34], showed no significant response to the HL treatment. This heterodimer binds several cofactors, including chlorophylls, pheophytin a molecules and the plastoquinones QA and QB involved in the electron transfer in PSII. y p g p Synthesis of carotenoids. A hypothetical carotenoid biosynthetic pathway is presented in Figure 2, according to Coesel et al. [32]. The genes identified and proposed to be involved in the synthesis of carotenoids in P. tricornutum by Coesel et al. are indicated on the figure. With a few intriguing exceptions the transcript levels of these genes were in general little affected by the exposure to HL. The most interesting and specific responses were the immediate regulation of genes that might be involved in controlling the forward reactions of the two xanthophyll cycles in P. tricornutum. The P. tricornutum genome has been found to contain three genes encoding zeaxanthin epoxidase (ZEP1-3), but it is not known whether these genes encode enzymes involved in the violaxanthin cycle, the diadinoxanthin cycle or possible both cycles. After exposure to HL for 0.5 h, the expression level of ZEP1 was clearly down-regulated, while ZEP3 gene expression was clearly up-regulated, indicating that these two genes encode enzymes with different functions. One possibility is that the ZEP1 gene encodes the enzyme responsible for converting zeaxanthin to violaxanthin in LL, while the ZEP3 gene encodes the enzyme that converts DD to DT in HL. ZEP1 and ZEP3 both showed little or no regulation during the intermediate phase. At the late acclimation phase, the ZEP1 gene expression was moderately increased, while the ZEP3 gene expression level was maintained at LL levels. Synthesis of carotenoids. A hypothetical carotenoid biosynthetic pathway is presented in Figure 2, according to Coesel et al. [32]. The genes identified and proposed to be involved in the synthesis of carotenoids in P. tricornutum by Coesel et al. are indicated on the figure. PLoS ONE \| www.plosone.org Transcriptional profiling of nuclear and plastid transcripts The genes encoding putative geranylgeranyl diphosphate synthase (GGPS) and geranylgeranyl reductase (CHLP), responsible for converting isopentenyl diphosphate through several possible intermediates to phytyl diphosphate, were also strongly down- regulated at the initial phase (Figure 1). During the intermediate acclimation phase, the transcript levels of the above mentioned nuclear encoded genes gradually recovered, and after 12 h most genes were back to LL levels. The majority of these genes were found to be moderately up-regulated in the late acclimation phase. handful of transcripts in the same families are strongly up- regulated, especially at the initial phase, indicating a role in photoprotection. The most strongly induced genes, LHCR6, LHCR8, LHCX2 and LHCX3, increased dramatically after exposure to HL and were up-regulated as much as 13-36 times already after 0.5 h. The expression of the LHCX2 gene remained at almost the same high level during the entire length of the experiment, while the expression of LHCR6 and LHCR8 dropped during the intermediate phase and stabilized at a lower level. The LHCX3 transcript level decreased gradually with time and was back to LL levels at the late acclimation phase. represented in the P. tricornutum genome by a single gene. The enzymes encoded by multi-gene families are glutamyl-tRNA synthetase (two genes, GLURS_1-2), uroporphyrinogen decar- boxylase(HEME_1-2), coproporphyrinogen III oxidase (HEMF_1- 3), protochlorophyllide oxidoreductase (POR_1-4), and the H subunit (CHLH_1-2) of Magnesium-chelatase (MgCh). All genes encoding enzymes in the Chl a pathway are found in the nucleus, with the exception of the CHLI gene encoding the MgCh I subunit, which is chloroplast-encoded. The expression of all nuclear single copy genes and at least one of each type of the nuclear-encoded multi-copy genes involved in Chl a biosynthesis dropped dramatically after 0.5 h (Figure 1), indicating a strong down-regulation of every step in the synthesis of Chl a at this initial response phase. The gene encoding CHLI was not significantly affected by the HL treatment. Phytyl diphosphate produced by the steroid biosynthesis pathway functions together with monovinyl chlorophyllide a from the Chl a biosynthesis pathway as a substrate for chlorophyll synthetase in the last step in the formation of Chl a. The genes encoding putative geranylgeranyl diphosphate synthase (GGPS) and geranylgeranyl reductase (CHLP), responsible for converting isopentenyl diphosphate through several possible intermediates to phytyl diphosphate, were also strongly down- regulated at the initial phase (Figure 1). Transcriptional profiling of nuclear and plastid transcripts With a few intriguing exceptions the transcript levels of these genes were in general little affected by the exposure to HL. The most interesting and specific responses were the immediate regulation of genes that might be involved in controlling the forward reactions of the two xanthophyll cycles in P. tricornutum. The P. tricornutum genome has been found to contain three genes encoding zeaxanthin epoxidase (ZEP1-3), but it is not known whether these genes encode enzymes involved in the violaxanthin cycle, the diadinoxanthin cycle or possible both cycles. After exposure to HL for 0.5 h, the expression level of ZEP1 was clearly down-regulated, while ZEP3 gene expression was clearly up-regulated, indicating that these two genes encode enzymes with different functions. One possibility is that the ZEP1 gene encodes the enzyme responsible for converting zeaxanthin to violaxanthin in LL, while the ZEP3 gene encodes the enzyme that converts DD to DT in HL. ZEP1 and ZEP3 both showed little or no regulation during the intermediate phase. At the late acclimation phase, the ZEP1 gene expression was moderately increased, while the ZEP3 gene expression level was maintained at LL levels. Light harvesting antenna proteins. The P. tricornutum genome is predicted to encode at least 40 genes belonging to the light-harvesting complex (LHC) superfamily. These gene transcripts were all detected by the whole-genome array, and 37 out of the 40 genes were found to be significantly regulated at least at one time point (Figure 3). The diatom light harvesting genes are divided into three main groups [33]: the LHCF’s, encoding the major fucoxanthin Chl a/c proteins, the red algal-like LHCR’s and the LI818-like LHCX’s. Among the chloroplast-localized psa genes encoding subunits of PSI, the psaE gene was confirmed by qRT-PCR to be continuously repressed when subjected to HL. PsaE is one of the subunits of the PSI core complex and functions as a binding site for soluble ferredoxin, and is also involved in cyclic electron transport [41]. From the results presented in Figure 3 it is evident that most of the transcripts encoding putative light harvesting antenna proteins are continuously down-regulated during all three phases, reflecting the expected acclimation to higher light irradiances. However, a PLoS ONE \| www.plosone.org November 2009 \| Volume 4 \| Issue 11 \| e7743 4 Genomics of Light Acclimation Figure 2. Hypothesized carotenoid biosynthetic pathway in P. tricornutum according to Coesel et al. [32]. Transcriptional profiling of nuclear and plastid transcripts The color code indicates expression values. Squares with a diagonal line inside indicate genes with an expression ratio (log2 transformed) greater than +/20.5 that are not significantly regulated. Genes where at least one of the probes representing the genes were significantly regulated by .2-fold at least at one time point during the acclimation period, were included in the figure. The expression patterns of genes marked with an asterisk are chloroplast encoded. The scale on the right represents gene expression ratio values, log2 transformed. The abbreviations used are LHCF: major fucoxanthin Chl a/c proteins; LHCR: red algal-like proteins; LHCX: LI818-like proteins; LHC#: unclassified light harvesting proteins; Psa: PSI proteins; PETJ: cytochrome c6; Psb: PSII proteins; HCF: high Chl fluorescence; FtsH: Filamentation temperature sensitive H; AUR: aureochrome; CRYL: cryptochrome-like protein; CPF: cryptochrome; SKP3: Sensor Kinase Protein 3; PPDK: pyruvate-phosphate dikinase; PEPCase: phosphoenolpyruvate carboxylase; MDH: malate dehydrogenase; PEPCK: phosphoenolpyruvate carboxykinase; PK: pyruvate kinase; PYC: pyruvate carboxylase; CA: carbonic anhydrase; SLC4A: bicarbonate transporter; OMT: oxoglutarate/malate transporter; FBPC: fructose-1,6-bisphosphatase; FBAC: fructose-1,6-bisphosphate aldolase; GPI: glucose-6-phosphate isomerase; TPI: triosephosphate isomerase; GLRXC: glutaredoxin; TRX: thioredoxin; TRXLl: thioredoxin-like; PRX: peroxiredoxin; GPX: glutathione peroxidase; APX: ascorbate peroxidise; SOD: superoxide dismutase; GST: glutathione S-transferase; TMT: gamma-tocopherol methyltransferase; TYPA: tyrosine phosphorylation protein A. doi:10.1371/journal.pone.0007743.g003 Figure 3. Regulation pattern of HL affected genes during the acclimation period. The differentially regulated genes encode proteins involved in light sensing, antenna proteins, photoreceptors, components involved in oxidative photosynthesis, carbon metabolism, Calvin cycle and ROS scavenging systems after exposure to HL for 0.5 h, 3 h, 6 h, 12 h, 24 h and 48 h. The color code indicates expression values. Squares with a diagonal line inside indicate genes with an expression ratio (log2 transformed) greater than +/20.5 that are not significantly regulated. Genes where at least one of the probes representing the genes were significantly regulated by .2-fold at least at one time point during the acclimation period, were included in the figure. The expression patterns of genes marked with an asterisk are chloroplast encoded. The scale on the right represents gene expression ratio values, log2 transformed. Transcriptional profiling of nuclear and plastid transcripts Colored squares indicate the regulation pattern of genes encoding putative enzymes involved in the synthesis of carotenoids after exposure to HL for 0.5 h, 3 h, 6 h, 12 h, 24 h and 48 h. Squares with a diagonal line inside indicate genes with an expression ratio (log2 transformed) greater than +/20.5 that are not significantly regulated. The scale on the right represents gene expression ratio values, log2 transformed. The violaxanthin cycle (A) and the diadinoxanthin cycle (B) are boxed. Dashed arrows indicate the hypothetical conversion of violaxanthin to diadinoxanthin and the formation of fucoxanthin from diadinoxanthin, as proposed by Lohr and Wilhelm [52,53]. The abbreviations used are PSY: phytoene synthase; PDS: phytoene desaturase; ZDS: f- carotene desaturase, CRTISO: carotenoid isomerase; crtI: bacterial-like desaturase; LCYB: lycopene b-cyclase; LUT: lutein deficient-like; ZEP: zeaxanthin epoxidase; VDE: violaxanthin de de-epoxidase; VDL: violaxanthin de de-epoxidase-like; VDR: violaxanthin de de-epoxidase related. doi:10.1371/journal.pone.0007743.g002 PLoS ONE \| www.plosone.org 5 November 2009 \| Volume 4 \| Issue 11 \| e7743 Figure 2. Hypothesized carotenoid biosynthetic pathway in P. tricornutum according to Coesel et al. [32]. Colored squares indicate the regulation pattern of genes encoding putative enzymes involved in the synthesis of carotenoids after exposure to HL for 0.5 h, 3 h, 6 h, 12 h, 24 h and 48 h. Squares with a diagonal line inside indicate genes with an expression ratio (log2 transformed) greater than +/20.5 that are not significantly regulated. The scale on the right represents gene expression ratio values, log2 transformed. The violaxanthin cycle (A) and the diadinoxanthin cycle (B) are boxed. Dashed arrows indicate the hypothetical conversion of violaxanthin to diadinoxanthin and the formation of fucoxanthin from diadinoxanthin, as proposed by Lohr and Wilhelm [52,53]. The abbreviations used are PSY: phytoene synthase; PDS: phytoene desaturase; ZDS: f- carotene desaturase, CRTISO: carotenoid isomerase; crtI: bacterial-like desaturase; LCYB: lycopene b-cyclase; LUT: lutein deficient-like; ZEP: zeaxanthin epoxidase; VDE: violaxanthin de de-epoxidase; VDL: violaxanthin de de-epoxidase-like; VDR: violaxanthin de de-epoxidase related. doi:10.1371/journal.pone.0007743.g002 November 2009 \| Volume 4 \| Issue 11 \| e7743 PLoS ONE \| www.plosone.org 5 Genomics of Light Acclimation Figure 3. Regulation pattern of HL affected genes during the acclimation period. The differentially regulated genes encode proteins involved in light sensing, antenna proteins, photoreceptors, components involved in oxidative photosynthesis, carbon metabolism, Calvin cycle and ROS scavenging systems after exposure to HL for 0.5 h, 3 h, 6 h, 12 h, 24 h and 48 h. Transcriptional profiling of nuclear and plastid transcripts In higher plants, the PsaF protein binds the electron donor plastocyanin and an antenna protein dimer, whereas PsaI stabilizes another core subunit [41]. Photoreceptors. Genes predicted to encode photoreceptors like blue-light sensing aureochromes and cryptochromes, and red/ far-red perceiving phytochromes have been identified in P. tricornutum [5]. As indicated on Figure 3, most of the regulated photoreceptor genes displayed a moderate response to the HL treatment, with the exception of one of the cryptochromes (CPF2). CPF2 expression levels increased up to 4–5 fold in HL compared to LL cultures cells at the beginning of the intermediate phase and also during the late acclimation phase. Carbon metabolism and Calvin cycle. Kroth et al. [42] has identified 16 genes putatively involved in a C4-like photosynthesis in P. tricornutum. The protein products of these genes were predicted to be localized to three different cell compartments; endoplasmatic reticulum (ER), mitochondria and plastid. In our experiment, 13 of the mentioned genes were found to be significantly regulated by HL at one or several time points (Figure 3). Among the most pronounced down-regulated genes were PYC1, encoding a mitochondrial-localized pyruvate carboxylase, and SLC4A_1, encoding a bicarbonate transporter predicted to be localized to the plastid [42]. PYC1 and SLC4A_1 displayed a decrease in expression levels during the entire acclimation period as a response to the HL treatment. The gene encoding phosphoenolpyruvate carboxylase (PEPCase_2), predicted to be targeted to the ER, mitochondrial-localized malate dehydrogenase (MDH), a pyruvate kinase (PK6), an oxoglutarate/malate transporter (OMT1), and the chloroplast- targeted pyruvate carboxylase (PYC2) all showed a similar regulation pattern after being subjected to HL conditions. These genes were all unaffected or only slightly regulated during the first hours of HL exposure, and up-regulated 2–11 times at the late acclimation phase. y g [ ] p Based on experiments with the halophytic plant Suaeda salsa, Wang et al. [48] has suggested that a member of the TypA/BipA GTPase family named SsTypA1 might play a crucial role in the defence against ROS damage, possibly functioning as a transla- tional regulator of the stress-responsive proteins involved in ROS scavenging in chloroplast. Two genes identified in P. tricornutum (designated TypA1-2) encoding putative TypA GTP-binding proteins were up-regulated for all, or all but one time-point during the HL experiment. Transcriptional profiling of nuclear and plastid transcripts The transcriptional analyses of the TypA genes revealed that the expression of the TypA1 gene reached its maximum level during the late acclimation phase, where the transcription was up-regulated almost 15 times in the HL-cultures compared to the LL-cultures, whereas the TypA2 gene peaked after only 0.5 h in HL (Figure 3). Pigment analysis Kroth et al. [42] has also identified 26 genes possibly encoding Calvin cycle enzymes, which are predicted to be distributed between the plastid, mitochondria and the cytosol. These genes, including the chloroplast-encoded subunits of ribulose-bispho- sphate carboxylase oxygenase (RUBISCO), showed in general little or no response to the HL treatment. A gene presumed to encode a triosephosphate isomerase (TPI_2) predicted to be targeted to the plastid, was among the most regulated Calvin cycle genes. The TPI_2 gene displayed a 3–6 fold up-regulation in HL at the late acclimation phase (Figure 3). The pigmentation in P. tricornutum comprises the major light harvesting pigments (LHPs) Chl a, Chl c1 and c2, Fucoxanthin (Fuco), the photoprotective carotenoid diadinoxanthin (DD) which can be de-epoxidized to diatoxanthin (DT) in addition to the ubiquitous b -carotene found in all photosynthetic organisms [49]. As expected, the HPLC analyses showed that Chls a and c, Fuco, DD and DT (the latter not found in the LL cultures) were the dominating pigments in the algae. Violaxanthin, bb-carotene and a few derivatives of Chl a and Fuco were present in trace amounts. ROS scavenging systems. Reactive oxygen species (ROS) form as by-products during photosynthesis and can result in significant damage to cellular components [43]. Genes encoding known enzymatic antioxidants like catalases, superoxide dismutases (SOD), ascorbate peroxidases (APX), gluthatione S- transferases (GST) and glutathione peroxidases (GPX) were unaffected, slightly up-regulated or even down-regulated during the two first response and acclimation phases (Figure 3). At the late acclimation phase, only a few of these genes showed a significant increase in expression levels. SOD3, encoding a CuZn superoxide dismutase, APX2, GPX1, GST3 and GST4 displayed a 2–3 fold up- regulation at these time points. A gene encoding a putative gamma-tocopherol methyltransferase (TMT) responsible for converting gamma-tocopherol to the non-enzymatic antioxidant alpha-tocopherol [44] was also moderately up-regulated at the latest time points. Chlorophyll and Fucoxanthin. The concentration of Chl a per cell ([Chl a]) remained unchanged during the initial response phase, after which [Chl a] decreased gradually during the next two acclimation phases (Figure 4). Although present in much lower concentrations, the measurements of Chl c per cell ([Chl c]) showed a similar pattern. Concentrations of Fuco per cell ([Fuco]) showed an immediate decline from the onset of HL and throughout the experiment. The ratio between the LHPs [Fuco+Chl c] and [Chl a] was stable during the experiment, ranging between 0.8 and 0.9. Transcriptional profiling of nuclear and plastid transcripts The abbreviations used are LHCF: major fucoxanthin Chl a/c proteins; LHCR: red algal-like proteins; LHCX: LI818-like proteins; LHC#: unclassified light harvesting proteins; Psa: PSI proteins; PETJ: cytochrome c6; Psb: PSII proteins; HCF: high Chl fluorescence; FtsH: Filamentation temperature sensitive H; AUR: aureochrome; CRYL: cryptochrome-like protein; CPF: cryptochrome; SKP3: Sensor Kinase Protein 3; PPDK: pyruvate-phosphate dikinase; PEPCase: phosphoenolpyruvate carboxylase; MDH: malate dehydrogenase; PEPCK: phosphoenolpyruvate carboxykinase; PK: pyruvate kinase; PYC: pyruvate carboxylase; CA: carbonic anhydrase; SLC4A: bicarbonate transporter; OMT: oxoglutarate/malate transporter; FBPC: fructose-1,6-bisphosphatase; FBAC: fructose-1,6-bisphosphate aldolase; GPI: glucose-6-phosphate isomerase; TPI: triosephosphate isomerase; GLRXC: glutaredoxin; TRX: thioredoxin; TRXLl: thioredoxin-like; PRX: peroxiredoxin; GPX: glutathione peroxidase; APX: ascorbate peroxidise; SOD: superoxide dismutase; GST: glutathione S-transferase; TMT: gamma-tocopherol methyltransferase; TYPA: tyrosine phosphorylation protein A. p doi:10.1371/journal.pone.0007743.g003 PLoS ONE \| www.plosone.org November 2009 \| Volume 4 \| Issue 11 \| e7743 6 Genomics of Light Acclimation consistent up-regulated genes of the entire data set. PRX Q showed an immediate response to the HL treatment and was up-regulated as much as 24 times after just 0.5 h in HL. During the intermediate phase the expression levels dropped, but increased again after 12 hours and stayed high at the late acclimation phase. The A. thaliana PRX Q is targeted to the plastid and has been suggested to be involved in protection of ROS generated in photosynthesis [45]. PRX Q reduces H2O2 using a thiol and is reactivated by thioredoxins. Other peroxiredoxins have been shown to be reactivated by glutaredoxins [46]. Genes encoding a glutaredoxin (GLRXC2), a thioredoxin (TRX Y) and several genes possibly encoding thioredoxin-like (TRXL_1-3) proteins were differentially regulated as a response to HL. GLRXC2 are predicted to be targeted to the plastid [42], and showed a similar regulation pattern as PRX Q. GLRXC2 displayed a 20-fold increase in expression level at the initial response phase, a drop in the expression level during the next hours and a subsequent increase at the late acclimation phase. TRXL_2 and TRXL_3 were also up- regulated at several time points. TRX Y, predicted to be plastid localized [42], and TRXL_1 were down-regulated and might be involved in enzyme regulation [47] instead of stress response. The microarray data indicated that the expression of the psaF and psaI genes, encoding two additional core proteins, was also down- regulated in the HL cultures. PLoS ONE \| www.plosone.org Pigment analysis The late acclimation phase showed a new peak in cellular DD after 24 h, indicating cell division. As the algae acclimated to HL (48 h) [DT] per cell was reduced. From onset of HL the cellular Fuco fraction started to decrease, and continued to do so during the experimental period. This suggests that DD might be the precursor of both DT and Fuco. shift from the intermediate to the late acclimation phase there was a decrease in DES, after which the algae seemed to have acclimated to the increased irradiance, reflected by the small decrease in DES from 24 to 48 h. The changes in the fractions of cellular DD, DT and Fuco from the original LL pool sizes as a function of HL exposure time is shown in Figure 5B. From the decrease in the DD fraction and the increase of the DT fraction measured in cultures that had been subjected to HL for 0.5 h, it is evident that these two pigments are in an inverse relationship with each other (Figure 5B). In the intermediate acclimation face the 3 h HL exposure time resulted in increased production of DD as a response to an increased need for photoprotection. From 3 h to 12 h the cellular [DD] pool size decreased while cellular [DT], from de-epoxidation of DD, increased simultaneously. The late acclimation phase showed a new peak in cellular DD after 24 h, indicating cell division. As the algae acclimated to HL (48 h) [DT] per cell was reduced. From onset of HL the cellular Fuco fraction started to decrease, and continued to do so during the experimental period. This suggests that DD might be the precursor of both DT and Fuco. Figure 5. De-epoxidation state index (DES) and change in [Fuco], [DD] and [DT] per cell. A) De-epoxidation state index (DES) index calculated from the HPLC pigment data. The 0 h sample value is the mean of the 18 LL control samples (blue symbol). HL exposure values are the mean of three biological parallels. Incubations as in Figure 4. Values are presented with6SD bars. (B) Change in Fucoxanthin, Diadinoxanthin and Diatoxanthin cell concentration as a function of high light exposure time. Change in pigment concentration for Fuco (normalized to LL, t = 0 h), DD (as for Fuco) and DT (normalized to HL at 0.5 h) as a function of time after HL exposure. Pigment analysis Values are average of three parallel HPLC samples. Incubations as in Figure 4. doi:10.1371/journal.pone.0007743.g005 markedly increased during the late acclimation phase (.12 h) compared to the control (LL treatment, Figure 7A). The maximum rETR express the maximum amount of electrons generated in PSII that is available to the ATP and NADPH2 synthesis at ambient light, and thus is an estimate of the maximum photosynthetic capacity. At 48 h, the maximum rETR had increased .2 fold in the HL treatment culture compared to the LL acclimated culture. PLoS ONE \| www.plosone.org Pigment analysis These results imply a highly effective acclimation to changed light climate, as the concentration of LHPs are down-regulated to adjust to the Chl a concentration. Chlorophyll and Fucoxanthin. The concentration of Chl a per cell ([Chl a]) remained unchanged during the initial response phase, after which [Chl a] decreased gradually during the next two acclimation phases (Figure 4). Although present in much lower concentrations, the measurements of Chl c per cell ([Chl c]) showed a similar pattern. Concentrations of Fuco per cell ([Fuco]) showed an immediate decline from the onset of HL and throughout the experiment. The ratio between the LHPs [Fuco+Chl c] and [Chl a] was stable during the experiment, ranging between 0.8 and 0.9. These results imply a highly effective acclimation to changed light climate, as the concentration of LHPs are down-regulated to adjust to the Chl a concentration. Diadinoxanthin, diatoxanthin and NPQ. 0.5 h after HL exposure of LL acclimated cells, de-epoxidation of DD to form DT had started to take place, thereby facilitating the dissipation of excess light energy by NPQ. The increasing de-epoxidation state (DES) index during the initial and intermediate acclimation phases describes the rapid conversion of DD to DT (Figure 5A). At the In contrast, a gene similar to the antioxidant peroxiredoxin Q (PRX Q) in Arabidopsis thaliana was among the strongest and most PLoS ONE \| www.plosone.org November 2009 \| Volume 4 \| Issue 11 \| e7743 7 Genomics of Light Acclimation shift from the intermediate to the late acclimation phase there was a decrease in DES, after which the algae seemed to have acclimated to the increased irradiance, reflected by the small decrease in DES from 24 to 48 h. The changes in the fractions of cellular DD, DT and Fuco from the original LL pool sizes as a function of HL exposure time is shown in Figure 5B. From the decrease in the DD fraction and the increase of the DT fraction measured in cultures that had been subjected to HL for 0.5 h, it is evident that these two pigments are in an inverse relationship with each other (Figure 5B). In the intermediate acclimation face the 3 h HL exposure time resulted in increased production of DD as a response to an increased need for photoprotection. From 3 h to 12 h the cellular [DD] pool size decreased while cellular [DT], from de-epoxidation of DD, increased simultaneously. Pigment analysis The late acclimation phase showed a new peak in cellular DD after 24 h, indicating cell division. As the algae acclimated to HL (48 h) [DT] per cell was reduced. From onset of HL the cellular Fuco fraction started to decrease, and continued to do so during the experimental period. This suggests that DD might be the precursor of both DT and Fuco. markedly increased during the late acclimation phase (.12 h) d h l (LL Fi A) Th Figure 4. Main [LHPs] per cell and their ratio as a function of HL exposure time. The Chl a, Chl c and Fuco concentrations per cell and the ratio of Fuco plus Chl c to Chl a as a function of high-light (500 mmol m22s21) exposure time. Incubation was performed in a LL (35 mmol m22s21) exponentially growing 10L batch culture 3 weeks prior to HL exposure. The 0 h sample value is the mean of the 18 LL control samples (blue symbol). HL exposure values are the mean of three biological parallels. Values are presented with6SD bars. doi:10.1371/journal.pone.0007743.g004 Figure 5. De-epoxidation state index (DES) and change in [Fuco], [DD] and [DT] per cell. A) De-epoxidation state index (DES) index calculated from the HPLC pigment data. The 0 h sample value is the mean of the 18 LL control samples (blue symbol). HL exposure values are the mean of three biological parallels. Incubations as in Figure 4. Values are presented with6SD bars. (B) Change in Fucoxanthin, Diadinoxanthin and Diatoxanthin cell concentration as a function of high light exposure time. Change in pigment concentration for Fuco (normalized to LL, t = 0 h), DD (as for Fuco) and DT (normalized to HL at 0.5 h) as a function of time after HL exposure. Values are average of three parallel HPLC samples. Incubations as in Figure 4. doi:10.1371/journal.pone.0007743.g005 Figure 4. Main [LHPs] per cell and their ratio as a function of HL exposure time. The Chl a, Chl c and Fuco concentrations per cell and the ratio of Fuco plus Chl c to Chl a as a function of high-light (500 mmol m22s21) exposure time. Incubation was performed in a LL (35 mmol m22s21) exponentially growing 10L batch culture 3 weeks prior to HL exposure. The 0 h sample value is the mean of the 18 LL control samples (blue symbol). HL exposure values are the mean of three biological parallels. November 2009 \| Volume 4 \| Issue 11 \| e7743 Pigment analysis Values are presented with6SD bars. doi:10.1371/journal.pone.0007743.g004 Figure 5. De-epoxidation state index (DES) and change in [Fuco], [DD] and [DT] per cell. A) De-epoxidation state index (DES) index calculated from the HPLC pigment data. The 0 h sample value is the mean of the 18 LL control samples (blue symbol). HL exposure values are the mean of three biological parallels. Incubations as in Figure 4. Values are presented with6SD bars. (B) Change in Fucoxanthin, Diadinoxanthin and Diatoxanthin cell concentration as a function of high light exposure time. Change in pigment concentration for Fuco (normalized to LL, t = 0 h), DD (as for Fuco) and DT (normalized to HL at 0.5 h) as a function of time after HL exposure. Values are average of three parallel HPLC samples. Incubations as in Figure 4. doi:10.1371/journal.pone.0007743.g005 Figure 4. Main [LHPs] per cell and their ratio as a function of HL exposure time. The Chl a, Chl c and Fuco concentrations per cell and the ratio of Fuco plus Chl c to Chl a as a function of high-light (500 mmol m22s21) exposure time. Incubation was performed in a LL (35 mmol m22s21) exponentially growing 10L batch culture 3 weeks prior to HL exposure. The 0 h sample value is the mean of the 18 LL control samples (blue symbol). HL exposure values are the mean of three biological parallels. Values are presented with6SD bars. doi:10.1371/journal.pone.0007743.g004 shift from the intermediate to the late acclimation phase there was a decrease in DES, after which the algae seemed to have acclimated to the increased irradiance, reflected by the small decrease in DES from 24 to 48 h. The changes in the fractions of cellular DD, DT and Fuco from the original LL pool sizes as a function of HL exposure time is shown in Figure 5B. From the decrease in the DD fraction and the increase of the DT fraction measured in cultures that had been subjected to HL for 0.5 h, it is evident that these two pigments are in an inverse relationship with each other (Figure 5B). In the intermediate acclimation face the 3 h HL exposure time resulted in increased production of DD as a response to an increased need for photoprotection. From 3 h to 12 h the cellular [DD] pool size decreased while cellular [DT], from de-epoxidation of DD, increased simultaneously. Discussion In this study, we set out to investigate the mechanisms behind photo-acclimation to high-light conditions in diatoms by combin- ing molecular and physiological methods. Physiological methods have been utilized for several decades to investigate how diatoms process the sudden changes in irradiance that they are exposed to in nature. Small scale analyses of photoregulated gene expression have previously been performed [33,50,51]. However, this is the first time global transcriptional profiling has been utilized to investigate these mechanisms in a diatom. The results show that exposure to HL results in dramatic changes in the transcriptional profiles. The measurements indicated that the responses could be divided into three distinct phases; an initial response phase (0– 0.5 h), an intermediate acclimation phase (3–12 h) and a late acclimation phase (12–48 h), which will be discussed below. A summary of the most important processes in protection and acclimation to HL in Phaeodactylum tricornutum is given in Table 1. Figure 7. The photosynthetic capacity and the light-saturation index. The A) maximum photosynthetic capacity ( i.e. maximum light- saturated rETR) and B) the light-saturation index versus time after exposure to HL (500 mmol m22 s21, red squares, solid line) and LL (35 mmol m22 s21, blue circles, dashed line), calculated from the photosynthesis vs. irradiance relationship measured using variable Chl a fluorescence (PAM). Bars are S.D. (n = 3). doi:10.1371/journal.pone.0007743.g007 NPQ is the most important short-term photoprotection mecha- nism in diatoms, where it involves the conversion of DD into DT under conditions of HL [14] and is recognized by the increasing DES index at this initial response phase. In our study, a marked decrease of DD and an increase of DT occurred together with a decrease in Fuco content after 0.5 h in HL (Figure 5B), supporting earlier suggestions that DD is also a precursor of Fuco [52,53]. In this way, the conversion of DD to DT not only leads to dissipation of excess energy, but the decrease in cellular [Fuco] also suppresses light harvesting and thereby light energy transfer for photosynthesis. The P. tricornutum genome contains three candidate genes that might encode the enzyme responsible for the conversion of DD to DT in HL. Based on the specific up-regulation seen only for the ZEP3 gene at this early stage, we suggest that the product from this gene performs the de-epoxidation reaction (Figure 2). In higher plants, a PSII protein, PsbS, is important for NPQ [8]. Variable Chl fluorescence The maximum quantum yield after HL exposure. The maximum quantum yield (Fv/Fm) as a function of time after exposure to HL (500 mmol m22 s21, red squares, solid line) and LL (35 mmol m22 s21, blue circles, dashed line) measured using variable fluorescence (PAM) after keeping the samples for 3 min in the dark. Bars are S.D. (n = 3). doi:10 1371/journal pone 0007743 g006 doi:10.1371/journal.pone.0007743.g006 Variable Chl fluorescence The Chl a variable fluorescence illustrate the overall physiolog- ical response of P. tricornutum, and the data can be read as a proxy for the photosynthetic efficiency and capacity of the cell. The photosynthetic (PSII) efficiency, measured from the maximum quantum yield, Fv/Fm, showed a decrease in the initial and intermediate phase (,12 h) after exposure to HL, illustrating that the ratio of electrons generated in PSII to photons absorbed by light-harvesting pigments decrease (Figure 6). During the late acclimation phase (.12 h), Fv/Fm increased again to a level similar to the initial phase. The HL treated culture overall showed a lower Fv/Fm than the LL samples. The light-saturation index, which is an indicator for the photo- acclimation status of the cell, showed a linear increasing trend as a function of time after HL exposure (Figure 7B). Data indicated that the physiological acclimation status of P. tricornutum increased from the initial phase throughout the late acclimation phase, with no sign of reaching an upper limit within the investigated 48 h time frame. The light saturation index is a proxy for the irradiance that is needed to saturate photosynthesis, i.e. the threshold irradiance that separates light-limited and light-saturated photo- synthesis. The maximum photosynthetic capacity (i.e. the maximum relative light-saturated electron transfer rate, rETR) calculated from the P vs. E relationship was not significantly altered by HL treatment during the initial and intermediate phase; however, it November 2009 \| Volume 4 \| Issue 11 \| e7743 November 2009 \| Volume 4 \| Issue 11 \| e7743 8 Genomics of Light Acclimation Figure 6. The maximum quantum yield after HL exposure. The maximum quantum yield (Fv/Fm) as a function of time after exposure to HL (500 mmol m22 s21, red squares, solid line) and LL (35 mmol m22 s21, blue circles, dashed line) measured using variable fluorescence (PAM) after keeping the samples for 3 min in the dark. Bars are S.D. (n = 3). doi:10.1371/journal.pone.0007743.g006 Figure 7. The photosynthetic capacity and the light-saturation index. The A) maximum photosynthetic capacity ( i.e. maximum light- saturated rETR) and B) the light-saturation index versus time after exposure to HL (500 mmol m22 s21, red squares, solid line) and LL (35 mmol m22 s21, blue circles, dashed line), calculated from the photosynthesis vs. irradiance relationship measured using variable Chl a fluorescence (PAM). Bars are S.D. (n = 3). doi:10.1371/journal.pone.0007743.g007 Figure 6. Discussion No homolog for this gene has been identified in diatoms [4,5], but one or several of the antenna proteins classified as LHCX’s might serve the role of PsbS in photoprotecion [33]. The LHCX’s are the diatom homologs of the LI818 proteins first identified in Chlamydomonas [54]. Members of this group of proteins are known to be induced in response to HL stress and are suggested to have photoprotective properties PLoS ONE \| www.plosone.org The initial response phase Level Initial response phase Intermediate acclimation phase Late acclimation phase Molecular Genes encoding antenna proteins possibly involved in photoprotection + + + + + + + Genes encoding proteins involved in degrad- ation, assembly and repair of PSII components + Genes encoding specific proteins involved in ROS scavenging + + + + + + Genes encoding additional proteins involved in ROS scavenging + Zeaxanthin epoxidase gene possibly encoding the enzyme responsible for conversion of DD to DT + + Genes encoding enzymes involved in Chl a biosynthesis 4 4 4 + Genes encoding components of the light harvesting and photosynthetic apparatus 4 4 4 4 Metabolic DES index + + + + + + Fuco content per cell 4 4 4 4 4 4 Chl a content per cell 4 4 4 4 4 Physiological Photosynthetic efficiency and capacity + + Summary Photoprotection mechanisms Execution of photoprotection mechanisms Photoprotection mechanisms still active, but not as pronounced as in the early phase Photoprotection mechanisms still active, but not as pronounced as in the early phase HL acclimation processes HL acclimation processes initiated at the transcrip- tional level. HL acclimation processes observed at the transcriptional and metabolic level, but not at the physiological level HL acclimation processes observed at all levels. Changes in the com- position of the photosynthetic apparatus enable the diatoms to perform highly efficient photosynthesis in HL Plus (+) and minus (4) symbols indicate an increase or decrease in HL compared to LL levels. The abbreviations used are PSII: photosystem II; ROS: reactive oxygen species; DD: diadinoxanthin; DT: diatoxanthin; Chl: chlorophyll; DES de-epoxidation state; Fuco: fucoxanthin. doi:10.1371/journal.pone.0007743.t001 Photoprotection mechanisms still active, but not as pronounced as in the early phase Plus (+) and minus (4) symbols indicate an increase or decrease in HL compared to LL levels. The abbreviations used are PSII: photosystem II; ROS: reactive oxygen species; DD: diadinoxanthin; DT: diatoxanthin; Chl: chlorophyll; DES de-epoxidation state; Fuco: fucoxanthin. doi:10.1371/journal.pone.0007743.t001 proteases functioning in degradation of photodamaged D1 protein [21], and also of two genes (HCF136 and PSB27) encoding proteins involved in assembly and repair of PSII [37–40] implies that the HL treatment has caused photodamage to the complex and that mechanisms necessary for the PSII to recover have been executed. Another protection mechanism initiated in the HL subjected cells is the severe induction of the H2O2 peroxidase gene PRX Q, encoding a protein similar to the A. The initial response phase thaliana peroxiredoxin Q that is suggested to be involved in protection of PSII [45]. The up-regulation of the PRX Q gene was accompanied with an almost equally strong increase in the transcription level of a glutaredoxin (GLRXC2) predicted to be targeted to the chloroplast [42]. The expression of PRX Q and GLRXC2 displays a similar regulation pattern throughout the entire length of the experiment. This observation indicates that the diatom PRX Q might be reduced and thereby reactivated by glutaredoxin instead of thioredoxins, which is the case in A. thaliana [45]. [33,50,55]. The transcription of the P. tricornutum LHCX2 and LHCX3 genes were found to be strongly induced after only 0.5 h in HL, suggesting that there might be a connection between these results and the other mechanisms involved in NPQ in diatoms (Figure 3). The expression level of both LHCX’s peaked at this initial response phase. The transcription of the LHCX2 gene stayed high throughout the experiment, whereas the LHCX3 expression decreased with time. The fast and drastic up-regulation of two of the LHCX’s and also two of the LHCR’s at this early time-point are in sharp contrast to the decrease in transcription level observed for almost all other genes encoding diatom antenna proteins, implying different roles for these proteins in protection and acclimation to HL (Figure 3). [33,50,55]. The transcription of the P. tricornutum LHCX2 and LHCX3 genes were found to be strongly induced after only 0.5 h in HL, suggesting that there might be a connection between these results and the other mechanisms involved in NPQ in diatoms (Figure 3). The expression level of both LHCX’s peaked at this initial response phase. The transcription of the LHCX2 gene stayed high throughout the experiment, whereas the LHCX3 expression decreased with time. The fast and drastic up-regulation of two of the LHCX’s and also two of the LHCR’s at this early time-point are in sharp contrast to the decrease in transcription level observed for almost all other genes encoding diatom antenna proteins, implying different roles for these proteins in protection and acclimation to HL (Figure 3). Although photosynthetic organisms are in the possession of numerous light sensing and acclimation mechanisms [56], excess light can generate ROS, causing damage especially to the PSII, where the D1 core protein is particularly vulnerable [20]. Excess light usually leads to an enhanced synthesis of D1 protein to replace the damaged ones [19]. The initial response phase At the molecular level, the initial response phase is character- ized by a fast and strong regulation of several genes encoding members of the light harvesting machinery and a few components of the ROS scavenging systems. One striking feature of this initial phase is a severe repression of nuclear-encoded genes involved in all steps of the Chl a metabolism, suggesting a light dependent and synchronised regulation of the transcription of these genes (Figure 1). The regulation of Chl a biosynthesis is only partly understood in plants, and it is generally believed that the regulation of enzyme activity is more important than a transcriptional regulation in terms of controlling the amount of Chl a produced [30]. Chl a and most intermediate molecules of the Chl a synthesis produce ROS under illumination if present in excess amounts [30], and it is therefore critical to accurately control the levels of these compounds. The down-regulation of Chl a metabolism at the transcriptional level was not reflected by the measurements of Chl a concentration at this early stage (Figure 4). PLoS ONE \| www.plosone.org November 2009 \| Volume 4 \| Issue 11 \| e7743 PLoS ONE \| www.plosone.org 9 Genomics of Light Acclimation Table 1. Summary of the most important processes in protection and acclimation to high light (HL) in P. tricornutum. Table 1. Summary of the most important processes in protection and acclimation to high light (HL) in P. tricornutum. The initial response phase In our study, several genes encoding subunits of PSII was continuously down-regulated as a response to HL, whereas the psbA gene encoding the D1 protein was maintained at LL levels, indicating a greater demand for this PSII transcript. In green algae the increase in D1 protein synthesis far exceeded the accumulation of the corresponding mRNA [22], suggesting that the D1 protein synthesis might also be up-regulated by HL in our study despite the lack of regulation at the transcriptional level. The early phase specific up-regulation of the two ftsH genes encoding At the initial response phase, neither the maximum rETR (Figure 7A) nor the light-saturation index (Figure 7B) changed significantly in the HL exposed cultures compared to the LL cultures. This shows that the P. tricornutum cells are able to maintain their photosynthetic efficiency despite the HL stress and that their capability to utilise incoming photons remain unchanged. PLoS ONE \| www.plosone.org Materials and Methods An axenic culture of P. tricornutum Bohlin clone Pt 1 8.6 (CCMP632) was obtained from the culture collection of the Provasoli-Guillard National Center for Culture of Marine Phytoplankton, Bigelow Laboratory for Ocean Sciences, USA. Cultures were grown in f/2 medium [58] made with 0.2 mm- filtered and autoclaved local seawater supplemented with f/2 vitamins and inorganic nutrients [58], filter sterilized and added after autoclaving. Cultures were incubated at 15uC under cool white fluorescent light at scalar irradiance (EPAR) of approximately 35 mmol m22s21 (LL) on a rotary table in continuous light (control conditions), and were kept in exponential growth phase under these conditions for 3 weeks to ensure that all cells were acclimated. Scalar irradiance (Photosynthetic Active Radiance, 400–700 nm) in culture flasks was measured with a Biospherical QSL-100 irradiance sensor (San Diego, US). According to the growth curve based on cell counting and in vivo Chl a fluorescence with and without addition of DCMU (3-(3,4-dichlorophenyl)-1,1- dimethylurea), the cells divided once a day under these conditions. Sterility was monitored by occasional inoculation into peptone- enriched f/2 medium to check for bacterial growth [59]. Cells for the experiments were grown in batch cultures, and growth was monitored by cell counting using a Bu¨rker-Tu¨rk counting chamber, counting 4–500 cells per volume-unity. The cells were first grown axenically in a 10-litre batch culture to reach an approximate density of 106 cells mL21, then volumes of 250 ml were transferred to 75 cm2 sterile Falcon polystyrene flasks to reach cell densities of 0.15–1.06106 cells mL21 on the day of the experiment. The cultures were transferred to EPAR irradiance conditions of 500 mmol m22s21 (HL = high light) and sampled at incubation times of 0.5 h, 3 h, 6 h, 12 h, 24 h and 48 h. In addition, LL control cultures were kept for each of the HL exposures and parallels. Three biological replica and two parallels for each treatment and control culture were harvested (6 samples) to ensure statistical validation. The two parallels for each of the biological replicas destined to be used for isolation of RNA were merged during harvesting to get enough starting material for the microarray analyses. The maximum cell density of 1.06106 cells mL21 was chosen and carefully monitored and diluted to minimize effects like intercellular shading, rapid depletion of nutrients and increase in pH above 9. Materials and Methods Material from the same cell cultures were utilized in the different analyses described below. The continuous up-regulation of the LHCX’s that might function in the role of psbS and the still increasing DES index implies that the diatoms continues to dissipate the excess light energy as heat dissipation by NPQ at this intermediate phase. The intermediate acclimation phase The global transcriptional profiling revealed a strong and rapid response to the change in irradiance already after 0.5 h, whereas November 2009 \| Volume 4 \| Issue 11 \| e7743 10 Genomics of Light Acclimation most physiological responses first became apparent at the intermediate acclimation phase (Figure 7A and B). This phase is characterised by a marked decline in cellular LHPs, where the decrease in Fuco levels observed after 0.5 h in HL continues, and is now also followed by a significant fall in Chl a level. The expression of the genes involved in the synthesis of Chl a return to LL levels during this period (Figure 1). This observation suggests that the reduction of the amount of Chl a might be caused not only by the down-regulation at the transcriptional level seen at the initial response phase, but also by other mechanisms like repression of the corresponding enzyme activity [30] and regulation of Chl a degradation. The decrease in LHPs also lowers the need for the antenna proteins that bind Chls and Fuco and thereby anchor them to the thylakoid membranes. This is reflected at the transcriptional level by the repression of most genes encoding antenna proteins seen throughout the experiment (Figure 3). Down-regulation of members of the LHC family have also been observed in several other HL experiments with diatoms, green algae and higher plants [33,56,57]. Several of the continuously repressed genes involved in the light harvesting machinery, including components of the photosystems, display the strongest down-regulation at the beginning of this phase. genes believed to be involved in the Calvin cycle does not bring greater insight into this question, especially since most genes of the Calvin cycle are also involved in other pathways. The late acclimation phase is recognized by low levels of light- harvesting pigments (Fuco and Chl a+c) occurring together with a pronounced increase in the photosynthetic capacity compared to the LL acclimated cultures (Figure 7). These measurements indicate that although the light-harvesting machinery has been downsized during the acclimation period, the adjustments made at the transcriptional and metabolic levels facilitate highly effective photosynthesis in HL-acclimated diatoms. The late acclimation phase During the first 12 hours of the HL treatment, the diatom cells seem to protect and adjust the photosynthetic apparatus to the new light regime without being able to make use of the increased amounts of light energy available for photosynthesis and growth. After 12 hours in HL, the acclimation mechanisms observed at the molecular level are replaced or supplemented with changes supporting the shorter generation time (data not shown) and thereby an accelerated protein synthesis, and responses charac- teristic for the late acclimation phase. The characteristic responses include an increase of the transcript levels of genes involved in the removal of potentially harmful ROS (Figure 3), in particular a strong induction of TypA1 encoding a member of the TypA/BipA GTPase family suggested to function as a translational regulator of stress-responsive proteins involved in ROS scavenging in chloro- plast [48]. ROS are inevitable by-products of photosynthesis, and the increased demand of antioxidants might be explained by the strongly increasing maximum rETR measured in this late phase of acclimation (Figure 7A). A moderate increase in expression levels of several genes encoding enzymes functioning in xanthophyll and Chl a metabolism was also observed during the late acclimation phase (Figure 1). The rise in expression levels of the genes connected to the formation of pigments is not reflected at the metabolic level, but might be explained by an increased demand of newly synthesized pigments in the HL cultures due to the shorter generation time at these light conditions (data not shown). The vast majority of the genes predicted to be involved in carbon metabolism were affected by HL, and showed the strongest regulation during the late phase. Kroth et al. [42] suggested that a futile and energy demanding C4-like cycle might occur in P. tricornutum that possibly functions as a way to dissipate excess light energy. The results achieved in this experiment support a light regulated carbon metabolism; however, the complexity of the proposed model and the uncertainties connected to the localiza- tion of the proteins involved make interpretation of single gene regulation difficult. The regulation mechanisms of the Calvin cycle in diatoms are largely unknown. The modest regulation of a few PLoS ONE \| www.plosone.org Quantitative real-time PCR A two-step quantitative real-time PCR (qRT-PCR) was performed on total RNA isolated from three biological replicas of diatom cultures grown in LL and HL at time points 0.5 h, 3 h, 12 h and 24 h. Reverse transcription of the RNA was performed with the PrimeScriptTM 1st strand cDNA Synthesis Kit (TaKaRa) following the recommended protocol for synthesis of real-time PCR template using random primers. 300 ng of total RNA was used in each reaction. 20 ml qRT-PCR mixtures were prepared containing forward and reverse primers listed in Supplementary Table S2, with a final concentration of 0.5 mM each, 5 ml cDNA template diluted 1:10 and 2x LightCyclerH 480 SYBR Green I Master mix (Roche). The qRT-PCR reactions were run in a LightCyclerH 480 Multiwell Plate 96 (Roche) in a LightCycler 480 instrument (Roche). No template controls, where the cDNA template was replaced with PCR-grade water, were included in each run to ensure that no reagents were contaminated with DNA. To detect the level of genomic DNA still present in the 24 RNA samples after the DNase I treatment, qRT-PCR was performed using 7.5 ng of isolated RNA as template, and three different primer pairs listed in Supplementary Table S2. The PCR parameters were programmed according to the manufacturer’s instructions for a LightCycler 480 System PCR run with the LightCyclerH 480 SYBR Green I Master: 5 min preincubation at 95uC, followed by 35 cycles with 10 s at 95uC, 10 s at 55uC and 10 s at 72uC. After 35 cycles the specificity of the amplified PCR products was tested by heating from 65uC up to 95uC with a ramp rate of 2.2uC/s, resulting in melting curves. The Second Derivative Maximum Method of the LightCycler 480 software was used to identify the crossing points (CPs) of the samples. LinRegPCR software [62] was used to determine the PCR efficiency for each sample. The primer set efficiency was determined by calculating the mean of the efficiency values obtained from the individual samples. Relative expression ratios of the target genes normalized to a reference gene encoding a putative hiv-1 rev binding protein (Phatr2_42776) were calculated using the REST 2005 software [63]. The primer efficiencies determined by LinRegPCR were included in the calculations. REST 2005 was also used to test significance of the expression ratio results of the investigated transcripts by a Pair Wise Fixed Reallocation Randomisation Test. Pigment analysis HPLC pigment analyses were performed as described in Rodriguez et al. [25] using a Hewlett-Packard HPLC 1100 Series system. The pigment values from the HPLC analyses were calculated as femtogram (fg) pigment per cell. De-epoxidation state index (DES) was calculated as in Ruban et al. [14]: DES = (DT)/ ((DD)+(DT)). Quantitative real-time PCR The gene encoding the hiv-1 rev binding protein that was chosen to function as a reference gene is represented by five different probes on the microarray and showed no response to the HL treatment at any time point. RNA isolation and processing Diatom cultures were centrifuged at 4000 g for 10 min at 15uC. The supernatant was removed and the cell pellet was resuspended in 1 ml f/2 medium. The suspension was transferred to 2 ml tubes and centrifuged at 18000 g for 1 min at 4uC. Supernatants were removed and the cell pellets were flash frozen in liquid nitrogen and stored at 280uC. Precooled (280uC) 5 mm stainless steel beads (QIAGEN) were added to the tubes with frozen cell pellets, November 2009 \| Volume 4 \| Issue 11 \| e7743 11 Genomics of Light Acclimation and the samples were mechanically disrupted and homogenized using the TissueLyser system (QIAGEN). Disruption was carried out for 262 min at 25 Hz. The samples were placed in a precooled (280uC) adapter set for the first shaking step. Before the second shaking step, the samples were transferred to a room temperate adapter set and 0.5 ml lysis buffer (SpectrumTM Plant Total RNA kit, Sigma-Aldrich) was added to each tube. Total RNA was isolated from the homogenized lysate using the SpectrumTM Plant Total RNA kit (Sigma-Aldrich). On-column digestion of DNA with DNase I (QIAGEN) was included for all RNA preparations. The concentration of the RNA was deter- mined by measuring the absorbance at 260 nm with the NanoDrop ND-1000 Spectrophotometer (NanoDrop Technolo- gies), and the purity of the RNA was estimated by the OD260/ OD280 nm absorption ratio (all ratios were .2.0). The integrity of the RNA was verified by denaturing agarose gel electrophoresis and ethidium bromide staining following the protocol described in the RNeasy Mini handbook (QIAGEN). representing the genes in question. Supplementary information on the P. tricornutum genes are given in Table S1. The study is MIAME compliant. Raw data has been deposited in GEO (accession GSE 17237). DNA microarray experiments Total RNA was isolated from three biological replicas of diatoms cultured under LL and HL conditions at time points 0.5 h, 3 h, 6 h, 12 h, 24 h and 48 h. The RNA (0.36–1 mg) was reverse transcribed, amplified and labelled using the Quick Amp Labelling Kit, Two-Color (Agilent p/n 5190-0444). Hybridization was performed with the Gene Expression Hybridization Kit (Agilent p/n 5188-5242). 825 ng cRNA from HL exposed cells were mixed with 825 ng cRNA from low light acclimated cells from the corresponding time point. The cRNA mixture was fragmented and hybridized on 4x44K Phaeodactylum tricornutum whole-genome 60-mer oligonucleotide microarrays (Agilent Tech- nologies) in a rotary oven (10 rpm, 65uC, 17 h). The slides were washed with Gene Expression Wash Buffer 1 (Agilent p/n 5188- 5325), Gene Expression Wash Buffer 2 (Agilent p/n 5188-5326), acetonitrile (VWR International) and Stabilization and Drying Solution (Agilent Technologies) according to the manufacturer’s instructions. The slides were scanned at 5 mm resolution on an Agilent DNA microarray scanner (Agilent Technologies). The resulting images were processed using GenePix 5.1 software (Axon Instruments, Union City, USA). Statistical analysis. The GenePix processed data were filtered to remove spots that had been flagged ‘Absent’, ‘Not Found’ or ‘Bad’, by the GenePix program or by manual flagging. Spots with more than 50% saturated pixels, or had median foreground intensity less than the local median background intensity were excluded from the analysis. No background subtraction was performed. For making statistical inference about differentially regulated genes the limma package [60] and the R statistical data analysis program package (R 2.7.1) was used. The limma approach is based on fitting a linear model to the expression data of each probe on a microarray. In this experiment each time point consists of 3 sets of biological replicates (test and control samples) which are dye swapped and adjusted for probe- specific dye effects. Due to some scale differences in the responses between samples, identified as differences in spread of M-values between the arrays, scale normalization between the arrays was performed. The Benjamini and Hochberg’s method to control the false discovery rate (fdr) was used to identify differentially regulated genes [61]. Genes with adjusted P-values less than 0.05 were regarded as statistical significantly differentially expressed. If a gene was identified by less than 3 spots it was excluded from the result tables. Genes are represented by 1–5 different probes on each microarray. DNA microarray experiments Expression ratios discussed in the text are an average of values obtained from all probes Statistical analysis. The GenePix processed data were filtered to remove spots that had been flagged ‘Absent’, ‘Not Found’ or ‘Bad’, by the GenePix program or by manual flagging. Spots with more than 50% saturated pixels, or had median foreground intensity less than the local median background intensity were excluded from the analysis. No background subtraction was performed. For making statistical inference about differentially regulated genes the limma package [60] and the R statistical data analysis program package (R 2.7.1) was used. The limma approach is based on fitting a linear model to the expression data of each probe on a microarray. In this experiment each time point consists of 3 sets of biological replicates (test and control samples) which are dye swapped and adjusted for probe- specific dye effects. Due to some scale differences in the responses between samples, identified as differences in spread of M-values between the arrays, scale normalization between the arrays was performed. The Benjamini and Hochberg’s method to control the false discovery rate (fdr) was used to identify differentially regulated genes [61]. Genes with adjusted P-values less than 0.05 were regarded as statistical significantly differentially expressed. If a gene was identified by less than 3 spots it was excluded from the result tables. Genes are represented by 1–5 different probes on each microarray. Expression ratios discussed in the text are an average of values obtained from all probes References 13. Brunet C, Johnsen G, Lavaud J, Roy S (2009) Pigments and photoacclimation processes. In: Roy S, Egeland E, Johnsen G, Llewellyn C, eds. Pigments in Oceanography. Cambridge: Cambridge University Press, In press. 1. Field CB, Behrenfeld MJ, Randerson JT, Falkowski P (1998) Primary production of the biosphere: integrating terrestrial and oceanic components. Science 281: 237–240. 14. Ruban AV, Lavaud J, Rousseau B, Guglielmi G, Horton P, Etienne AL (2004) The super-excess energy dissipation in diatom algae: comparative analysis with higher plants. Photosynth Res 82: 165–175. 2. Long S, Humphries S, Falkowski P (1994) Photoinhibition of photosynthesis in nature. Annu Rev Plant Physiol Plant Mol Biol 45: 633–662. 3. Raven JA, Geider RJ (2003) Adaptation, acclimation and regulation in algal photosynthesis. In: Larkum AW, Douglas SE, Raven JA, eds. Photosynthesis in algae. Dordrecht: Kluwer academic publ. pp 386–412. 15. Owens TG (1986) Light-harvesting function in the diatom Phaeodactylum tricornutum. II. Distribution of excitation energy between the photosystems. Plant Physiol 80: 739–746. 4. Armbrust V, Berges JA, Bowler C, Green B, Martinez D, et al. (2004) The genome of the diatom Thalassiosira pseudonana: ecology, evolution, and metabolism. Science 306: 79–85. y 16. Horton P, Ruban AV (1992) Regulation of photosystem II. Photosynth Res 34: 375–385. 17. Barber J (1995) Molecular basis of the vulnerability of photosystem-II to damage by light. Aust J Plant Physiol 22: 201–208. 17. Barber J (1995) Molecular basis of the vulnerabilit by light. Aust J Plant Physiol 22: 201–208. 5. Bowler C, Allen AE, Badger JH, Grimwood J, Jabbari K, et al. (2008) The Phaeodactylum genome reveals the evolutionary history of diatom genomes. Nature 456: 239–244. y g J y 18. Niyogi KK (1999) Photoprotection revisited: genetic and molecular approaches. Annu Rev Plant Physiol Lant Mol Biol 50: 333–359. y g y 18. Niyogi KK (1999) Photoprotection revisited: genetic a Annu Rev Plant Physiol Lant Mol Biol 50: 333–359. Annu Rev Plant Physiol Lant Mol Biol 50: 333–359 6. Genty B, Briantais JM, Baker NR (1989) The relationship between the quantum yield of photosynthetic electron-transport and quenching of chlorophyll fluorescence. Biochim Biophys Acta 990: 87–92. 19. Adir N, Zer H, Shochat S, Ohad I (2003) Photoinhibition – a historical perspective. Photosynth Res 76: 343–370. 20. Ohad I, Adir N, Koike H, Kyle DJ, Inoue Y (1990) Mechanism of photoinhibition in vivo. Variable Chl fluorescence Found at: doi:10.1371/journal.pone.0007743.s001 (0.69 MB EPS) Found at: doi:10.1371/journal.pone.0007743.s001 (0.69 MB EPS) Table S1 Information on P. tricornutum genes discussed in the text. Parameters given are the protein identification number (ID), chromosome location, best NCBI hit (BLASTP search) outside the Bacillariophyceae, the identity/similarity with respect to the corresponding P. tricornutum protein and respective NCBI accession numbers. Found at: doi:10.1371/journal.pone.0007743.s002 (0.13 MB XLS) Found at: doi:10.1371/journal.pone.0007743.s002 (0.13 MB XLS) Table S2 Primers used for quantitative real-time PCR. Table S2 Primers used for quantitative real-time PCR. Found at: doi:10.1371/journal.pone.0007743.s003 (0.03 MB XLS) Author Contributions Figure S1 Regulation pattern of eight chloroplast-encoded genes during the HL acclimation period. Expression ratios are investigated by both microarray analyses (A) and qRT-PCR (B). The color code indicates expression values and squares with a diagonal line inside indicate genes with an expression ratio (log2 transformed) greater than +/2 0.5 that are not significantly regulated. The scale on the right represents gene expression ratio Figure S1 Regulation pattern of eight chloroplast-encoded genes during the HL acclimation period. Expression ratios are investigated by both microarray analyses (A) and qRT-PCR (B). Conceived and designed the experiments: MN KCV TB GJ AMB. Performed the experiments: MN KCV TB KH KA GJ. Analyzed the data: MN KCV KH PW KA AMB. Contributed reagents/materials/analysis tools: GJ AMB. Wrote the paper: MN KCV TB KH PW AMB. Initiated and started the project: AMB. The color code indicates expression values and squares with a diagonal line inside indicate genes with an expression ratio (log2 transformed) greater than +/2 0.5 that are not significantly regulated. The scale on the right represents gene expression ratio Acknowledgments The custom made Agilent microarrays was made available by Dr. Chris Bowler and colleagues, which we also thank for generously sharing methods and materials used in their laboratories. The authors would also like to thank Torfinn Sparstad and Bente U. Halvorsen for help with the microarray hybridization and Mari-Ann Østensen for great help with the axenic cultures. Variable Chl fluorescence Variable Chl a fluorescence was measured using a PhytoPAM (System I, Walz, Germany), equipped with a sensitive Photo- multiplier-Detector (PM-101P, Walz). Fluorescence was excited by a weak and nonactinic modulated light supplied by a LED (light emitting diode, Array-Cone PHYTO-ML, Walz, Germany) and a PLoS ONE \| www.plosone.org November 2009 \| Volume 4 \| Issue 11 \| e7743 12 Genomics of Light Acclimation saturating flash by a strong red LED (.2000 mmol m22 s21, Actinic LED-Array-Cone PHYTO-AL, Walz) to ensure that all reaction centres of PSII were closed during the flash period. The instrument light source excites fluorescence at four different wavelengths; however, only results from 665 nm excitation were used. Nomenclature of van Kooten and Snell [64] was used. The minimum (F0) and maximum fluorescence (Fm) was measured at the end of a dark-acclimation period (3 min) and the maximum quantum yield (the PSII efficiency) was calculated from Fv/Fm, where Fv is the variable part of fluorescence emission and equal to Fm–F0. The photosynthesis vs. irradiance (P vs. E) relationship was obtained by exposing the samples (after the 3 min dark- acclimation) to 12 step-wise increasing irradiances (1 and 1200 mmol photons m22 s21) at intervals of 30 s each. The operational quantum yield of PSII (WPSII) was calculated from the steady-state fluorescence (Fs) at each irradiance and the maximum fluorescence measured after a saturation pulse (Fm9) at the end of each irradiance interval, from (Fm9–Fs)/Fm9 [6]. The relative electron transport rates (rETR) was then calculated from multiplying WPSII with the incubation irradiance, and the relationship was fitted using the build-in hyperbolic fitting routine in the PhytoWin software package (ver. 2.00a) to determine the maximum light utilization coefficient (a) and the maximum rETR. The light saturation index was calculated from rETR divided by a [65,66]. A Peltier cell (US-T/S, Walz) kept the temperature constant (60.2uC) during incubations. values, log2 transformed. The abbreviations used are NA: not assessed; ChlI: subunit I of magnesium chelatase; FtsH: Filamen- tation temperature sensitive H; Psa: PSI proteins; Psb: PSII proteins; RbcS: ribulose-bisphosphate carboxylase oxygenase small subunit; RbcL: ribulose-bisphosphate carboxylase oxygenase large subunit. values, log2 transformed. 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https://openalex.org/W3146275690	https://bmcgenomics.biomedcentral.com/track/pdf/10.1186/s12864-021-07512-6	English	null	Telomere-to-telomere assembly of the genome of an individual Oikopleura dioica from Okinawa using Nanopore-based sequencing	BMC genomics	2,021	cc-by	13,497	Telomere-to-telomere assembly of the genome of an individual Oikopleura dioica from Okinawa using Nanopore-based sequencing Aleksandra Bliznina1* , Aki Masunaga1, Michael J. Mansfield1, Yongkai Tan1, Andrew W. Liu1, Charlotte West1,2, Tanmay Rustagi1, Hsiao-Chiao Chien1, Saurabh Kumar1, Julien Pichon1, Charles Plessy1* and Nicholas M. Luscombe1,2,3 Bliznina et al. BMC Genomics (2021) 22:222 https://doi.org/10.1186/s12864-021-07512-6 Bliznina et al. BMC Genomics (2021) 22:222 https://doi.org/10.1186/s12864-021-07512-6 Open Access * Correspondence: aleksandra.bliznina2@oist.jp; charles.plessy@oist.jp 1Genomics and Regulatory Systems Unit, Okinawa Institute of Science and Technology Graduate University, Okinawa, Japan Full list of author information is available at the end of the article Abstract Background: The larvacean Oikopleura dioica is an abundant tunicate plankton with the smallest (65–70 Mbp) non- parasitic, non-extremophile animal genome identified to date. Currently, there are two genomes available for the Bergen (OdB3) and Osaka (OSKA2016) O. dioica laboratory strains. Both assemblies have full genome coverage and high sequence accuracy. However, a chromosome-scale assembly has not yet been achieved. Results: Here, we present a chromosome-scale genome assembly (OKI2018_I69) of the Okinawan O. dioica produced using long-read Nanopore and short-read Illumina sequencing data from a single male, combined with Hi-C chromosomal conformation capture data for scaffolding. The OKI2018_I69 assembly has a total length of 64.3 Mbp distributed among 19 scaffolds. 99% of the assembly is contained within five megabase-scale scaffolds. We found telomeres on both ends of the two largest scaffolds, which represent assemblies of two fully contiguous autosomal chromosomes. Each of the other three large scaffolds have telomeres at one end only and we propose that they correspond to sex chromosomes split into a pseudo-autosomal region and X-specific or Y-specific regions. Indeed, these five scaffolds mostly correspond to equivalent linkage groups in OdB3, suggesting overall agreement in chromosomal organization between the two populations. At a more detailed level, the OKI2018_I69 assembly possesses similar genomic features in gene content and repetitive elements reported for OdB3. The Hi-C map suggests few reciprocal interactions between chromosome arms. At the sequence level, multiple genomic features such as GC content and repetitive elements are distributed differently along the short and long arms of the same chromosome. (Continued on next page) (Continued on next page) Background chromosome counts, ranging between n = 3 [14] and n = 8 [15]. In preparation for this study, we karyotyped the Okinawan O. dioica by staining centromeres with anti- bodies targeting phosphorylated histone H3 serine 28 [16], and determined a count of n = 3. This is also in agreement with the physical map of OdB3 [9]. Larvaceans (synonym: appendicularians) are among the most abundant and ubiquitous taxonomic groups within animal plankton communities [1, 2]. They live inside self-built “houses” which are used to trap food particles [3]. The animals regularly replace houses as filters be- come damaged or clogged and a proportion of discarded houses with trapped materials eventually sink to the ocean floor. As such larvaceans play a significant role in global vertical carbon flux [4]. Currently, the method of choice for producing chromosome-scale sequences is to assemble contigs using long reads (~ 10 kb or more) produced by either the Oxford Nanopore or PacBio platforms, and to scaf- fold them using Hi-C contact maps [17, 18]. To date, there have been no studies of chromosome contacts in Oikopleura or any other larvaceans. Oikopleura dioica is the best documented species among larvaceans. It possesses several invaluable fea- tures as an experimental model organism. It is abundant in coastal waters and can be easily collected from the shore. Multigenerational culturing is possible [5]. It has a short lifecycle of 4 days at 23 °C and remains free- swimming throughout its life [6]. As a member of the tunicates, a sister taxonomic group to vertebrates, O. dioica offers insights into their evolution [7]. Here, we present a chromosome-length assembly of the Okinawan O. dioica genome sequence generated with datasets stemming from multiple genomic tech- nologies and data types, namely long-read sequencing data from Oxford Nanopore, short-read sequences from Illumina and Hi-C chromosomal contact maps (Fig. 1). O. dioica’s genome size is 65–70 Mbp [8, 9], making it one of the smallest among all sequenced animals. Inter- estingly, genome-sequencing of other larvacean species uncovered large variations in genome sizes, which corre- lated with the expansion of repeat families [10]. O. dioica is distinguished from other larvaceans as the only reported dioecious species [11] with sex determination system using an X/Y pair of chromosomes [9]. The first published genome assembly of O. © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Page 2 of 18 Bliznina et al. BMC Genomics (2021) 22:222 (Continued from previous page) (Continued from previous page) Conclusions: We show that a hybrid approach of integrating multiple sequencing technologies with chromosome conformation information results in an accurate de novo chromosome-scale assembly of O. dioica’s highly polymorphic genome. This genome assembly opens up the possibility of cross-genome comparison between O. dioica populations, as well as of studies of chromosomal evolution in this lineage. Keywords: Oikopleura dioica, Oxford Nanopore sequencing, Hi-C, Telomere-to-telomere, Chromosome-scale assembly, Single individual Background dioica (OdB3, B stands for Bergen) was performed with Sanger sequencing which allowed for high sequence accuracy but limited coverage [9]. The OdB3 assembly was scaffolded with a physical map produced from BAC-end sequences, which revealed two autosomal linkage groups and a sex chromosome with a long pseudo-autosomal region (PAR) [9]. Recently, a genome assembly for a mainland Japanese population of O. dioica (OSKA2016, OSKA de- notes Osaka) was published, which displayed a high level of coding sequence divergence compared with the OdB3 reference [12, 13]. Although OSKA2016 was sequenced with single-molecule long reads produced with the Pac- Bio RSII technology, it does not have chromosomal resolution. Genome sequencing and assembly b The assembly was generated using Nanopore and Illumina data, followed by scaffolding using Hi-C chromosomal capture information data 0.5 mm a Collection of a single Oikopleura dioica male gDNA extraction and library preparation Illumina MiSeq sequencing Canu Contig assembly Self-polishing Racon Error-correction Quality check Filtration and trimming FASTX-toolkit Pilon Redundancy removal HaploMerger2 Scaffold assembly Juicer, 3D-DNA Hi-C library preparation Illumina MiSeq sequencing Quality check and filtration Hi-C Dovetail Kit FASTX-toolkit Quality assessment QUAST, BUSCO LG assignment Collection of 50 males Genome assembly Nanopore sequencing LAST, Circlize b b a Fig. 1 Genome assembly and annotation workflow used to generate the OKI2018_I69 genome assembly. a Life images of adult male (top) and female (bottom) O. dioica. b The assembly was generated using Nanopore and Illumina data, followed by scaffolding using Hi-C chromosomal capture information data Racon, and then with Illumina reads using Pilon. The initial Okinawa O. dioica assembly length was 99.3 Mbp, or ~ 1.5 times longer than the OdB3 genome at 70.4 Mbp. Merging haplotypes with HaploMerger2 resulted in two sub-assemblies (reference and allelic) of 64.3 Mbp with an N50 of 4.7 Mbp. Repeating the procedure on a second individual from the same culture showed overall agreement in assembly lengths, sequences and structures (Fig. 2c). Racon, and then with Illumina reads using Pilon. The initial Okinawa O. dioica assembly length was 99.3 Mbp, or ~ 1.5 times longer than the OdB3 genome at 70.4 Mbp. Merging haplotypes with HaploMerger2 resulted in two sub-assemblies (reference and allelic) of 64.3 Mbp with an N50 of 4.7 Mbp. Repeating the procedure on a second individual from the same culture showed overall agreement in assembly lengths, sequences and structures (Fig. 2c). for the remaining 663 kbp (lengths ranging from 2.9 to 131.6 kbp). One of the small scaffolds is a draft assembly of the mitochondrial genome that we discuss below. Most of the other smaller scaffolds are highly repetitive and might represent unplaced fragments of centromeric or telomeric regions. We annotated telomeres by search- ing for the TTAGGG repeat sequence and found that most of the megabase-scale scaffolds have single telo- meric regions: therefore, we reasoned that they represent chromosome arms. Indeed, pairwise genome alignment to OdB3 identified two syntenic scaffolds for each auto- somal linkage group, two for the pseudo-autosomal re- gion (PAR) and one for each sex-specific region. Genome sequencing and assembly O. dioica’s genome is highly polymorphic [9], making as- sembly of its complete sequence challenging. To reduce the level of variation, we sequenced genomic DNA from a single O. dioica male. The low amount of extracted DNA is an issue when working with small-size organ- isms like O. dioica. Therefore, we optimized the extrac- tion and sequencing protocols to allow for low-template input DNA yields of around 200 ng and applied a hybrid sequencing approach using Oxford Nanopore reads to span repeat-rich regions and Illumina reads to correct individual nucleotide errors. The Nanopore run gave 8.2 million reads (221× coverage) with a median length of 840 bp and maximum length of 166 kb (Fig. 2a). Based on k-mer counting of the Illumina reads, the genome was estimated to contain ~ 50 Mbp (Fig. 2b) – compar- able in size to the OdB3 and OSKA2016 assemblies – and a relatively high heterozygosity of ~ 3.6%. We used the Canu pipeline [19] to correct, trim and assemble Nanopore reads, yielding a draft assembly comprising 175 contigs with a weighted median N50 length of 3.2 Mbp. We corrected sequencing errors and local misas- semblies of the draft contigs with Nanopore reads using Historical attempts at karyotyping O. dioica by traditional histochemical stains arrived at different Page 3 of 18 Bliznina et al. BMC Genomics (2021) 22:222 Bliznina et al. BMC Genomics RepeatModeler, RepeatMasker Intron hints STAR BLAT, GMAP PASA AUGUSTUS AUGUSTUS Genome browser ZENBU Repeat identification and masking Genome annotation Collection of a single Oikopleura dioica male gDNA extraction and library preparation Illumina MiSeq sequencing Canu Contig assembly Self-polishing Racon Error-correction Quality check Filtration and trimming FASTX-toolkit Pilon Redundancy removal HaploMerger2 Scaffold assembly Juicer, 3D-DNA Hi-C library preparation Illumina MiSeq sequencing Quality check and filtration Hi-C Dovetail Kit FASTX-toolkit Quality assessment QUAST, BUSCO LG assignment Collection of 50 males Genome assembly Nanopore sequencing LAST, Circlize Quality check and filtration Transcriptome assembly Redundancy removal Assembly quality check FASTX-toolkit Trinity CD-HIT rnaQUAST, BUSCO Collection of 3 developm. stages cDNA library preparation Illumina MiSeq sequencing b 0.5 mm a Exon hints Gene structure annotation and analysis Training AUGUSTUS Gene model prediction Fig. 1 Genome assembly and annotation workflow used to generate the OKI2018_I69 genome assembly. a Life images of adult male (top) and female (bottom) O. dioica. Genome sequencing and assembly Since we had previously inferred a karyotype of n = 3 by im- munohistochemistry [16], we completed the assembly by pairing the megabase-scale scaffolds into chromosome arms based on their synteny with the OdB3 physical map (Fig. 3b). The final assembly named OKI2018_I69 To scaffold the genome, we sequenced Hi-C libraries from a pool of ~ 50 individuals from the same culture. More than 99% of the Hi-C reads could be mapped to the contig assembly. After removing duplicates, Hi-C contacts were passed to the 3D-DNA pipeline to correct major misassemblies, as well as order and orient the contigs. The resulting assembly consisted of 8 megabase-scale scaffolds containing 99% of the total se- quence (Fig. 3a), and 14 smaller scaffolds that account Page 4 of 18 Bliznina et al. BMC Genomics (2021) 22:222 Bliznina et al. BMC Genomics Genomescope estimated size (Mb) 20 30 40 50 Repetitive genome Unique genome Haploid genome b Read Length Histogram a Estimated Read Length in Bases (kb) 0 32.4 16.2 48.6 64.2 81 97.2 113.4 ) b G ( s e s a B d e t a m its E la t o T 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 c I69 contigs I28 contigs Fig. 2 Quality control checks implemented on different steps of genome sequencing and assembly. a Graph showing length distribution of raw Nanopore reads used to generate the OKI2018_I69 assembly. b Estimated total and repetitive genome size based on k-mer counting of the Illumina paired-end reads used for polishing the OKI2018_I69 assembly. c Pairwise genome alignment of the contig assemblies of I69 and I28 O. dioica individuals Genomescope estimated size (Mb) 20 30 40 50 Repetitive genome Unique genome Haploid genome b Read Length Histogram a Estimated Read Length in Bases (kb) 0 32.4 16.2 48.6 64.2 81 97.2 113.4 ) b G ( s e s a B d e t a m its E la t o T 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 Genomescope estimated size (Mb) 20 30 40 50 Repetitive genome Unique genome Haploid genome b b Estimated Read Length in Bases (kb) Genomescope estimated size (Mb) Genomescope estimated size (Mb) c I69 contigs I28 contigs Fig. 2 Quality control checks implemented on different steps of genome sequencing and assembly. a Graph showing length distribution of raw Nanopore reads used to generate the OKI2018_I69 assembly. Genome sequencing and assembly b Estimated total and repetitive genome size based on k-mer counting of the Illumina paired-end reads used for polishing the OKI2018_I69 assembly. c Pairwise genome alignment of the contig assemblies of I69 and I28 O. dioica individuals c Fig. 2 Quality control checks implemented on different steps of genome sequencing and assembly. a Graph showing length distribution of raw Nanopore reads used to generate the OKI2018_I69 assembly. b Estimated total and repetitive genome size based on k-mer counting of the Illumina paired-end reads used for polishing the OKI2018_I69 assembly. c Pairwise genome alignment of the contig assemblies of I69 and I28 O. dioica individuals (Table 1; Suppl. Table 1) comprises telomere-to- telomere assemblies of the autosomal chromosomes 1 (chr 1) and 2 (chr 2). The sex chromosomes are split into pseudo-autosomal region (PAR) and X-specific re- gion (XSR) or Y-specific region (YSR; Fig. 3). We assume that the sex-specific regions belong to the long arm of the PAR, as the long arm does not contain any telomeric repeats (Fig. 4a). Alignment of the Illumina polishing reads to the OKI2018_I69 assembly estimated an error rate of 1.3% showing high sequence accuracy. (Table 1; Suppl. Table 1) comprises telomere-to- telomere assemblies of the autosomal chromosomes 1 (chr 1) and 2 (chr 2). The sex chromosomes are split into pseudo-autosomal region (PAR) and X-specific re- gion (XSR) or Y-specific region (YSR; Fig. 3). We assume that the sex-specific regions belong to the long arm of the PAR, as the long arm does not contain any telomeric repeats (Fig. 4a). Alignment of the Illumina polishing reads to the OKI2018_I69 assembly estimated an error rate of 1.3% showing high sequence accuracy. haploid status in males. The chromosome arms them- selves show few interactions between each other, even when they are part of the same chromosome. Chromosome-level features The genome contains between 1.4 and 2.6 Mbp of tan- dem repeats (detected using the tantan and ULTRA al- gorithms respectively with maximum period lengths of 100 and 2000). Subtelomeric regions tend to contain ret- rotransposons or tandem repeats with longer periods. We also found telomeric repeats in smaller scaffolds. A possible explanation is that subtelomeric regions display high heterozygosity, leading to duplicated regions that fail to assemble with the chromosomes. Alternatively, these scaffolds could be peri-centromeric regions con- taining interstitial telomeric sequences. In some species, high-copy tandem repeats can be utilized to discover the position of centromeric regions [20]; however, we could not find such regions. Additional experimental The genome-wide contact matrix from the Hi-C data (Fig. 3c) shows bright, off-diagonal spots that suggest spatial clustering of the telomeres and centromeres both within the same and across different chromosomes [18]. The three centromeric regions are outside the sex- specific regions, dividing the PAR and both autosomes into long and short arms. The two sex-specific regions have lower apparent contact frequencies compared with the rest of the assembly which is consistent with their Bliznina et al. BMC Genomics (2021) 22:222 Page 5 of 18 Bliznina et al. BMC Genomics OKI2018_I69 contigs OKI2018_I69 Size (Mbp) <1 2 4 6 8 10 12 14 16 a chr 1 chr 2 PAR XSR YSR b chr 1 chr 2 PAR YSR XSR N LG 1 N LG 2 N Un d N Un a N Un f N Un f N Un e N Un b N YSR N XSR N PAR OKI2018_I69 OdB3 XSR PAR chr 2 chr 1 YSR c Fig. 3 OKI2018_I69 assembly of the Okinawan O. dioica. a Treemap comparison between the contig (left) and scaffold (right) assemblies of the O. dioica genome. Each rectangle represents a contig or a scaffold in the assembly with the area proportional to its length. b Comparison between the OKI2018_I69 (left) and OdB3 (right) linkage groups. The Sankey plot shows what proportion of each chromosome in the OKI2018_I69 genome is aligned to the OdB3 linkage groups. c Contact matrix generated by aligning Hi-C data set to the OKI2018_I69 assembly with Juicer and 3D-DNA pipelines. Chromosome-level features Pixel intensity in the contact matrices indicates how often a pair of loci collocate in the nucleus OKI2018 I69 contigs OKI2018 I69 Size (Mbp) <1 2 4 6 8 10 12 14 16 a chr 1 chr 2 PAR XSR YSR a _ g b chr 1 chr 2 PAR YSR XSR N LG 1 N LG 2 N Un d N Un a N Un f N Un f N Un e N Un b N YSR N XSR N PAR OKI2018_I69 OdB3 b XSR PAR chr 2 chr 1 YSR c c Fig. 3 OKI2018_I69 assembly of the Okinawan O. dioica. a Treemap comparison between the contig (left) and scaffold (right) assemblies of the O. dioica genome. Each rectangle represents a contig or a scaffold in the assembly with the area proportional to its length. b Comparison between the OKI2018_I69 (left) and OdB3 (right) linkage groups. The Sankey plot shows what proportion of each chromosome in the OKI2018_I69 genome is aligned to the OdB3 linkage groups. c Contact matrix generated by aligning Hi-C data set to the OKI2018_I69 assembly with Juicer and 3D-DNA pipelines. Pixel intensity in the contact matrices indicates how often a pair of loci collocate in the nucleus Fig. 3 OKI2018_I69 assembly of the Okinawan O. dioica. a Treemap comparison between the contig (left) and scaffold (right) assemblies of the O. dioica genome. Each rectangle represents a contig or a scaffold in the assembly with the area proportional to its length. b Comparison between the OKI2018_I69 (left) and OdB3 (right) linkage groups. The Sankey plot shows what proportion of each chromosome in the OKI2018_I69 genome is aligned to the OdB3 linkage groups. c Contact matrix generated by aligning Hi-C data set to the OKI2018_I69 assembly with Juicer and 3D-DNA pipelines. Pixel intensity in the contact matrices indicates how often a pair of loci collocate in the nucleus Table 1 Comparison of the OKI2018_I69 assembly with the previously published O. Chromosome-level features dioica genomes OdB3 OSKA2016 OKI2018_I69 Geographical origin Bergen, Norway (North Atlantic) Hyogo, Japan (Western Pacific) Okinawa, Japan (Ryukyu archipelago) Assembly length (Mbp) 70.4 65.6 64.3 Number of scaffolds 1260 576 19 Longest scaffold (Mbp) 3.2 6.8 17.1 Scaffold N50 (Mbp) 0.4 1.5 16.2 Number of contigs 5917 746 42 Contig N50 (Mbp) 0.02 0.6 4.7 GC content (%) 39.77 41.34 41.06 Gap rate (%) 5.589 0.585 0.034 Complete BUSCOs (%) 70.8 71.7 73.01 Table 1 Comparison of the OKI2018_I69 assembly with the previously published O. dioica genomes OdB3 OSKA2016 Bliznina et al. BMC Genomics (2021) 22:222 Page 6 of 18 a b chr 1 short arm chr 2 short arm chr 2 long arm PAR short arm PAR long arm chr 1 long arm GC Cov. Rep. Genes DpnII 0 100 200 300 0 10 20 30 0 40 80 200 600 1000 35 45 40 50 GC chr 1 . p e R .v o C Genes 44 40 36 250 100 80 40 0 20 0 10 0 Mbp 4.8 Mbp 9.59 Mbp 14.53 Mbp chr 2 .v o C C G Rep. Genes 40 38 36 150 250 80 40 0 0 10 20 0 Mbp 5.33 Mbp 10.66 Mbp 16.16 Mbp PAR .v o C C G Rep. Genes 46 38 36 200 600 1000 80 40 0 5 15 0 Mbp 5.64 Mbp 11.28 Mbp 17.09 Mbp XSR .v o C C G Rep. Genes 0 Mbp 4.28 Mbp 8.55 Mbp 12.96 Mbp 50 44 38 120 160 200 80 0 40 0 10 20 YSR .v o C C G Rep. Genes 44 40 36 500 300 100 20 50 80 10 0 0 Mbp 2.92 Mbp Fig. 4 Chromosome-level features of the Okinawan O. dioica genome. a Visualization of sequence properties across chromosomes in the OKI2018_I69 assembly. For each chromosome, 50 kbp windows of GC (orange), Nanopore sequence coverage (blue), the percent of nucleotides masked by RepeatMasker (purple), and the number of genes (yellow) are indicated. Differences in these sequence properties occur near predicted sites of centromeres and telomeres, as well as between the short and long arms of each non-sex-specific chromosome. Telomeres and gaps in the assembly are indicated with black and grey rectangles, respectively. Chromosome-level features b Long and short chromosome arms exhibit significant differences sequence properties, including GC content, repetitive sequence content, and the number of restriction sites recognized by the DpnII enzyme used to generate the Hi-C library XSR .v o C C G Rep. Genes 0 Mbp 4.28 Mbp 8.55 Mbp 12.96 Mbp 50 44 38 120 160 200 80 0 40 0 10 20 YSR .v o C C G Rep. Genes 44 40 36 500 300 100 20 50 80 10 0 0 Mbp 2.92 Mbp a Genes b chr 2 PAR Fig. 4 Chromosome-level features of the Okinawan O. dioica genome. a Visualization of sequence properties across chromosomes in the OKI2018_I69 assembly. For each chromosome, 50 kbp windows of GC (orange), Nanopore sequence coverage (blue), the percent of nucleotides masked by RepeatMasker (purple), and the number of genes (yellow) are indicated. Differences in these sequence properties occur near predicted sites of centromeres and telomeres, as well as between the short and long arms of each non-sex-specific chromosome. Telomeres and gaps in the assembly are indicated with black and grey rectangles, respectively. b Long and short chromosome arms exhibit significant differences sequence properties, including GC content, repetitive sequence content, and the number of restriction sites recognized by the DpnII enzyme used to generate the Hi-C library centered on their centromeric regions. Most strikingly, there is a clear difference in sequence content between chromosome arms (Fig. 4; Supp. Table 3). The short arms consistently display depleted GC content and ele- vated repetitive content compared with the correspond- ing long arms. Although GC content tends to be weakly negatively correlated with repeat content, it is not cur- rently possible to ascertain causality and the mechanism techniques such as chromatin immunoprecipitation and sequencing with centromeric markers might be neces- sary to resolve the centromeres precisely. Therefore, the current assembly skips over centromeric regions, repre- sented as gaps of arbitrary size of 500 bp in the chromo- somal scaffolds. We studied genome-scale features by visualizing them along whole chromosomes, from the short to long arm, Page 7 of 18 Page 7 of 18 Bliznina et al. BMC Genomics (2021) 22:222 Bliznina et al. BMC Genomics (2021) 22:222 interspersed nuclear elements (SINEs) make up a smaller portion of the OKI2018_I69 sequence (< 0.1%) compared with the OdB3 (0.6%). Chromosome-level features It has been suggested that SINEs contribute significantly to genome size variation in other oikopleurids [10], but further analysis is required to de- termine whether that is the case at shorter evolutionary distances. Non-LTR LINE/Odin and Penelope-like ele- ments are large components of most oikopleurid ge- nomes [10], but they are almost absent from the OKI2018_I69 assembly. Indeed, 44% of the predicted re- peats in the Okinawan O. dioica could not be classified through searches against repeat databases and may ei- ther represent highly divergent relatives of known repeat classes, or novel repeats specific to Okinawan O. dioica. behind the marked difference in sequence content be- tween the short and long chromosome arms remains un- known. It should be noted that the differences in GC contents affects the density of the GATC DpnII restric- tion enzyme recognition sites used for Hi-C library prep- aration; however, this bias is insufficient to explain the low degree of intra-chromosomal interaction observed in the Hi-C contact maps. behind the marked difference in sequence content be- tween the short and long chromosome arms remains un- known. It should be noted that the differences in GC contents affects the density of the GATC DpnII restric- tion enzyme recognition sites used for Hi-C library prep- aration; however, this bias is insufficient to explain the low degree of intra-chromosomal interaction observed in the Hi-C contact maps. Gene annotation We annotated the OKI2018_I69 assembly using RNA- Seq-based gene prediction. RNA-Seq reads mapped to the assembly showed 99.14% agreement between the genome and transcriptome indicating high sequence ac- curacy. Annotation of the genome yielded 18,794 tran- script isoforms distributed among 17,260 protein-coding genes. The number of predicted genes for the OKI2018_ I69 is slightly lower than what was reported for OdB3 (18,020) [9] and OSKA2016 (18,743) [13] (Table 2). The rest of the genes are either lost from the Okinawan O. dioica genome or were not assembled and/or annotated with our pipeline. On the other hand, the higher number of genes might be artifacts of the OdB3 and OSKA2016 annotations. The completeness of the annotation com- pares to the genome: BUSCO recovered 75.3% complete and 4.8% fragmented metazoan genes (Fig. 5a). Like the OdB3 assembly, gene density is very high at one gene per 3.7 kbp. OKI2018_I69 has similar gene and exon length distributions, and very short introns with a me- dian length of only 49 bp (Table 2). Indeed, we found a high frequency of the non-canonical (non-GT/AG) in- trons in the OKI2018_I69 (11%). Previously, Denoeud et al. reported that 12% of the introns were non- canonical in the OdB3 [9]. Some of those non-canonical introns were found in the same genes as in the OdB3. However, more close examination is required to under- stand if it is the case for the rest of the genes. Therefore, overall genomic features seem to be conserved among O. dioica population despite large geographic distance. Quality assessment using BUSCO y g To assess the completeness of our assembly, we searched for 978 metazoan Benchmarking Universal Single-Copy Orthologs (BUSCOs) provided with the BUSCO tool [21–23]. To increase sensitivity, we trained BUSCO’s gene prediction tool, AUGUSTUS [24], with transcript models generated from RNA-Seq data collected from the same laboratory culture (see below). We detected 73.0% of BUSCOs (Table 1), which is similar to OdB3 and OSKA2016 (Fig. 5a; Suppl. Table 4). All detected BUSCOs except one reside on the chromosomal scaf- folds. As the reported fraction of detected genes is lower than for other tunicates such as Ciona intestinalis HT (94.6%) [25] or Botrylloides leachii (89%) [26], we searched for BUSCO genes in the transcriptomic train- ing data (83.0% present) and confirmed the presence of all but one by aligning the transcript sequence to the genome. We then inspected the list of BUSCO genes that were found neither in the genome nor in the tran- scriptome. Bibliographic analysis confirmed that BUSCO genes related to the peroxisome were lost from O. dioica [27, 28]. There are two possible explanations for the remaining missing genes: first is that protein sequence divergence [29] or length reduction [30] in Oikopleura complicate detection by BUSCO, and second is gene loss. In line with the possibility of gene loss, most BUSCO genes missing from our assembly are also un- detectable in OdB3 and OSKA2016 (Fig. 5b; Suppl. Table 5). To summarize, the Okinawa assembly achieved comparable detection of universal single-copy conserved orthologs compared with previous O. dioica assemblies, and consistently undetectable genes may have been lost or diverged extensively in Oikopleura. Repeat annotation b Venn diagram showing the number of BUSCO genes missing in OKI2018_I69, OdB3 and/or OSKA2016 genomes the real number of the tandem rDNA copies could range between 20 (MiSeq) and 100 (Nanopore) copies. Be- tween or flanking the rDNA genes, we also found short tandem repeats made of two to three copies of a 96-bp sequence. This tandem repeat is unique to the rDNA genes and to our reference and draft genomes, and was not found in the OdB3 reference nor in other larvacean genomes. The 5S rRNA is transcribed from loci distinct to the rDNA gene tandem arrays. In Oikopleura, they have the particularity of being frequently associated with the spliced leader (SL) gene and to form inverted repeats present in more than 40 copies [31]. We found 27 copies of these genes on every chromosomal scaffold except YSR, 22 of which were arranged in inverted tandem re- peats. Altogether, we found in our reference genome one rDNA gene repeat region assembled at the end of a chromosome short arm. This sequence might provide useful markers for phylogenetic studies in the future. the real number of the tandem rDNA copies could range between 20 (MiSeq) and 100 (Nanopore) copies. Be- tween or flanking the rDNA genes, we also found short tandem repeats made of two to three copies of a 96-bp sequence. This tandem repeat is unique to the rDNA genes and to our reference and draft genomes, and was not found in the OdB3 reference nor in other larvacean genomes. The 5S rRNA is transcribed from loci distinct to the rDNA gene tandem arrays. In Oikopleura, they have the particularity of being frequently associated with the spliced leader (SL) gene and to form inverted repeats present in more than 40 copies [31]. We found 27 copies of these genes on every chromosomal scaffold except YSR, 22 of which were arranged in inverted tandem re- peats. Altogether, we found in our reference genome Repeat annotation In order to identify repetitive elements in the OKI2018_ I69 genome, we combined the results of several de novo repeat detection algorithms and used this custom library as an input to RepeatMasker to identify repeat se- quences. Interspersed repeats make up 14.4% of the as- sembly (9.25 Mbp; Fig. 6), comparable to the 15% reported for OdB3 [9]. Of the annotated elements, the most abundant type is the long terminal repeat (LTRs; ~ 4.6%) with Ty3/gypsy Oikopleura transposons (TORs) dominating 2.97 Mbp of the sequence. Short The ribosomal DNA gene encoding the precursor of the 18S, 5.8S and 28S rRNAs occurs as long tandem re- peats that form specific chromatin domains in the nucle- olus. We identified 4 full tandem copies of the rDNA gene at the tip of the PAR’s short arm, separated by 8738 bp (median distance). As this region has excess coverage of raw reads, and assemblies of tandem repeats are limited by the read length (99% of Nanopore reads in our data are shorter than 42,842 bp), we estimate that Page 8 of 18 Bliznina et al. BMC Genomics (2021) 22:222 b 33 20 21 20 9 11 177 OdB3 OSKA2016 OKI2018_I69 a Complete and duplicated BUSCOs (D) Fragmented BUSCOs (F) Complete and single−copy BUSCOs (S) Missing BUSCOs (M) BUSCO Assessment Results OKI2018_I69 OdB3 OSKA2016 OKI2018_I28 OKI2018_I69 (no training) OKI2018_I69 (gene models) Okinawa Osaka Oikobase 0% 20% 40% 60% 80% 100% % BUSCOs Transcriptome Genome Fig. 5 Quality assessment of the OKI2018_I69 genome assembly. a Proportion of BUSCO genes detected or missed in Oikopleura genomes and transcriptomes. The search on the OKI2018_I69 assembly was repeated with default parameters (“no training”) to display the effect of AUGUSTUS training. b Venn diagram showing the number of BUSCO genes missing in OKI2018_I69, OdB3 and/or OSKA2016 genomes a Complete and duplicated BUSCOs (D) Fragmented BUSCOs (F) Complete and single−copy BUSCOs (S) Missing BUSCOs (M) BUSCO Assessment Results OKI2018_I69 OdB3 OSKA2016 OKI2018_I28 OKI2018_I69 (no training) OKI2018_I69 (gene models) Okinawa Osaka Oikobase 0% 20% 40% 60% 80% 100% % BUSCOs Transcriptome Genome BUSCO Assessment Results a 11 20 Fig. 5 Quality assessment of the OKI2018_I69 genome assembly. a Proportion of BUSCO genes detected or missed in Oikopleura genomes and transcriptomes. The search on the OKI2018_I69 assembly was repeated with default parameters (“no training”) to display the effect of AUGUSTUS training. Draft mitochondrial genome scaffold We identified a draft mitochondrial genome among the smaller scaffolds, chrUn_12, by searching for mitochon- drial sequences using the Cox1 protein sequence and the ascidian mitochondrial genetic code [32]. Automated annotation of this scaffold using the MITOS2 server de- tected the coding genes cob, cox1, nad1, cox3, nad4, cox2, and atp6 (Fig. 7a), which are the same as in Denoeud et al., 2010 [9] except for the nd5 gene that is missing from our assembly. The open reading frames are often interrupted by T-rich regions, in line with Bliznina et al. BMC Genomics (2021) 22:222 Page 9 of 18 e m o n e g f o t n e c r e P Kimura substitution level (CpG adjusted) Interspersed Repeat Landscape SINE LINE/Odin1 LINE/Penelope-like RC/Helitron DNA/MuDr DNA/MITE Unknown LTR/TOR5 LTR/TOR4B LTR/TOR4A LTR/TOR3A LTR/TOR3B LTR/TOR2 LTR/TOR1 LTR 0 5 10 15 20 25 30 35 40 45 50 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 85.8% Non-repetitive 6.3% Fig. 6 Analysis of repetitive elements. The repeat landscape and proportions of various repeat classes in the genome are indicated and color- coded according to the classes shown on the right side of the figure. The non-repetitive fraction of the genome is shown in black Kimura substitution level (CpG adjusted) ig. 6 Analysis of repetitive elements. The repeat landscape and proportions of various repeat classes in the genome are ind oded according to the classes shown on the right side of the figure. The non-repetitive fraction of the genome is shown in etitive elements. The repeat landscape and proportions of various repeat classes in the genome are indicated and color- he classes shown on the right side of the figure. The non-repetitive fraction of the genome is shown in black Fig. 6 Analysis of repetitive elements. The repeat landscape and proportions of various repeat classes in the genome are indicated and color- coded according to the classes shown on the right side of the figure. The non-repetitive fraction of the genome is shown in black Denoeud et al. (2010) [9]. However, we cannot rule out the possibility that these regions represent sequencing errors, as homopolymers are difficult to resolve with the Nanopore technology available in 2019. The cob gene is interrupted by a long non-coding region, but this might be a missassembly. Draft mitochondrial genome scaffold Indeed, an independent assembly using the flye software [33] with the --meta option to ac- count for differential coverage also produced a draft mitochondrial genome, but its non-coding region was ~ 2 kbp longer. Moreover, a wordmatch dotplot shows tandem repeats in this region (Fig. 7b), and thus this re- gion is prone to assembly errors, especially with respect to the number of repeats. Altogether, the draft contig produced in our assembly shows as a proof of principle that sequencing reads covering the mitochondrial gen- ome alongside the nuclear genome can be produced from a single individual, although it may need support- ing data such as targeted resequencing in order to be properly assembled. OKI2018_I69 assembly quality BMC Genomics b chrUn_12 chrUn_12 1 1001 2001 3001 4001 5001 6001 7001 8001 9001 1 1001 2001 3001 4001 5001 6001 7001 8001 9001 Gene RNA Origin of replication 0kb 6kb 8kb 2kb 4kb 9.225kb OH 1−c OH 4 OH 2 nad4 0−a nad4 0−b nad4 1 rrnL OH 0 trnA(agc) OH 1−a atp6 OH 3 OH 1−b OH 1−d cob 2 cox1 0−d cox1 1 cox1 0−c lagli cox1 0−b cox1 0−a nad1−b trnH(gtg) nad1−a trnM(tat) cox2 cob 1 cob 0−b cob 0−a trnW(tca) cox3 1 cox3 0−b cox3 0-a Okinawa O. dioica draft mitochondrial genome a Fig. 7 Draft scaffold of the mitochondrial genome in the OKI2018_I69 assembly. a Predicted gene annotation of the draft mitochondrial genome sequence. b Self-similarity plot of the draft mitochondrial genome sequence. A tandem repeat can be seen, which complicates the complete assembly of the mitochondrial genome from whole-genome sequencing data Gene RNA Origin of replication 0kb 6kb 8kb 2kb 4kb 9.225kb OH 1−c OH 4 OH 2 nad4 0−a nad4 0−b nad4 1 rrnL OH 0 trnA(agc) OH 1−a atp6 OH 3 OH 1−b OH 1−d cob 2 cox1 0−d cox1 1 cox1 0−c lagli cox1 0−b cox1 0−a nad1−b trnH(gtg) nad1−a trnM(tat) cox2 cob 1 cob 0−b cob 0−a trnW(tca) cox3 1 cox3 0−b cox3 0-a Okinawa O. dioica draft mitochondrial genome a Gene RNA Origin of replication a b chrUn_12 chrUn_12 1 1001 2001 3001 4001 5001 6001 7001 8001 9001 1 1001 2001 3001 4001 5001 6001 7001 8001 9001 2kb he mitochondrial genome in the OKI2018 I69 assembly a Predicted gene annotation of the draft mitochondrial genome b chrUn 12 chrUn_12 1 1001 2001 3001 4001 5001 6001 7001 8001 9001 1 1001 2001 3001 4001 5001 6001 7001 8001 9001 b Fig. 7 Draft scaffold of the mitochondrial genome in the OKI2018_I69 assembly. a Predicted gene annotation of the draft mitochondrial genome sequence. b Self-similarity plot of the draft mitochondrial genome sequence. A tandem repeat can be seen, which complicates the complete assembly of the mitochondrial genome from whole-genome sequencing data assembly errors in OdB3 would not be transferred to our assembly. Apart from these syntenic relationships, our karyotyping results and the count of three centro- meres on the Hi-C contact map supports the presence of three pairs of chromosomes in the Okinawan O. dioica. OKI2018_I69 assembly quality Previously, different techniques have been used to se- quence and assemble O. dioica genomes which have produced assemblies of varying quality. The Sanger- based OdB3 sequence was published in 2010 [9]. Due to limitations in sequencing technologies at the time, it is highly fragmented, comprising 1260 scaffolds with an N50 of 0.4 Mbp. The recently released OSKA2016 as- sembly was generated from long-read PacBio data and, therefore, has a larger N50 and fewer scaffolds (Table 1) [13]. Both assemblies have high sequence quality and nearly full genome coverage, but neither of them con- tains resolved chromosomes. However, Denoeud et al. (2010) [9] released a physical map calculated for OdB3 from BAC end sequences that comprises five linkage groups (LGs): two autosomal LGs, one pseudo- autosomal region of sex chromosomes, and two sex spe- cific regions (X and Y). The use of reference chromosome information from a closely related species to order contigs or scaffolds into chromosome-length sequences is a common way to gen- erate final genome assemblies [34]. However, this ap- proach precludes discovery of structural variants. In our study, we first assembled long Nanopore reads de novo into contigs that we ordered and joined into megabase- scale scaffolds using long-range Hi-C data. The synteny- based approach with OdB3’s linkage groups as a refer- ence was only required to guide final pairing of chromo- some arms into single scaffolds of chr 1, chr 2 and PAR, as we found that these scaffolds mostly align to one of the autosomal LGs or PAR. Therefore, any potential Table 2 Comparison of the annotations of the three O. dioica genome assemblies OdB3 OSKA2016 OKI2018_I69 Masked sequence (%) 15.0 – 14.4 Number of genes 18,020 18,743 17,260 Median gene length (bp) 1488 1483 1505 Median exon length (bp) 159 155 152 Median intron length 48 51 49 Table 2 Comparison of the annotations of the three O. dioica genome assemblies Page 10 of 18 Bliznina et al. BMC Genomics (2021) 22:222 Bliznina et al. Isolation and sequencing of DNA Staged fully mature males were collected prior to spawn- ing. Each male was washed with 5 ml filtered autoclaved seawater (FASW) for 10 min three times before resus- pension in 50 μl 4 M guanidium isothiocyanate, 0.5% SDS, 50 mM sodium citrate and 0.05% v/v 2- mercaptoethanol. This was left on ice for 30 min before being precipitated with 2 volumes of ice-cold ethanol and centrifuged at 14,000 rpm 4 °C for 20 min. The pellet OKI2018_I69 assembly quality However, there is a possibility that chromosome arms might have been exchanged between chromosomes in the Okinawan population. Additional experimental evidence is needed to confirm the pairing of chromo- some arms, such as data generated by the Omni-C method which does not rely on restriction enzyme fragmentation. Our synteny-based scaffolding is based on the simplest definition of synteny meaning “on the same chromo- some”. It does not make assumptions on gene order, which is why we report our results with a position- independent Sankey plot in Fig. 3b. We initially assumed that animals collected from the Atlantic and Pacific oceans are from the same species and conserve these chromosomal properties. However, there are visible Page 11 of 18 Page 11 of 18 Bliznina et al. BMC Genomics (2021) 22:222 models, in silico-predicted features such as repeats and non-coding RNAs, or syntenies with other Oikopleura genomes. To facilitate the study of known genes, we screened the literature for published sequences (Suppl. Table 6) and mapped them to the genome with a trans- lated alignment. The ZENBU track for these alignments is searchable by gene name, accession number and PubMed identifier. Chromosome-level visualization of this track shows that the genes studied so far are distrib- uted evenly on each chromosome, except for the repeat- rich YSR (Fig. 8). In line with the observed loss of syn- teny in the Hox genes noted in Oikopleura [40], we did not see apparent clustering of genes by function or re- latedness. The view of the OKI2018_I69 genome assem- bly can be found here: differences in gene number, gene order and repeat content compared with the OdB3 and OSKA2016. O. dioica is dis- tributed all over the world, and all the populations are classified as a single species owing to the lack of obvious morphological differences and limited understanding of population structure. However, the short life span of O. dioica combined with limited mobility and high mutation rate contribute to an accelerated genome evolution that might have led to multiple speciation events. Sequence polymorphism was previously noted when comparing the OdB3 genome to genomic libraries of a laboratory strain collected on the North American Pacific coast [9], and more recently when comparing OdB3 to OSKA2016 [12, 13]. The chromosome-scale OKI2018_I69 assembly opens up the possibility for further work on cross-comparison among O. Conclusions The sequence of O. dioica’s chromosomes and their con- tact map suggest that chromosome arms may be the fundamental unit of synteny in larvaceans. Hi-C contact matrices in vertebrates typically display greater intra- chromosomal than inter-chromosomal interactions. A similar pattern was reported in the tunicate Ciona ro- busta (also known as intestinalis type A) [25] and the lancelet Branchiostoma floridae [35]. By comparison, in flies and mosquitoes, the degree of contacts between two arms of the same chromosome appear to be reduced but nonetheless more frequent than between different chromosomes [18]. Indeed, in Drosophila, the chromo- some arms – which are termed Muller elements owing to studies with classical genetics [36] – are frequently exchanged between chromosomes across speciation events. O. dioica’s genome shares with fruit flies its small size and small number of chromosomes. However, small chromosome size is also seen in the tunicate Ciona ro- busta, which has 14 meta- or sub-meta-centric pairs [37], with an average length of ~ 8 Mbp [25] that exhibit a more extensive degree of contacts, particularly for intra-chromosomal interactions across the centromeres [25]. As we prepared our Hi-C libraries from adult ani- mals, where polyploidy is high [38], we cannot rule out that it could be a possible cause of the low inter-arm in- teractions in our contact matrix. Further studies such as investigations of other developmental stages will be needed to elucidate the mechanism at work for the similarity between O. dioica and insect’s chromosome contact maps. We demonstrated that a combination of long- and short-read sequencing data from a single animal, to- gether with the long-range Hi-C data and the use of various bioinformatic approaches can result in a high- quality de novo chromosome-scale assembly of O. dioica’s highly polymorphic genome. However, further work is needed to properly resolve the polymorphisms into separated haplotypes using a different approach, such as trio-binning. We believe that the current version of the assembly will serve as an essential resource for a broad range of biological studies, including genome- wide comparative studies of Oikopleura and other spe- cies, and provides insights into chromosomal evolution. OKI2018_I69 assembly quality dioica populations that will elucidate the rela- tion of the Okinawan populations to the North Atlantic and North Pacific ones. https://fantom.gsc.riken.jp/zenbu/gLyphs/#config= 0tPT7vwSO1Vm5QV9iKqfAC;loc=OKI2018_I69_1.0:: chr1:677717..880998+ (ZENBU view “OKI2018_I69_1.0 view with tracks (updated)”). Oikopleura sample and culture Wild live specimens were collected from Ishikawa Har- bor (26°25′39.3″N 127°49′56.6″E) by a hand-held plankton net and returned to the lab for culturing [5]. A typical generation time from hatchling to fully mature adult is 4 days at 23 °C for the Okinawan O. dioica. Indi- viduals I28 and I69 were collected at generation 44 and 47, respectively. Visualization and access We prepared a public view of our reference genome in the ZENBU browser [39], displaying tracks for our gene Page 12 of 18 Bliznina et al. BMC Genomics (2021) 22:222 0 5000000 10000000 15000000 odMT2 Bmp3 hox11 pum1 od-ARNT Bmp.a oik16 od-CesA1 otxa Bmp.b oik27 otxc otxb Aldh2 od-Noto9b od-PCNA oik29a oik29b od-ASAK pitx od-laminin a1 pax6 od-Calumenin2 oik45 oik26 oik5 oik48 oik12 oik44 oik31b oik20 oik42 oik7 octB PAR 0 5000000 10000000 15000000 cdk1a od-prickle pax2/5/8a hox1 Cyclin B3a SoxBb six3/6b catenin alpha-1 odiT od-Noto9c Aldh8a1 eya Tis11b oik22 Adh3 od-CaMK rpa-interacting protein a cse1l six3/6a od-cdc45 octA2 octA1 hox13 engrailed ChAT od-ACL oik39 hox4 oik3 oik25 oik1 oik11 oik10 od-Noto15a od-netrin chr 2 0 5000000 10000000 oik35 od-FCol1 hox2 od-quaking od-leprecan gad six1/2 od-Noto17 hox12 Wnt11 cadherin-6 precursor od-ERM oik31a od-CesA2 alpha_1b_tubulin Glcm oik47 oik50 oik13 oik43 oik2 oik21b oik17b oik14 oik9 od-thrombospondin3 chr 1 0 1000000 2000000 3000000 YSR 0 5000000 10000000 od-Calumenin1 Tis11a oik8 SoxBa oik15 oik23 Nkx2.3/5/6 od-b4-GalT od-Zipper od-IQGAP tubulin_beta_2c oik32 oik21a od-Noto9a actin ActnM1 Cyclin_Ba pax2/5/8b oik37 od-tensin od-Noto10 oik46 vas4 hox10 oik18 XSR Fig. 8 Genomic locations of various oikopleurid gene homologs in the OKI2018_I69. The genes are searchable by name and PubMed identifiers in the ZENBU genome browser. Colours indicate genes from the same family 0 5000000 10000000 15000000 odMT2 Bmp3 hox11 pum1 od-ARNT Bmp.a oik16 od-CesA1 otxa Bmp.b oik27 otxc otxb Aldh2 od-Noto9b od-PCNA oik29a oik29b od-ASAK pitx od-laminin a1 pax6 od-Calumenin2 oik45 oik26 oik5 oik48 oik12 oik44 oik31b oik20 oik42 oik7 octB PAR 0 5000000 10000000 15000000 cdk1a od-prickle pax2/5/8a hox1 Cyclin B3a SoxBb six3/6b catenin alpha-1 odiT od-Noto9c Aldh8a1 eya Tis11b oik22 Adh3 od-CaMK rpa-interacting protein a cse1l six3/6a od-cdc45 octA2 octA1 hox13 engrailed ChAT od-ACL oik39 hox4 oik3 oik25 oik1 oik11 oik10 od-Noto15a od-netrin chr 2 0 5000000 10000000 oik35 od-FCol1 hox2 od-quaking od-leprecan gad six1/2 od-Noto17 hox12 Wnt11 cadherin-6 precursor od-ERM oik31a od-CesA2 alpha_1b_tubulin Glcm oik47 oik50 oik13 oik43 oik2 oik21b oik17b oik14 oik9 od-thrombospondin3 chr 1 0 1000000 2000000 3000000 YSR 0 5000000 10000000 od-Calumenin1 Tis11a oik8 SoxBa oik15 oik23 Nkx2.3/5/6 od-b4-GalT od-Zipper od-IQGAP tubulin_beta_2c oik32 oik21a od-Noto9a actin ActnM1 Cyclin_Ba pax2/5/8b oik37 od-tensin od-Noto10 oik46 vas4 hox10 oik18 XSR Fig. 8 Genomic locations of various oikopleurid gene homologs in the OKI2018_I69. The genes are searchable by name and PubMed identifiers in the ZENBU genome browser. Hi-C library preparation 50 fully matured males were rinsed three times for 10 min each by transferring from well to well in a 6-well plate filled with 5 ml FASW. Rinsed animals were com- bined in a 1.5 ml microcentrifuge tube. Tissues were pel- leted for 10 min at 12,000 rpm and leftover FASW was discarded. A Hi-C library was then prepared by follow- ing the manufacturer’s protocol (Dovetail, 21,004). Briefly, tissues were cross-linked for 20 min by adding 1 ml 1× PBS and 40.5 μl 37% formaldehyde to the pellet. The tubes were kept rotating to avoid tissue settle dur- ing incubation. Cross-linked DNA was then blunt-end digested with DpnII (Dovetail) to prepare ends for ligation. After ligation, crosslinks were reversed, DNA was purified by AMPure XP Beads (Beckman, A63880) and quantified by Qubit 3 Fluorometer (Thermo Fisher Scientific, Q10210). The purified DNA was sheared to a size of 250–450 bp by sonication using a Covaris M220 instrument (Covaris, Woburn, MA) with peak power 50 W, duty factor 20, and cycles/burst 200 times for 65 s. DNA end repair, adapter ligation, PCR enrichment, and size selection were carried out by using reagents pro- vided with the kit (Dovetail, 21,004). Finally, the library was checked for quality and quantity on an Agilent 4200 TapeStation (Agilent, 5067–5584) and a Qubit 3 Fluorometer. The library was sequenced on a MiSeq (Illumina, SY-410-1003) platform using a 300 cycles V2 sequencing kit (Illumina, MS-102-2002), yielding 20,832, 357 read pairs. Contigs were joined into scaffolds based on long-range Hi-C Dovetail™data using Juicer v1.6 [49] and 3D de novo assembly (3D-DNA) [18] pipelines. The megabase- scale scaffolds were joined into pairs of chromosome arms based on their synteny with the OdB3 physical map (see below). The candidate assembly was visualized and reviewed with Juicebox Assembly Tools (JBAT) v1.11.08 [50]. Whole-genome alignment between OKI2018_I69 and OdB3 assemblies was performed using LAST v1066 [51]. The sequence of OdB3 linkage groups were recon- structed as defined in the Supplementary Fig. 2 (“Draft chromosome scale assembly based on scaffolds of the reference genome sequence”) in Denoeud et al. 2010 [9]. The resulting alignments were post-processed in R with a c u s t o m s c r i p t ( h t t p s : / / g i t h u b . Genome assembly bl Genome assembly was conducted with the Canu pipeline v1.8 [19] and 32.3 Gb (~ 221.69×) raw Nanopore reads (correctedErrorRate = 0.105, minReadLength = 1000). The resulting contig assembly was polished three times with Racon v1.2.1 [45] using Canu-filtered Nanopore reads. Nanopore-specific errors were corrected with Pilon v1.22 [46] using filtered 150-bp paired-end Illu- mina reads (~ 99.7×). Illumina reads were aligned to the Canu contig assembly with BWA v0.7.17 [47] and the corresponding alignments were provided as input to Pilon. Next, one round of the HaploMerger2 processing pipeline [48] was applied to eliminate redundancy in contigs and to merge haplotypes. Genome size estimation Genome size estimation Jellyfish [41] was used to generate k-mer count profiles for various values of k (17, 21, 25, 29, 33, 37, and 41) based on the genome-polishing Illumina MiSeq reads, with a maximum k-mer count of 1000. These k-mer profiles were subsequently used to estimate heterozygos- ity and genome size parameters using the GenomeScope web server [42]. Isolated genomic DNA used for long-reads on Nano- pore MinION platform were processed with the Ligation Sequencing Kit (Nanopore LSK109) according to manu- facturer’s protocol, loading approximately 200 ng total sample per R9.4 flow-cell. Raw signals were converted to sequence files with the Guppy proprietary software (model “template_r9.4.1_450bps_large_flipflop”, version 2.3.5). Approximately 5 ng was set aside for whole gen- ome amplification to perform sequencing on Illumina MiSeq platform, using the TruePrime WGA Kit (Sygnis, 370,025) according to manufacturer’s protocol. Magnetic bead purification (Promega, NG2001) was employed for all changes in buffer conditions required for enzymatic reactions and for final buffer suitable for sequencing sys- tem. Approximately 1 μg of amplified DNA was se- quenced by our core sequencing facility with a 600-cycle MiSeq Reagent Kit v3 (Illumina, MS-102-3003) following the manufacturer’s instructions. These Illumina runs were used for polishing and error checking of Nanopore runs. Filtering of Illumina MiSeq raw reads Before using at different steps, all raw Illumina reads were quality-filtered (−q 30, −p 70) and trimmed on both ends with the FASTX-Toolkit v0.0.14 [43]. The quality of the reads before and after filtering were checked with FASTQC v0.11.5 [44]. Read pairs that lacked one of the reads after the filtering were discarded in order to preserve paired-end information. Visualization and access Colours indicate genes from the same family 0 1000000 2000000 3000000 YSR 0 5000000 10000000 od-Calumenin1 Tis11a oik8 SoxBa oik15 oik23 Nkx2.3/5/6 od-b4-GalT od-Zipper od-IQGAP tubulin_beta_2c oik32 oik21a od-Noto9a actin ActnM1 Cyclin_Ba pax2/5/8b oik37 od-tensin od-Noto10 oik46 vas4 hox10 oik18 XSR Fig. 8 Genomic locations of various oikopleurid gene homologs in the OKI2018_I69. The genes are searchable by name and PubMed identifiers in the ZENBU genome browser. Colours indicate genes from the same family 0 10000000 and pooled. The pooled aqueous phase was subjected to a final extraction with chloroform and spun down as previously described. The aqueous fraction was then re- moved and precipitated by centrifugation with two vol- umes of cold ethanol and 10 μg/ml glycogen; washed with 1 ml of cold 70% ethanol and centrifuged once more as previously described. The resulting pellet was allowed to air-dry for 5 min and finally resuspended in molecular biology grade H2O for quantitation using a Qubit 3 Fluorometer (Thermo Fisher Scientific, was washed with 1 ml of 70% cold-ethanol, centrifuged at 14,000 rpm 4 °C for 5 min and air dried briefly before resuspension in 200 μl 100 mM NaCl, 25 mM EDTA, 0.5% SDS and 10 μg/ml proteinase K. The lysates were incubated overnight at 50 °C. The next morning, the total nucleic acids were first extracted and then back- extracted once more with chloroform:phenol (1:1). Or- ganic and aqueous phases were resolved by centrifuga- tion at 13,000 rpm for 5 min for each extraction; both first and back-extracted aqueous phases were collected Page 13 of 18 Bliznina et al. BMC Genomics (2021) 22:222 Page 13 of 18 Page 13 of 18 Q32850), and the integrity of the genomic DNA was val- idated using Agilent 4200 TapeStation (Agilent, 5067– 5365). Gene prediction and annotation p Gene models were predicted using AUGUSTUS v3.3 [62]. AUGUSTUS was trained following the Hoff and Stanke protocol [24] with the initial RNA-Seq reads and transcriptome assembly used as intron and exon hints, correspondingly. Transcript models were generated with the PASA pipeline v20140417 [63] using BLAT v36 and GMAP v2018-02-12 to align transcripts to the genome. RNA-Seq reads were mapped to the genome with STAR v2.0.6a [64]. Running AUGUSTUS using hints resulted in a set of 17,277 protein-coding genes and 18,811 tran- script models. Chromosomal coordinates were ported to our final assembly using the Liftoff tool [65] filtering out 17 genes and corresponding transcripts. The quality of the predicted gene models was assessed with BUSCO. Hi-C library preparation c o m / o i s t / oikGenomePaper) and visualized using the R package “networkD3” (“sankeyNetwork” function). The color scheme for chromosomes was adopted from R Package RColourBrewer, “Set2”. The final assembly was checked for contamination by BLAST searches against the NCBI non-redundant Page 14 of 18 Page 14 of 18 Bliznina et al. BMC Genomics (2021) 22:222 were placed in 100 μl of fresh FASW and allowed to spawn naturally. Staged embryos were initiated by gently mixing 10 μl of the spawned male sperm to the awaiting eggs in FASW at 23 °C. Generation 30 developing em- bryos at 1 h and 3 h post-fertilization were visually veri- fied by dissecting microscope and collected as a pool for the mixed staged embryo time point. Immature adults at generation 31 and sexually differentiated adults at gener- ation 30 were used for the two adult staged time points. All individuals for each time point were pooled and washed with FASW three times for 10 min. Total RNA was extracted and isolated with RNeasy Micro Kit (Qia- gen, 74,004) and quantitated using Qubit 3 Fluorometer (Thermo Fisher Scientific, Q10210). Additional quality control and integrity of isolated total RNA was checked using Agilent 4200 TapeStation (Agilent, 5067–5576). Further processing for mRNA selection was performed with Oligo-d(T)25 Magnetic Beads (NEB, E7490) and the integrity of the RNA was validated once more with Agilent 4200 TapeStation (Agilent, 5067–5579). Adapters for the creation of DNA libraries for the Illu- mina platform were added per manufacturer’s guidance (NEB, E7805) as were unique indexed oligonucleotides (NEB, E7600) to each of the three staged samples. Each cDNA library was sequenced paired-end with a 300- cycle MiSeq Reagent Kit v2 (Illumina, MS-102-2002) loaded at approximately 12 pM. sequence database. 12 smaller scaffolds were found to have strong matches to bacterial DNA (Suppl. Table 2), as well as possessing significantly higher Nanopore se- quence coverage (> 500×) than the rest of the assembly, and were therefore removed from the final assembly. The completeness and quality of the assembly were checked with QUAST v5.0.2 [52] and by searching for the set of 978 highly conserved metazoan genes (OrthoDB version 9.1) [23] using BUSCO v3.0.2 [21, 22]. The --sp option was set to match custom AUGUSTUS parameters [24] trained using the Trinity transcriptome assembly (see below) split 50% / 50% for training and testing. Repeat masking and transposable elements A custom library of repetitive elements (RE) present in the genome assembly was built with RepeatModeler v2.0.1 that uses three de novo repeat finding programs: RECON v1.08, RepeatScout v1.0.6 and LtrHarvest/Ltr_ retriever v2.8. In addition, MITE-Hunter v11–2011 [53] and SINE_Finder [54] were used to search for MITE and SINE elements, respectively. The three libraries were pooled together as input to RepeatMasker v4.1.0 [55] to annotate and soft-mask these repeats in the genomic se- quence. Resulting sets of REs were annotated by BLAST searches against RepeatMasker databases and sequences of transposable elements published for different oiko- pleurids [10]. After quality assessment and data filtering (see Filter- ing of Illumina MiSeq raw reads), Illumina RNA-Seq reads were pooled together and de novo assembled with Trinity v2.8.2 [59]. Redundancy in the transcriptome as- sembly was removed by CD-HIT v4.8.1 [60] with a cut- off value of 95% identity. The quality and completeness of the transcriptome assembly was verified with rna- QUAST v1.5.1 [61] and BUSCO. Tandem repeats were detected using two different pro- grams, tantan [56] and ULTRA [57] using two different maximal period lengths (100 and 2000). Version 23 of tantan was used with the parameters -f4 (output repeats) and -w100 or 2000 (maximum period length). ULTRA version 0.99.17 was used with -mu 2 (minimum number of repeats) -p 100 or 2000 (maximum period length) and -mi 5 -md 5 (maximum consecutive insertions or dele- tions). ULTRA detected more tandem repeats than tan- tan, but its predictions include more than 90% of tantan’s. Both tools detected O. dioica’s telomeric tan- dem repeat sequence, which is TTAGGG as in other chordates [58]. Developmental staging, isolation and sequencing of mRNA, transcriptome assembly Mixed stage embryos, immature adults (3 days after hatching) and adults (4 days after hatching) were col- lected separately from our on-going laboratory culture for RNA-Seq analysis. Eggs were washed three times for 10 min by moving eggs along with micropipette from well to well in a 6-well dish each containing 5 ml of FASW and left in a fresh well of 5 ml FASW in the same dish. These were stored at 17 °C and set aside for fertilization. Matured males, engorged with sperm, were also washed 3 times in FASW. Still intact mature males A draft annotation of the mitochondrial genome was obtained by submitting the corresponding scaffold (chr_ Un12) as input to the MITOS2 mitochondrial genome Bliznina et al. BMC Genomics (2021) 22:222 Page 15 of 18 Page 15 of 18 annotation server [66] (accessed May 28, 2020) with the ascidian mitochondrial translation table specified [9, 32]. annotation server [66] (accessed May 28, 2020) with the ascidian mitochondrial translation table specified [9, 32]. regions using LAST’s command maf-convert -J 200000. We then collated syntenic region blocks (sequence ontology term SO:0005858) that map to the same se- quence landmark (chromosome, scaffold, contigs) on the query genome with a distance of less than 500,000 bp with the custom script syntenic_regions.sh (https:// github.com/oist/oikGenomePaper). In contrast to the “match” regions, the syntenic ones are not necessarily colinear and can overlap with each other. The GFF3 file was then uploaded to the ZENBU genome browser. Nanopore read realignments Nanopore reads were realigned to the genome with the LAST software [51] as in the whole-genome alignments above. FASTQ qualities were discarded with the option –Q0 of lastal. Optimal split alignments were calculated with last-split. Alignment blocks belonging to the same read were joined with maf-convert -J 1e6 and the cus- tom script syntenic_regions_stranded.sh. The resulting GFF3 files were loaded in the ZENBU genome browser to visualize the alignments near gap regions in order to check for reads spanning the gaps. Detection of coding RNAs A translated alignment was used to detect known O. dioica genes available from GenBank using the TBLASTN software [67] with the options -ungapped -comp_based_stats F to prevent O. dioica’s small introns from being incorporated as alignment gaps, and -max_ intron_length 100,000 to reflect the compactness of O. dioica’s genome. The best hits were converted to GFF3 format using BioPerl’s bp_search2gff program [68] be- fore being uploaded to the ZENBU genome browser [39]. For some closely related pairs of genes that gave ambiguous results with that method, we searched for the protein sequence in our transcriptome assembly with TBLASTN, located the genomic region where the best transcript model hit was aligned, and selected the hit from the original TBLASTN search that matched this region. We summarized our results in Suppl. Table 6. For both searches, we used an E-value filter of 10−40. Genes marked as not found in the table might be present in the genome while failing to pass the filter. Analysis of sequence properties across chromosome-scale scaffolds To validate the results of cmscan on rRNAs, genomic re- gions were screened with a nucleotide BLAST search using the O. dioica isolate MT01413 18S ribosomal RNA gene, partial sequence (GenBank:KJ193766.1). 200- kbp windows surrounding the hits where then analysed with the RNAmmer 1.2 web service [69]. RNAmmer did not detect the 5.8S RNA, but we could confirm its pres- ence by a nucleotide BLAST search using the AF158726.1 reference sequence. The loci containing the 5S rRNA (AJ628166) and the spliced leader RNA (AJ628166) were detected with the exonerate 2.4 soft- ware [70], with its affine:local model and a score thresh- old of 1000 using the region chr1:8487589–8,879,731 as a query. Each chromosome-scale scaffold was separated into win- dows of 50 kbp and evaluated for GC content, repeat content, sequencing depth, and the presence of DpnII restriction sites. For chr 1, chr 2, and the PAR, windows corresponding to long and short chromosome arms were separated based on their positioning relative to a central gap region (chr 1 short arm: 1–5,191,657 bp, chr 1 long arm: 5,192,156-14,533,022 bp; chr 2 short arm: 1–5,707, 009, chr 2 long arm: 5,707,508-16,158,756 bp; PAR short arm: 1–6,029,625 bp, PAR long arm: 6,030,124-17,092, 476). Since none of our assemblies or sequencing reads spanned both the PAR and either sex-specific chromo- some, the X and Y chromosomes were excluded from this analysis. For each of GC content, sequencing depth, repeat content, gene count, and DpnII restriction sites, the significance of the differences between long and short arms was assessed with Welch’s two-sided T test as well as a nonparametric Mann-Whitney test imple- mented in R (Suppl. Table 3). The results of the two tests were largely in agreement, but groups were only in- dicated as significantly different if they both produced significance values below 0.05 (p < 0.05). Abbreviations BUSCOs: Benchmarking Universal Single-Copy Orthologs; chr 1: Autosomal chromosome 1; chr 2: Autosomal chromosome 2; kbp: Kilobase pairs; LGs: Linkage groups; Mbp: Megabase pairs; PAR: Pseudo-autosomal regions; XSR: X-specific region; YSR: Y-specific region Whole-genome alignments Pairs of genomes were mapped to each other with the LAST software [51] version 1066. When indexing the reference genome, we replaced the original lowercase soft masks with ones for simple repeats (lastdb -R01) and we selected a scoring scheme for near-identical matches (−uNEAR). Substitution and gap frequencies were determined with last-train [71], with the alignment options -E0.05 -C2 and forcing symmetry with the op- tions --revsym --matsym --gapsym. 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https://openalex.org/W3136470808	https://www.researchsquare.com/article/rs-121170/latest.pdf	English	null	Motivating antibiotic stewardship in Bangladesh: identifying audiences and target behaviours using the behaviour change wheel	BMC public health	2,021	cc-by	11,697	Motivating antibiotic stewardship in Bangladesh: Identifying audiences and target behaviours using the Behaviour Change Wheel Leanne Unicomb (  leanne.unicomb60@gmail.com ) International Centre for Diarrhoeal Disease Research Motivating antibiotic stewardsh Identifying audiences and targe the Behaviour Change Wheel Leanne Unicomb (  leanne.unicomb60@gmail.com ) International Centre for Diarrhoeal Disease Research Fosiul Nizame International Centre for Diarrhoeal Disease Research Mohammad Ro¦ Uddin International Centre for Diarrhoeal Disease Research Papreen Nahar University of Sussex Patricia Lucas University of Bristol Nirnita Khisa International Centre for Diarrhoeal Disease Research S. M. Salim Akter International Centre for Diarrhoeal Disease Research Mohammad Aminul Islam Washington State University Mahbubur Rahman International Centre for Diarrhoeal Disease Research Emily Rousham Loughborough University Research Article K d I t ti tibi ti t d hi B l d h Motivating antibiotic stewardsh Identifying audiences and targe the Behaviour Change Wheel Leanne Unicomb (  leanne.unicomb60@gmail.com ) International Centre for Diarrhoeal Disease Research Fosiul Nizame International Centre for Diarrhoeal Disease Research Mohammad Ro¦ Uddin International Centre for Diarrhoeal Disease Research Papreen Nahar University of Sussex Patricia Lucas University of Bristol Nirnita Khisa International Centre for Diarrhoeal Disease Research S. M. Salim Akter International Centre for Diarrhoeal Disease Research Mohammad Aminul Islam Washington State University Mahbubur Rahman International Centre for Diarrhoeal Disease Research Emily Rousham Loughborough University Research Article K d I t ti tibi ti t d hi B l d h Research Article Keywords: Intervention, antibiotic stewardship, Bangladesh, antibiotic resistance Posted Date: December 17th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-121170/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Motivating antibiotic stewardship in Bangladesh: Identifying audiences and target behaviours using the Behaviour Change Wheel Motivating antibiotic stewardship in Bangladesh: Identifying audiences and target behaviours using the Behaviour Change Wheel Leanne E. Unicomb1, Fosiul Alam Nizame1, Mohammad Rofi Uddin1, Papreen Nahar2, Patricia J. Lucas3, Nirnita Khisa1, S. M. Salim Akter1, Mohammad Aminul Islam4, Mahbubur Rahman1, Emily K. Rousham5 1. International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b). 1. International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b). 1. International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b). 2. Department of Global Health and Infection, Brighton and Sussex Medical School, Sussex University, UK 2. Department of Global Health and Infection, Brighton and Sussex Medical School, Sussex University, UK 2. Department of Global Health and Infection, Brighton and Sussex Medical School, Sussex University, UK 3. School for Policy Studies, University of Bristol, UK. E-mail: Patricia.lucas@bristol.ac.uk 4 Paul G. Allen School for Global Animal Health, Washington State University, Pullman, Washington, USA. 3. School for Policy Studies, University of Bristol, UK. E-mail: Patricia.lucas@bristol.ac.uk 4 Paul G. Allen School for Global Animal Health, Washington State University, Pullman, Washington, USA. 5. Centre for Global Health and Human Development, School of Sport, Exercise and Health Sciences, Loughborough University, UK. 1 ABSTRACT Background: South Asia is a hotspot for antimicrobial resistance due largely to over-the-counter antibiotic sales for humans and animals, a lack of compliance with policy among healthcare providers, high population density and high infectious disease burden. This paper describes development of social and behavioural change communication (SBCC) to increase appropriate use of antibiotics. Methods: We used formative research to explore contextual drivers of antibiotic sales, purchase, consumption/use and promotion among four groups: 1) households, 2) drug shop staff, 3) qualified physicians and 4) pharmaceutical companies/medical representatives. We used formative research findings and an intervention design workshop with stakeholders to select target behaviours, to prioritize audiences and develop SBCC messages, in consultation with a creative agency, and through pilots and feedback. The behaviour change wheel was used to summarise findings. Results: Workshop participants identified behaviours considered amenable to change for all four groups. Household members and drug shop staff were prioritized as target audiences, both of which could be reached at drug shops. Among household members, there were two behaviours to change; suboptimal health seeking and ceasing antibiotic courses early. Thus, SBCC target Results: Workshop participants identified behaviours considered amenable to change for all four groups. Household members and drug shop staff were prioritized as target audiences, both of which could be reached at drug shops. Among household members, there were two behaviours to change; suboptimal health seeking and ceasing antibiotic courses early. Thus, SBCC target behaviours included: seek qualified physician consultations; ask whether the medicine provided is an antibiotic; ask for instructions on use and timing. Among drug shop staff, several antibiotic dispensing practices needed to change. SBCC target behaviours included: asking customers for prescriptions; referring them to qualified physicians and increasing customer awareness by instructing that they were receiving antibiotics to take as a full course. behaviours included: seek qualified physician consultations; ask whether the medicine provided is an antibiotic; ask for instructions on use and timing. Among drug shop staff, several antibiotic dispensing practices needed to change. SBCC target behaviours included: asking customers for prescriptions; referring them to qualified physicians and increasing customer awareness by instructing that they were receiving antibiotics to take as a full course. 2 2 Conclusions: We prioritized drug shops for intervention delivery to drug shop staff and their customers to improve antibiotic stewardship. ABSTRACT Knowledge deficits among these groups were notable and considered amenable to change using a SBCC intervention addressing improved health seeking behaviours, improved health literacy on antibiotic use, and provision of information on policy governing shops. Further intervention refinement should consider using participatory methods and should address the impact on profit and livelihoods for drug shop staff for optimal compliance. Keywords: Intervention, antibiotic stewardship, Bangladesh, antibiotic resistance Keywords: Intervention, antibiotic stewardship, Bangladesh, antibiotic resistance 3 BACKGROUND: South and South East Asia are hotspots for antimicrobial resistance (AMR) [1] and newly evolved AMR organisms from other regions have spread rapidly across the world [2]. South and South East Asia are hotspots for antimicrobial resistance (AMR) [1] and newly evolved AMR organisms from other regions have spread rapidly across the world [2]. Contributing factors include availability of cheap, locally manufactured over-the-counter antibiotics for humans and animals, a lack of compliance with standards among healthcare providers, weak regulatory system, and high population density [3]. While per capita antibiotic consumption rates in low- and middle-income countries (LMICs) have been lower than those in high income countries, these are increasing dramatically in line with increases in gross domestic consumption [4]. Human antibiotic consumption rates are predicted to increase globally by 200% from 2015-2030 [5]. In Bangladesh, community members access antibiotics for themselves and their animals through a healthcare system that is pluralistic and less institution-based than elsewhere [6, 7]. Unqualified healthcare providers are a major source of health care for the poor and disadvantaged [8, 9]. These providers can include drug shop staff that have no recognized qualification; it is estimated that Bangladesh has 200,000 retail drug shops and approximately 50% of those are unlicensed [10]. While little is known about quantities of antibiotics dispensed through the various channels in the healthcare system, a recent study reported that 29% of antibiotic prescriptions came from qualified doctors and 63% from unqualified healthcare providers [11]. Recent studies conducted by our group suggest that there is a considerable volume of antibiotics dispensed without a prescription, often by unqualified providers [12]. Antibiotics administered by these groups are shorter courses and not appropriate for the illness [13], which can contribute to AMR. Antibiotics administered by these groups are shorter courses and not appropriate for the illness [13], which can contribute to AMR. 4 From the supply-side, previous studies on antibiotic dispensing in Bangladesh have reported polypharmacy, detected among 25% of prescriptions from rural hospital outpatient 4 clinic doctors [11]. Overprescribing, using unnecessarily expensive drugs and dispensing drugs without a prescription are common [10, 14]. Additionally, drug sellers and healthcare providers are exposed to aggressive marketing strategies, especially through pharmaceutical company representatives [15]. From the demand side, limited data are available on household-level antibiotic use for humans and their animals/livestock [11, 16]. BACKGROUND: Bangladesh has taken steps to address poor compliance with accepted standards for antibiotic prescription with the launch of the Bangladesh Pharmacy Model Initiative (BPMI) for all medication categories in 2016, which also forms a key part of the national action plan on AMR. It includes standards for drug outlet personnel, premises, dispensing, storage, hygiene, record keeping, disposal and allowable products. To date there have been 193 model pharmacies and 154 model medicine shops developed and 154 model medicine shops developed (http://www.dgda.gov.bd/index.php/pharmacies/whole-sale-pharmacy-view-2/319-list-of-model- pharmacy; http://www.dgda.gov.bd/index.php/pharmacies/whole-sale-pharmacy-view-2/320-list- of-model-medicine-shop; accessed December, 2020). The BPMI requires retail outlets to provide medications only to customers with a prescription, dispensed by staff with pharmacy qualifications and training (http://www.dgda.gov.bd/index.php/2013-03-31-05-16-29/guidance- documents/175-guideline-for-model-pharmacy). The National Drug Policy, 2016 [17] provides legal requirements on drug dispensing: it “prohibits sales and distribution of drugs without prescription from registered physician to ensure rational use of drugs” and “Retail sales of drugs is prohibited without prescription by registered physicians/ veterinarians other than the over the counter drugs”. pharmacy; http://www.dgda.gov.bd/index.php/pharmacies/whole-sale-pharmacy-view-2/320-list- of-model-medicine-shop; accessed December, 2020). The BPMI requires retail outlets to provide medications only to customers with a prescription, dispensed by staff with pharmacy Antibiotic stewardship, programmes to optimize antibiotic dispensing and consumption, have been predominantly implemented in high income countries and in hospitals [18]. A review 5 of programmes/interventions to reduce antibiotic prescribing in LMICs reported that the majority (n=36) took place in hospitals and 9 in pharmacies, with mixed success [19]. In the review, authors noted that few stewardship interventions included more than one healthcare provider group or setting [19]. In Tanzania, a programme to improve antibiotic stewardship targeted drug shops as part of accreditation in as many as 9,000 premises which included training staff in appropriate antibiotic dispensing [20]. Ten years after the programme began audit studies have detected residual needless antibiotic dispensing; however, this was only among one-third of the shops [21]. Understanding the behaviours, service and economic priorities of healthcare providers, and the needs of consumers is therefore central to developing an effective strategy to engage in antimicrobial stewardship to reduce antibiotic resistance [12, 22]. This study is part of a larger project that aimed to inform government policy and identify pathways to behaviour change among groups from the antibiotic supply and demand sides [23]. The specific objective of this paper is to describe the development of social and behavioural change communication (SBCC) messages aimed to increase appropriate use of antibiotics. BACKGROUND: We explored antibiotic sales, Understanding the behaviours, service and economic priorities of healthcare providers, and the needs of consumers is therefore central to developing an effective strategy to engage in antimicrobial stewardship to reduce antibiotic resistance [12, 22]. This study is part of a larger project that aimed to inform government policy and identify pathways to behaviour change among groups from the antibiotic supply and demand sides [23]. The specific objective of this paper is to describe the development of social and behavioural change communication (SBCC) messages aimed to increase appropriate use of antibiotics. We explored antibiotic sales, purchase, use/consumption, and promotion to identify contextual drivers among four groups: 1) households, 2) drug shop staff, 3) qualified physicians and 4) pharmaceutical companies/medical representatives. We used formative study findings from this research in conjunction with outcomes from an intervention design workshop with stakeholder to identify target behaviours, and in consultation with a creative agency, to prioritize audiences and refine SBCC messages. purchase, use/consumption, and promotion to identify contextual drivers among four groups: 1) households, 2) drug shop staff, 3) qualified physicians and 4) pharmaceutical companies/medical representatives. We used formative study findings from this research in conjunction with outcomes from an intervention design workshop with stakeholder to identify target behaviours, and in consultation with a creative agency, to prioritize audiences and refine SBCC messages. purchase, use/consumption, and promotion to identify contextual drivers among four groups: 1) households, 2) drug shop staff, 3) qualified physicians and 4) pharmaceutical companies/medical representatives. We used formative study findings from this research in conjunction with METHODS: The study aim was development of SBCC to increase appropriate use of antibiotics in Bangladesh and to do this we collected data for integration into the first four steps of the 6 behaviour change wheel, used to formulate behaviour change interventions [24, 25]. These steps include 1) defining the problem in behavioural terms, identifying who performs the behaviours and listing all other behaviours that might influence the problem behaviour; 2) selecting the target behaviour, 3) specifying the target behaviour, and 4) identifying what needs to change [25]. Formative data collection provided information for step 1 [12, 22] on current key behaviours. Following the formative research, steps 2 through 4 were undertaken through an intervention design workshop and the co-creation of intervention resources with a creative agency. Formative study The objective of the formative study was to a) determine drivers of household decision making on healthcare consultations and antibiotic purchase and consumption; b) determine practices among qualified and unqualified healthcare providers (doctors, drug shops); and examine interactions of drug shops and doctors with pharmaceutical company representatives. We selected one rural community in Tangail district and one urban community in Gazipur district. Sites were selected where households had access to a range of drug shops and health care facilities. Formative data collection and data analysis details have been described previously [12, 23]. In brief, between May 2017 to January 2018 data were collected through face-to-face and in-depth interviews with groups that were identified as key actors in community-level antibiotic dispensing, use, consumption, and promotion based on review of existing literature. The key actors for data collection comprised household members (n=48); drug shop staff who were either unqualified (n=13) or had up to 12 months training (n=14); qualified physicians in human and 7 7 veterinary medicine (n=12); auxiliary healthcare providers (n=7) and key informants including pharmaceutical company representatives and non-government organisations (e.g. Bangladesh Druggist and Chemist Association) (n=13). Interviews were audio-recorded and transcribed in Bangla. A third of all transcripts were translated in full into English by native Bangla-speaking researchers. The remaining Bangla transcripts were coded in Bangla, and then translated into English and shared with the research team. Intervention design workshop The objective of the workshop was to obtain stakeholder expert input in selection of key behaviours and audiences for an intervention. In conjunction with a local creative agency, Visual Communications Ltd. (VISCOM, https://www.viscombd.com), we invited 60 people to participate in a one day workshop in February, 2018 including stakeholders from the Government Directorate General of Health Services, Directorate General of Drug Administration and the Department of Livestock Services; pharmaceutical industry and medical representatives; the Bangladesh Chemist and Druggist Association, local non-governmental organisations (NGOs), drug shop owners, implementing partners, and the research team; 33 attended. We shared emerging findings from the formative studies with the workshop participants relating to healthcare seeking behaviours and antibiotic supply, dispensing and consumption. Based on research findings and input from the workshop participants the study team, in association with the design agency, discussed different specific content, target audiences and behaviour options. Workshop attendees participated in group work focusing on each of the four possible target audiences (householders, drug shop staff, qualified physicians, pharmaceutical companies/medical representatives) to a) define the problem in behavioural terms (who performs behaviours, other behaviours that might influence the problem behaviour); b) select target 8 8 behaviour, specifying target behaviour, identify what needs to change and c) propose SBCC messages. Each group had a facilitator from the research team and a rapporteur who recorded discussions on template PowerPoint slides which were presented to workshop participants. Suggestions provided during the group work feedback session were recorded. The workshop concluded with a voting exercise where each attendee was provided with stickers to vote for the first and second priority among the four groups/ target audiences. Working with a creative agency to develop key messages in collaboration with the research VISCOM collaborated with the research team to collate the suggested target behaviours we wished to change and supporting messages from the workshops. Draft intervention resources were designed in English and Bangla and the research team provided feedback. Further feedback was obtained through a display session in the Environmental Interventions Unit at the International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b) to solicit suggestions from experienced researchers who have worked on a wide range of SBCC interventions. After incorporating initial feedback, intervention resources were piloted in two drug shops each from one rural and one urban setting (total 4) in July 2018. Interviews with one drug shop staff and one customer per drug shop were used to explore understanding of messages and anticipated impact of the materials on behaviour. This feedback was used to revise intervention materials. Revised intervention resources were subsequently distributed to 95 drug shops in September 2018 from the same two geographic areas and remained for a month. From the 95 pharmacies, 50 were randomly selected and interviews conducted with one drug shop staff and one customer from each drug shop one month after intervention resource delivery. Interviews 9 9 followed an open-ended questionnaire like that used in the earlier pilot among 4 shops, collected demographic information, and sought suggested further changes on appearance or content. followed an open-ended questionnaire like that used in the earlier pilot among 4 shops, collected demographic information, and sought suggested further changes on appearance or content. Data analysis Data collected from formative studies were analysed using framework analysis, as described previously [12]. In addition to guiding data collection, data were mapped using the Behaviour Change Wheel [24] for steps 1 to 4, as described by Munir et al [25]; these are mapped against study components in Figure 1. For steps 1-3 data analysed from formative studies and data collected during the workshop were summarised. For step 4, for the prioritised audiences, we used the Capability, Opportunities, Motivation-Behaviour (Com-B) framework [24] to summarise data to provide detail on the behaviours that need to change. 10 Figure 1: Schematic of study components Behaviour change wheel components Figure 1: Schematic of study components Behaviour change wheel components Study components Findings from formative study; 4 population groups Step 1 Define the problem in behavioural terms Design Workshop: presentation of summary data and group work Suggest intervention messages Identify priority audiences Step 2 Select the target behaviour Step 3 Specify the target behaviour Working with a creative agency to develop key messages: for household members and drug shop staff Step 4 Identify what needs to change (Com-B model) for household members and drug shop staff Study components Findings from formative study; 4 population groups Step 1 Define the problem in behavioural terms Step 2 Select the target behaviour Step 4 Identify what needs to change (Com-B model) for household members and drug shop staff Step 4 Identify what needs to change (Com-B model) for household members and drug shop staff 11 Defining the problem in behavioural terms among four groups from formative studies (Step 1) Defining the problem in behavioural terms among four groups from formative studies (Step 1) Contextual drivers of antibiotic use among household members, and inappropriate prescribing and dispensing practices of healthcare professionals and drug shop staff have been published [12, 22]. Among household members, an important contextual driver was that most did not what know an antibiotic was or what antibiotics were for. Household members and healthcare providers reported that antibiotics were stopped when symptoms disappeared. Antibiotics were often purchased by proxies for ill household members/patients. Underage children and adolescents were also able to purchase antibiotics from drug shops. Drug shop staff, regardless of training and qualification, in addition to dispensing antibiotics prescribed by qualified physicians, dispensed antibiotics as they would over-the-counter medications. Counter to common assumptions, households did not report storing or re-using old antibiotics. A contextual driver of antibiotic dispensing among drug shop staff was that they regularly sold prescription drugs including antibiotics without a government license, in conflict with government policy. Drug shop staff advised and dispensed drugs to patients and their proxies, who were usually family members. Unqualified drug shop staff reported that they followed antibiotic prescribing patterns of qualified physicians, referred to as elite doctors/boro (big) doctors. Patients were more likely to consult qualified physicians for more severe diseases, or after initial treatment had failed. Travel costs and distance to health facilities or clinics were barriers to seeking earlier consultation with qualified professionals [12]. However, doctors were reported to give little time during consultation, prescribe drugs including antibiotics over the 12 12 telephone and patients perceived that the additional costs of consulting a doctor or undergoing recommended tests were to enhance profits and were often viewed as unnecessary [22]. telephone and patients perceived that the additional costs of consulting a doctor or undergoing recommended tests were to enhance profits and were often viewed as unnecessary [22]. Pharmaceutical companies were reported to provide incentives to doctors for prescribing their company’s antibiotics. In contrast with qualified doctors and drug shop staff, pharmaceutical representatives were fully aware of the BPMI policy and had a thorough understanding of antibiotic resistance. Pharmaceutical companies were reported to provide incentives to doctors for prescribing their company’s antibiotics. In contrast with qualified doctors and drug shop staff, pharmaceutical representatives were fully aware of the BPMI policy and had a thorough • Consult qualified doctors or health workers • Ask the healthcare provider if they have been given antibiotics and why • Buy full course of antibiotic and continue according to the prescription • Ask about dose frequency and duration/instruction on use • Choose a pharmacy that maintains quality drugs Selecting and specifying target key behaviours (Steps 2 and 3) Based on data from formative studies on contextual drivers, participants in the intervention design workshop selected and specified behaviours that were amenable to change (Table 1). Selecting and specifying target key behaviours (Steps 2 and 3) 13 Table 1: Behaviour change and intervention components for the four groups from the intervention design workshop Behaviours Proposed intervention messages* Current practice/contextual drivers* Selected behaviours to change Table 1: Behaviour change and intervention components for the four groups from the intervention design w Behaviours Proposed interventi Current practice/contextual drivers* Selected behaviours to change Households • Don't know what an antibiotic is • Don't know what an antibiotic is for • Drug shops common first point for health advice • Purchase drug without prescription/ without doctor’s advice; sometimes ask for antibiotics by name • Underage purchaser and proxy for patient • Use peer group prescribed antibiotic • Antibiotics are stopped when symptoms disappear or if when patients consider that they don't work • Don't reuse drugs for subsequent illnesses • Have high level knowledge of medicine expiry • Consult qualified doctors or health workers • Ask the healthcare provider if they have been given antibiotics and why • Buy full course of antibiotic and continue according to the prescription • Ask about dose frequency and duration/instruction on use • Choose a pharmacy that maintains quality drugs • Consult with regis prescription; (who ar patients know if doc • Buy full course of the course; Follow y recover • If you don’t follow cost you more ultima • If you suffer from consult your doctor i • Buy medicines fro Drug shops • Most common source of medical advice; minor illness • Dispensing without government license • Unqualified staff dispensing antibiotics, with short (6 weeks) or no training • Dispense antibiotic without seeing the patient/animal, prescribe over phone • Follow elite doctor prescriptions when asked for advice • Do not always give a name to the type of illness • Often advise and sell drugs for livestock • Ask for prescription before dispensing antibiotics • Refer customers to qualified doctors • Increase awareness among patients; tell purchasers which is an antibiotic- stress importance of taking full course • Recruit qualified staff • Do not change the medicine when prescribed by a qualified physician • Stop selling antib registered doctor’s p • Tell customer tha antibiotic • Dispense full anti instructions for cons • Unnecessary antib • Check expiry date drugs Table 1: Behaviour change and intervention components fo Behaviours Current practice/contextual drivers* Selected be Households • Don't know what an antibiotic is • Don't know what an antibiotic is for • Drug shops common first point for health advice • Purchase drug without prescription/ without doctor’s advice; sometimes ask for antibiotics by name • Underage purchaser and proxy for patient • Use peer group prescribed antibiotic • Antibiotics are stopped when symptoms disappear or if when patients consider that they don't work • Don't reuse drugs for subsequent illnesses • Have high level knowledge of medicine expiry • Consult workers • Ask the have been g • Buy full continue ac • Ask abo duration/in • Choose quality dru Drug shops • Most common source of medical advice; minor illness • Dispensing without government license • Unqualified staff dispensing antibiotics, with short (6 weeks) or no training • Dispense antibiotic without seeing the patient/animal, prescribe over phone • Follow elite doctor prescriptions when asked for advice • Do not always give a name to the type of illness • Often advise and sell drugs for livestock • Ask for dispensing • Refer cu • Increase tell purchas stress impo • Recruit q • Do not c prescribed Proposed intervention messages** • Consult with registered physician for prescription; (who are they and how do patients know if doctor is registered) • Consult with registered physician for prescription; (who are they and how do patients know if doctor is registered) • Consult qualified doctors or health workers • Drug shops common first point for health advice • Ask the healthcare provider if they have been given antibiotics and why p g • Buy full course of drugs and complete the course; Follow your prescription to recover • Buy full course of antibiotic and continue according to the prescription • If you don’t follow instructions, it will cost you more ultimately • Ask about dose frequency and duration/instruction on use • Choose a pharmacy that maintains quality drugs • If you suffer from any adverse effects, consult your doctor immediately • Buy medicines from reputable company • Stop selling antibiotic without registered doctor’s prescription • Tell customer that you are providing an antibiotic • Dispense full antibiotic course and give instructions for consumption • Unnecessary antibiotic use is harmful • Check expiry date before dispensing drugs • Stop selling antibiotic without registered doctor’s prescription g p p • Tell customer that you are providing an antibiotic • Refer customers to qualified doctors • Increase awareness among patients; tell purchasers which is an antibiotic- stress importance of taking full course • Recruit qualified staff • Dispense full antibiotic course and give instructions for consumption • Unnecessary antibiotic use is harmful • Do not change the medicine when prescribed by a qualified physician • Check expiry date before dispensing drugs • Do not always give a name to the type of illness • Often advise and sell drugs for livestock 14 • Some drug sellers know antibiotic generation no. p Pharmaceutical companies/medical representatives • Have regional offices and numerous staff for product promotion and distribution • Are aware of government policy • Have thorough understanding of antibiotic resistance • Purported provision of incentives to doctors Companies • Modify business strategy • Deliver quality training • Package antibiotics according to course • Include warning messages on packet • Promote full courses of antibiotic for better health • Ensure the proper use of antibiotics • Ensure profit, protect yourself and others • Do quality business for community Selecting and specifying target key behaviours (Steps 2 and 3) • Few drug sellers are familiar with antibiotic resistance” • No clear idea on consumer rights • Qualified physicians • See patients with more severe disease • Give little time during consultation • Prescribe over telephone • Overprescribe antibiotics and provide unnecessary tests, to make profit • Prescribe to keep patients happy • Prescribe poor quality antibiotics • Follow prescriptions of senior/renowned doctors • Limited knowledge of antibiotic generations • Don’t request tests before prescribing antibiotics/prescribe for viral infections • Don’t counsel on antibiotic use or the consequences of overuse • Not up to date on current literature • Take incentives from pharmaceutical companies • Motivated by medical representatives to prescribe antibiotics • Do not provide antibiotic prescription when not necessary; consult recent recommendation information • Resist prescribing later generation antibiotics • Reinforce that the full course is important to complete • Prescribe only when the patient presents at the consultation • Follow knowledge an date information on anti • Do not provide antibi when not necessary • Provide instructions f include the need for a fu Pharmaceutical companies/medical representatives • Have regional offices and numerous staff for product promotion and distribution • Are aware of government policy • Have thorough understanding of antibiotic resistance • Purported provision of incentives to doctors Companies • Modify business strategy • Deliver quality training • Package antibiotics according to course • Include warning messages on packet • Promote full courses better health • Ensure the proper use • Ensure profit, protect others • Do quality business f • Follow knowledge and practice on up to date information on antibiotic resistance • Follow knowledge and practice on up to date information on antibiotic resistance • Do not provide antibiotic prescription when not necessary • Provide instructions for consumption, include the need for a full course • Ensure the proper use of antibiotics • Ensure profit, protect yourself and others 15 to prescribe, drug shop staff to sell • Range of product quality • Representatives have monthly visit quotas for doctors to promote products, distribute sample medicines to physicians • Provide products to drug shops sometimes on credit, some with incentives (e.g. Selecting and specifying target key behaviours (Steps 2 and 3) one free box) • Review prescriptions for marketing strategy Medical representatives • Motivate the drug seller to sell full courses of antibiotic • Don’t review prescriptions at doctors’ offices, drug shops • Don’t motivate patients to purchase drugs in front of doctor’s offices, drug shops health data from formative studies; data from workshop working groups; †DGDA: Directorate General Drug Administration; Medical representatives • Representatives have monthly visit quotas for doctors to promote products, distribute sample medicines to physicians ‡BPMI: Bangladesh Pharmacy Model Initiative 16 Household members: should be encouraged to consult qualified physicians and be pro-active in obtaining information about dispensed medications such as whether they are antibiotics. For those receiving antibiotics, the dose, frequency and duration of the course should be explained. Patients/consumers should be encouraged to take a full course of antibiotics obtained from a drug shop that sells quality medicines. Drug shop staff: should be encouraged to ask the customer for a prescription before dispensing antibiotics; referring them to doctors when they do not provide a prescription. Drug shop staff were considered to have a responsibility to increase awareness among patients, particularly on the importance of taking a full course of antibiotics. Stakeholders thought that there should be incentives for drug shops to recruit qualified staff. Qualified physicians: should be encouraged to base practices on current recommendations to reduce unnecessary prescribing, especially avoiding prescribing multiple and higher generation antibiotics than is necessary. Doctors were considered as important information sources that should reinforce the importance of completing a full course of antibiotics. Pharmaceutical companies/medical representatives: Stakeholders acknowledged the potential for companies and their representatives to maintain a viable business whilst playing a role in antibiotic stewardship. Suggestions included: modifications to the business strategy of companies; delivering quality training to all representatives and designing antibiotic packaging in a way that would encourage sale of a full course. Identifying priority audiences Among 28 participants who voted for first and second priority audiences to target. Nineteen participants voted for household members, 12 of which considered this population as 17 their number one priority and 16 voted for drug shop staff, 8 of which voted for this group as their first priority. Doctors were third with 13 votes and pharmaceutical companies/representatives were the lowest priority with 8 votes. During the post-workshop discussions, the research team and creative agency members concluded that using drug shops as a venue for intervention delivery had potential to address the first and second priority audiences thereby providing an opportunity to maximize intervention impact. their number one priority and 16 voted for drug shop staff, 8 of which voted for this group as their first priority. Doctors were third with 13 votes and pharmaceutical their number one priority and 16 voted for drug shop staff, 8 of which voted for this group as their first priority. Doctors were third with 13 votes and pharmaceutical companies/representatives were the lowest priority with 8 votes. During the post-workshop discussions, the research team and creative agency members concluded that using drug shops as a venue for intervention delivery had potential to address the first and second priority audiences, thereby providing an opportunity to maximize intervention impact. Capability, Opportunity and Motivation to improve antibiotic stewardship among priority audiences (Com-B Model, step 4). In line with government policy and guidelines [17, 26], to improve antibiotic stewardship, the target behaviours were to sell and purchase fewer antibiotics and to sell and consume antibiotics as full courses only. Among households, there were two main behaviours that needed to change: suboptimal health seeking and early cessation of antibiotic treatment. When assessing capabilities, a recurring theme was knowledge. Most household members could not distinguish a qualified from an unqualified provider and made decisions on who to visit primarily on disease severity considerations. They also had limited knowledge about antibiotics and their mode of action. Thus, opportunities exist for developing an SBCC that strengthens knowledge that can empower household members with potential to impact responsible antibiotic consumption. These include encouraging household members to ask about the medicines that they receive, and ask about timing, dosage and course duration. Motivation for this group can be encouraging them to seek appropriate healthcare advice and medicines by appealing to potential financial burden and accessibility to qualified physicians. ations. Identifying priority audiences They also had limited knowledge about antibiotics and their mode of action 18 Identifying drug shop staff behaviours that needed to change, there were similar capability issues evident as limited knowledge of government policy including the BPMI on licensing, staff qualification and adequate provision of information on antibiotics to customers. They lacked knowledge about antibiotic resistance. Some of these knowledge gaps can be filled using an educational SBCC. Drug shop staff are likely to remain the first line of access to health care in many communities, presenting an opportunity to have them serve as an information source on antibiotic use/dose/timing for customers. For an educational campaign that is located at drug shops, it must motivate drug shop staff by addressing financial concerns of potential lost business by encouraging household members to seek care from qualified physicians and by acknowledging their status in the community. While the BPMI is clear about staff qualifications, there does not appear to be evidence of enforcement, which could act as a motivator (Table 2). resource development and pilots for household members and drug shop staff The National Drug Policy, 2016 states ‘To prohibit sales and distribution of drugs without prescription from registered physician to ensure rational use of drugs”. During the pilot phase of intervention resource development, there was considerable discussion on how to convey ‘qualified physician’. Household members often referred to their local drug shop staff as a doctor or ‘small doctor’ [12]. Village doctor is also a common term used for a rural health practitioner, a post that is not considered a qualified physician. To overcome existing ambiguity, we decided to pilot ‘(Bachelor of Medicine, Bachelor of Surgery) MBBS doctor’ for the intervention resources and measure understanding of this term among drug shop staff and customers. Among the messages suggested during the intervention design workshop, the research team and VISCOM team prioritised those that we thought would resonate with the two selected audiences. For drug shop staff, these were related to asking for prescriptions, referral to qualified physicians, increase in client/customer awareness when they were receiving an antibiotic, and the need for a full course. Identifying priority audiences 19 Table 2: Using the Com-B model to inform households/customer and drug shop staff intervention to improve antibiotic stewardship Behaviour to change Capability Opportunity Motivation Households Suboptimal health seeking • Purchase drugs without prescription; sometimes ask for antibiotics by name since unqualified healthcare providers first point for health • Use peer group prescribed antibiotic Psychological • Limited knowledge of difference between qualified and unqualified physicians • Self-prescription • Accessibility, cost, symptom severity drive health seeking behaviour Physical • Easy access to (free) health advice from drug shop staff Physical • Need information on who and where are registered physicians • Registered physician/population is low Social • Males are decision makers on expenditure, visit drug shop • Social norm to visit drug shop first Reflective • Want quality healthcare for the family at reasonable cost • Want adequate information Automatic • Sometimes question advice Antibiotics are stopped • When symptoms disappear • When patients consider that they don't work Psychological • Don't know what an antibiotic is/ its use • Limited understanding of how antibiotics work Physical • Full course not purchased Physical • Need information about the importance of why drug is prescribed • Need information on dosage and timing, need for full course • Cost barrier to full course Social • Social norm to stop medications when disease is ‘cured’ Reflective • Empower to ask about treatment, cost Automatic • Trust drug shop staff Drug shop staff Antibiotic dispensing • Without government license Psychological • Limited knowledge of the Physical • Educate drug sellers on Reflective • Respected in the community Table 2: Using the Com-B model to inform households/customer and drug shop staff intervention to improve antibiotic stewardship 20 • Without prescription from registered physician** • Without seeing the patient/animal, prescribe over phone • By unqualified staff/ with short (6 weeks) or no training* • Follow elite doctor prescriptions when asked for advice • Do not always give dosing instructions* • Not familiar with antibiotic resistance policies, rules and penalties • Need information on policy for prescribing and minimum staff qualification antibiotic resistance • Address financial implications on their businesses • Intervention can replace medical representatives as a source of trusted, unbiased information on antibiotics • Policy is specific about staff qualification Automatic • Dispense multiple times during longer illnesses as outlined in the Bangladesh Model Pharmacy initiative (ref); as outlined in the Bangladesh National Drug Policy, 2016 Automatic • Dispense multiple times during longer illnesses 21 Pilot feedback 23 Figure 2: Example intervention resource, English translation of the Bengali version * produced as a paper bag for customers to carry dispensed antibiotics and other medicines, provided by drug retail shops – to raise awareness of when customers were receiving an antibiotic : Example intervention resource, English translation of the Bengali version* glish translation of the Bengali version* * produced as a paper bag for customers to carry dispensed antibiotics and other medicines, provided by drug retail shops – to raise awareness of when customers were receiving an antibiotic * produced as a paper bag for customers to carry dispensed antibiotics and other medicines, provided by drug retail shops – to raise awareness of when customers were receiving an antibiotic Pilot feedback Pilot drug shop staff respondents were all male, between 20 and 62 years of age and education status ranged from Secondary School Certificate (approximately grade 10) to Master’s degree, with most having a Higher School Certificate (approximately grade 12) qualification. All 50 respondents received and displayed the intervention materials and found them simple, clear and easy to read. Among the recommendations on antibiotics, 5 thought they would be difficult, 5 thought they would need time to integrate recommendations and the remainder thought the recommended behaviours were reasonable. There were suggestions on format (color, number of messages and font size). 22 Among the customers, 40 were male, 10 were female, ranging in age from 20 to 65 years. Some had no education, and most had attended between grade 5 and Higher School Certificate with one completing a Master’s degree. The vast majority (45) found that the messages were simple and easy to understand and were able to repeat the key messages. However, they found recommendations were different from their usual practices reporting that they buy antibiotics directly as it is easier and saves money and they did not see a justification for visiting a qualified physician for simple diseases. The majority (40) reported that medicines were recommended to them by the drug shop staff. Similar to drug shop staff materials, customers recommended some format revisions such as using more color, including fewer messages and using a larger font. Revised intervention resources included the following messages (example in Figure 2), for drug shop staff: tell the customer when you sell them antibiotics, remind them of timing and completing the full course, always sell antibiotics prescribed by a doctor (MBBS), refer patients to doctors for appropriate treatment, tell customers to report side effects to doctors. For customers messages included: antibiotics cure illness by killing germs, take a full course and follow dose and timing to be cured, not taking a full course may cause your disease to return and cost more money, antibiotics are not needed for all diseases, only a MBBS doctor can prescribe antibiotics. DISCUSSION Among the four groups examined, each contributed to poor antibiotic stewardship. Drivers of over-the-counter antibiotic dispensing came from the demand side, such as customers sometimes asking for specific antibiotics and from the supply side where important influences included product promotion by pharmaceutical company representatives. Most antibiotic stewardship programmes have centered around institutional settings where more control can be exerted [19]. However, in Bangladesh and other LMICs, institution prescriptions for antibiotics likely constitute a small proportion of those consumed [11-13]. 24 24 Based on expert input from stakeholders, for an intervention to address antibiotic stewardship in Bangladesh, the two priority audiences were household members (customers) and drug shop staff; delivering an intervention at drug shops can reach both audiences. In Bangladesh and other LMICs, drug shops are the first point of healthcare contact [27]. A barrier to encourage community members to obtain a prescription is an inability to convey ‘registered physicians’, the term used in the National Drug Policy, 2016 [17] in simple terms, largely due to the community’s inability to distinguish between the various labels of ‘doctors’. There are many groups that are referred to as some type of doctor in the absence of formal qualification in both urban [6] and rural settings [7]. Despite this, community members likely understand that their local drug shop staff is not a fully qualified, practicing, medical practitioner that has completed medical school training. Moreover, there is confusion over training levels for people working in drug shops and almost no knowledge among consumers on policy. While access and cost remain important motivators to seek health care and relevant medications from the local drug shop, it seems impossible for drug shop staff and consumers to adhere to the current policy. Further advocacy among drug policy stakeholders to convey that several steps are needed to bridge the vast gulf between obtaining medical advice and antibiotics from drug shops (current practice) and obtaining prescriptions from a registered physician (policy targeted practice) is imperative for the country to move towards improved antibiotic stewardship. We detected poor knowledge of relevant policies, including penalties for non- compliance, especially among drug shop staff, who are those predominantly affected. An international analysis found that poor governance and poor community infrastructure were both associated with higher AMR prevalence [28]. In Asia, rates of antibiotic dispensing without a 25 25 prescription have changed little over the last 30 years [29], suggesting weak regulation. DISCUSSION Thus, interventions that can improve policy knowledge and in turn can impact AMR should be considered along with lobby and advocacy efforts with relevant government agencies. Policy on antibiotic prescribing needs to be considered along with the lack of an alternate route to medicines; restricting access to antibiotics purchased through unlicensed drug shops will have a negative effect on human health in communities where infectious disease rates remain high [12]. Thus, our expert group suggested that drug shop staff should be included when addressing antibiotic stewardship, even though government policy specifies that antibiotic prescriptions are acceptable from ‘registered physicians’ only. Access to registered physicians is not currently possible for large sectors of the population because the number of qualified doctors is insufficient to meet demand [30, 31]. Cost and convenience are also important barriers for patients to seek care from qualified physicians [12, 22], echoed in the findings from piloting the SBCC materials. These challenges are similar in other settings with over the counter provision of antibiotics [32, 33]. Drug shop staff have little incentive to comply with government policy. Drug shop staff need motivation to comply with licensing, the prescribing policy and minimum staff qualification, all of which are beyond the scope of an education based SBCC. The accredited drug dispensing outlet (ADDO) programme from Africa includes business incentives [34] which have the potential to offset perceived loss from compliance with a stewardship intervention. Knowledge deficits were notable, and these are amendable to change using a SBCC intervention to improve health seeking behaviours, improve health literacy on antibiotic use and action and to inform drug shop staff about legal requirements. Training based programmes for drug shops have been conducted in Asia on dispensing medications for specific illnesses, with 26 mixed results [29]. The ADDO programme that permits groups other than registered physicians to prescribe and dispense antibiotics may be a model that the Government of Bangladesh could consider. The cadre of government employed auxiliary healthcare providers (persons with 1–4 years of medical-related training) who are permitted to prescribe a limited number of drugs, some of which include antibiotics, could be further trained and mobilised to meet needs that fall between qualified physicians and unqualified providers. This study has several limitations. Here we describe data collection and synthesis to complete steps 1-4 outlined by Munir et al. [25] of the behaviour wheel [24] and the Com-B model for intervention development. CONCLUSIONS This study drew on expert opinion and formative studies to guide development of an SBCC with priorities audiences; these need further collaboration with target audiences to refine messages and delivery methods. The study identified the likely poor penetration of relevant policy and penalties. Policies had limitations of flexibility to respond to the vast gap between current health seeking and prescribing practices at drug shops compared to recommendation of dispensing antibiotic only against prescription from qualified physicians. Research that includes BPMI audit studies similar to those conducted in Africa [20] will further aid collaborative and advocacy efforts with relevant agencies in the Government of Bangladesh to improve antibiotic stewardship. DISCUSSION The remaining aspects of step 5 and subsequent steps: identify behavioural change techniques; use APEASE (Affordability, Practicability, This study has several limitations. Here we describe data collection and synthesis to complete steps 1-4 outlined by Munir et al. [25] of the behaviour wheel [24] and the Com-B model for intervention development. The remaining aspects of step 5 and subsequent steps: identify behavioural change techniques; use APEASE (Affordability, Practicability, Effectiveness and cost-effectiveness, Acceptability, Side-effects and safety, Equity) criteria to grade these; identify mode of delivery, need to be explored in future research. Further refinement of intervention messages to improve antibiotic stewardship among drug shop staff and their customers could benefit from more interactive development such as using co-design methods. Effectiveness and cost-effectiveness, Acceptability, Side-effects and safety, Equity) criteria to grade these; identify mode of delivery, need to be explored in future research. Further refinement of intervention messages to improve antibiotic stewardship among drug shop staff and their customers could benefit from more interactive development such as using co-design methods. Effectiveness and cost-effectiveness, Acceptability, Side-effects and safety, Equity) criteria to grade these; identify mode of delivery, need to be explored in future research. Further refinement of intervention messages to improve antibiotic stewardship among drug shop staff and their customers could benefit from more interactive development such as using co-design methods. Drug shop staff will be unable to comply with an intervention that severely impacts profit and their livelihoods and further exploration of the economic aspects of an antibiotic stewardship intervention, relevant to this context, is necessary. This study focuses on intervention development for two of the four groups explored, based on priorities suggested by expert opinion. It may be necessary to involve the remaining groups that may either have greater affect or be easier to engage with on antibiotic stewardship in the future. Drug shop staff will be unable to comply with an intervention that severely impacts profit and their livelihoods and further exploration of the economic aspects of an antibiotic stewardship intervention, relevant to this context, is necessary. This study focuses on intervention development for two of the four groups explored, based on priorities suggested by expert opinion. It may be necessary to involve the remaining groups that may either have greater affect or be easier to engage with on antibiotic stewardship in the future. 27 DECLARATIONS DECLARATIONS Ethical approval and consent to participate Written informed consent was obtained from study respondents. Workshop participants registered and provided information on their job title and organization. Ethical approval was obtained through the IRB at icddr,b (PR-16100), Loughborough University Ethics committee (R17-P081) and Durham University Research Ethics and Data Protection committee. All methods were performed in accordance with the guidelines and regulations of these committees. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. ABBREVIATIONS ADDO: accredited drug dispensing outlet ADDO: accredited drug dispensing outlet AMR: antimicrobial resistance APEASE: Affordability, Practicability, Effectiveness and cost-effectiveness, Acceptability, Side- effects and safety, Equity MBBS: Bachelor of Medicine, Bachelor of Surgery BPMI: Bangladesh Pharmacy Model Initiative Com-B: Capability, Opportunities, Motivation-Behaviour icddr,b: International Centre for Diarrhoeal Disease Research, Bangladesh LMICs: low- and middle-income countries NGOs: non-governmental organisations SBCC: social and behavioural change communication VISCOM: Visual Communications Ltd. 28 Competing interests The authors declare that they have no competing interests. Funding This study was funded by the Antimicrobial Resistance Cross-Council Initiative supported by the seven UK research councils in partnership with the Department of Health, the UK Department of Environment Food and Rural Affairs and the Global Challenges Research Fund (Economic and Social Research Council grant ES/ P004563/1 awarded to Dr Emily Rousham). The funding bodies did not play a role in the design of the study and collection, analysis, and interpretation of data or in writing the manuscript. Authors’ contributions LEU, EKR, MAI developed the research concept, LEU, EKR, MAI, FAN, PN, PJL, MR contributed to writing grant applications, LEU, EKR, MAI, FAN, MRU, PN, PJL, NK, SMSA, 29 analysed and interpreted data and all authors made a major contribution and read and approved the final manuscript. analysed and interpreted data and all authors made a major contribution and read and approved the final manuscript. Acknowledgements icddr,b acknowledges with gratitude the commitment of the Antimicrobial Resistance Cross- Council Initiative supported by the seven UK research councils in partnership with the Department of Health, the UK Department of Environment Food and Rural Affairs and the Global Challenges Research Fund in its research efforts. icddr,b is also grateful to the Governments of Bangladesh, Canada, Sweden and the UK for providing core/unrestricted support.. The authors acknowledge the assistance of Mahbub-ul Alam, Muhammed Asaduzzaman and Abdullah Al-Masud for workshop facilitation. We acknowledge the contribution of study participants including local experts who attended the intervention design Workshop. References: Lucas PJ, Uddin MR, Khisa N, Akter SMS, Unicomb L, Nahar P, Islam MA, Nizame FA, Rousham EK: Pathways to antibiotics in Bangladesh: A qualitative study investigating how and when households access medicine including antibiotics for humans or animals when they are ill. PLoS One 2019, 14(11):e0225270. 13. Morgan DJ, Okeke IN, Laxminarayan R, Perencevich EN, Weisenberg S: Non-prescription antimicrobial u worldwide: a systematic review. Lancet Infect Dis 2011, 11(9):692-701. 14. Ahmed SM, Islam QS: Availability and rational use of drugs in primary healthcare facilities following th national drug policy of 1982: is Bangladesh on right track? J Health Popul Nutr 2012, 30(1):99-108. 15. Mohiuddin M, Rashid SF, Shuvro MI, Nahar N, Ahmed SM: Qualitative insights into promotion of pharmaceutical products in Bangladesh: how ethical are the practices? BMC Med Ethics 2015, 16(1):80 16. Roess AA, Winch PJ, Akhter A, Afroz D, Ali NA, Shah R, Begum N, Seraji HR, El Arifeen S, Darmstadt GL et al: Household Animal and Human Medicine Use and Animal Husbandry Practices in Rural Bangladesh: Risk Factors for Emerging Zoonotic Disease and Antibiotic Resistance. Zoonoses Public Health 2015, 62(7):569-578. 17. Directorate General Drug Administration: National Drug Policy. In. Dhaka, Bangladesh; 2016. 18. Davey P, Marwick CA, Scott CL, Charani E, McNeil K, Brown E, Gould IM, Ramsay CR, Michie S: Interventions to improve antibiotic prescribing practices for hospital inpatients. Cochrane Database Sy Rev 2017, 2:CD003543. 19. Wilkinson A, Ebata A, MacGregor H: Interventions to Reduce Antibiotic Prescribing in LMICs: A Scoping Review of Evidence from Human and Animal Health Systems. Antibiotics (Basel) 2018, 8(1). 20. Chalker JC, Vialle-Valentin C, Liana J, Mbwasi R, Semali IA, Kihiyo B, Shekalaghe E, Dillip A, Kimatta S, Valimba R et al: What roles do accredited drug dispensing outlets in Tanzania play in facilitating access to antimicrobials? Results of a multi-method analysis. Antimicrob Resist Infect Control 2015, 4:33. 1. Gandra S, Joshi J, Trett A, Lamkang AS, Laxminarayan R: Scoping Report on Antimicrobial Resistance in India. . In. Washington, DC, USA: Center for Disease Dynamics, Economics & Policy. ; 2017. 2. Wang R, van Dorp L, Shaw LP, Bradley P, Wang Q, Wang X, Jin L, Zhang Q, Liu Y, Rieux A et al: The global distribution and spread of the mobilized colistin resistance gene mcr-1. Nat Commun 2018, 9(1):1179. 3. WHO: Worldwide country situation analysis: response to antimicrobial resistance. In. References: Geneva, S it l d W ld H lth O i ti 2015 distribution and spread of the mobilized colistin resistance gene mcr-1. Nat Commun 2018, 9(1):1179 3. WHO: Worldwide country situation analysis: response to antimicrobial resistance. In. Geneva, Switzerland: World Health Organization; 2015. 4. Laxminarayan R, Van Boeckel T, Frost I, Kariuki S, Khan EA, Limmathurotsakul D, Larsson DGJ, Levy-Hara Mendelson M, Outterson K et al: The Lancet Infectious Diseases Commission on antimicrobial resistan 6 years later. Lancet Infect Dis 2020, 20(4):e51-e60. 5. Klein EY, Van Boeckel TP, Martinez EM, Pant S, Gandra S, Levin SA, Goossens H, Laxminarayan R: Global increase and geographic convergence in antibiotic consumption between 2000 and 2015. Proc Natl Ac Sci U S A 2018, 115(15):E3463-E3470. 6. Adams AM, Islam R, Ahmed T: Who serves the urban poor? A geospatial and descriptive analysis of health services in slum settlements in Dhaka, Bangladesh. Health Policy Plan 2015, 30 Suppl 1:i32-45. 7. Ahmed SM, Hossain MA: Knowledge and practice of unqualified and semi-qualified allopathic provide in rural Bangladesh: implications for the HRH problem. Health Policy 2007, 84(2-3):332-343. 8. Ahmed SM, Hossain MA, Chowdhury MR: Informal sector providers in Bangladesh: how equipped are they to provide rational health care? Health Policy Plan 2009, 24(6):467-478. 9. Chowdhury F, Sturm-Ramirez K, Mamun AA, Iuliano AD, Bhuiyan MU, Chisti MJ, Ahmed M, Haider S, Rahman M, Azziz-Baumgartner E: Factors driving customers to seek health care from pharmacies for acute respiratory illness and treatment recommendations from drug sellers in Dhaka city, Bangladesh Patient Prefer Adherence 2017, 11:479-486. 10. SIAPS: Baseline Study of Private Drug Shops in Bangladesh: Findings and Recommendations. In. Arlington, VA, USA: Management Sciences for Health.; 2015. 11. Biswas M, Roy DN, Tajmim A, Rajib SS, Hossain M, Farzana F, Yasmen N: Prescription antibiotics for outpatients in Bangladesh: a cross-sectional health survey conducted in three cities. Ann Clin Microbio Antimicrob 2014, 13:15. 12. Lucas PJ, Uddin MR, Khisa N, Akter SMS, Unicomb L, Nahar P, Islam MA, Nizame FA, Rousham EK: Pathways to antibiotics in Bangladesh: A qualitative study investigating how and when households access medicine including antibiotics for humans or animals when they are ill. PLoS One 2019, 4. Laxminarayan R, Van Boeckel T, Frost I, Kariuki S, Khan EA, Limmathurotsakul D, Larsson DGJ, Levy-Hara G, Mendelson M, Outterson K et al: The Lancet Infectious Diseases Commission on antimicrobial resistance: 6 years later. Lancet Infect Dis 2020, 20(4):e51-e60. Authors’ information Leanne Unicomb, International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b). E- mail: leanne.unicomb60@gmail.com Leanne Unicomb, International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b). E- mail: leanne.unicomb60@gmail.com Fosiul Alam Nizame, International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b). E-mail: fosiul@icddrb.org Fosiul Alam Nizame, International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b). E-mail: fosiul@icddrb.org Mohammad Rofi Uddin, International Centre for Diarrhoeal Disease Research, Bangladesh (icddr, b). E-mail: rofi.uddin@icddrb.org Mohammad Rofi Uddin, International Centre for Diarrhoeal Disease Research, Bangladesh (icddr, b). E-mail: rofi.uddin@icddrb.org Papreen Nahar, Primary Care and Public Health, Brighton and Sussex Medical School, University of Sussex, UK. E-mail address: P.Nahar@bsms.ac.uk Papreen Nahar, Primary Care and Public Health, Brighton and Sussex Medical School, University of Sussex, UK. E-mail address: P.Nahar@bsms.ac.uk Papreen Nahar, Primary Care and Public Health, Brighton and Sussex Medical School, University of Sussex, UK. E-mail address: P.Nahar@bsms.ac.uk Patricia J Lucas, School for Policy Studies, University of Bristol, UK. E-mail: Patricia.lucas@bristol.ac.uk Patricia J Lucas, School for Policy Studies, University of Bristol, UK. E-mail: Patricia.lucas@bristol.ac.uk Patricia J Lucas, School for Policy Studies, University of Bristol, UK. E-mail: Patricia.lucas@bristol.ac.uk 30 Nirnita Khisa, International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b). E- mail: nirnikhisa@gmail.com S. M. Salim Akter, International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b). E-mail: bappi2008@yahoo.com Mohammad Aminul Islam, Paul G. Allen School for Global Animal Health, Washington State University, Pullman, Washington, USA and International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b). E-mail: maislam@icddrb.org Mahbubur Rahman, International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b). E-mail: mahbubr@icddrb.org Emily K Rousham, Centre for Global Health and Human Development, School of Sport, Exercise and Health Sciences, Loughborough University, UK. E-mail: E.K.Rousham@lboro.ac.uk 31 References: References: References: 1. Gandra S, Joshi J, Trett A, Lamkang AS, Laxminarayan R: Scoping Report on Antimicrobial Resistance in India. . In. Washington, DC, USA: Center for Disease Dynamics, Economics & Policy. ; 2017. 2. Wang R, van Dorp L, Shaw LP, Bradley P, Wang Q, Wang X, Jin L, Zhang Q, Liu Y, Rieux A et al: The global distribution and spread of the mobilized colistin resistance gene mcr-1. Nat Commun 2018, 9(1):1179. 3. WHO: Worldwide country situation analysis: response to antimicrobial resistance. In. Geneva, Switzerland: World Health Organization; 2015. 4. Laxminarayan R, Van Boeckel T, Frost I, Kariuki S, Khan EA, Limmathurotsakul D, Larsson DGJ, Levy-Hara Mendelson M, Outterson K et al: The Lancet Infectious Diseases Commission on antimicrobial resistanc 6 years later. Lancet Infect Dis 2020, 20(4):e51-e60. 5. Klein EY, Van Boeckel TP, Martinez EM, Pant S, Gandra S, Levin SA, Goossens H, Laxminarayan R: Global increase and geographic convergence in antibiotic consumption between 2000 and 2015. Proc Natl Aca Sci U S A 2018, 115(15):E3463-E3470. 6. Adams AM, Islam R, Ahmed T: Who serves the urban poor? A geospatial and descriptive analysis of health services in slum settlements in Dhaka, Bangladesh. Health Policy Plan 2015, 30 Suppl 1:i32-45. 7. Ahmed SM, Hossain MA: Knowledge and practice of unqualified and semi-qualified allopathic provider in rural Bangladesh: implications for the HRH problem. Health Policy 2007, 84(2-3):332-343. 8. Ahmed SM, Hossain MA, Chowdhury MR: Informal sector providers in Bangladesh: how equipped are they to provide rational health care? Health Policy Plan 2009, 24(6):467-478. 9. Chowdhury F, Sturm-Ramirez K, Mamun AA, Iuliano AD, Bhuiyan MU, Chisti MJ, Ahmed M, Haider S, Rahman M, Azziz-Baumgartner E: Factors driving customers to seek health care from pharmacies for acute respiratory illness and treatment recommendations from drug sellers in Dhaka city, Bangladesh Patient Prefer Adherence 2017, 11:479-486. 10. SIAPS: Baseline Study of Private Drug Shops in Bangladesh: Findings and Recommendations. In. Arlington, VA, USA: Management Sciences for Health.; 2015. 11. Biswas M, Roy DN, Tajmim A, Rajib SS, Hossain M, Farzana F, Yasmen N: Prescription antibiotics for outpatients in Bangladesh: a cross-sectional health survey conducted in three cities. Ann Clin Microbio Antimicrob 2014, 13:15. 12. References: 5. Klein EY, Van Boeckel TP, Martinez EM, Pant S, Gandra S, Levin SA, Goossens H, Laxminarayan R: Global increase and geographic convergence in antibiotic consumption between 2000 and 2015. Proc Natl Acad Sci U S A 2018, 115(15):E3463-E3470. 6. Adams AM, Islam R, Ahmed T: Who serves the urban poor? A geospatial and descriptive analysis of health services in slum settlements in Dhaka, Bangladesh. Health Policy Plan 2015, 30 Suppl 1:i32-45. 7. Ahmed SM, Hossain MA: Knowledge and practice of unqualified and semi-qualified allopathic providers in rural Bangladesh: implications for the HRH problem. Health Policy 2007, 84(2-3):332-343. 8. Ahmed SM, Hossain MA, Chowdhury MR: Informal sector providers in Bangladesh: how equipped are they to provide rational health care? Health Policy Plan 2009, 24(6):467-478. 9. Chowdhury F, Sturm-Ramirez K, Mamun AA, Iuliano AD, Bhuiyan MU, Chisti MJ, Ahmed M, Haider S, Rahman M, Azziz-Baumgartner E: Factors driving customers to seek health care from pharmacies for acute respiratory illness and treatment recommendations from drug sellers in Dhaka city, Bangladesh. Patient Prefer Adherence 2017, 11:479-486. 10. SIAPS: Baseline Study of Private Drug Shops in Bangladesh: Findings and Recommendations. In. Arlington, VA, USA: Management Sciences for Health.; 2015. 11. Biswas M, Roy DN, Tajmim A, Rajib SS, Hossain M, Farzana F, Yasmen N: Prescription antibiotics for outpatients in Bangladesh: a cross-sectional health survey conducted in three cities. Ann Clin Microbiol Antimicrob 2014, 13:15. 12. Lucas PJ, Uddin MR, Khisa N, Akter SMS, Unicomb L, Nahar P, Islam MA, Nizame FA, Rousham EK: Pathways to antibiotics in Bangladesh: A qualitative study investigating how and when households access medicine including antibiotics for humans or animals when they are ill. PLoS One 2019, 14(11):e0225270. 13. Morgan DJ, Okeke IN, Laxminarayan R, Perencevich EN, Weisenberg S: Non-prescription antimicrobial use worldwide: a systematic review. Lancet Infect Dis 2011, 11(9):692-701. 14. Ahmed SM, Islam QS: Availability and rational use of drugs in primary healthcare facilities following the national drug policy of 1982: is Bangladesh on right track? J Health Popul Nutr 2012, 30(1):99-108. national drug policy of 1982: is Bangladesh on right track? J Health Popul Nutr 2012, 30(1):99-108. 15. Mohiuddin M, Rashid SF, Shuvro MI, Nahar N, Ahmed SM: Qualitative insights into promotion of pharmaceutical products in Bangladesh: how ethical are the practices? BMC Med Ethics 2015, 16(1):80. 16. References: Munir F, Biddle SJH, Davies MJ, Dunstan D, Esliger D, Gray LJ, Jackson BR, O'Connell SE, Yates T, Edwardson CL: Stand More AT Work (SMArT Work): using the behaviour change wheel to develop an intervention to reduce sitting time in the workplace. BMC Public Health 2018, 18(1):319. 26. Ministry of Health and Family Welfare: Standards for the establishment and operations of model pharmacies and model medicine shops. In. Dhaka: Directorate General of Drug Administration, Ministry of Health and Family Welfare; 2016. 27. Khan MM, Grubner O, Kramer A: Frequently used healthcare services in urban slums of Dhaka and adjacent rural areas and their determinants. J Public Health (Oxf) 2012, 34(2):261-271. 28. Collignon P, Beggs JJ, Walsh TR, Gandra S, Laxminarayan R: Anthropological and socioeconomic factors contributing to global antimicrobial resistance: a univariate and multivariable analysis. Lancet Planet Health 2018, 2(9):e398-e405. 29. Miller R, Goodman C: Performance of retail pharmacies in low- and middle-income Asian settings: a systematic review. Health Policy Plan 2016, 31(7):940-953. 30. Mahmood SS, Iqbal M, Hanifi SM, Wahed T, Bhuiya A: Are 'Village Doctors' in Bangladesh a curse or a blessing? BMC Int Health Hum Rights 2010, 10:18. 31. WHO: Working together for health: The world health report 2006. In. Geneva, Switzerland: World Healt Organization; 2006. 32. Barker AK, Brown K, Ahsan M, Sengupta S, Safdar N: What drives inappropriate antibiotic dispensing? A mixed-methods study of pharmacy employee perspectives in Haryana, India. BMJ Open 2017, 7(3):e013190. 33. Mbonye AK, Buregyeya E, Rutebemberwa E, Clarke SE, Lal S, Hansen KS, Magnussen P, LaRussa P: Prescription for antibiotics at drug shops and strategies to improve quality of care and patient safety: a cross-sectional survey in the private sector in Uganda. BMJ Open 2016, 6(3):e010632. 34. Rutta E, Liana J, Embrey M, Johnson K, Kimatta S, Valimba R, Lieber R, Shekalaghe E, Sillo H: Accrediting retail drug shops to strengthen Tanzania's public health system: an ADDO case study. J Pharm Policy Pract 2015, 8:23. 21. Embrey M, Vialle-Valentin C, Dillip A, Kihiyo B, Mbwasi R, Semali IA, Chalker JC, Liana J, Lieber R, Johnson K et al: Understanding the Role of Accredited Drug Dispensing Outlets in Tanzania's Health System. PLoS One 2016, 11(11):e0164332. 21. 22. Nahar P, Unicomb L, Lucas PJ, Uddin MR, Islam MA, Nizame FA, Khisa N, Akter SMS, Rousham EK: What contributes to inappropriate antibiotic dispensing among qualified and unqualified healthcare providers in Bangladesh? A qualitative study. References: BMC Health Serv Res 2020, 20(1):656. 23. Rousham EK, Islam MA, Nahar P, Lucas PJ, Naher N, Ahmed SM, Nizame FA, Unicomb L antibiotic use in Bangladesh: qualitative protocol for the PAUSE study. BMJ Open 201 24. Michie S, van Stralen MM, West R: The behaviour change wheel: a new method for characterising and designing behaviour change interventions. Implement Sci 2011, 6:42. 25. Munir F, Biddle SJH, Davies MJ, Dunstan D, Esliger D, Gray LJ, Jackson BR, O'Connell SE, Yates T, Edwardson CL: Stand More AT Work (SMArT Work): using the behaviour change wheel to develop an intervention to reduce sitting time in the workplace. BMC Public Health 2018, 18(1):319. 26 Ministry of Health and Family Welfare Standards for the establishment and operations of model 26. Ministry of Health and Family Welfare: Standards for the establishment and operations of model pharmacies and model medicine shops. In. Dhaka: Directorate General of Drug Administration, Ministry of Health and Family Welfare; 2016. 27. Khan MM, Grubner O, Kramer A: Frequently used healthcare services in urban slums of Dhaka and adjacent rural areas and their determinants. J Public Health (Oxf) 2012, 34(2):261-271. 28. Collignon P, Beggs JJ, Walsh TR, Gandra S, Laxminarayan R: Anthropological and socioeconomic factors contributing to global antimicrobial resistance: a univariate and multivariable analysis. Lancet Planet Health 2018, 2(9):e398-e405. 29. Miller R, Goodman C: Performance of retail pharmacies in low- and middle-income Asian settings: a systematic review. Health Policy Plan 2016, 31(7):940-953. 30. Mahmood SS, Iqbal M, Hanifi SM, Wahed T, Bhuiya A: Are 'Village Doctors' in Bangladesh a curse or a blessing? BMC Int Health Hum Rights 2010, 10:18. 31. WHO: Working together for health: The world health report 2006. In. Geneva, Switzerland: World Health Organization; 2006. 32. Barker AK, Brown K, Ahsan M, Sengupta S, Safdar N: What drives inappropriate antibiotic dispensing? A mixed-methods study of pharmacy employee perspectives in Haryana, India. BMJ Open 2017, 7(3):e013190. 33. Mbonye AK, Buregyeya E, Rutebemberwa E, Clarke SE, Lal S, Hansen KS, Magnussen P, LaRussa P: Prescription for antibiotics at drug shops and strategies to improve quality of care and patient safety: a cross-sectional survey in the private sector in Uganda. BMJ Open 2016, 6(3):e010632. 34. Rutta E, Liana J, Embrey M, Johnson K, Kimatta S, Valimba R, Lieber R, Shekalaghe E, Sillo H: Accrediting retail drug shops to strengthen Tanzania's public health system: an ADDO case study. References: Roess AA, Winch PJ, Akhter A, Afroz D, Ali NA, Shah R, Begum N, Seraji HR, El Arifeen S, Darmstadt GL et al: Household Animal and Human Medicine Use and Animal Husbandry Practices in Rural Bangladesh: Risk Factors for Emerging Zoonotic Disease and Antibiotic Resistance. Zoonoses Public Health 2015, 62(7):569-578. g p y g g p 15. Mohiuddin M, Rashid SF, Shuvro MI, Nahar N, Ahmed SM: Qualitative insights into promotion of pharmaceutical products in Bangladesh: how ethical are the practices? BMC Med Ethics 2015, 16(1):80. 16. Roess AA, Winch PJ, Akhter A, Afroz D, Ali NA, Shah R, Begum N, Seraji HR, El Arifeen S, Darmstadt GL et al: Household Animal and Human Medicine Use and Animal Husbandry Practices in Rural Bangladesh: 15. Mohiuddin M, Rashid SF, Shuvro MI, Nahar N, Ahmed SM: Qualitative insights into promotion of pharmaceutical products in Bangladesh: how ethical are the practices? BMC Med Ethics 2015, 16(1):80. 16. Roess AA, Winch PJ, Akhter A, Afroz D, Ali NA, Shah R, Begum N, Seraji HR, El Arifeen S, Darmstadt GL et al: Household Animal and Human Medicine Use and Animal Husbandry Practices in Rural Bangladesh: Risk Factors for Emerging Zoonotic Disease and Antibiotic Resistance. Zoonoses Public Health 2015, 62(7):569-578. Directorate General Drug Administration: National Drug Policy. In. Dhaka, Bangladesh; 2016. g g y , g ; 18. Davey P, Marwick CA, Scott CL, Charani E, McNeil K, Brown E, Gould IM, Ramsay CR, Michie S: Interventions to improve antibiotic prescribing practices for hospital inpatients. Cochrane Database Syst Rev 2017, 2:CD003543. 32 21. Embrey M, Vialle-Valentin C, Dillip A, Kihiyo B, Mbwasi R, Semali IA, Chalker JC, Liana J, Lieber R, Johnson et al: Understanding the Role of Accredited Drug Dispensing Outlets in Tanzania's Health System. PLoS One 2016, 11(11):e0164332. 22. Nahar P, Unicomb L, Lucas PJ, Uddin MR, Islam MA, Nizame FA, Khisa N, Akter SMS, Rousham EK: What contributes to inappropriate antibiotic dispensing among qualified and unqualified healthcare provide in Bangladesh? A qualitative study. BMC Health Serv Res 2020, 20(1):656. 23. Rousham EK, Islam MA, Nahar P, Lucas PJ, Naher N, Ahmed SM, Nizame FA, Unicomb L: Pathways of antibiotic use in Bangladesh: qualitative protocol for the PAUSE study. BMJ Open 2019, 9. 24. Michie S, van Stralen MM, West R: The behaviour change wheel: a new method for characterising and designing behaviour change interventions. Implement Sci 2011, 6:42. 25. Figures Figure 1 Schematic of study components References: J Pharm Policy Pract 2015, 8:23. 33 Figure 1 Schematic of study components Figure 2 Example intervention resource, English translation of the Bengali version* * produced as a paper bag for customers to carry dispensed antibiotics and other medicines, provided by drug retail shops – to raise Figure 2 Example intervention resource, English translation of the Bengali version* * produced as a paper bag for customers to carry dispensed antibiotics and other medicines, provided by drug retail shops – to raise awareness of when customers were receiving an antibiotic
https://openalex.org/W4361932128	https://figshare.com/articles/journal_contribution/Table_S1_from_A_Population_of_Heterogeneous_Breast_Cancer_Patient-Derived_Xenografts_Demonstrate_Broad_Activity_of_PARP_Inhibitor_in_BRCA1_2_Wild-Type_Tumors/22469043/1/files/39920508.pdf	English	null	Table S2 from A Population of Heterogeneous Breast Cancer Patient-Derived Xenografts Demonstrate Broad Activity of PARP Inhibitor in BRCA1/2 Wild-Type Tumors	null	2,023	cc-by	346	Mouse antibody assessment shown in parenthesis. Supplementary Table 1. Reassessment of hormone receptor status in surgical samples and PDX with rabbit and mouse antibodies. Supplementary Table 1. Reassessment of hormone receptor status in surgical samples and PDX with rabbit and mouse antibodies. Patient PDX ER (%) PR (%) HER2 ER (%) PR (%) HER2 BCX.006 0 (0) 0 (0) 0 <1 (<1) 0 (0) 0 BCX.009 0 (0) 0 (0) 0 3 (3) 0 (0) 0 BCX.010 0 (<1) <1 (<1) 0 0 (<1) 40 (0) 0 BCX.011 0 (0) 0 (0) 0 0 (0) <1 (0) 0 BCX.017 <1 (5) <1 (0) 1 0 (1) 0 (0) 1 BCX.022 3 (2) 0 (0) 0 <1 (<1) 0 (0) 0 BCX.024 <1 (5) <1 (<1) 0 <1 (<1) <1 (<1) 1 BCX.042 0 (<1) <1 (<1) 0 0 (<1) 30 (<1) 0 BCX.051 0 (<1) 0 (0) 0 0 (0) 0 (0) 0 BCX.055 <1 (<1) 0 (<1) 0 <1 (<1) 0 (0) 0 BCX.060 0 (<1) 0 (0) 0 0 (5) 0 (0) 0 BCX.065 0 (<1) 0 (<1) 1 0 (0) 0 (0) 0 BCX.066 1 (1) 0 (0) 0 7 (8) 40 (<1) 0 BCX.070 0 (<1) 0 (0) 0 0 (0) 0 (0) 1 BCX.080 0 (0) 0 (0) 0 2 (2) <1 (<1) 1 BCX.084 0 (0) 0 (<1) 0 0 (<1) 7 (0) 0 BCX.087 0 (<1) 0 (0) 0 0 (3) 0 (0) 1 BCX.092 0 (0) 0 (0) 0 0 (8) 0 (<1) 0 BCX.094 <1 (10) 0 (0) 0 0 (8) 0 (0) 0 BCX.095 <1 (1) 0 (0) 0 8 (15) 0 (0) 0 BCX.096 10 (20) 1 (0) 0 8 (35) 10 (0) 0 BCX.099 30 (30) 0 (0) 0 80 (85) 0 (0) 0 BCX.100 0 (<1) 0 (0) 0 1 (4) 0 (0) 0 BCX.102 <1 (4) 0 (0) 1 0 (4) 0 (0) 0 BCX.104 5 (15) <1 (1) 1 5 (11) 1 (2) 1 BCX.105 0 (<1) 7 (0) 1 0 (0) 0 (0) 1 Mouse antibody assessment shown in parenthesis.
https://openalex.org/W2972769596	https://europepmc.org/articles/pmc7206116?pdf=render	English	null	βarrestin-1 regulates DNA repair by acting as an E3-ubiquitin ligase adaptor for 53BP1	Cell death and differentiation	2,019	cc-by	11,579	Cell Death & Differentiation (2020) 27:1200–1213 https://doi.org/10.1038/s41418-019-0406-6 ARTICLE ARTICLE Abstract Cellular DNA is constantly under threat from internal and external insults, consequently multiple pathways have evolved to maintain chromosomal ﬁdelity. Our previous studies revealed that chronic stress, mediated by continuous stimulation of the β2-adrenergic-βarrestin-1 signaling axis suppresses activity of the tumor suppressor p53 and impairs genomic integrity. In this pathway, βarrestin-1 (βarr1) acts as a molecular scaffold to promote the binding and degradation of p53 by the E3- ubiquitin ligase, MDM2. We sought to determine whether βarr1 plays additional roles in the repair of DNA damage. Here we demonstrate that in mice βarr1 interacts with p53-binding protein 1 (53BP1) with major consequences for the repair of DNA double-strand breaks. 53BP1 is a principle component of the DNA damage response, and when recruited to the site of double-strand breaks in DNA, 53BP1 plays an important role coordinating repair of these toxic lesions. Here, we report that βarr1 directs 53BP1 degradation by acting as a scaffold for the E3-ubiquitin ligase Rad18. Consequently, knockdown of βarr1 stabilizes 53BP1 augmenting the number of 53BP1 DNA damage repair foci following exposure to ionizing radiation. Accordingly, βarr1 loss leads to a marked increase in irradiation resistance both in cells and in vivo. Thus, βarr1 is an important regulator of double strand break repair, and disruption of the βarr1/53BP1 interaction offers an attractive strategy to protect cells against high levels of exposure to ionizing radiation. Ainhoa Nieto 1 ●Makoto R. Hara2,5 ●Victor Quereda3 ●Wayne Grant4 ●Vanessa Saunders4 ●Kunhong Xiao2 ● Patricia H. McDonald1 ●Derek R. Duckett3 Received: 5 October 2018 / Revised: 6 August 2019 / Accepted: 8 August 2019 / Published online: 10 September 2019 © The Author(s) 2019. This article is published with open access βarrestin-1 regulates DNA repair by acting as an E3-ubiquitin ligase adaptor for 53BP1 Ainhoa Nieto 1 ●Makoto R. Hara2,5 ●Victor Quereda3 ●Wayne Grant4 ●Vanessa Saunder Patricia H. McDonald1 ●Derek R. Duckett3 Ainhoa Nieto 1 ●Makoto R. Hara2,5 ●Victor Quereda3 ●Wayne Grant4 ●Vanessa Saunders4 ●Kunhong Xiao2 ● Patricia H. McDonald1 ●Derek R. Duckett3 Makoto R. Hara2,5 ●Victor Quereda3 ●Wayne Grant4 ●Vanessa Saunders4 ●Kunhong Xiao2 ● ld1 ●Derek R. Duckett3 Introduction Mammalian cells are continuously bombarded by DNA damaging insults which threaten cell viability and genomic integrity [1]. Amongst the most toxic lesions are DNA double-strand breaks (DSBs), as failure to properly repair even a single DSB has dire consequences triggering cell death, cell cycle arrest, or gross chromosomal rearrange- ments such as deletions, translocations, and ampliﬁcations; all causal drivers of tumorigenesis [2]. Breaks in both strands of DNA can be induced by numerous sources such as, the collapse of replication forks, reactive products of oxidative metabolism, as well as from external sources such as, ionizing radiation (IR). To repair DSBs cells utilize two major pathways namely, nonhomologous end joining (NHEJ), which occurs throughout the cell cycle and directly rejoins the broken ends with minimal processing [3], and homologous recombination (HR), which requires large stretches of sequence homology necessary for repairing lesions with high ﬁdelity. Typically, HR requires the sister chromatid to serve as a template and thus is restricted to S/G2 phases of the cell cycle [4]. Coordination of these Edited by V. DʼAngiolella Supplementary information The online version of this article (https:// doi.org/10.1038/s41418-019-0406-6) contains supplementary material, which is available to authorized users. * Patricia H. McDonald patsy.McDonald@mofﬁtt.org * Patricia H. McDonald patsy.McDonald@mofﬁtt.org * Patricia H. McDonald patsy.McDonald@mofﬁtt.org * Derek R. Duckett derek.Duckett@mofﬁtt.org 1 Department of Cancer Physiology, Mofﬁtt Cancer Center, Tampa, FL 33612, USA 2 Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA 3 Department of Drug Discovery, Mofﬁtt Cancer Center, Tampa, FL 33612, USA 4 Department of Molecular Medicine, The Scripps Research Institute, Jupiter, FL 33458, USA 5 Present address: Novartis Institutes for Biomedical Research, Cambridge, MA 02139, USA βarrestin-1 regulates DNA repair by acting as an E3-ubiquitin ligase adaptor for 53BP1 1201 Millipore); Anti-mouse IgG-HRP and anti-rabbit IgG-HRP (GE Healthcare); Alexa Fluor 647 Donkey anti-rabbit IgG (Invitrogen); Alexa Fluor 555 Donkey anti-rabbit IgG (Invitrogen); Alexa Fluor 488 Donkey anti-mouse IgG (Invitrogen). Rabbit polyclonal βarr1 antibody (A1CT) was generated as previously described [24]. Millipore); Anti-mouse IgG-HRP and anti-rabbit IgG-HRP (GE Healthcare); Alexa Fluor 647 Donkey anti-rabbit IgG (Invitrogen); Alexa Fluor 555 Donkey anti-rabbit IgG (Invitrogen); Alexa Fluor 488 Donkey anti-mouse IgG (Invitrogen). Rabbit polyclonal βarr1 antibody (A1CT) was generated as previously described [24]. repair processes is essential, and tightly regulated to ensure that the appropriate counter is taken to ameliorate the threat to the cell [5]. Co-immunoprecipitation Immunoprecipitation analyses were performed as described [29]. Input samples were run with 5% of the IP lysate. To detect polyubiquitylation of 53BP1, 20 μM MG132 was added to plates 4 h prior to harvesting cells, and 10 mM N-ethylmaleimide and 20 μM MG132 were added to the lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 0.1% Chaps, 0.1 mg/ml BSA, 1 mM PMSF, 1 mM EDTA, with Halt protease and phosphatase inhibitor cocktail (Pierce)). Introduction The tumor suppressor protein p53-binding protein 1 (53BP1) is an important modulator of DSB repair [6]. This is exempliﬁed in mice deﬁcient in 53BP1, which are sen- sitive to IR and have signiﬁcant defects in adaptive immune response due to defective repair of programmed DSBs such as, class switch recombination and V(D)J recombination in lymphocytes [7, 8]. Furthermore, 53BP1 knockout mice are growth retarded and tumor prone [9, 10]. 53BP1 plays opposing roles to BRCA1 (breast cancer susceptibility gene-1) during the initial stages of DSB repair by inhibiting accumulation of BRCA1 at the break site and prevents degradation of DNA at stalled replication forks [11–15]. This is particularly revealing in the setting of BRCA1 deﬁciency where HR is markedly reduced and DSBs that occur during S phase are funneled through 53BP1 for repair by NHEJ. Accordingly, silencing 53BP1 rescues the chro- mosomal instability, embryonic lethality and tumorigenicity in brca1−/−deﬁcient mice [16–18]. Reagents Unless otherwise noted, chemicals were purchased from Sigma Aldrich. Puriﬁed Rad18 and Rad6 proteins were purchased from Abcam and synthetic siRNAs were obtained from Dharmacon RNA Technologies. The antibodies used in this study were obtained from the following sources: 53BP1 (Santa Cruz; Abcam); ubiquitin (Santa Cruz, K4D1); βarr1 (Cell Signaling); βarr2 (Abcam); LDH (BD Biosciences); α- tubulin (Cell Signaling); γ-H2AX (Cell Signaling; Cell culture conditions and treatments Wild-type mouse embryo ﬁbroblasts (MEFs), βarr1−/− MEFs, and βarr2−/−MEFs were prepared according to the 3T3 protocol [25, 26]. Established MEFs were maintained in Dulbecco’s Modiﬁed Eagle Medium with 10% FBS and 2 mM L-glutamine at 37 °C with a 5% CO2 atmosphere in a humidiﬁed incubator. U2OS and HEK293 cells were maintained in Modiﬁed Eagle Medium with 10% FBS and 2 mM L-glutamine with the same conditions above. For knockdown of βarr1 and Rad18, cells were transfected with GeneSilencer (Gene Therapy Systems, San Diego) as described [27]. Two different types of control siRNA were used in this study: CTL1: siGLO control RNAi (catalog #: D-001620-02) from Thermo Scientiﬁc and CTL2 (5′-UUC UCCGAACGUGUCACGU-3′) [28]. siRNA for Rad18 was from Thermo Scientiﬁc (SMARTpool: ON-TARGETplus RAD18 siRNA). Lentiviral pLKO1 constructs containing shRNA sequences directed against βarr1 (CCGGGCCAG TAGATACCAATCTCATCTCGAGATGAGATTGGTAT CTACTGGCTTTTTG), 53BP1 (CCGGCGCGTCATCAC AGATGTTTATCTCGAGATAAACATCTGTGATGACG CGTTTTTG), Rad18 (GGTTAACATTCCAGAAAGTCA) and CTL (CCGGCAACAAGATGAAGAGCACCAACTC GAGTTGGTGCTCTTCATCTTGTTGTTTTT) were pur- chased from Sigma Aldrich. Retroviral N-Myc-53BP1 WT pLPC-Puro construct for 53BP1 overexpression was pur- chased from Addgene (#19836). Lentiviral CRISPR-Cas9 construct containing sgRNA against βarr1(CATCGACCT CGTGGACCCTG). The molecular events mediated by 53BP1, its recruitment to sites of DNA damage, and its interactions with other proteins are tightly controlled [19, 20]. Here, we report a novel mechanism of 53BP1 regulation. We show that βarrestin-1 (βarr1), originally identiﬁed as a molecule that desensitizes G protein-coupled receptor signaling but now known to also have roles in regulating MAPK, PI3K/AKT, and c-Src signaling [21, 22], plays an essential role in controlling the levels of basal and DNA damage-inducible 53BP1. Akin to its described function as an MDM2 ubi- quitin ligase adaptor for controlling p53 destruction [23], we now show that βarr1 functions as an E3-ubiquitin ligase adaptor that controls 53BP1 levels and thus inﬂuences the mode of repair of DNA DSBs. Importantly, our studies establish that disabling βarr1-directed 53BP1 degradation confers remarkable resistance to IR, both in vitro and in vivo, underscoring the physiological roles of βarr1 as a mediator of the DNA damage response (DDR). Real-Time PCR (RT-PCR) MEF and U2OS cells were grown on eight-well chamber slides (Labtek), coverslips or 96-well Viewplates (Perkin Elmer) and exposed to increasing levels of γ-irradiation. Cells were allowed to recover for the indicated times, washed in PBS, ﬁxed with 4% paraformaldehyde for 15 min at room temperature, permeabilized with 0.1% Triton X-100/PBS, and blocked for 30 min in blocking buffer (1% goat serum, 0.3% BSA in PBS). Cells were incubated overnight at room temperature in primary antibody diluted as per manufacturer’s recommendation in blocking buffer overnight at 4 °C, washed three times with 0.05% Tween- 20/PBS, incubated with Alexa Fluor 647 Donkey anti-rabbit IgG, Alexa Fluor 555 Donkey anti-rabbit IgG, and/or Alexa Fluor 488 Donkey anti-mouse IgG secondary antibody diluted 1:1000 in blocking buffer for 1 h at room tempera- ture. Nuclei were stained for 20 min at room temperature with 4,6-diamidino-2-phenylindole (Sigma/AppliChem) in PBS, diluted at 1:5000 from a 1 mg/ml stock. MEF cells were analyzed by mounting coverslips on microscope slides in SlowFade (Molecular Probes) and viewed with an Olympus IX81 Fluoview FV1000 confocal microscope ﬁt- ted with a 100× objective. To quantify 53BP1 foci, 96-well samples were imaged with an automated high-content Incell 1000 microscope (GE Healthcare) using a 20× Nikon objective and analyzed with the Incell Developer Toolbox version 1.6 software. U2OS cells were imaged using a Leica SP8 Laser Scanning Confocal Microscope ﬁtted with a 60× objective to view the cells and the Cytation 5 cell imaging multimode reader (Biotek) was used for imaging and For RT-PCR, WT, and βarr1−/−MEFs were exposed to IR (4 Gy). RNA was harvested 1 h post IR as well as from untreated control samples, using the Qiagen RNeasy Mini Kit as per manufacturer’s recommendations. Quantiﬁcation of RNA was performed using the Nanodrop 1000 Spec- trophotometer. RNA (2 μg) was reverse transcribed using the High Capacity Reverse Transcription Kit (Applied Biosystems) as per manufacturer’s recommendations. RT- PCR detection with SYBR green was performed with the resulting cDNA and a 50/50 forward primer: reverse primer ratio using the ABI 7900HT Fast Real-Time PCR System. The 53BP1 primer sequences were as follows: 53BP1F— ATTGAACGGTTACCTCAGCCA, 53BP1R—CCCAACT GTGATGAAGCAGAAT. GAPDH primer sequences were as follows: GAPDHF—ACACATTGGGGGTAGGAACA, GAPDHR—AACTTTGGCATTGTGGAAGG. Analysis of 53BP1 as compared with GAPDH was performed using the delta Ct equation [31]. Real-Time PCR (RT-PCR) The relative expression of 53BP1 in cells exposed to IR versus untreated controls was deter- mined using the ΔΔCt method [31] and is expressed as arbitrary units. For RT-PCR, WT, and βarr1−/−MEFs were exposed to IR (4 Gy). RNA was harvested 1 h post IR as well as from untreated control samples, using the Qiagen RNeasy Mini Kit as per manufacturer’s recommendations. Quantiﬁcation of RNA was performed using the Nanodrop 1000 Spec- trophotometer. RNA (2 μg) was reverse transcribed using the High Capacity Reverse Transcription Kit (Applied Biosystems) as per manufacturer’s recommendations. RT- PCR detection with SYBR green was performed with the resulting cDNA and a 50/50 forward primer: reverse primer ratio using the ABI 7900HT Fast Real-Time PCR System. The 53BP1 primer sequences were as follows: 53BP1F— ATTGAACGGTTACCTCAGCCA, 53BP1R—CCCAACT GTGATGAAGCAGAAT. GAPDH primer sequences were as follows: GAPDHF—ACACATTGGGGGTAGGAACA, GAPDHR—AACTTTGGCATTGTGGAAGG. Analysis of 53BP1 as compared with GAPDH was performed using the delta Ct equation [31]. The relative expression of 53BP1 in cells exposed to IR versus untreated controls was deter- mined using the ΔΔCt method [31] and is expressed as arbitrary units. Immunoblotting Western blot analyses were conducted as previously described [29]. Brieﬂy, SDS-PAGE gel electrophoresis was 1202 A. Nieto et al. performed using NuPAGE 4–12% Bis-Tris gels (Invitro- gen) and transferred to nitrocellulose membranes by semi- dry transfer, using trans-blot transfer medium (Bio-Rad). Blots were blocked with blocking buffer (5% skimmed milk in PBS with 0.02% Tween-20) then incubated at 4 °C overnight with primary antibodies, diluted in blocking buffer. Blots were washed three times in PBS with 0.02% Tween-20, and then incubated with secondary antibodies diluted in blocking buffer for 1 h at room temperature. Blots were then washed three times in PBS with 0.02% Tween- 20, and developed by SuperSignal West Pico/Femto solu- tion (Pierce). Immunoblots were quantiﬁed by densitometry using the ImageJ [30]. Alternatively, membranes were blocked in Odyssey blocking buffer (LI-COR Biosciences) and incubated overnight at 4 °C with primary antibodies. After repeated washes with TBS-T [20 mM tris (pH 7.6), 140 mM NaCl, and 0.1% Tween-20], blots were incubated with the appropriate IRDye-conjugated secondary antibody (LI-COR Biosciences) and imaged using the LI-COR Odyssey. Bands were quantiﬁed using the Odyssey soft- ware (LI-COR Biosciences). quantitative analysis. We determined the frequency of nuclei with more than ﬁve 53BP1 foci. At least 100 nuclei were analyzed for each sample. Clonogenic cell survival assay MEF cells (500) were plated in six-well dishes (n = 6) and irradiated with graded doses (0, 1, 2, 4, or 8 Gy) of γ- radiation using a 137Cs source (Gammacell 40 irradiator; 3.7 Gy/min). After 7–10 days of cell growth, during which media was changed every 2–3 days, cell colonies were ﬁxed with 4% paraformaldehyde/PBS, stained with 0.5% crystal violet in 25% methanol for 20 min at room temperature, and washed with water. Once the wells had dried, colony for- mation was document by photography and 10% acetic acid was used to resolubilize crystal violet for spectro- photometry. Absorbance was measured in duplicate at 590 nm. The fraction of surviving colonies was calculated as the ratio of the absorbance of irradiated cells to that of non- irradiated cells. Survival curves were constructed by ﬁtting the average survival levels to a linear quadratic equation using GraphPad Prism. Mass spectrometry analysis LC/MS/MS analyses were performed on a Thermo Scien- tiﬁc LTQ Orbitrap XL (Thermo Scientiﬁc) with a Finnigan Nanospray II electrospray ionization source. Peptides were injected onto a 75 μm × 150 mm BEH C18 column (particle size 1.7 μm, Waters) and separated using a Waters nano ACQUITY Ultra Performance LC™(UPLC™) System (Waters, Milford, MA). The LTQ Orbitrap XL was operated in the data dependent mode using the TOP10 strategy. In brief, each scan cycle was initiated with a full MS scan of high mass accuracy [375–1800 m/z; acquired in the Orbi- trap XL at 6 × 104 resolution setting and automatic gain control (AGC) target of 106], which was followed by MS/ MS scans (AGC target 5000; threshold 3000) in the linear ion trap on the ten most abundant precursor ions. Selected ions were dynamically excluded for 30 s. Singly charged ions were excluded from MS/MS analysis. MS/MS spectra were searched against a composite database containing the IPI Homo sapiens (human) protein sequences and their reverse sequences using the SEQUEST algorithm. Search parameters allowed two missed tryptic cleavages, a mass tolerance of ±10 ppm for precursor ion, a mass tolerance of ±0.02 D for product ion, a static modiﬁcation of 57.02146 D (carboxyamidomethylation) on cysteine, and a dynamic modiﬁcation of 15.99491 D (oxidation) on methionine. Cycloheximide chase assay New protein synthesis was blocked by the addition of 25 µg/ mL of cycloheximide to tissue culture media. Whole cell lysates were collected at different time points and analyzed by immunoblotting. Lentiviral and retroviral transduction Lentiviral particles were produced using HEK293T cells and a third-generation packaging system, MISSION® Len- tiviral Packaging Mix, per the manufacturer’s recommen- dations (Sigma Aldrich). To stably express speciﬁc βarrestin-1 regulates DNA repair by acting as an E3-ubiquitin ligase adaptor for 53BP1 1203 shRNAs, MEF, or U2OS cells were transduced with opti- mized titers of lentiviruses. Next day medium was changed, and cells were allowed to recover for 24 h before antibiotic selection (2 µg/mL of puromycin) for 3–6 days. A lentiviral CRISPR-Cas9 [32–34] construct containing sgRNA against βarr1 was used to generate U2OS βarr1 KO cell lines, mRNA expression measured by real-time PCR and western blot analysis conﬁrmed deletion of βarr1 expression in the selected clones. reduced by dithiothreitol, alkylated by iodoacetamide, and then subjected to overnight trypsin (working concentration 10 ng/µL) digestion at 37 °C. Tryptic peptides were extracted, lyophilized, resuspended in 40 μL of 5% formic acid, and then further processed on C18 resin, using handmade StageTips. Peptides were eluted with 5% formic acid, 50% acetonitrile, lyophilized with a speed-vac, reconstituted in 0.1% triﬂuoroacetic acid, 2% acetonitrile, and then subjected to LC-MS/MS analysis. Retroviral transduction was performed with ecotropic viruses. In brief, for the production of virus HEK293T cells were transiently transfected with pcl ECO vector (which expresses the viral genes gag, pol, and envEco) and the vector of interest (1:1 ratio). After 48 h, the supernatant containing the viral particles was diluted 1:2 with fresh medium, ﬁltered, and polybrene was added to a ﬁnal concentration of 5 µg/mL; MEF cells were transduced with this mix. This procedure was repeated 3 times, every 4 h. Next day medium was changed, and cells allowed to recover for 24 h before antibiotic selec- tion (2 µg/mL of puromycin) for 3–6 days. GST pulldown assay Rat βarr1 was subcloned into pGEX4T1 vector and pre- pared according to the manufacturer’s recommendations (Amersham Biosciences). 53BP1-myc (2.3 nM), Rad18 (9 nM) and Rad6 (30 nM) were co-incubated with 17 nM of GST-βarr1 or 17 nM of GST at 4 °C overnight in 1 ml binding buffer (50 mM Tris pH = 7.4, 150 mM NaCl, 0.1 mg/mL BSA, 10 μM D-myo-inositol 1,2,3,4,5,6-hexaki- sphosphate), and then 20 μl of 50% glutathione-sepharose were added to the mixture. The mixture was further incu- bated at 4 °C for 1 h with rotation. The beads were washed ﬁve times with 1 ml binding buffer and separated by SDS- PAGE and analyzed by immunoblotting. In vivo study Wild-type (C57B/6), and Arrb1knockout(βarr1−/−) mice were exposed to whole-body irradiation (8.75 Gy). Weight loss and overall survival was monitored for 40 days. Ani- mals were euthanized and endpoint apoptosis and DNA damage was assessed in the intestine. All animals used in these studies were adult male mice of 8–12 weeks of age. Animals were handled according to approved protocols and animal welfare regulations of the Institutional Review Board at The Scripps Research Institute. In-gel trypsin digestion Immunoprecipitated proteins in the βarr1 signaling com- plexes were separated by SDS-PAGE (4–20% gradient gel; Invitrogen). The gel lane was excised into six fractions. Each gel fraction was chopped into small pieces and transferred to 1.7 ml Maximum-recovery microcentrifuge tubes and subjected to in-gel trypsin digestion. In brief, the gel pieces were destained by 25 mM ammonium bicarbo- nate in 50% acetonitrile. The proteins in the gel pieces were βarr1 forms a speciﬁc complex with 53BP1 To deﬁne signaling cascades controlled by βarrestins the Lefkowitz laboratory (Duke University, NC) previously conducted global proteomic analyses to identify important binding partners and protein phosphorylation alterations induced speciﬁcally by βarrestin signaling [35, 36]. Nucleic acid binding emerged as a large functional cate- gory of βarrestin interacting proteins and 53BP1 was identiﬁed as a βarr1 interacting partner. To corroborate these results, we performed co-immunoprecipitation experiments from whole cell lysates derived from MEF cells and indeed observed that these proteins interacted or were present within the same complex (Fig. 1a). By sequential isolation of proteins associated with the cytosol, membranes, nucleus, and cytoskeleton from cell lysates, we observed that βarr1 colocalized with 53BP1 in the cytosol (Fig. 1b), which occurs via scaf- folding by βarr1 into a multiprotein complex (Supple- mentary Fig. 1). Loss of βarr1 increases 53BP1 foci and radiation resistance placed on a Leica BondMax Immunostainer and stained with the antibody previously optimized. Slides were dehy- drated and cover-slipped with Cytoseal 60 (Richard-Allan Scientiﬁc) mounting medium. placed on a Leica BondMax Immunostainer and stained with the antibody previously optimized. Slides were dehy- drated and cover-slipped with Cytoseal 60 (Richard-Allan Scientiﬁc) mounting medium. Following detection of a DSB, phosphorylation of the his- tone variant H2AX (γ-H2AX) in chromatin surrounding the break site initiates a cascade of recruiting repair proteins into microscopically visible aggregates known as DNA- repair “foci”. 53BP1 is one member of a large cast of proteins recruited into repair foci upon DNA damage [6]. Accordingly, βarr1 knockout (βarr1−/−) MEF cells display an increase in 53BP1 foci size and intensity that results in a signiﬁcant increase in overlap with γ-H2AX foci in βarr1−/− cells compared with WT cells (Fig. 2a). This observation correlates with signiﬁcantly increased numbers of 53BP1 foci compared with paired wild type (WT) in response to DNA damage induced by IR (Fig. 2b, c). Importantly, this increase in 53BP1 repair foci observed in βarr1−/−MEFs translates into a remarkably enhanced cell survival follow- ing exposure to irradiation (Fig. 2d, Supplementary Table 1), suggesting that loss of βarr1 leads to an increased capacity to repair or tolerate damaged DNA. Statistics Each experiment was repeated at least three times unless indicated otherwise. P values were calculated using Student’s t test (two-tailed), for survival data, log- rank (Mantel–Cox) test was used (GraphPad, San Diego, CA). Immunohistochemistry Parafﬁn sections (3 µm) of intestinal tissue were mounted on Plus slides and dried in a 60 °C oven. The slides were 1204 A. Nieto et al. Loss of βarr1 increases 53BP1 foci and radiation resistance Loss of βarr1 increases 53BP1 foci and radiation resistance Fig. 1 βarr1 binds to 53BP1. a Co-immunoprecipitation (co-IP) experiments were performed from cell lysates derived from MEF cells endogenously expressing βarr1 and 53BP1. Clariﬁed lysates were incubated with either, anti-βarr1 (K-16) antibody, anti-53BP1 antibody, or IgG and the resulting products resolved by SDS-PAGE and probed by western blot analysis using antibodies directed against 53BP1 and βarr1. b 53BP1 and βarr1 expression in cytosol and nucleus 53BP1 protein levels correlate with cell survival after irradiation To test whether 53BP1 is required and responsible for the increased survival of βarr1−/−cells after exposure to IR (4 Gy), we overexpressed (53BP1 OE) or downregulated (Sh 53BP1) 53BP1 in WT MEFs (Fig. 3a). Notably, the 53BP1 protein levels in the 53BP1 OE MEFs were 1.5-fold versus WT (not as high as those observed in βarr1−/−MEFs), signiﬁcantly improving the survival rate after IR (4 Gy) compared with that of WT MEFs (Fig. 3b, c). Conversely, knockdown of 53BP1 increased the sensitivity of MEFs to IR (Fig. 3b, c). Importantly, these data support the hypothesis that the βarr1-53BP1 signaling axis plays a key A B IgG 53BP1 IgG βarr1 53BP1 βarr1 53BP1 βarr1 GAPDH 190 50 190 50 30 kD Inputs Inputs IP IP WT Cytosol WT Nucleus 53BP1 βarr1 GAPDH βarr1-/- βarr1-/- Histone 190 50 10 30 kD Fig. 1 βarr1 binds to 53BP1. a Co-immunoprecipitation (co-IP) experiments were performed from cell lysates derived from MEF cells endogenously expressing βarr1 and 53BP1. Clariﬁed lysates were incubated with either, anti-βarr1 (K-16) antibody, anti-53BP1 antibody, or IgG and the resulting products resolved by SDS-PAGE and probed by western blot analysis using antibodies directed against 53BP1 and βarr1. b 53BP1 and βarr1 expression in cytosol and nucleus A IgG 53BP1 190 50 kD IP IgG βarr1 53BP1 βarr1 IP B B WT Cytosol WT Nucleus 53BP1 βarr1 GAPDH βarr1-/- βarr1-/- Histone 190 50 10 30 kD Fig. 1 βarr1 binds to 53BP1. a Co-immunoprecipitation (co-IP) experiments were performed from cell lysates derived from MEF cells endogenously expressing βarr1 and 53BP1. Clariﬁed lysates were incubated with either, anti-βarr1 (K-16) antibody, anti-53BP1 antibody, or IgG and the resulting products resolved by SDS-PAGE and probed by western blot analysis using antibodies directed against 53BP1 and βarr1. b 53BP1 and βarr1 expression in cytosol and nucleus 53BP1 βarr1 GAPDH Inputs 190 50 30 Inputs βarrestin-1 regulates DNA repair by acting as an E3-ubiquitin ligase adaptor for 53BP1 1205 B C D Radiation dose (Gy) Fractional Survival 0 1 2 3 4 0.01 0.1 1 βarr1-/- WT 0.0 0.2 0.4 0.6 0.8 1.0 βarr1-/- WT No IR IR Ratio (53BP1 foci / γ-H2AX foci) * WT βarr1-/- γ-H2AX 53BP1 DAPI Merge IR (4Gy) No IR γ-H2AX 53BP1 DAPI Merge βarr1-/- WT * * No IR IR 53BP1 foci / nucleus 20 40 60 0 A Fig. 53BP1 protein levels correlate with cell survival after irradiation 2 Loss of βarr1 augments 53BP1 foci formation and cell survival following ionizing radiation (IR). a Representative confocal immuno- ﬂuorescence microscopy images of WT and βarr1−/−MEF cells 10 min after 4 Gy IR, probed with anti-53BP1 or anti-γ-H2AX antibodies. Merged image reveals colocalization of 53BP1 foci with γ-H2AX foci and increased 53BP1-containing DNA-repair foci formation in βarr1−/−MEFs compared with WT MEFs in response to IR. Scale bar, 5 µm. b Confocal images were used to quantify the number of 53BP1 foci per nucleus in both βarr1−/−and WT MEF cells (p < 0.0001). c, d Clonogenic cell survival analysis of WT and βarr1−/−MEFs following exposure to increasing doses of IR. Experiments (n = 3) were carried out in triplicate (p < 0.05 at all doses of IR tested) WT βarr1-/- γ-H2AX 53BP1 DAPI Merge IR (4Gy) No IR γ-H2AX 53BP1 DAPI Merge A A Merge B βarr1-/- WT * No IR IR 53BP1 foci / nucleus 20 40 60 0 C 0.0 0.2 0.4 0.6 0.8 1.0 βarr1-/- WT No IR IR Ratio (53BP1 foci / γ-H2AX foci) * D Radiation dose (Gy) Fractional Survival 0 1 2 3 4 0.01 0.1 1 βarr1-/- WT D C B Fig. 2 Loss of βarr1 augments 53BP1 foci formation and cell survival following ionizing radiation (IR). a Representative confocal immuno- ﬂuorescence microscopy images of WT and βarr1−/−MEF cells 10 min after 4 Gy IR, probed with anti-53BP1 or anti-γ-H2AX antibodies. Merged image reveals colocalization of 53BP1 foci with γ-H2AX foci and increased 53BP1-containing DNA-repair foci formation in βarr1−/−MEFs compared with WT MEFs in response to IR. Scale bar, 5 µm. b Confocal images were used to quantify the number of 53BP1 foci per nucleus in both βarr1−/−and WT MEF cells (p < 0.0001). c, d Clonogenic cell survival analysis of WT and βarr1−/−MEFs following exposure to increasing doses of IR. Experiments (n = 3) were carried out in triplicate (p < 0.05 at all doses of IR tested) between WT and βarr1−/−MEFs (Fig. 4b), suggesting that βarr1 plays a posttranscriptional role. role in the observed IR tolerance. Furthermore, we observed a direct correlation between 53BP1 protein levels and resistance of MEFs (Fig. 3d), Regulation of 53BP1 by βarr1 occurs post translationally In order to discern if the accumulation of 53BP1 that is observed in the βarr1−/−MEF cells is due to improved stability of 53BP1 or an altered steady state rate of turnover, we blocked new protein synthesis by the addition of cycloheximide [38]. Comparison of the 53BP1 levels over time, between the WT and βarr1−/−MEF cells post cyclo- heximide addition, reveals that 53BP1 protein levels have a slower rate of turnover in βarr1−/−MEFs cells (Fig. 5). This is consistent with the proposal that βarr1 controls 53BP1 degradation. To gain mechanistic insight into the radiation resistance associated with βarr1 silencing, we examined 53BP1 expression levels in WT, βarr1−/−, βarr2−/−, and double knockout (DKO: βarr1−/−βarr2−/−) MEFs. Both βarr1−/− and DKO MEFs had increased levels of 53BP1 protein compared with the WT and βarr2−/−MEFs (Fig. 4a), indicating a potential role of βarr1 in regulating 53BP1. Interestingly, Kim et al. demonstrated that activation of the nuclear factor-erythroid 2-related factor 2 by the synthetic triterpenoid bardoxolone methyl, upregulates 53BP1 gene expression and acts as an IR protectant [37]. To evaluate whether regulation of 53BP1 by βarr1 is pre- or post translational, the levels of 53BP1 mRNA in WT and βarr1 −/−MEFs were assessed by qRT-PCR. There were no signiﬁcant differences in the levels of 53BP1 transcripts βarr1 regulates DNA repair by acting as an E3- ubiquitin ligase adaptor for 53BP1 through a complex with Rad18/Rad6 d Fractional survival plot as a function of 53BP1 protein expression levels (r = 0.85) A 0 1 2 3 4 53BP1 βarr1 Relative protein expression WT βarr1-/- 53BP1 OE Sh 53BP1 WT βarr1-/- Sh 53BP1 53BP1 OE 53BP1 βarr1 GAPDH kD 190 50 30 A WT βarr1-/- Sh 53BP1 53BP1 OE 53BP1 βarr1 GAPDH kD 190 50 30 0 1 2 3 4 53BP1 βarr1 Relative protein expression WT βarr1-/- 53BP1 OE Sh 53BP1 B WT βarr1-/- 53BP1 OE Sh 53BP1 B C D 53BP1 OE βarr1-/- Sh 53BP1 WT Fractional Survival 0.01 1 0.1 1 2 3 4 0 Radiation Dose (Gy) 0.0 0.2 0.4 0.6 0.8 1.0 Relative 53BP1 protein expression 2 0 1 3 4 Fractional Survival WT βarr1-/- 53BP1 OE Sh 53BP1 0.0 0.2 0.4 0.6 1.0 Fractional Survival 0.8 * Fi 3 53BP1 i i di l l i h ll i l 53BP1 i h d d l d i MEF ll b i i WT βarr1-/- 53BP1 OE Sh 53BP1 0.0 0.2 0.4 0.6 1.0 Fractional Survival 0.8 C 53BP1 OE βarr1-/- Sh 53BP1 WT Fractional Survival 0.01 1 0.1 1 2 3 4 0 Radiation Dose (Gy) D 0.0 0.2 0.4 0.6 0.8 1.0 Relative 53BP1 protein expression 2 0 1 3 4 Fractional Survival C C D 53BP1 OE βarr1-/- Sh 53BP1 WT Fractional Survival 0.01 1 0.1 1 2 3 4 0 Radiation Dose (Gy) 0.0 0.2 0.4 0.6 0.8 1.0 Relative 53BP1 protein expression 2 0 1 3 4 Fractional Survival WT βarr1-/- 53BP1 OE Sh 53BP1 0.0 0.2 0.4 0.6 1.0 Fractional Survival 0.8 Fig. 3 53BP1 protein expression directly correlates with cell survival. a 53BP1 was either overexpressed or downregulated in MEF cells obtaining a range of different expression levels of the protein. Data are mean ± s.e.m of three independent experiments. b The clonogenicity assay images corresponding to 4 Gy irradiation. c Clonogenic cell survival analysis in MEF cells revealed a signiﬁcant difference in survival after exposure to IR in the conditions tested; bar graph for 4 Gy (p < 0.01 compare with WT). Data are mean ± s.e.m of an experiment run n = 6. d Fractional survival plot as a function of 53BP1 protein expression levels (r = 0.85) Relative 53BP1 protein expression Fig. 3 53BP1 protein expression directly correlates with cell survival. βarr1 regulates DNA repair by acting as an E3- ubiquitin ligase adaptor for 53BP1 through a complex with Rad18/Rad6 a 53BP1 was either overexpressed or downregulated in MEF cells obtaining a range of different expression levels of the protein. Data are mean ± s.e.m of three independent experiments. b The clonogenicity assay images corresponding to 4 Gy irradiation. c Clonogenic cell survival analysis in MEF cells revealed a signiﬁcant difference in survival after exposure to IR in the conditions tested; bar graph for 4 Gy (p < 0.01 compare with WT). Data are mean ± s.e.m of an experiment run n = 6. d Fractional survival plot as a function of 53BP1 protein expression levels (r = 0.85) in the level of 53BP1 protein (Fig. 6b); indicating that Rad18 is an important regulator of steady state levels of 53BP1. Moreover, Rad18 knockdown decreased levels of polyubiquitylated 53BP1 (Fig. 6c). Finally, consistent with the notion that βarr1 facilitates Rad18/53BP1 interaction, βarr1−/−MEFs had reduced levels of polyubiquitylated 53BP1 compared with WT MEFs (Fig. 6d). Collectively, these ﬁndings support a role for βarr1 as an E3-ubiquitin ligase adaptor for 53BP1. stability by facilitating MDM2-dependent p53 degradation in response to stress induced by chronic β2AR signaling [29]. Mass spectrometry of proteins that bind to βarr1 in cells in response to IR revealed that the E3-ubiquitin ligase Rad18, which has previously been shown to mono-ubiquitylate 53BP1 and lead to its retention with chromatin [40–44], associates with βarr1 (Supplementary Fig. 1). Subsequent in vitro binding studies with puriﬁed recombinant proteins demonstrated that 53BP1 and βarr1 do not directly associate, but rather require the presence of Rad18 and its E2-ubiquitin- conjugating enzyme Rad6 (Fig. 6a). Thus, βarr1 may form a ternary complex comprised of the E3/E2-ubiquitin ligase Rad18/Rad6 heterodimer and 53BP1. βarr1 regulates DNA repair by acting as an E3- ubiquitin ligase adaptor for 53BP1 through a complex with Rad18/Rad6 βarr1 is known to function as an E3-ubiquitin ligase adaptor [39], which we have demonstrated inﬂuences genomic A. Nieto et al. 1206 A 0 1 2 3 4 53BP1 βarr1 Relative protein expression WT βarr1-/- 53BP1 OE Sh 53BP1 B C D 53BP1 OE βarr1-/- Sh 53BP1 WT Fractional Survival 0.01 1 0.1 1 2 3 4 0 Radiation Dose (Gy) 0.0 0.2 0.4 0.6 0.8 1.0 Relative 53BP1 protein expression 2 0 1 3 4 Fractional Survival WT βarr1-/- 53BP1 OE Sh 53BP1 WT βarr1-/- 53BP1 OE Sh 53BP1 0.0 0.2 0.4 0.6 1.0 Fractional Survival 0.8 WT βarr1-/- Sh 53BP1 53BP1 OE 53BP1 βarr1 GAPDH kD 190 50 30 Fig. 3 53BP1 protein expression directly correlates with cell survival. a 53BP1 was either overexpressed or downregulated in MEF cells obtaining a range of different expression levels of the protein. Data are mean ± s.e.m of three independent experiments. b The clonogenicity assay images corresponding to 4 Gy irradiation. c Clonogenic cell survival analysis in MEF cells revealed a signiﬁcant difference in survival after exposure to IR in the conditions tested; bar graph for 4 Gy (p < 0.01 compare with WT). Data are mean ± s.e.m of an experiment run n = 6. Discussion Fig. 5 Turnover of 53BP1 is dependent on βarr1. Lysates derived from WT and βarr1−/−MEF cells treated with 25 µg/ml of cycloheximide, were subjected to western blot analysis and analyzed for 53BP1 levels. Rel. Exp. relative expression The diverse signaling functions of βarr1 were recently revealed using a global mass spectrometry approach, which identiﬁed numerous βarr1 binding partners [35]. Included within the cast of proteins that βarr1 interacts with are those with known functions in DNA repair. We have previously demonstrated that βarr1 controls p53 levels in response to catecholamine-induced chronic stress, by acting as a scaf- fold for the E3-ubiquitin ligase MDM2 that targets p53 for degradation. This stress-induced genomic instability is βarr1-mediated, as genetic deletion of βarr1 dramatically reduces DNA damage in vivo in response to treatment with the β2-adrenergic receptor agonist, isoproterenol [29]. Fur- ther, βarr1−/−mice subjected to 2 weeks of chronic restraint had signiﬁcantly less DNA damage in the frontal cortex compared with their wild-type counterparts [46]. Here we establish an additional role for βarr1 in the response to IR, where we show βarr1 also controls the destruction of the DNA-repair protein 53BP1. to model acute radiation syndrome (ARS) [45]. Six days post IR, WT mice exhibited augmented weight loss (8–16%) versus βarr1−/−mice (6–11%), and by 8 days post IR weight loss of βarr1−/−mice (average of 13%) was half of that of WT mice (26%, Fig. 7a). This was followed by deaths of the WT mice as early as 6 days after exposure, and all WT animals were dead by day 9 (Fig. 7b). In contrast, all the βarr1−/−mice were still alive at day 9 (Fig. 7b). Thus, the βarr1 deﬁciency is associated with radiation resistance in vivo. to model acute radiation syndrome (ARS) [45]. Six days post IR, WT mice exhibited augmented weight loss (8–16%) versus βarr1−/−mice (6–11%), and by 8 days post IR weight loss of βarr1−/−mice (average of 13%) was half of that of WT mice (26%, Fig. 7a). This was followed by deaths of the WT mice as early as 6 days after exposure, and all WT animals were dead by day 9 (Fig. 7b). In contrast, all the βarr1−/−mice were still alive at day 9 (Fig. 7b). Thus, the βarr1 deﬁciency is associated with radiation resistance in vivo. ARS is associated with severe toxicity and failure of the hematopoietic, gastrointestinal, and cerebrovascular sys- tems [45]. βarr1 deﬁciency increases survival after irradiation in vivo To test if βarr1-dependent control of 53BP1 inﬂuences the DDR in vivo, WT and βarr1−/−mice were exposed to whole-body irradiation (8.75 Gy); levels that are sufﬁcient To test if Rad18 inﬂuences 53BP1 protein levels in cells, we performed knockdown studies using Rad18-speciﬁc siRNAs. Efﬁcient knockdown of Rad18 led to an increase βarrestin-1 regulates DNA repair by acting as an E3-ubiquitin ligase adaptor for 53BP1 1207 53BP1 mRNA Relative fold change WT βarr1-/- B A WT βarr1-/- DKO βarr2 tubulin βarr1 53BP1 βarr2-/- 1 2.35 1.38 2.64 53BP1 Rel. Exp. kD 190 50 50 50 Fig. 4 βarr1 post transcriptionally regulates expression levels of 53BP1. a Cell lysates derived from WT, βarr1−/−, βarr2−/−MEFs, and βarr1−/−; βarr2−/−double knockout MEFs (DKO) were immuno- blotted and analyzed using the indicated antibodies. b Real-time PCR analysis of 53BP1 mRNA levels in paired WT and βarr1−/−MEFs following exposure to IR A WT βarr1-/- DKO βarr2 tubulin βarr1 53BP1 βarr2-/- 1 2.35 1.38 2.64 53BP1 Rel. Exp. kD 190 50 50 50 53BP1 mRNA Relative fold change WT βarr1-/- B A intensity of γ-H2AX staining (Fig. 7c). This correlated with a reduction in apoptosis in the intestinal crypt cells of IR- treated βarr1−/−mice compared with IR-treated WT mice (Fig. 7d). These ﬁndings are consistent with the notion that increased levels of 53BP1 that are manifest in βarr1-deﬁcient cells augment DNA repair in IR-treated βarr1−/−mice. βarr1 forms a complex with 53BP1 in human cells Co-immunoprecipitation experiments performed using cell lysates from human osteosarcoma (U2OS) cells demon- strate that 53BP1 is also an interacting partner of βarr1 in human cells (Fig. 8a), where both proteins colocalize in the cytosol (Fig. 8b). CRISPR/ Cas9 technology was used to knockout βarr1 in U2OS cells (Fig. 8c). Immuno- ﬂuorescence studies show increased 53BP1 foci (Fig. 8d, e) after irradiation in βarr1−/−cells compare with WT cells. Moreover, βarr1−/−cells showed a signiﬁcant increase of 53BP1 protein levels (Fig. 8f), which correlates with improved survival after IR (4 Gy) compared with that of WT U2OS cells (Fig. 8g), suggesting that post- transcriptional regulation of 53BP1 by βarr1 may be a general response. While further studies are needed, the data suggest the βarr1 may also play an upstream role in the DDR in human cells. Fig. 4 βarr1 post transcriptionally regulates expression levels of 53BP1. a Cell lysates derived from WT, βarr1−/−, βarr2−/−MEFs, and βarr1−/−; βarr2−/−double knockout MEFs (DKO) were immuno- blotted and analyzed using the indicated antibodies. b Real-time PCR analysis of 53BP1 mRNA levels in paired WT and βarr1−/−MEFs following exposure to IR 0 0.5 2 4 Time (hrs) 0 0.5 2 4 53BP1 βarr1 α-tubulin MEFs WT MEFs βarr1-/- 1 0.72 1 0.39 1 0.9 0.83 0.6 53BP1 Rel. Exp. kD 190 50 50 Fig. 5 Turnover of 53BP1 is dependent on βarr1. Lysates derived from WT and βarr1−/−MEF cells treated with 25 µg/ml of cycloheximide, were subjected to western blot analysis and analyzed for 53BP1 levels. Rel. Exp. relative expression Discussion of three independent experiments A GST GST-βarr1 53BP1-myc Rad18/Rad6 + + + + + + + + + + wb:53BP1 wb:Rad18 wb:GST-βarr1 wb:GST kD 190 80 80 25 B D 0 0.2 0.4 0.6 0.8 1 1.2 53BP1 ubiquitination WT βarr1-/- * Total wb: 53BP1 wb: βarr1 IP: 53BP1 wb: ubiquitin WT βarr1-/- kD 190 80 1 8 βarr1 53BP1 Rad18 GAPDH Ctrl 2 Rad18 RNAi - + + - kD 190 30 80 with either control (Ctrl) siRNA or siRNA directed against Rad18 and treated with 2 µg/mL bleomycin (BLM). Cell lysates were immuno- precipitated with anti-53BP1 antibody and levels of polyubiquitylated 53BP1 were assessed by western blot using an anti-ubiquitin (P4D1) antibody. All bars represent mean ± s.e.m of three independent experiments. d Lysates derived from WT and βarr1−/−MEFs treated with 2 µg/mL BLM were immunoprecipitated using an anti-53BP1 antibody, immunoblotted, and probed with an anti-ubiquitin (P4D1) antibody. All bars represent mean ± s.e.m. of three independent experiments B 53BP1 Rad18 GAPDH Ctrl 2 Rad18 RNAi - + + - kD 190 30 80 B A C D 0 0.2 0.4 0.6 0.8 1 1.2 Ctrl Rad18 RNAi 53BP1 ubiquitination IP: 53BP1 wb: ubiquitin Ctrl 2 Rad18 RNAi wb: 53BP1 wb: Rad18 Total kD 190 80 Total kD 190 80 C D 0 0.2 0.4 0.6 0.8 1 1.2 Ctrl Rad18 RNAi 53BP1 ubiquitination IP: 53BP1 wb: ubiquitin Ctrl 2 Rad18 RNAi wb: 53BP1 wb: Rad18 Total kD 190 80 0 0.2 0.4 0.6 0.8 1 1.2 53BP1 ubiquitination WT βarr1-/- * Total wb: 53BP1 wb: βarr1 IP: 53BP1 wb: ubiquitin WT βarr1-/- kD 190 80 C IP: 53BP1 wb: ubiquitin Ctrl 2 Rad18 RNAi wb: 53BP1 wb: Rad18 Total kD 190 80 D C Fig. 6 βarr1 forms a complex with Rad18/Rad6 promoting poly- ubiquitylation of 53BP1. a Immunoblot showing complex formation of βarr1, Rad18/Rad6, and 53BP1 in vitro. Puriﬁed 53BP1-myc was incubated with either GST or GST-βarr1 in the presence or absence of Rad18/Rad6. Proteins residing in the precipitates were subjected to western blot analysis and probed with the indicated antibodies. Pre- sented data was cropped from the same blot image exposed for an equal time-period. b HEK293 cells were transfected with either control (Ctrl 2) siRNA or siRNA directed against Rad18 and 53BP1 levels were determined by immunoblotting. Discussion Since we observed signiﬁcant weight loss pre- ceding the IR-induced death of WT mice, we examined apoptosis in intestinal crypt cells, which are one of the most highly replicative cell types and are known to be exquisitely sensitive to the acute effects of IR [45]. Consistent with the IR resistant phenotype of βarr1−/−mice, there were sig- niﬁcant reductions in DNA damage as shown by lower Mass spectrometry and immunoprecipitation experi- ments demonstrate that βarr1 functions as an E3-ubiquitin ligase adaptor for 53BP1, promoting ubiquitination and degradation of 53BP1 by the E3-ubiquitin ligase Rad18 in complex with the E2-ubiquitin-conjugating enzyme Rad6. 1208 A. Nieto et al. A B C D 0 0.2 0.4 0.6 0.8 1 1.2 Ctrl Rad18 RNAi 53BP1 ubiquitination ** IP: 53BP1 wb: ubiquitin Ctrl 2 Rad18 RNAi wb: 53BP1 wb: Rad18 Total kD 190 80 0 0.2 0.4 0.6 0.8 1 1.2 53BP1 ubiquitination WT βarr1-/- * Total wb: 53BP1 wb: βarr1 IP: 53BP1 wb: ubiquitin WT βarr1-/- kD 190 80 GST GST-βarr1 53BP1-myc Rad18/Rad6 + + + + + + + + + + wb:53BP1 wb:Rad18 wb:GST-βarr1 wb:GST kD 190 80 80 25 53BP1 Rad18 GAPDH Ctrl 2 Rad18 RNAi - + + - kD 190 30 80 6 βarr1 forms a complex with Rad18/Rad6 promoting poly- uitylation of 53BP1. a Immunoblot showing complex formation arr1, Rad18/Rad6, and 53BP1 in vitro. Puriﬁed 53BP1-myc was bated with either GST or GST-βarr1 in the presence or absence of 18/Rad6. Proteins residing in the precipitates were subjected to ern blot analysis and probed with the indicated antibodies. Pre- ed data was cropped from the same blot image exposed for an al time-period. b HEK293 cells were transfected with either control 2) siRNA or siRNA directed against Rad18 and 53BP1 levels e determined by immunoblotting. c HEK293 cells were transfected with either control (Ctrl) siRNA or siRNA directed against Rad18 and treated with 2 µg/mL bleomycin (BLM). Cell lysates were immuno- precipitated with anti-53BP1 antibody and levels of polyubiquitylated 53BP1 were assessed by western blot using an anti-ubiquitin (P4D1) antibody. All bars represent mean ± s.e.m of three independent experiments. d Lysates derived from WT and βarr1−/−MEFs treated with 2 µg/mL BLM were immunoprecipitated using an anti-53BP1 antibody, immunoblotted, and probed with an anti-ubiquitin (P4D1) antibody. All bars represent mean ± s.e.m. Discussion c HEK293 cells were transfected with either control (Ctrl) siRNA or siRNA directed against Rad18 and treated with 2 µg/mL bleomycin (BLM). Cell lysates were immuno- precipitated with anti-53BP1 antibody and levels of polyubiquitylated 53BP1 were assessed by western blot using an anti-ubiquitin (P4D1) antibody. All bars represent mean ± s.e.m of three independent experiments. d Lysates derived from WT and βarr1−/−MEFs treated with 2 µg/mL BLM were immunoprecipitated using an anti-53BP1 antibody, immunoblotted, and probed with an anti-ubiquitin (P4D1) antibody. All bars represent mean ± s.e.m. of three independent experiments The interaction between βarr1 and 53BP1 is indirect, where it appears that βarr1 scaffolds the Rad18/Rad6 heterodimer, which then binds to and polyubiquitylates 53BP1 (Fig. 6). Rad18 plays an important role in several DDR processes, where it monoubiquitylates proliferating cell nuclear anti- gen at replication forks that are stalled by UV-induced DNA lesions [47] and facilitates chromatin retention of 53BP1 in response to IR-induced DSB [40]. In our studies, immu- noﬂuorescence analyses demonstrate that formation of the βarr1-Rad18-Rad6-53BP1 ternary complex is not observed within the DSB-repair foci (data not shown). Thus, we hypothesize that this complex maintains steady state levels of 53BP1, whereby silencing βarr1 or Rad18 raises 53BP1 levels which would be available to promote 53BP1 IR- induced DNA-repair foci formation. Further, the increase in the number and duration of 53BP1-containing foci correlates with in vitro cellular resistance to IR with a sig- niﬁcant dose modifying factor observed at each irradiation dose tested (Supplementary Table 1). This is highlighted by the correlation comparing 53BP1 protein levels and the resistance of MEF cells with IR (Fig. 3). Reduction of 53BP1 partially increases the sensitivity to IR while con- versely, overexpression of this protein provides protection against it. These data suggest that βarr1 plays an important role in DNA repair in mice through both modulating p53 [29] as well as post translational control of 53BP1 levels. Moreover, this observations translates in vivo, were βarr1 deﬁciency promotes prolonged survival after whole- organism irradiation, consistent with reduced IR-induced γ-H2AX staining and apoptosis in these mice (Fig. 7). Two major pathways of DSB repair are HR and NHEJ. HR requires a homologous DNA template and the process βarrestin-1 regulates DNA repair by acting as an E3-ubiquitin ligase adaptor for 53BP1 1209 of gene conversion is a high-ﬁdelity process that occurs during late S and G2 phases of the cell cycle. Discussion By contrast, NHEJ involves the direct ligation of the break, and depending upon the extent of processing at the break site, th f NHEJ h hi h b bilit f multilevel control of 53BP1 regulation in human cells. Since 53BP1 protein levels and chromatin binding differ in mouse and human systems, control of 53BP1 protein levels by βarr1-Rad18 might be more important in mouse than h ll F l th 53BP1 bi di t TIRR B 0 5 10 15 20 25 30 35 40 0 20 40 60 80 100 Time after IR (Days) Live Animals (%) WT (n = 6) βarr1-/- (n=8) A WT (n = 6) βarr1-/- (n=8) Time after IR (Days) % Weight Loss 0 2 4 6 8 0 5 10 15 20 25 30 35 ** 0 2 4 6 % Tunel Positive Cells WT * D TUNEL DAPI WT βarr1-/- γ-H2AX WT βarr1-/- 0 10 20 30 40 50 % Score 3 % Score 2 % Score 1 % Score 0 % of Cells γ-H2AX Level WT βarr1-/- C βarr1 -/- Fig. 7 βarr1 deﬁciency augments survival and reduces intestinal injury in mice exposed to lethal doses of IR. a Weight loss in βarr1−/−mice is decreased as compared with WT mice in response to 8.75 Gy total-body irradiation (TBI) (p < 0.001). b Increased survival rate of βarr1−/−mice in response to 8.75 Gy TBI (p < 0.0001). c IR-induced γ- H2AX staining showed lower intensity in tissue from βarr1−/− mice as compared with WT. Pathological scores were given using Aperio Image Scope software. d TUNEL analysis showing reduced apoptosis in the intestinal crypts of βarr1−/− mice as compared with WT mice 5 h after 8.75 Gy TBI. Scale bar, 200 µm. High-content image quantiﬁcation of TUNEL staining in WT and βarr1−/− crypt cells. (p < 0.05) βarrestin-1 regulates DNA repair by acting as an E3-ubiquitin ligase adaptor for 53BP1 1209 B 0 5 10 15 20 25 30 35 40 0 20 40 60 80 100 Time after IR (Days) Live Animals (%) WT (n = 6) βarr1-/- (n=8) A WT (n = 6) βarr1-/- (n=8) Time after IR (Days) % Weight Loss 0 2 4 6 8 0 5 10 15 20 25 30 35 ﬁciency al and reduces in mice exposed of IR. Discussion a Weight mice is mpared with WT e to 8.75 Gy ation (TBI) Increased βarr1−/−mice in Gy TBI (p induced γ- A WT (n = 6) βarr1-/- (n=8) Time after IR (Days) % Weight Loss 0 2 4 6 8 0 5 10 15 20 25 30 35 * B 0 5 10 15 20 25 30 35 40 0 20 40 60 80 100 Time after IR (Days) Live Animals (%) WT (n = 6) βarr1-/- (n=8) B B Time after IR (Days) Time after IR (Days) Time after IR (Days) γ-H2AX WT βarr1-/- 0 10 20 30 40 50 % Score 3 % Score 2 % Score 1 % Score 0 % of Cells γ-H2AX Level WT βarr1-/- C γ-H2AX Level γ-H2AX Level D 0 2 4 6 % Tunel Positive Cells WT * D TUNEL DAPI WT βarr1-/- βarr1 -/- βarr1-/- multilevel control of 53BP1 regulation in human cells. Since 53BP1 protein levels and chromatin binding differ in mouse and human systems, control of 53BP1 protein levels by βarr1-Rad18 might be more important in mouse than human cells. For example, the 53BP1 binding partner TIRR (Tudor-interacting repair regulator) regulates DNA repair by sequestering 53BP1 and preventing its loading onto chromosomes [20]. Moreover, 53BP1 is recruited to DNA double-strand breaks by histone marks including those catalyzed and regulated by the RNF8/RNF168/TRIP12/ UBR5 signaling complex [51]. Further studies will be required to determine whether βarr1 alters the dynamics and activities of proteins required for 53BP1 chromatin loading. of gene conversion is a high-ﬁdelity process that occurs during late S and G2 phases of the cell cycle. By contrast, NHEJ involves the direct ligation of the break, and depending upon the extent of processing at the break site, the process of NHEJ may have a higher probability for reduced ﬁdelity. DNA DSBs are repaired via both fast and slow kinetics in chromatin, and the majority of IR-induced repair in mammalian cells occurs via the more efﬁcient NHEJ pathway [48, 49]. The utilization of NHEJ may be particularly necessary when a highly proliferative cell suf- fers an excessive number of DSBs as occurs following IR exposure [50]. Discussion B 53BP1 βarr1 GAPDH H4 WT Cytosol WT Nucleus βarr1-/- βarr1-/- kD 190 50 30 10 IgG 53BP1 IgG βarr1 53BP1 βarr1 53BP1 βarr1 GAPDH 190 50 190 50 30 kD Inputs Inputs 190 50 30 A C 50 50 WT βarr1-/- #1 kD βarr1-/- #11 βarr1 βactin B B IgG 53BP1 IgG βarr1 53BP1 βarr1 53BP1 βarr1 GAPDH 190 50 190 50 30 kD Inputs Inputs 190 50 30 A 53BP1 βarr1 GAPDH H4 WT Cytosol WT Nucleus βarr1-/- βarr1-/- kD 190 50 30 10 WT βarr1-/- IR (4Gy) 53BP1 DAPI γ-H2AX Merge No IR 53BP1 DAPI γ-H2AX Merge C 50 50 WT βarr1-/- #1 kD βarr1-/- #11 βarr1 βactin D C A βactin WT βarr1-/- IR (4Gy) 53BP1 DAPI γ-H2AX Merge No IR 53BP1 DAPI γ-H2AX Merge D D D F E βarr1-/- WT * Ratio (53BP1 foci / γ-H2AX foci) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 R 53BP1 βarr1 GAPDH WT βarr1-/- Sh 53BP1 kD 190 50 30 Relative protein expression T 1-/- P1 53BP1 βarr1 0.00 0.25 0.50 0.75 1.00 1.50 1.25 1.75 F 53BP1 βarr1 GAPDH WT βarr1-/- Sh 53BP1 kD 190 50 30 Relative protein expression WT βarr1-/- Sh 53BP1 53BP1 βarr1 0.00 0.25 0.50 0.75 1.00 1.50 1.25 1.75 E βarr1-/- WT * Ratio (53BP1 foci / γ-H2AX foci) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 IR E IR G WT βarr1-/- Sh 53BP1 0.0 0.2 0.4 0.6 1.0 0.8 Fractional Survival * * IR G 0.0 0.2 0.4 0.6 0.8 1.0 Relative 53BP1 protein expression 1 0.0 0.5 1.5 2 Fractional Survival WT βarr1-/- Sh 53BP1 0.0 0.2 0.4 0.6 1.0 0.8 Fractional Survival * * WT βarr1 Sh 53BP 0.0 0.2 0.4 0.6 0.8 1.0 Relative 53BP1 protein expression 1 0.0 0.5 1.5 2 Fractional Survival G may also account for the reduced GI toxicity observed during IR-induced ARS in βarr1−/−mice (Fig. 7). These studies suggest that pharmacological blockade of the βarr1- 53BP1 signaling cascade provides a novel strategy for developing therapeutic agents with radiation protection properties through enhanced repair of IR-induced DNA may also account for the reduced GI toxicity observed during IR-induced ARS in βarr1−/−mice (Fig. 7). These studies suggest that pharmacological blockade of the βarr1- 53BP1 signaling cascade provides a novel strategy for developing therapeutic agents with radiation protection properties through enhanced repair of IR-induced DNA DSBs. Discussion We have demonstrated that silencing βarr1 protects against IR-induced DNA damage in both mouse and human cells, although the magnitude of the observed response seems to be larger in mouse cells, likely reﬂecting the Our ex vivo studies demonstrate that silencing βarr1 substantially protects cells from IR-induced toxicity. As mentioned before, this protection translates in vivo and B IgG 53BP1 IgG βarr1 53BP1 βarr1 53BP1 βarr1 GAPDH 190 50 190 50 30 kD Inputs Inputs 190 50 30 A F 53BP1 βarr1 GAPDH H4 WT Cytosol WT Nucleus βarr1-/- βarr1-/- kD 190 50 30 10 E WT βarr1-/- IR (4Gy) 53BP1 DAPI γ-H2AX Merge No IR 53BP1 DAPI γ-H2AX Merge βarr1-/- WT * Ratio (53BP1 foci / γ-H2AX foci) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 IR 53BP1 βarr1 GAPDH WT βarr1-/- Sh 53BP1 kD 190 50 30 Relative protein expression WT βarr1-/- 53BP1 53BP1 βarr1 0.00 0.25 0.50 0.75 1.00 1.50 1.25 1.75 C 50 50 WT βarr1-/- #1 kD βarr1-/- #1 D 1210 A. Nie A. Nieto et al. 1210 may also account for the reduced GI toxicity observed during IR-induced ARS in βarr1−/−mice (Fig. 7). These studies suggest that pharmacological blockade of the βarr1- 53BP1 signaling cascade provides a novel strategy for DSBs. There are several radiation countermeasures under clinical development which are supported by the Depart- ment of Defense and the Armed Forces Radiation Research Institute [52]. While each approach is promising, the B IgG 53BP1 IgG βarr1 53BP1 βarr1 53BP1 βarr1 GAPDH 190 50 190 50 30 kD Inputs Inputs 190 50 30 A F 53BP1 βarr1 GAPDH H4 WT Cytosol WT Nucleus βarr1-/- βarr1-/- kD 190 50 30 10 E WT βarr1-/- IR (4Gy) 53BP1 DAPI γ-H2AX Merge No IR 53BP1 DAPI γ-H2AX Merge βarr1-/- WT * Ratio (53BP1 foci / γ-H2AX foci) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 IR G 53BP1 βarr1 GAPDH WT βarr1-/- Sh 53BP1 kD 190 50 30 0.0 0.2 0.4 0.6 0.8 1.0 Relative 53BP1 protein expression 1 0.0 0.5 1.5 2 Fractional Survival WT βarr1-/- Sh 53BP1 0.0 0.2 0.4 0.6 1.0 0.8 Fractional Survival * * Relative protein expression WT βarr1-/- Sh 53BP1 53BP1 βarr1 0.00 0.25 0.50 0.75 1.00 1.50 1.25 1.75 C 50 50 WT βarr1-/- #1 kD βarr1-/- #11 βarr1 βactin D 1210 A. Nieto et al. Discussion There are several radiation countermeasures under clinical development which are supported by the Depart- ment of Defense and the Armed Forces Radiation Research Institute [52]. While each approach is promising, the mechanisms of action of the current agents act upstream of the IR-induced DNA break itself. We anticipate that βarrestin-1 regulates DNA repair by acting as an E3-ubiquitin ligase adaptor for 53BP1 1211 Fig. 8 βarr1 forms a complex with 53BP1 and inﬂuences the DNA damage response also in human cells. a Co-immunoprecipitation (co- IP) experiments were performed from cell lysates derived from U2OS cells endogenously expressing βarr1 and 53BP1. Clariﬁed lysates were incubated with either anti-βarr1 (K-16) antibody, or anti-53BP1 anti- body or IgG and the resulting products resolved by SDS-PAGE and probed by western blot analysis using antibodies directed against 53BP1 and βarr1. b 53BP1 and βarr1 expression in cytosol and nucleus. c Cell lysates derived from WT U2OS, U2OS/Cas9/ARRB1- gRNA1 clone 1 (βarr1−/−#1) and U2OS/Cas9/ARRB1-gRNA1 clone 11 (βarr1−/−#11) were immunoblotted and analyzed using the indi- cated antibodies. βarr1−/−#11 was selected for further assays. d Representative confocal immunoﬂuorescence microscopy images of WT and βarr1−/−U2OS cells 10 min after 4 Gy IR, probed with anti- 53BP1 or anti-γ-H2AX antibodies. Merged image reveals colocaliza- tion of 53BP1 foci with γ-H2AX foci and increased 53BP1-containing DNA-repair foci formation in βarr1−/−U2OS compared with WT U2OS in response to IR. e High-content imaging quantiﬁcation comparing the ratio of 53BP1 foci to γ-H2AX foci in both βarr1−/− and WT U2OS cells (p < 0.05). f βarr1 and 53BP1 were down- regulated in U2OS cells obtaining a range of different expression levels of 53BP1. Data are mean ± s.e.m of three independent experi- ments. g Clonogenic cell survival analysis in U2OS cells revealed a signiﬁcant difference in survival after exposure to IR in the conditions tested; bar graph for 4 Gy (**p < 0.0001 compare with WT). Data are mean ± s.e.m of an experiment run n = 3. Fractional survival plot as a function of 53BP1 protein expression levels (r = 0.93) in U2OS cells Goldstein and L. Wingler for experimental guidance. We also thank Dr John L. Cleveland and members of the Duckett and McDonald lab for input and editing of the paper. Goldstein and L. Wingler for experimental guidance. We also thank Dr John L. Cleveland and members of the Duckett and McDonald lab for input and editing of the paper. Discussion Funding This work was supported by funds from the State of Florida to The Scripps Research Institute, Florida, and institutional funds provided by Mofﬁtt Cancer Center and Research Institute. Author contributions AN and MRH were the primary authors of the paper and designed and executed experiments, collected, analyzed, and interpreted data. VS, VQ, and KX collected and contributed data with contributions from WG. PHM and DRD oversaw the study, con- tributed experimental design, and interpreted the data. AN, PHM, and DRD cowrote the paper. Conﬂict of interest The authors declare that they have no conﬂict of interest. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. 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To view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/. pharmacologically mimicking the βarr1 knockout pheno- type would act as an effective countermeasure to IR and may also augment the efﬁcacy of the current molecules under investigation. BRCA1 is an essential component of HR-mediated repair, and tumors arising in patients with mutated BRCA1 have evidence of genomic instability and defects in DNA repair [53–55]. Indeed, this repair defect has been exploited to develop “synthetic lethal” treatment strategies using PARP inhibitors. However, the efﬁcacy of PARP inhibitors has been plagued with resistance mechanisms that restore the repair capacity of HR, including secondary mutations in BRCA1, loss of 53BP1, or silencing of the subunits com- promising the multiprotein Shieldin complex which can reverse many aspects of the BRCA1 deﬁcient phenotype [16–18, 56]. 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https://openalex.org/W2903135009	https://www.frontiersin.org/articles/10.3389/fneur.2018.00941/pdf	English	null	The Method of Silent Substitution for Examining Melanopsin Contributions to Pupil Control	Frontiers in neurology	2,018	cc-by	8,290	The Method of Silent Substitution for Examining Melanopsin Contributions to Pupil Control Manuel Spitschan 1* and Tom Woelders 2 1 Department of Experimental Psychology, University of Oxford, Oxford, United Kingdom, 2 Chronobiology Unit, Groningen Institute for Evolutionary Life Sciences, University of Groningen, Groningen, Netherlands The human pupillary light response is driven by all classes of photoreceptors in the human eye—the three classes of cones, the rods, and the intrinsically photosensitive retinal ganglion cells (ipRGCs) expressing the photopigment melanopsin. These photoreceptor classes have distinct but overlapping spectral tuning, and even a monochromatic light with a wavelength matched to the peak spectral sensitivity of a given photoreceptor will stimulate all photoreceptors. The method of silent substitution uses pairs of lights (“metamers”) to selectively stimulate a given class of photoreceptors while keeping the activation of all others constant. In this primer, we describe the method of silent substitution and provide an overview of studies that have used it to examine inputs to the human pupillary light response. Keywords: pupil, melanopsin, silent substitution, color vision, pupillometry, ipRGC (intrinsically photosensitive retinal ganglion cell), metamers Edited by: Andrew J. Zele, Queensland University of Technology, Australia INTRODUCTION Reviewed by: Sei-ichi Tsujimura, Kagoshima University, Japan Dingcai Cao, University of Illinois at Chicago, United States Reviewed by: Sei-ichi Tsujimura, Kagoshima University, Japan Dingcai Cao, University of Illinois at Chicago, United States Correspondence: Manuel Spitschan manuel.spitschan@psy.ox.ac.uk At the input level, the size of the pupil is controlled by the activity of the diﬀerent photoreceptors in the human eye (1). These diﬀerent photoreceptors diﬀer in many respects: their wavelength tuning (spectral sensitivity), their temporal properties, their operating range and their distribution across the retina. The goal of this primer is to describe the method of silent substitution for examining photoreceptor-speciﬁc pupil responses. We start with the fundamentals underlying the method of silent substitution, provide an overview of studies that have used this method, provide a practical guide and R code to implement silent substitution and highlight a few challenges to the method of silent substitution. Correspondence: Manuel Spitschan manuel.spitschan@psy.ox.ac.uk Specialty section: This article was submitted to Neuro-Ophthalmology, a section of the journal Frontiers in Neurology METHODS published: 27 November 2018 doi: 10.3389/fneur.2018.00941 METHODS published: 27 November 2018 doi: 10.3389/fneur.2018.00941 FUNDAMENTALS Overlapping Spectral Sensitivities of the Human Photoreceptors Photoreception in the human retina is based on the signals produced by the three types of cones—the long[L]-wavelength-sensitive cones, the medium[M]-wavelength-sensitive cones, and the short[S]-wavelength-sensitive cones—, the rods, and the intrinsically photosensitive retinal ganglion cells (ipRGCs), which contain the photopigment melanopsin (2–6). ipRGCs receive synaptic input from cones and rods but, in the absence of those inputs, these cells themselves are photosensitive due to the expression of the melanopsin photopigment in the cell membrane. The peak spectral sensitivities (λmax) of the human photoreceptors are distinct. The photopigments (cone opsins) in the L, M, and S cones peak around 420, 530, and 558 nm, respectively; rhodopsin, the pigment in rods, has a peak at around 495 nm. Finally, the melanopsin photopigment has a Received: 04 September 2018 Accepted: 17 October 2018 Published: 27 November 2018 Wavelength Exchange peak spectral sensitivity at around 480 nm. Even though these peaks are spectrally distinct and distant, the spectral sensitivities overlap quite extensively due to the relative broadband tuning of photopigments (Figure 1A). One challenge in targeting the operation of a single class of photoreceptor is that the spectral sensitivities of the photoreceptors in vivo does not necessarily correspond to the spectral sensitivity of a pigment. All light that reaches the retina is ﬁltered by the lens and ocular media (7), thereby shifting the eﬀective spectral sensitivity. Typically, this pre-receptoral ﬁltering is accounted for in the spectral sensitivities for cones, rods, and melanopsin-containing ipRGCs. Because photoreceptors weight input light by their spectral sensitivity function, in the case of two photoreceptors, it is possible to ﬁnd two lights and scale them such that the excitation of one of the photoreceptors remains constant in this wavelength exchange, while the other one “sees” a diﬀerence. This is shown in Figure 1D: The peak emissions of lights E1 and E2 have been chosen to match the two 50% points of the S-cone spectral sensitivity, thereby eliciting the same responses. This is called silencing the S cones. Because the spectral sensitivity of melanopsin is diﬀerent from that of the S cones, our two lights E1 and E2 necessarily produce a diﬀerent response, and in this case we call melanopsin the stimulated photoreceptor. Wavelength exchange for two photopigments is the most simple case of silent substitution. But, with the exception of certain classes of color-blindness such as rod monochromacy, the human retina contains ﬁve photoreceptors. Fortunately, the same principle can be extended to more than two photoreceptors. Fundamentals To introduce the method of silent substitution we begin with an example from human color vision. Human color vision is trichromatic under daylight conditions, i.e., when rods do not participate: A color-normal observer can match the color appearance of any light using a combination of three primary lights (15). Under these conditions, it is assumed that only the three classes of cones participate in the color match; it follows that because three photoreceptors participate, three independent primary lights need to be used. It is impossible to match the activation of three photoreceptors in one condition using just two primary lights. Non-speciﬁcity of Single-Wavelength Lights An important desideratum for examining how the diﬀerent photoreceptors contribute to the human pupillary light response is that stimuli produce responses speciﬁc to a given photoreceptor class. One consequence of the extensive spectral overlap of the photoreceptors is that most light sources activate all photoreceptors, and therefore, the responses elicited are largely nonspeciﬁc. For example, monochromatic light with a peak spectral output of 490 nm will activate melanopsin maximally relative to the other photoreceptors, but it will also lead to substantial activation of rods and the cones (Figure 1B). The relative amounts by which a monochromatic light of a given wavelength activates all photoreceptors is directly predicted from the relative spectral sensitivity of the photoreceptors at that wavelength. Monochromatic lights have been of great use in determining the spectral sensitivity of the sustained pupil constrictions that match that of melanopsin (8–11). This speciﬁc type of measurement is called the “post-illumination pupil response,” abbreviated PIPR, in which the pupil response is typically measured in response to a non-speciﬁc short- wavelength light ﬂash and a non-speciﬁc long-wavelength light ﬂash against a dim or no background. THE METHOD OF SILENT SUBSTITUTION In the method of silent substitution, pairs of light are found that have the property that they stimulate the targeted photoreceptor class (or classes) whilst not changing the excitation of the other photoreceptors, the silenced ones. The method has a long history for determining the properties of the mechanisms of human color vision (13, 14). Citation: Spitschan M and Woelders T (2018) The Method of Silent Substitution for Examining Melanopsin Contributions to Pupil Control. Front. Neurol. 9:941. doi: 10.3389/fneur.2018.00941 November 2018 \| Volume 9 \| Article 941 Frontiers in Neurology \| www.frontiersin.org Method of Silent Substitution in Pupillometry Spitschan and Woelders Frontiers in Neurology \| www.frontiersin.org Principle of Univariance One property of photoreceptors is the principle of univariance (12), which states that a given photoreceptor has only scalar output, namely its photocurrent: It cannot distinguish between changes in intensity and changes in wavelength. This is shown in Figure 1C using theoretical lights containing power at only a single wavelength (monochromatic light): Lights E1, E2, and E3 all nominally elicit the same photoreceptor excitation. Lights E1 and E3 have their peak emission at the 50% point of spectral sensitivity on either side of the peak; light E2 is scaled to be 50% of the emission of lights E1 and E3. To the photoreceptor (in this case melanopsin), which weights the input light by its spectral sensitivity, the lights are equally eﬀective. The key insight is that photoreceptors integrate light of diﬀerent wavelengths, weighting the input spectrum by their spectral sensitivity and summing it up. A consequence of the principle of univariance is that single photoreceptors are color-blind: Whether two lights diﬀer in wavelength or intensity cannot be determined from the photoreceptor output alone. In general, to stimulate one class of photoreceptor classes out of NR photoreceptor classes while leaving the activation of the other NR−1 unchanged, at least NR primary lights (Np) are necessary. When NR = NP (i. e. there are as many primary lights as photoreceptor classes under consideration), there is only one algebraic solution to match the activation of the NR−1 photoreceptors under one set of settings for the NP lights to another other setting that will only stimulate the remaining photoreceptor class. For the case of four photoreceptor classes in the human retina (three classes of cones and melanopsin), four lights are necessary to match the activation of cones and stimulate melanopsin. When including the rods, ﬁve lights are necessary to match the activation of cones and rods and stimulate melanopsin. November 2018 \| Volume 9 \| Article 941 2 Method of Silent Substitution in Pupillometry Spitschan and Woelders A B C D \| (A) Overlapping spectral sensitivities of the human photoreceptors. (B) Non-speciﬁcity of single-wavelength lights. Right panel: Pattern of photoreceptor to the single-wavelength light at 490 nm. (C) Principle of univariance. Right panel: Pattern of photoreceptor responses to the single-wavelength lights E1, 3 designed to elicit the same response in melanopsin. (D) Wavelength exchange between two short-wavelength lights E1 and E2 which stimulate S cones at evel but yield different photoreceptor responses for melanopsin. Principle of Univariance Right panel: Pattern of excitations for lights E1 and E2. B C D FIGURE 1 \| (A) Overlapping spectral sensitivities of the human photoreceptors (B) Non-speciﬁcity of single-wavelength lights Right panel: Pattern of photoreceptor B C B D FIGURE 1 \| (A) Overlapping spectral sensitivities of the human photoreceptors. (B) Non-speciﬁcity of single-wavelength lights. Right panel: Pattern of photoreceptor responses to the single-wavelength light at 490 nm. (C) Principle of univariance. Right panel: Pattern of photoreceptor responses to the single-wavelength lights E1, E2, and E3 designed to elicit the same response in melanopsin. (D) Wavelength exchange between two short-wavelength lights E1 and E2 which stimulate S cones at the same level but yield different photoreceptor responses for melanopsin. Right panel: Pattern of excitations for lights E1 and E2. unstimulated ones, and enforcing additional constraints on the optimisation. It is possible to have more primary lights than photoreceptors under consideration, i.e., NR < NP. This would for example be the case when there were, e.g., eight independent primaries and the retina to be studied was a human one. In that case, there are inﬁnitely many solutions to match the activation of the NR−1 photoreceptors under one set of settings for the NP lights to any other setting that will only stimulate the remaining photoreceptor class. In practice, this is typically solved by implementing a numerical optimization which maximizes the contrast seen by the stimulated photoreceptor while setting a constraint to have no contrast on the Frontiers in Neurology \| www.frontiersin.org An Intuitive Example We now describe an example case for the method of silent substitution corresponding to the stimuli used in Spitschan, Jain, Brainard and Aguirre (16). These authors used a calibrated spectrally tuneable light source that modiﬁed the output of a broadband Xenon arc lamp using a digital micromirror device (DMD) to produce, eﬀectively, arbitrary spectral power distributions. While this is a special case of light sources, most experimenters have used a set of discrete lights, the intensities of which are controlled to produce silent substitution stimuli. The goal is to produce two lights with spectral power distributions that do not diﬀer in the amount they activate the cones, and only yield a change in the amount they activate melanopsin. Such pairs of stimuli are also called metamers– they are indistinguishable to the cones, despite having diﬀerent spectral power distributions. In this example, we ignore the rods. 5. g 5. Inverting the melanopsin activation: The modulation spectrum shown in Figure 2E (red line) produces a signiﬁcant increase in melanopsin excitation. By “mirroring” the modulation spectrum around the background spectrum (i.e., an increase in emitted light in the positive modulation spectrum becomes a decrease by the same amount in emitted light in the negative modulation spectrum), we can also generate a negative (rather than a positive) melanopsin stimulus (Figure 2F, blue line), thereby producing negative, or decremental, contrast on melanopsin (Figure 2F, right panel). In practice, negative and positive modulation spectra are alternated to yield the highest diﬀerential activation possible. 5. Inverting the melanopsin activation: The modulation spectrum shown in Figure 2E (red line) produces a signiﬁcant increase in melanopsin excitation. By “mirroring” the modulation spectrum around the background spectrum (i.e., an increase in emitted light in the positive modulation spectrum becomes a decrease by the same amount in emitted light in the negative modulation spectrum), we can also generate a negative (rather than a positive) melanopsin stimulus (Figure 2F, blue line), thereby producing negative, or decremental, contrast on melanopsin (Figure 2F, right panel). In practice, negative and positive modulation spectra are alternated to yield the highest diﬀerential activation possible. 1. Background spectrum: In the ﬁrst instance, we begin with a background spectrum of known spectral power distribution (Figure 2A). An Intuitive Example We call this the background spectrum because the observer is typically light-adapted to this spectrum, and the silent-substitution stimuli are shown to the observer “around” this background in the form of pulses or temporal modulations. This background spectrum elicits a pattern of photoreceptors responses (Figure 2B, right panel). The activation of photoreceptors is calculated by weighting the spectrum by the spectral sensitivities and summing it up for each photoreceptor class. p p 2. Increasing melanopsin activation: Pragmatically, we can increase the amount of light seen by melanopsin by simply increasing the amount of light emitted near the melanopsin peak. This is shown in Figure 2B. However, this is only partly successful: Because of the overlapping peak spectral sensitivities of the human photoreceptors, such an increase in emitted light leads to an increase in activation of all photoreceptors (Figure 2B, middle panel). Rather than considering the absolute amount of activation of the photoreceptors (which is also dependent on the exact light level), it is customary to speak of contrast (Figure 2B, right panel). Contrast here refers to the percentage diﬀerence in activation of photoreceptors between the modulation spectrum (red line in Figure 2B) and the background spectrum (Figure 2A and dashed line in Figures 2B–F). As can be seen in the right panel in Figure 2B, the increase in light near the melanopsin peak leads to an increase in contrast to all photoreceptors. To reiterate, the desideratum here is to have no contrast seen by L, M and S cones, and positive contrast seen by melanopsin. 2. Increasing melanopsin activation: Pragmatically, we can increase the amount of light seen by melanopsin by simply increasing the amount of light emitted near the melanopsin peak. This is shown in Figure 2B. However, this is only partly successful: Because of the overlapping peak spectral sensitivities of the human photoreceptors, such an increase in emitted light leads to an increase in activation of all photoreceptors (Figure 2B, middle panel). Rather than considering the absolute amount of activation of the photoreceptors (which is also dependent on the exact light level), it is customary to speak of contrast (Figure 2B, right panel). Contrast here refers to the percentage diﬀerence in activation of photoreceptors between the modulation spectrum (red line in Figure 2B) and the background spectrum (Figure 2A and dashed line in Figures 2B–F). A Quantitative Example We provide a quantitative example along with code in Appendices A1, A2, respectively. We use the stimuli from Woelders et al. (17) for this example. Contrast The term contrast refers to a speciﬁc quantity, which is the fractional diﬀerence of activation of a photopigment around a background: I I I = Imodulation −Ibackground Ibackground Intuitively, when the light-adapted background activates a given photoreceptor by some amount, e.g., 100 (arbitrary units), November 2018 \| Volume 9 \| Article 941 Frontiers in Neurology \| www.frontiersin.org 3 Method of Silent Substitution in Pupillometry Spitschan and Woelders and the modulation activates it by a higher amount, e.g., 120 (arbitrary units), the contrast in that case would be 0.2 or 20%. Contrast can be speciﬁed either as fractions or as percentages. cones (Figure 2C, middle panel). There is no diﬀerence in the absolute activation of S cones, and consequently, the S cone contrast is zero–they are silent. y 4. Silencing L and M cones: To silence the L and M cones, a similar trick is applied: Light near the peak spectral sensitivity of L and M cones is decreased to reduce the overall absolute activation of L and M cones (Figure 2D). However, we note that there has been an “overshoot” in the decrease in L and M cones activation (Figure 2D, middle panel): The modulation spectrum is now producing less activation in the L and M cones than the background spectrum. This translates into a small amount of negative contrast seen by the L and M cones (Figure 2D, right panel). This can be overcome by again increasing the amount of long-wavelength light in the modulation spectrum (Figure 2E), thereby equalizing the activation of L and M cones relative to the background spectrum (Figure 2D, middle panel). The L, M and S cones are now silent (Figure 2E, right panel), and melanopsin is stimulated at 50%. Because no attempt was taken to silence the rods, they are also stimulated by this spectral exchange. 4. An Intuitive Example As can be seen in the right panel in Figure 2B, the increase in light near the melanopsin peak leads to an increase in contrast to all photoreceptors. To reiterate, the desideratum here is to have no contrast seen by L, M and S cones, and positive contrast seen by melanopsin. History of Silent Substitution The method of silent substitution has enjoyed use in empirical work well before the discovery of melanopsin and the ipRGCs. We point the reader to Estévez and Spekreijse (13) for an exposé of the early history of the method, which indeed dates back to experiments involving wavelength exchanges performed in 1906 (18) (see above section “Wavelength exchange”). From the insight that metameric lights such as those obtained in color matching experiments are silent substitution stimuli (i.e., matching the activation of the three cone types), the extension to experimentally control more photoreceptors is conceptually straightforward. In the 1990s, methods to manipulate four photoreceptors independently (three cone classes and rods) using mixtures of four primary lights were developed (18, 19). These methods were then expanded to examining melanopsin function either by 3. Silencing S cones: To zero, or silence, the S cones, we decrease the amount of short-wavelength light, to which the S cones are most sensitive (Figure 2C). This indeed leads to a silencing of S November 2018 \| Volume 9 \| Article 941 Frontiers in Neurology \| www.frontiersin.org 4 Method of Silent Substitution in Pupillometry Spitschan and Woelders A B C D E F FIGURE 2 \| (A) Background spectrum (left panel) to which the observer is light-adapted, eliciting a pattern of responses in the photoreceptors (right panel). (B) Increase in emitted light near the melanopsin peak relative to the background spectrum (left panel; dashed line = background spectrum, red line = modulation spectrum) leads to an increase in the excitation of all photoreceptors (middle panel), or equivalently, positive contrast on the photoreceptors (right panel). (C) To balance the excitation of the S cones, a decrease in emitted short-wavelength light (left panel) leads to silencing of the S cones (middle panel), or equivalently, zero contrast on the S cones (right panel). (D) To balance the excitation of the L and M cones, a decrease in emitted medium-wavelength light (left panel) leads to a reduction in L and M cone activity (middle panel) but not yet zero contrast on the L and M cones (right panel); indeed, the contrast seen by the L and M cones is now negative. (E) To silence the excitation of the L and M cones, a decrease in emitted long-wavelength light (left panel) leads to balancing of the L and M cones (middle panel), or equivalently, zero contrast on the L and M cones (right panel). History of Silent Substitution The contrast seen by melanopsin is 50%. (F) The modulation spectrum shown in (E) yields positive contrast relative to the background spectrum but the spectrum can also be “mirrored” around the background spectrum, thereby leading to a negative modulation of melanopsin (and rods). A B C D E F C FIGURE 2 \| (A) Background spectrum (left panel) to which the observer is light-adapted, eliciting a pattern of responses in the photoreceptors (right panel). (B) Increase in emitted light near the melanopsin peak relative to the background spectrum (left panel; dashed line = background spectrum, red line = modulation spectrum) leads to an increase in the excitation of all photoreceptors (middle panel), or equivalently, positive contrast on the photoreceptors (right panel). (C) To balance the excitation of the S cones, a decrease in emitted short-wavelength light (left panel) leads to silencing of the S cones (middle panel), or equivalently, zero contrast on the S cones (right panel). (D) To balance the excitation of the L and M cones, a decrease in emitted medium-wavelength light (left panel) leads to a reduction in L and M cone activity (middle panel) but not yet zero contrast on the L and M cones (right panel); indeed, the contrast seen by the L and M cones is now negative. (E) To silence the excitation of the L and M cones, a decrease in emitted long-wavelength light (left panel) leads to balancing of the L and M cones (middle panel), or equivalently, zero contrast on the L and M cones (right panel). The contrast seen by melanopsin is 50%. (F) The modulation spectrum shown in (E) yields positive contrast relative to the background spectrum but the spectrum can also be “mirrored” around the background spectrum, thereby leading to a negative modulation of melanopsin (and rods). assuming rod saturation at high light levels (20), or using ﬁve primaries (21). authors of this article have published papers using the method of silent substitution which are included in the table [M.S.: (16, 28), T.W.: (17)]. The table shows that there is a set of experimental parameters that are subject to the experimenters’ discretion. We summarise these here. Number of Primaries (21) 5 473 ± 25 nm 511 ± 33 nm 530 ± 36 nm 595 ± 15 nm 627 ± 20 nm Light booth with white paint Ganzfeld Melanopsin-only (cone and rod silent) [3.4%] Multiple mixed modulations 35 cd/m2 10 Measurement after 1 min of continuous exposure Cones: CIEPO2006 Rods: V’(λ) Melanopsin: Stockman-Sharpe shifted to peak at 482 nm; optical density 0.1; lens from Stockman & Sharpe Tsijumura and Tokuda, (22) 4 468, 524, 599, and 633 nm (test) 466 nm, 500 nm, 517 nm, 596 nm (background) ±15–38 nm Diffusing screen in front of integrating sphere Annulus id 5 od 18◦ Total ﬁeld 23◦ 8% 612 cd/m2 background 1,109 cd/m2 test ﬁeld 6 Sinusoidal & square wave stimuli Cones: CIEPO2006, 10◦ Melanopsin: Dartnall nomogram at 480 nm Assumes macular and lens ﬁltering from CIEPO2006 Optical density 0.4 Spitschan et al. (16) 128 n/a Viewing of surface through lens 27.5◦circular, central 5◦blocked S, (L+M), melanopsin, (L+M+melanopsin) [50%] 382–1,033 cd/m2 16 Sinusoidal, 0.01 – 2 Hz 10◦ Stockman–Sharpe/CIE cone fundamentals, melanopsin estimated by shifting tudies examining human pupil responses with silent substitution. Number of primaries Primary wavelengths [nm] Viewing geometry Field size Modulations [max. Number of Primaries We provide an overview of extant studies examining speciﬁcally melanopsin photoreception using the method of silent substitution in Table 1 and hope that it serves to the reader as an orientation to the literature. This overview includes literature available in early September 2018. We note that both As described above (The method of silent substitution– Fundamentals), when stimulating melanopsin, at least four (for matching the cones) or ﬁve (for matching both cones and rods) independent primary lights are necessary. Most silent November 2018 \| Volume 9 \| Article 941 Frontiers in Neurology \| www.frontiersin.org 5 Method of Silent Substitution in Pupillometry Spitschan and Woelders p p g (same as cones, optical density 0.3). Barrionuevo et al. (23) 4 442, 516, 594, and 634 nm (one set) 466, 514, 590, and 634 nm (second set) Ganzfeld 54◦ﬁeld Mixed joint modulations, no melanopsin-isolaing modulation 0.002–100 cd/m2 3 (authors) Sinusoidal, 0.5–8 Hz Smith–Pokorny cone fundamentals Enezi et al. melanopsin function (Continued) N b 2018 \| V l 9 \| A ti l 941 TABLE 1 \| Studies examining human pupil responses with silent substitution. References Number of primaries Primary wavelengths [nm] Viewing geometry Field size Modulations [max. contrast] Background light level N observers Temporal properties Spectral sensitivities assumed Tsujimura et al. (20) 4 470, 500, 525, 615 nm ± 20–36 nm Diffusing screen in front of integrating sphere 20◦ﬁeld size Melanopsin [−53%] Luminance [−53%] Isochromatic [−53%] 301 cd/m2 to 642 cd/m2 to 982 cd/m2 6 10 min stimuli 5 min background Cones: CIEPO2006 Melanopsin: Dartnall nomogram at 482 nm Assumes macular and lens ﬁltering from CIEPO2006 Optical density 0.5 Viénot et al. Frontiers in Neurology \| www.frontiersin.org November 2018 \| Volume 9 \| Article 941 Number of Primaries contrast] Background light level N observers Temporal properties Spectral sensitivities assumed 4 470, 500, 525, 615 nm ± 20–36 nm Diffusing screen in front of integrating sphere 20◦ﬁeld size Melanopsin [−53%] Luminance [−53%] Isochromatic [−53%] 301 cd/m2 to 642 cd/m2 to 982 cd/m2 6 10 min stimuli 5 min background Cones: CIEPO2006 Melanopsin: Dartnall nomogram at 482 nm Assumes macular and lens ﬁltering from CIEPO2006 Optical density 0.5 5 473 ± 25 nm 511 ± 33 nm 530 ± 36 nm 595 ± 15 nm 627 ± 20 nm Light booth with white paint Ganzfeld Melanopsin-only (cone and rod silent) [3.4%] Multiple mixed modulations 35 cd/m2 10 Measurement after 1 min of continuous exposure Cones: CIEPO2006 Rods: V’(λ) Melanopsin: Stockman-Sharpe shifted to peak at 482 nm; optical density 0.1; lens from Stockman & Sharpe 4 468, 524, 599, and 633 nm (test) 466 nm, 500 nm, 517 nm, 596 nm (background) ±15–38 nm Diffusing screen in front of integrating sphere Annulus id 5 od 18◦ Total ﬁeld 23◦ 8% 612 cd/m2 background 1,109 cd/m2 test ﬁeld 6 Sinusoidal & square wave stimuli Cones: CIEPO2006, 10◦ Melanopsin: Dartnall nomogram at 480 nm Assumes macular and lens ﬁltering from CIEPO2006 Optical density 0.4 128 n/a Viewing of surface through lens 27.5◦circular, central 5◦blocked S, (L+M), melanopsin, (L+M+melanopsin) [50%] 382–1,033 cd/m2 16 Sinusoidal, 0.01 – 2 Hz 10◦ Stockman–Sharpe/CIE cone fundamentals, melanopsin estimated by shifting Stockman-Sharpe nomogram to λmax = 480 nm, corrected for prereceptoral ﬁltering (same as cones, optical density 0.3). 4 442, 516, 594, and 634 nm (one set) 466, 514, 590, and 634 nm (second set) Ganzfeld 54◦ﬁeld Mixed joint modulations, no melanopsin-isolaing modulation 0.002–100 cd/m2 3 (authors) Sinusoidal, 0.5–8 Hz Smith–Pokorny cone fundamentals Enezi et al. melanopsin function TABLE 1 \| Studies examining human pupil responses with silent substitution. Frontiers in Neurology \| www.frontiersin.org November 2018 \| Volume 9 \| Article 941 Method of Silent Substitution in Pupillometry Spitschan and Woelders \| References Number of primaries Primary wavelengths [nm] Viewing geometry Field size Modulations [max. contrast] Background light level N observers Spectral sensitivities assumed Cao et al. November 2018 \| Volume 9 \| Article 941 Frontiers in Neurology \| www.frontiersin.org Number of Primaries (25) 5 456, 488, 540, 592, 633 Maxwellian view 30◦circular, central 10.5◦blocked Experiment 1: S, M, L, Rod, Melanopsin [16%] Experiment 2: CSF Experiment 1: 200 Photopic Td Experiment 2: 2,000 Photopic Td 3 Sinusoidal, 1 Hz Smith–Pokorny cone fundamentals applied for the CIE 1964 10◦ Standard Observer CIE 1951 scotopic luminosity function Enezi et al. melanopsin function Barrionuevo and Cao, (24) 5 456, 488, 540, 592, 633 Maxwellian view 30◦circular, central 10.5◦blocked Experiment 1: S, M, L, Rods, Melanopsin, (L+M+S) [17%], red-green [4% M,−4% L] Experiment 2: LMS [17% each], L+M [17% each], LMS + melanopsin [16% each], Rods + melanopsin [9% each], S + melanopsin [16% each], red-green + melanopsin [2% M,−2% L, 8% melanopsin] 2–20,000 Photopic Td 3 (2 authors) Sinusoidal, 1 Hz Smith–Pokorny cone fundamentals applied for the CIE 1964 10◦ Standard Observer CIE 1951 scotopic luminosity function Enezi et al. melanopsin function Spitschan et al. (28) 56 n/a Viewing of surface through lens 64◦circular, central 5◦ blocked 25–400% 100– 200 cd/m2 4 Tapered pulses (3 s, 14–16 s ISI) CIE 2006 parametric model Woelders et al. (17) 5 465, 500, 515, 595 Diffusing screen in front of LEDs 24.68◦horizontal, 12.13◦vertical S, M, L, Melanopsin [23%] Background or average of 8.5 melanopic lux 16 Square-wave (0.25–4 Hz) α-opic lux (Lucas et al.): Govardovski nomograms, λmax from Dartnall, optical densities 0.3, 0.38, 0.38 (S, M, L) Murray et al. (27) 4 460, 524, 590, 635 Ganzfeld Central 7◦of surface covered with disk of no reﬂective black material. L, M, (L+M+S) [11% Weber] 17 cd/m2 5 Square-wave (1 s increment/ decrement followed by ISI of 2 s) Stockman Sharpe cone fundamentals Zele et al. (26) 5 456, 488, 540, 592, 633 Maxwellian view 30◦circular, central 10.5◦blocked Color:[7%, 22% or 24% Weber] CFF and pupil: (L+M), S, melanopsin [17% Michelson] 2,000 photopic Td (detection thresholds and pupil) 200–5,000 Td (CFF) 4 (2 authors) Pupil: 1 Hz sinusoidal Smith–Pokorny cone fundamentals applied for the CIE 1964 108 Standard Observer CIE 1951 scotopic luminosity function Enezi et al. melanopsin function Frontiers in Neurology \| www.frontiersin.org November 2018 \| Volume 9 \| Article 941 Method of Silent Substitution in Pupillometry Spitschan and Woelders substitution studies that have examined pupil responses to photoreceptor-speciﬁc modulations have employed a ﬁnite set of LEDs (four or ﬁve), though using spectrally tuneable light sources, more eﬀective primaries are possible. Number of Primaries is currently no standard(ised) spectral sensitivity, though by using a template (also called nomogram) centered at 480 nm and assuming a low peak optical density, such a spectral sensitivity can easily be derived (33, 34). Individual Differences in the Cone Spectral Sensitivities As can be seen in the table, the ﬁeld sizes used in the ﬁeld vary somewhat, and will again depend on constraints set by the optical apparatus used to deliver the stimuli, as well as theoretical considerations such as the distribution of the photoreceptor types across the retina. There are individual diﬀerences in the spectral sensitivities of the cones and this biological variability will aﬀect the degree to which the cones are truly silenced in a melanopsin-directed modulation. Inter-observer diﬀerences have been a concern in the accurate speciﬁcation of cone signals well before the discovery of melanopsin (37–39). Biological variability arises from inter- observer variability in lens density, macular pigment density, taxial density of the pigment (32, 37–39); and the peak spectral sensitivity due to polymorphisms in the opsin genes (40–43). A given set of cone fundamentals only describes the average spectral sensitivities within a population and ignoring biological variability will introduce error. In the ﬁeld of melanopsin- mediated pupillometry, some experimenters correct the stimuli by having the observers perform a color matching procedure (25, 26), while others (16, 17, 28) simulate the variability of the stimuli using simulations based on estimates of the biological variability of parameters of the cones (32). Retinal Inhomogenities The human retina is inhomogeneous. One obvious feature of the retina making it inhomogeneous is the spatial location of the macular pigment around the fovea, with a drop-oﬀ toward the periphery. A consequence of macular pigment is that all light seen by the fovea is ﬁltered through the pigment, thereby shifting the eﬀective peak spectral sensitivity of the foveal cones vs. the peripheral cones. In addition, there are also diﬀerences in how much photopigment is expressed in foveal vs. peripheral cones—the optical densities are diﬀerent. Another source of retinal inhomogeneity is that cones that are in the partial shadow of retinal blood vessels—penumbral cones—have a diﬀerent spectral sensitivity than the open-ﬁeld cones (35). Eﬀectively, for the method of silent substitution, this means that that there are three additional photoreceptor classes that need to be silenced, and therefore, more primaries are necessary. Practically, penumbral cones can be desensitized using a white- noise stimulus (26), or silenced, though with a signiﬁcant drop in contrast (36). Viewing Geometry Typical viewing geometries include Ganzfeld viewing conditions (in which the stimulus is a homogenous ﬁeld in an integrating sphere) or Maxwellian view (in which an image is focused on the entrance pupil of the observer). These again depend on the type of design used when building the stimulation system. Modulations and Contrast Depending on the spectra of the primary lights, diﬀerent amounts of contrast are available to stimulate melanopsin. Typically, the highest contrast can be achieved when LEDs are chosen of which the distribution of peak wavelengths is as broad as possible. CHALLENGES TO SILENT SUBSTITUTION g In the case where the primary lights are discrete (such as LEDs), the peak emission wavelengths are subject to design considerations when building the apparatus. Both the choice of peak wavelengths and primary widths aﬀects the contrast available for the silent substitution modulations. The contrast available is also called the gamut. In principle, choosing broader primaries will reduce also the amount of susceptibility to individual diﬀerences in the cone spectral sensitivities (29). In practice, unless a spectrally tuneable light source is used allowing to create arbitrary spectral power distributions, the choices of primary wavelengths and widths is limited by what is commercially available. When building a system, we recommend ﬁrst estimating the gamut for a given conﬁguration of peak wavelengths and widths. There are various sources of uncertainty when using silent substitution stimuli. We highlight a few of these here. There are various sources of uncertainty when using silent substitution stimuli. We highlight a few of these here. Background Light The choice of background light level is again somewhat arbitrary in many situations, though experimenters typically strive to be well in photopic conditions, where rods are assumed to be saturated, and can therefore be ignored (but see Rod intrusion below). Spectral Sensitivities Assumed The extent to which a given melanopsin-stimulating modulation silences the cones depends on the spectral sensitivities assumed. Various spectral sensitivities are available (30). Choosing the wrong spectral sensitivities can lead to artefactual results, unless care is taken to correct the modulations. We recommend the use of the CIE 2006 “physiologically relevant” cone fundamentals (31) as it allows for ﬂexible extensions to simulate individual diﬀerences parametrically (32). For melanopsin, there FUNDING MS is supported by a Sir Henry Wellcome Trust Fellowship (Wellcome Trust 204686/Z/16/Z). MS is supported by a Sir Henry Wellcome Trust Fellowship (Wellcome Trust 204686/Z/16/Z). AUTHOR CONTRIBUTIONS All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. Scatter The human eye is an imperfect optical system. In cases where the stimulus is a spatially extended light source and there is light outside the primary stimulation area (both centrally, if the macular region is blocked, and in the far periphery), there will be undesired stimulation of potentially unadapted photoreceptors (such as the rods). This can be addressed by adding a light outside the primary stimulation area that light- adapts the photoreceptors outside of the primary stimulation area. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fneur. 2018.00941/full#supplementary-material ACKNOWLEDGMENTS We thank Prof. Hannah Smithson for comments on the manuscript. We have noted in the introduction that the photoreceptors contributing to pupillary control diﬀer not only in their spectral sensitivity (as is exploited in the method of silent substitution) but also in their temporal properties, their operating range and their distribution across the retina. These properties might also be exploited to selectively stimulate melanopsin. For example, the retinal location corresponding Melanopsin Bistability and Tristability The method of silent substitution assumes that melanopsin the spectral sensitivity of melanopsin can be described by a single function. There is ample evidence that melanopsin is a bistable (44–49) or tristable photopigment (50). While the cone and rod photopigment is regenerated in the retinal pigment epithelium (RPE), melanopsin, being expressed in November 2018 \| Volume 9 \| Article 941 Frontiers in Neurology \| www.frontiersin.org 8 Method of Silent Substitution in Pupillometry Spitschan and Woelders to the blind spot does not contain rods and cones, but light might stimulate melanopsin in the axons of ipRGCs. Delivering a stimulus only in the blind spot would therefore ensure that only melanopsin would be activated (58–60), but there could be scatter on rod and cone photoreceptors near the blind spot, and accidental displacement of a small circumscribed stimulus ﬁeld would need to be controlled for. In the temporal domain, melanopsin photoreception is much slower than cone- and rod-mediated photoreception, and thus, the temporal properties of a stimulus can be optimized to bias the measured response toward melanopsin-mediated properties, e.g., the steady-state pupil size under continuous light (61). ipRGCs in the inner retina, and therefore removed from the RPE is thought to rely on a diﬀerent mechanism for pigment regeneration. A bistable (or tristable) photopigment relies on light itself to regenerate the pigment, and that this regeneration process again is wavelength-dependent and therefore has a separate spectral sensitivity. It is controversial whether the multistable photochemistry of melanopsin has physiological consequences (51–53). Under conditions of adaptating to a constant background light as employed in silent substitution, melanopsin will be in photoequlibrium, i.e., the diﬀerent states of the pigment will exist in ﬁxed (though possibly unknown) proportions. Device Uncertainty The light source used may not be stable over time and change spectral output between operations, or throughout the sessions. These drifts in device output need to be either calibrated, or at least characterized. CONCLUSION Under daylight conditions, rods are typically thought to be saturated (54, 55), though the range of light levels in which both rods and cones are known to be active is substantial (56, 57). Using a ﬁve-primary stimulator [e.g., (25, 26)], it is possible to generate melanopsin-directed stimuli which not only silence the cones but also silence rods. Typically, when rods are silenced, the contrast available to melanopsin is typically only around 1/3 relative to a stimulus in which rods are ignored, though this will depend on the choice of background. The method of silent substitution is a powerful technique to stimulate a speciﬁc photoreceptor class or speciﬁc photoreceptor classes in the living human retina while leaving other classes un-stimulated. 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Absence of long-wavelength photic potentiation of murine intrinsically photosensitive retinal ganglion cell ﬁring in vitro. J Biol Rhythms (2008) 23:387–91. doi: 10.1177/0748730408323063 Conﬂict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. y 52. Rollag MD. Does melanopsin bistability have physiological consequences? J Biol Rhythms (2008) 23:396–9. doi: 10.1177/0748730408323067 53. Brown TM, Allen AE, al-Enezi J, Wynne J, Schlangen L, Hommes V, et al. The melanopic sensitivity function accounts for melanopsin-driven responses in mice under diverse lighting conditions. PLoS ONE (2013) 8:e53583. doi: 10.1371/journal.pone.0053583 Copyright © 2018 Spitschan and Woelders. Frontiers in Neurology \| www.frontiersin.org November 2018 \| Volume 9 \| Article 941 REFERENCES This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 54. Aguilar M, Stiles WS. Saturation of the rod mechanism of the retina at high levels of stimulation. Opt Acta (1954) 1:59–65. doi: 10.1080/713818657 55. Adelson EH. Saturation and adaptation in the rod system. Vision Res. (1982) 22:1299–312. doi: 10.1016/0042-6989(82)90143-2 55. Adelson EH. Saturation and adaptation in the rod system. Vision Res. 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https://openalex.org/W2889569425	https://revistas.uned.ac.cr/index.php/cuadernos/article/download/2167/2558	es		Depredación del escorpión Centruroides bicolor por la serpiente alacranera Stenorrhina degenhardtii (Squamata: Dipsadidae)	Cuadernos de Investigación UNED/Cuadernos de investigación UNED	2,018	cc-by	327	NOTA Depredación del escorpión Centruroides bicolor por la serpiente alacranera Stenorrhina degenhardtii (Squamata: Dipsadidae) Alejandro Solórzano Museo de Zoología, Universidad de Costa Rica, Ciudad Universitaria Rodrigo Facio, 2060 San José, Costa Rica; solorzano29@gmail.com Recibido 29-I-2018 • Corregido 03-II-2018 • Aceptado 02-IV-2018 ABSTRACT: Predation of the scorpion Centruroides bicolor by the Scorpion Eater Snake, Stenorrhina degenhardtii (Squamata: Dipsadidae). I present a photographic sequence of how the snake Stenorrhina degenhardtii captures and eats the scorpion Centruroides bicolor. RESUMEN: Presento una secuencia fotográfica de cómo la serpiente Stenorrhina degenhardtii captura y come al escorpión Centruroides bicolor. Palabras clave: alacrán, escorpión bútido, comportamiento depredador de una serpiente, documentación fotográfica. Key words: Scorpiones, Buthidae, snake predatory behavior, photographic documentation. Las serpientes alacraneras del género Stenorrhina (Dipsadidae), que habitan desde el sur de México hasta el norte de Sudamérica, se caracterizan por sus hábitos alimenticios especializados en organismos invertebrados, incluyendo principalmente arácnidos (tarántulas y escorpiones) y diversos tipos de insectos (Solórzano, 2004; Solórzano & Greene, 2011). En Costa Rica, la alacranera sureña Stenorrhina degenhardtii habita en bosques lluviosos de baja y mediana elevación en el Pacífico central y sur y en la vertiente del Caribe. Durante la mañana del 14 de noviembre del 2017 observé un adulto capturando un escorpión bicolor del Pacífico (Centruroides bicolor, Buthidae) en un parche de bosque en Tinamastes de Pérez Zeledón, al sur de la provincia de San José. La serpiente lo muerde y sostiene por el abdomen hasta que el veneno hace efecto, y luego lo traga (Fig. 1). Estas serpientes son inmunes a las repetidas picaduras defensivas del escorpión. REFERENCIAS Solórzano, A. (2004). Serpientes de Costa Rica: Distribución, Taxonomía e Historia Natural. Santo Domingo, Heredia: Editorial Instituto Nacional de Biodiversidad (INBio). Solórzano, A., & Greene, H. W. (2011). Predation in nature by a scorpion hunter Stenorrhina freminvillei (Serpentes: Colubridae). UNED Research Journal, 4(1), 31-32. doi: 10.22458/urj.v4i1.130 Fig. 1. Stenorrhina degenhardtii consumiendo un Centruroides bicolor. 386 UNED Research Journal (ISSN digital: 1659-441X) Vol. 10(2): 386, Diciembre, 2018
https://openalex.org/W3135473281	https://hal.science/hal-03274554/document	English	null	Modelling of damage of a liquid-core microcapsule in simple shear flow until rupture	Journal of fluid mechanics	2,021	cc-by	15,417	To cite this version: Nicolas Grandmaison, Delphine Brancherie, Anne-Virginie Salsac. Modelling of damage of a liquid- core microcapsule in simple shear flow until rupture. Journal of Fluid Mechanics, 2021, 914, 10.1017/jfm.2020.652. hal-03274554 Distributed under a Creative Commons Attribution 4.0 International License HAL Id: hal-03274554 https://hal.science/hal-03274554v1 Submitted on 30 Jun 2021 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License 1 This draft was prepared using the LaTeX style ﬁle belonging to the Journal of Fluid Mechanics (Received xx; revised xx; accepted xx) Capsules, composed of a liquid core protected by a thin deformable membrane, oﬀer high-potential applications in many ﬁelds of industry such as bioengineering. One of their limitations comes from the absence of models of capsule damage and/or rupture when they are subjected to an external ﬂow. To assess when rupture is initiated, we develop a ﬂuid-structure interaction (FSI) numerical model of a capsule in Stokes ﬂow that accounts for potential damage of the capsule membrane. We consider the framework of Continuum Damage Mechanics and model the membrane with an isotropic brittle damage model, in which the membrane damage state depends on the history of loading. The FSI problem is solved by coupling the ﬁnite element method, to solve for the membrane deformation, with the boundary integral method, to solve for the inner and outer ﬂuid ﬂows. The model is applied to an initially spherical capsule subjected to a simple shear ﬂow. Damage initiates at a critical value of the capillary number, ratio of the ﬂuid viscous forces to the membrane elastic forces, and rupture at a higher capillary number, when it reaches a threshold value. The material parameters introduced in the damage model do not inﬂuence the mode of damage but only the values of the critical and threshold capillary numbers. When the capillary number is larger than the critical value, damage develops in the two symmetric central regions containing the vorticity axis. It is indeed in these regions that the internal tensions are the highest on the membrane. : Email address for correspondence: a.salsac@utc.fr Nicolas Grandmaison1, Delphine Brancherie2 and Anne-Virginie Salsac1: Biomechanics and Bioengineering Laboratory (UMR 7338), Universit´e de technologie de Compi`egne – CNRS, CS 60319, 60203 Compi`egne cedex, France. 1Biomechanics and Bioengineering Laboratory (UMR 7338), Universit´e de technologie de Compi`egne – CNRS, CS 60319, 60203 Compi`egne cedex, France. 2Roberval Laboratory (FRE 2012), Universit´e de technologie de Compi`egne – CNRS, CS 60319, 60203 Compi`egne cedex, France. Roberval Laboratory (FRE 2012), Universit´e de technologie de Compi`egne – CNRS, CS 60319, 60203 Compi`egne cedex, France. Modelling of damage of a liquid-core microcapsule in simple shear ﬂow until rupture Nicolas Grandmaison1, Delphine Brancherie2 and Anne-Virginie Salsac1: 1Biomechanics and Bioengineering Laboratory (UMR 7338), Universit´e de technologie de Compi`egne – CNRS, CS 60319, 60203 Compi`egne cedex, France. 2Roberval Laboratory (FRE 2012), Universit´e de technologie de Compi`egne – CNRS, CS 60319, 60203 Compi`egne cedex, France. Nicolas Grandmaison1, Delphine Brancherie2 and Anne-Virginie Salsac1: 1. Introduction Increasing the shear rate, rupture typically occurred in the central region, close to the tips of the ﬂow vorticity axis, which correspond to the zones of maximum tension. This study corroborated the results by Husmann et al. (2005), who showed that spherical and non-spherical polysiloxane microcapsules bursted at the points of maximum elastic tensions, when placed in a spinning-drop apparatus. A similar breakup mechanism has been obtained in conﬁned environments by Abkarian et al. (2008) for red blood cells ﬂowing through a 5-µm wide channel, and by Le Goﬀet al. (2017) for artiﬁcial millimetric capsules and ﬁsh eggs trapped at a constriction within a cylindrical channel under a set pressure diﬀerence. In both studies, rupture initiated at the front of the capsule, where the tensile tension is the highest. Note that, in Le Goﬀet al. (2017), rupture could also occur at the point of contact between the capsule and the constriction, but this mode of rupture is diﬀerent, as it is induced by contact and not by deformation under ﬂow. What is currently lacking is a model of capsule deformation under ﬂow, capable of assessing when and where the initiation of rupture occurs. The objective of the present study is to develop the ﬁrst ﬂuid-structure interaction model accounting for membrane damage induced on a liquid-core microcapsule subjected to a simple shear ﬂow. We will use Continuum Damage Mechanics (CDM) to model the initiation and growth of microdiscontinuities (microcavities and microcracks), which lead to the local initiation of macrocracking as they accumulate and coalesce. Contrary to fracture mechanics, which accounts explicitly for the inherent geometrical discontinuity and the associated boundary conditions, the microdiscontinuities are not geometrically modelled in CDM. The local average damage state due to the microdiscontinuities is instead represented by a continuum variable: the damage variable. CDM has beneﬁted from numerous contributions to its theoretical development (e.g. Kachanov (1986); Lemaitre & Desmorat (2005)) since the pioneering work of Kachanov (1958). It is based on the thermodynamics of irreversible processes with internal variables (Coleman & Gurtin 1967), and has been applied to model the damage mechanisms of a large spectrum of materials, from engineering materials (an overview of applications is presented in Lemaitre & Desmorat (2005)) to biological tissues (Holzapfel & Fereidoonnezhad 2017; Hokanson & Yazdani 1997; Natali et al. 2005). 1. Introduction Capsules consisting of a liquid droplet enclosed by a thin elastic membrane are commonly encountered in nature in the form of red blood cells, ﬁsh eggs and vesicles, and in numerous industrial processes. The protection and controlled release of active agents is of great importance for diverse applications in the food, cosmetic, bioengineering and medical engineering industries, among others. In medicine, encapsulation has opened the way to new treatment techniques like targeted drug/gene therapy (Bhujbal et al. 2014). New-generation bioartiﬁcial organs/cells are being developed, for instance, by encapsu- lating islets of Langerhans to treat diabetic patients (Su et al. 2010) or hemoglobin to create artiﬁcial blood (Li et al. 2005). But when placed in suspension, capsules are subjected to intense stresses from the surrounding ﬂow, which may cause the mechanical degradation of the membrane. In vivo tests have shown that artiﬁcial blood cells could be easily damaged in circulation N. Grandmaison, D. Brancherie and A.-V. Salsac 2 depending on the particle shape and deformability (Li et al. 2005): this example illustrates the importance to control rupture. Depending on the applications, capsule damage is to be prevented to preserve the inner substance, or, on the contrary, fostered and directed to allow a targeted release of the encapsulated substance. This necessitates to gain a good understanding of the capsule damage mechanisms under low-inertia ﬂow conditions and of the parameters that control the initiation of rupture. p p Very few studies have been conducted on the rupture of capsules subjected to hydrody- namic stresses. The only results that currently exist are experimental. Early experimental studies showed the possibility of wrinkling formation at low shear rates (Walter et al. 2001), which could lead to fatigue mechanisms, and of capsule burst at high shear (Chang & Olbricht 1993). The results by Chang and Olbricht (Chang & Olbricht 1993) were obtained on macroscopic spherical capsules, produced through interfacial polymerization. Flow-induced rupture initiated from one of the major axis tips of the deformed ellipsoidal shape of the capsule, which corresponds to the point of minimum thickness. The crack then propagated in the shear plane. Rupture of microcapsules under simple shear ﬂow was observed by Koleva & Rehage (2012) on thin polysiloxane capsules having a high degree of crosslinking. It was reached at small deformations indicating a brittle behaviour of the capsule membrane. 1. Introduction We propose to incorporate a CDM model into a ﬂuid-structure interaction framework, in order to investigate the time-evolution of damage as the capsule deforms under ﬂow. Capsule damage in simple shear ﬂow O ez ex v8, 9γ Gs S ρ, µ ρ, µ Figure 1: Capsule suspended in the unbounded simple shear ﬂow. Capsule damage in simple shear ﬂow Capsule damage in simple shear ﬂow Capsule damage in simple shear ﬂow 3 Figure 1: Capsule suspended in the unbounded simple shear ﬂow. In this study, we focus on the damage process of a capsule under intense hydrodynamic stresses induced by an external ﬂow over a relatively short time. Due to the short time scale of the phenomena, fatigue or creep damage models are thus not presently relevant. Previous studies have shown that microcapsules may experience ductile (Ghaemi et al. 2016) or brittle (Koleva & Rehage 2012; Le Goﬀet al. 2017) damage depending on the material and history of loading (external thermo-mechanical stresses). We derive the damage model assuming a quasi-brittle behaviour of the capsule membrane, for which dissipation prior to cracking occurs with negligible irreversible strains (i.e. negligible plasticity). However, CDM provides a general framework: the present model will thus be straightforwardly extended to the other damage behaviours (ductile material, creep or fatigue). After having detailed the formulation of the damage model of a capsule in inﬁnite shear ﬂow in Section 2, we present the model discretization and numerical solver in Section 3. We ﬁrst investigate damage of a spherical capsule under isotropic inﬂation in Section 4, as it provides insight on capsule damage and allows us to validate the numerical method by comparison of the results with the corresponding analytical solution. We then study damage in simple shear ﬂow in section 5, and assess the eﬀect that the dimensionless parameters of the model have on damage evolution and rupture initiation. We ﬁnally discuss the model and results in Section 6 and analyze the potential of the model to identify the capsule membrane limit of elasticity by comparison with experiments. Internal and external ﬂows Internal and external ﬂows tial eﬀects being neglected, the ﬂuid problem is governed by the Stokes equations div ` σ ˘ “ 0, div pvq “ 0, (2.1) (2.1) where σ designates the Cauchy stress tensor, v is the velocity vector and divp.q is the divergence operator. At a given point x of the membrane S, the boundary integral formulation of the Stokes equations gives the relationship between the velocity vector v and the stress tensor σ (Pozrikidis 1992): @x P S, vpxq “ v8pxq ´ 1 8πµ ż S Jpx, yq ¨ vσw ¨ npyq dSy , (2.2) (2.2) where n is the unit vector normal to S pointing towards the external ﬂuid and vσw ¨ n “ pσext ´σintq¨n is the stress jump across the membrane. We denote as J the second order Oseen-Burgers tensor deﬁned by: where n is the unit vector normal to S pointing towards the external ﬂuid and vσw ¨ n “ pσext ´σintq¨n is the stress jump across the membrane. We denote as J the second order Oseen-Burgers tensor deﬁned by: Jpx, yq “ 1 r 1 ` 1 r3 r b r , (2.3) (2.3) where r “ x ´ y, r “ ∥r∥and 1 is the identity tensor. where r “ x ´ y, r “ ∥r∥and 1 is the identity tensor. 2. Formulation of the problem We consider a spherical microcapsule of radius a enclosed in an elastic envelope of very small thickness with respect to its radius. The capsule is thus modelled as a two- dimensional incompressible membrane with surface shear elastic modulus Gs. It is placed in an inﬁnite shear ﬂow of shear rate 9γ. The problem is studied in the reference frame of center O and Cartesian basis pex, ey, ezq corresponding to the barycentric reference frame of the capsule oriented such that the unperturbed velocity ﬁeld is given by v8pxq “ 9γzex (Figure 1). The inner and outer ﬂuids are the same incompressible Newtonian ﬂuids of dynamic viscosity µ and density ρ. Gravitational and inertial eﬀects being negligible due to the microscopic capsule size, the ﬂuid-structure interaction problem is governed by only one non-dimensional parameter: the capillary number Ca “ µ9γa{Gs, ratio of the viscous to the elastic characteristic forces. N. Grandmaison, D. Brancherie and A.-V. Salsac 4 Wall mechanics The capsule wall is modelled as a membrane of mid-surface S. The curvilinear coor- dinates pξ1, ξ2q describe the position xpξ1, ξ2, tq on S in the conﬁguration at time t. The position xpξ1, ξ2, 0q on the initial conﬁguration S0 of S is noted X. It is convenient to write the membrane equations in local tangent bases. In what follows, if not speciﬁed, indices written with Latin letters take values in t1, 2, 3u, while indices written with Greek letters are in t1, 2u. The covariant basis paiq attached to S is deﬁned by: aα “ Bx Bξα , a3 “ a1 ˆ a2 ∥a1 ˆ a2∥. (2.4) (2.4) The contravariant basis paiq is deﬁned by ai ¨ aj “ δj i , where δj i designates the Kronecker symbol. On S0, the covariant and contravariant bases are denoted pAiq and pAiq, re- spectively. The metric tensor is g on S and G on S0. The contravariant and covariant components of g are aαβ “ aα ¨ aβ and aαβ “ aα ¨ aβ, respectively (similar deﬁnitions for the components Aαβ and Aαβ of G). The contravariant basis paiq is deﬁned by ai ¨ aj “ δj i , where δj i designates the Kronecker symbol. On S0, the covariant and contravariant bases are denoted pAiq and pAiq, re- spectively. The metric tensor is g on S and G on S0. The contravariant and covariant components of g are aαβ “ aα ¨ aβ and aαβ “ aα ¨ aβ, respectively (similar deﬁnitions for the components Aαβ and Aαβ of G). The wall inertia being negligible (Walter et al. 2010), the motion of the membrane is governed by the local mechanical equilibrium: @x P S, ∇s ¨ T ` q “ 0, (2.5) (2.5) where q is the surface external load, T is the tension (resultant of the internal Cauchy stress over the thickness), ∇s¨ is the surface divergence operator. The dynamic boundary condition imposes that: where q is the surface external load, T is the tension (resultant of the internal Cauchy stress over the thickness), ∇s¨ is the surface divergence operator. The dynamic boundary condition imposes that: @x P S, q “ vσw ¨ n. Material behaviour The model of the capsule wall behaviour is developed in the standard framework of CDM (Lemaitre & Desmorat 2005) to account for the progressive degradation of the membrane while staying in the ﬁeld of continuum mechanics. More speciﬁcally, CDM is a branch of the thermodynamics of irreversible processes with internal variables, the focus of which is to model irreversible transformations associated with damage. The development of a damage model is thus based on four key concepts inherited from the thermodynamics of irreversible processes: state variables, state potential, damage criterion and damage evolution law. A short review of these concepts together with the details of how we developed the model are given hereafter. We specify them in the case of quasi-brittle damage which corresponds to the membrane deformation until the initiation of rupture without irreversible strains (see Table 1 for a summary). Wall mechanics (2.6) (2.6) The weak form of the membrane equilibrium equation is obtained applying the principle of virtual work: For any virtual displacement ˆu P H1pSq, ż ż ( For any virtual displacement ˆu P H1pSq, ż ż (2.7) ż S ˆu ¨ q dS “ ż S T : εpˆuq dS , (2.7) Capsule damage in simple shear ﬂow 5 Table 1: Summary of the key ingredients of the present associated damage model. State / Associated variables Green Lagrange strain e / Second Piola-Kirchhoﬀtension π Damage variable d / Energy release rate Y State potential Free energy φpe, dq “ p1 ´ dqφNHpeq with φNH the isochore neo-Hookean potential Damage threshold function f “ Y ´ κpdq with κpdq “ YD ` YCd, YD and YC being the model constants Table 1: Summary of the key ingredients of the present associated damage model Green Lagrange strain e / Second Piola-Kirchhoﬀtension π Damage variable d / Energy release rate Y Free energy φpe, dq “ p1 ´ dqφNHpeq with φNH the isochore neo-Hookean potential where H1pSq designates the Sobolev space associated to the Lebesgue space L2pSq and εpˆuq is the symmetric part of g ¨ ∇ˆu, the tensor ∇ˆu being the gradient of ˆu. where H1pSq designates the Sobolev space associated to the Lebesgue space L2pSq and εpˆuq is the symmetric part of g ¨ ∇ˆu, the tensor ∇ˆu being the gradient of ˆu. In terms of kinematics, the no-slip boundary condition holds on S and gives the relationship between the ﬂuid velocity and the position x of the corresponding point of the membrane: v “ dx dt . (2.8) (2.8) State variables We assume that the transformations of the capsule wall correspond to isothermal elastic deformation and damage. The damage variable represents the irreversible growth of microdefects in a representative volume element (RVE) (Figure 2). We assume that the transformations of the capsule wall correspond to isothermal elastic deformation and damage. The damage variable represents the irreversible growth of microdefects in a representative volume element (RVE) (Figure 2). To illustrate the deﬁnition of the damage variable, we consider a deformed RVE of the capsule wall containing microdefects in the form of microcavities and microcracks (Figure 2). We deﬁne damage in direction k as the surface ratio δSD{δS, with δSD the maximum intersection of microdefects in a cross-section δS of normal k of the RVE. The stresses on this cross-section are thus transmitted on δ ˜S “ δS ´ δSD. We assume that the microdefects have no preferential orientation: the δSD{δS ratio is thus independent of the direction k and corresponds to isotropic damage. The state variable is then the 6 N. Grandmaison, D. Brancherie and A.-V. Salsac S v8 O k δS δSD δ ˜S RVE Figure 2: Representation of a microcapsule of mid-surface S placed in an inﬁnite shear ﬂow (left). Zoom on a representative volume element (RVE) containing microcavities and microcracks (middle). Decomposition of the cross-section δS of normal vector k into the eﬀective load-bearing cross-section δ ˜S and the total surface of the microdefects δSD (right). N. Grandmaison, D. Brancherie and A.-V. Salsac N. Grandmaison, D. Brancherie and A.-V. Salsac 6 N. Grandmaison, D. Brancherie and A.-V. Salsac S v8 O k δS δSD δ ˜S RVE 6 Figure 2: Representation of a microcapsule of mid-surface S placed in an inﬁnite shear ﬂow (left). Zoom on a representative volume element (RVE) containing microcavities and microcracks (middle). Decomposition of the cross-section δS of normal vector k into the eﬀective load-bearing cross-section δ ˜S and the total surface of the microdefects δSD (right). scalar damage variable d deﬁned as scalar damage variable d deﬁned as d “ δSD δS “ 1 ´ δ ˜S δS . (2.9) (2.9) It ranges from 0, for the local sound (undamaged) state of the material, to 1, when a crack initiates having the size of the RVE. It ranges from 0, for the local sound (undamaged) state of the material, to 1, when a crack initiates having the size of the RVE. State variables The other state variable is the standard elastic deformation, used in all the mechanical models. The capsule incompressible wall being modelled as a membrane, the in-plane deformation tensor on the mid-surface S is given by the Green-Lagrange strain tensor e e “ 1 2pF T ¨ F ´ Gq. (2.10) (2.10) The tensor F is the gradient of the transformation of S F “ Bx BX “ aα b Aα, (2.11) (2.11) in which, as in what follows, we adopt the convention of summation over repeated indices. In conclusion, the state variables are d and e, which both depend only on x P S. State potential Following the standard framework of CDM, the constitutive law of the membrane and the deﬁnition of the variable controlling d are derived from a unique state potential function of the state variables. We note φpe, dq the speciﬁc membrane free energy per unit surface of S0. Knowing φ, one can derive the associated variables dual to e and d, using the state laws $ ’ & ’ % π “ Bφ Be , Y “ ´Bφ Bd, (2.12) (2.12) 7 Capsule damage in simple shear ﬂow Capsule damage in simple shear ﬂow 7 λ 2 λ 2 δS δ ˜S δFtrac δFtrac δFtrac δFtrac ˜σ “ δFtrac δ ˜S σ “ δFtrac δS ô Real RVE Equivalent RVE Undeformed RVE Real RVE Undeformed RVE Equivalent RVE Figure 3: Illustration of the homogenization principle on a representative volume element (RVE) under an uniaxial traction of intensity δFtrac and of the associated elongation λ. The heterogeneous damaged material (real RVE) is modelled as a homogeneous domain (equivalent RVE) with the same cross-section δS and subjected to the same loading/elongation. The force equilibrium leads to σ “ δ ˜S{δS˜σ “ p1 ´ dq˜σ, where the eﬀective stress ˜σ is the stress transmitted through the load-bearing cross-section δ ˜S and determined with the constitutive law of the undamaged material. where π is the second Piola-Kirchhoﬀtension tensor and Y the speciﬁc elastic energy release rate. The Cauchy tension tensor T is related to π through T “ 1 J F ¨ π ¨ F T . (2.13) (2.13) The undamaged wall is chosen to follow the neo-Hookean (NH) law, which was shown to model well the elastic behaviour of thin artiﬁcial proteic membranes (Chu et al. 2011; Gubspun et al. 2016). The corresponding speciﬁc free energy φNH (Barth`es-Biesel et al. 2002) is φNHpeq “ Gs 2 ˆ I1 ´ 1 ` 1 I2 ` 1 ˙ , (2.14) (2.14) where the two invariants of the transformation I1 and I2 are deﬁned by: where the two invariants of the transformation I1 and I2 are deﬁned by: I1 “ trpF T ¨ Fq ´ 2 “ Aαβaαβ ´ 2, I2 “ detpF T ¨ Fq ´ 1 “ detpAαβqdetpaαβq ´ 1. (2.15) (2.15) What is classical in CDM is to obtain the free energy φ in the damage state using homogenization, which is based on the principle of strain equivalence. State potential We propose to illustrate this concept on the 3D RVE shown in Figure 2, in the case of a uniaxial traction of intensity δFtrac which induces an elongation λ (Figure 3). We look for the 8 N G d i D B h i d A V S l 8 N. Grandmaison, D. Brancherie and A.-V. Salsac equivalent RVE (right) having the same cross-section δS, and being subjected to the same elongation λ and loading δFtrac as the real RVE. The stress in the equivalent RVE is thus σ “ δFtrac{δS, which is related to the eﬀective stress ˜σ “ δFtrac{δ ˜S through: equivalent RVE (right) having the same cross-section δS, and being subjected to the same elongation λ and loading δFtrac as the real RVE. The stress in the equivalent RVE is thus σ “ δFtrac{δS, which is related to the eﬀective stress ˜σ “ δFtrac{δ ˜S through: σpλ, dq “ δ ˜S δS ˜σpλq “ p1 ´ dq˜σpλq. (2.16) (2.16) where ˜σ is computed from the constitutive law of the undamaged material. where ˜σ is computed from the constitutive law of the undamaged material. The concept of equation (2.16) can be translated to our 2D membrane and generalized to any in-plane stress state with: The concept of equation (2.16) can be translated to our 2D membrane and generalized to any in-plane stress state with: Bφ Be pe, dq “ p1 ´ dqBφNH Be peq. (2.17) (2.17) We thus choose to express the speciﬁc free energy φ as: φpI1, I2, dq “ p1 ´ dqφNHpI1, I2q. (2.18) (2.18) Note that the present homogenization process preserves the membrane properties ob- served in the undamaged case. The state laws deﬁned by equations (2.12) and (2.13) then have the following expressions: $ ’ & ’ % T αβ “ p1 ´ dqGs ˆ 1 J Aαβ ´ 1 J3 aαβ ˙ Y “ φNH, (2.19) (2.19) where the Cauchy tension tensor is given through its contravariant components. where the Cauchy tension tensor is given through its contravariant components. Damage criterion and damage evolution law The last ingredients of the model are the damage criterion and the damage evolution law. We choose to adopt an associated model (Besson et al. 2010), which is numerically robust. State potential It only requires the introduction of the damage threshold function fpY ; dq (d acts as a parameter) to derive the damage criterion and the evolution law through the admissibility condition (i) and the principle of maximum dissipation (ii). (i) Admissibility condition (i) Admissibility condition To be admissible, the associated variable Y must satisfy the standard admissibility condition To be admissible, the associated variable Y must satisfy the standard admissibility condition fpY ; dq ď 0. (2.20) (2.20) nes a bounded domain for Y , illustrated in Figure 4a. It deﬁnes a bounded domain for Y , illustrated in Figure 4a. Principle of maximum dissipation (ii) Principle of maximum dissipation (ii) Principle of maximum dissipation The damage evolution is accompanied by dissipation. The associated governing laws are based on the principle of maximum dissipation The damage evolution is accompanied by dissipation. The associated governing laws are based on the principle of maximum dissipation DpY, 9dq “ max fpY ˚;dqď0 ! DpY ˚, 9dq ) , (2.21) (2.21) where DpY, 9dq “ Y 9d, and 9d designates the material temporal derivative of d. The solution of the maximization problem under constrain (2.21) is provided by the Kuhn-Tucker conditions B $ & % 9d “ 9η Bf BY f ď 0, 9η ě 0, 9ηf “ 0. (2.22) (2.22) Capsule damage in simple shear ﬂow 9 Table 2: Loading case possibilities as a function of the values of f, 9f and 9η. An illustration is given in Figure 4 for a loading/unloading cycle. Table 2: Loading case possibilities as a function of the values of f, 9f and 9η. An illustration is given in Figure 4 for a loading/unloading cycle. ①elastic loading/unloading f ă 0 9η “ 0 ②elastic unloading f “ 0 9f ă 0 9η “ 0 ③neutral loading f “ 0 9f “ 0 9η “ 0 ④loading with damage f “ 0 9f “ 0 9η ą 0 f ă 0 f ą 0 (non-admissible) f “ 0 f “ 0 f “ 0 9d 9η “ 0 9η ą 0 9η “ 0 (a) (b) (c) ① ④ ③ ② ① Figure 4: Illustration in 2D of (a) the admissible domain of the associated variable Y , deﬁned by fpY q ď 0, (b) the case of damage evolution ( 9η ą 0) where the yield surface f “ 0 moves due to hardening and where the rate of damage 9d is along the normal to the yield surface (normality rule), and (c) the case when damage ceases ( 9η “ 0). The thick black lines represent one example of loading cycle, which successively contains all the phases given in table 2: (a) elastic loading ①, (b) loading with damage ④, (c) neutral loading ③followed by elastic unloading ②+ ①. (ii) Principle of maximum dissipation Damage only occurs when 9η ‰ 0, the value of which is obtained by solving 9fpY ; dq “ 0 (imposed by equation (2.24)). Diﬀerent cases of loading may exist (see Table 2 and Figure 4). When 9η “ 0, no damage occurs regardless the values of f and 9f. Damage only occurs when 9η ‰ 0, the value of which is obtained by solving 9fpY ; dq “ 0 (imposed by equation (2.24)). y g fp q ( y ( )) Note that from the inequality of Clausius-Duhem D ě 0, and given that Y ě 0, the damage variable d can only grow in time 9d ě 0. (2.25) 9d ě 0. (2.25) Thus, during damage (f “ 0) Thus, during damage (f “ 0) Bf BY ě 0, (2.26) (2.26) which restrains the choice of f. Since most artiﬁcial and natural microcapsules have been shown to be brittle, we choose to follow the model developed by Marigo (1981) for quasi-brittle damage: fpY ; dq “ Y ´ κpdq ď 0. (2.27) (2.27) We presently deﬁne κ as a function of two parameters, the damage threshold YD ě 0 and the hardening modulus YC ě 0, such that: κpdq “ YD ` YCd. (2.28) (2.28) The size of the domain of admissible states f ď 0 increases with damage (Figure 4b). It is due to the hardening of the material and is controlled by the parameter YC. The damage evolution law eq. (2.22) can be written equivalently in an explicit form: d “ă ζpY maxq ą`, d “ă ζpY maxq ą`, (2.29) where ă . ą` designates the Macaulay brackets deﬁned by " ă x ą`“ x if x ě 0 ă x ą`“ 0 otherwise. (2.30) (2.30) The function ζpY q “ pY ´YDq{YC designates the reciprocal of the bijection κ, and Y max is deﬁned by The function ζpY q “ pY ´YDq{YC designates the reciprocal of the bijection κ, and Y max is deﬁned by tion ζpY q “ pY ´YDq{YC designates the reciprocal of the bijection κ, and Y max b Y maxptq “ max τďt tY pτqu . (2.31) (2.31) (ii) Principle of maximum dissipation ①elastic loading/unloading f ă 0 9η “ 0 ②elastic unloading f “ 0 9f ă 0 9η “ 0 ③neutral loading f “ 0 9f “ 0 9η “ 0 ④loading with damage f “ 0 9f “ 0 9η ą 0 f ă 0 f ą 0 (non-admissible) f “ 0 9η “ 0 (a) ① f “ 0 9d 9η ą 0 (b) ④ f “ 0 9η “ 0 (c) ③ ② ① (c) (b) (a) 9η “ 0 Figure 4: Illustration in 2D of (a) the admissible domain of the associated variable Y , deﬁned by fpY q ď 0, (b) the case of damage evolution ( 9η ą 0) where the yield surface f “ 0 moves due to hardening and where the rate of damage 9d is along the normal to the yield surface (normality rule), and (c) the case when damage ceases ( 9η “ 0). The thick black lines represent one example of loading cycle, which successively contains all the phases given in table 2: (a) elastic loading ①, (b) loading with damage ④, (c) neutral loading ③followed by elastic unloading ②+ ①. the four of which constitute the evolution law of damage, where 9η acts as a Lagrange multiplier. the four of which constitute the evolution law of damage, where 9η acts as a Lagrange multiplier. The three conditions within equation (2.22)2 are known as the loading/unloading conditions. They provide the damage criterion # fpY q ă 0 ñ 9η “ 0 fpY q “ 0 ñ 9η ě 0. (2.23) (2.23) The interior of the admissible domain corresponding to fpY q ă 0 (Figure 4a) is thus the elastic domain, in which damage remains constant ( 9d “ 0). The domain boundary corresponds to fpY q “ 0 and thus to cases where damage evolves. The damage evolution follows equation (2.22)1 which can be interpreted geometrically as 9d being along the normal to the yield surface f “ 0 (Figure 4b). It is thus referred to as the normality rule. Together, the admissibility condition (2.20) and the damage criterion (2.23) lead to 10 N. Grandmaison, D. Brancherie and A.-V. Salsac the consistency condition 9η 9f “ 0. (2.24) (2.24) 9η 9f “ 0. Diﬀerent cases of loading may exist (see Table 2 and Figure 4). When 9η “ 0, no damage occurs regardless the values of f and 9f. 3. Numerical method (2.8) is integrated with a second-order explicit Runge-Kutta scheme to solve for the position of the membrane nodes at the next time step. is computed explicitly at each node from eq. (2.2). Finally, eq. (2.8) is integrated with a second-order explicit Runge-Kutta scheme to solve for the position of the membrane nodes at the next time step. 3. Numerical method Knowing the current position of the material points of the membrane, we perform a Lagrangian tracking of the nodes of the capsule to solve the ﬂuid-structure interaction problem (eq. (2.2), (2.6), (2.7), (2.8), (2.29)). We use the strategy proposed by Walter et al. (2010) coupling the Finite Element Method to solve for the solid and the Boundary Integral Method to solve for the ﬂuid (Figure 5). The problem is solved using the dimensionless forms of the equations, in which the lengths are non-dimensionalized by a, time by 1{ 9γ and tensions by Gs. The two parameters YD and YC are thus also non- dimensionalized by Gs. The originality of our work consists in introducing a damage model in the solid problem. At the material level, the evolution of the damage variable d is determined for each integration point using the explicit equation (2.29). The external load q is then obtained by solving the global problem (2.7) and transferred to the ﬂuid problem. The velocity Capsule damage in simple shear ﬂow 11 Solid solver Finite Element Method 1) Find dn solving eq. (2.29) at the integration points. 2) Find qn at the nodes solving eq. (2.7) Fluid solver Boundary Integral Method Find vn solving eq. (2.2) at the nodes. Kinematical coupling Find the position xn`1 at the next time step solving eq. (2.8) at the nodes using an explicit scheme Dynamical coupling Find ´ vσw ¨ n ¯ n using eq. (2.6) Initial position x0 xn Ð xn`1 qn, dn vn ´ vσw ¨ n ¯ n Figure 5: Numerical method to solve the ﬂuid-structure interaction problem over a time step. 11 Solid solver Finite Element Method Fluid solver Boundary Integral Method Initial position Find vn solving eq. (2.2) at the nodes. Figure 5: Numerical method to solve the ﬂuid-structure interaction problem over a time step. (b) ez ex (a) (b) Figure 6: Projection on the shear plane of the mesh of a damaged capsule captured (a) in the initial conﬁguration and (b) at a steady deformed state. P2 elements, NE=1280, NN=2562. (a) (b) Figure 6: Projection on the shear plane of the mesh of a damaged capsule captured (a) in the initial conﬁguration and (b) at a steady deformed state. P2 elements, NE=1280, NN=2562. is computed explicitly at each node from eq. (2.2). Finally, eq. Fluid solver For a given deformed conﬁguration of the capsule and knowing the stress exerted by the membrane on the ﬂuid, the velocity ﬁeld v is given explicitly by eq. (2.2). The velocity ﬁeld v is computed at each node. The integral on the right-hand side of eq. (2.2) is computed with 12 Hammer points per element. To handle the singularity of the tensor J at node x, we use polar coordinates centred on x when integrating on the elements sharing this node (for more details see e.g. Lac et al. (2004)). We do not use penalty methods to impose the conservation of the volume of the ﬂuids. Still, the maximum relative variation of the capsule volume is limited to 0.1% of the initial volume. Solid solver For a given deformed conﬁguration of the capsule, the discrete solid problem consists in ﬁnding the external load q P L2 h and the damage d P L2 h that satisfy eq. (2.7) and eq. (2.29), where the subscript h indicates the ﬁnite element space. The position x and the virtual displacement ˆu are searched in H1 h. Using isoparametric elements, we restrict the solution for q in H1 h. A ﬁeld vpx, tq P H1 h writes: vpx, tq “ N ppqpxqvppqptq, p P r1, NNs, where N ppq and vppq are the shape function and the nodal coordinates of v associated to the node p, respectively. Noting vppq Xj the coordinates of vppq in a Cartesian basis peXjq, the left-hand side of the discretized form of eq. (2.7) writes: ÿ el ˆuppq Xj ż el N ppqN pqq dS qpqq Xi “ tˆuppq XjuT rMstqppq Xju, (3.1) (3.1) and the right-hand side writes: and the right-hand side writes: ÿ el ż el T αβpe, dqχppqXj αβ dS ˆuppq Xj “ tˆuppq XjuT tRupe, dq, (3.2) (3.2) where tqppq Xju and tˆuppq Xju are the vectors of size 3NN of the nodal coordinates, and χppqXj αβ is deﬁned by: χppqXj αβ “ 1 2 ˆBN ppq Bξβ aα ` BN ppq Bξα aβ ˙ ¨ eXj, p P r1, NNs. (3.3) (3.3) Eq. (2.7) being satisﬁed for any virtual displacement, the discrete solid problem writes: Find q and d, such that, # rMs tqu “ tRupe, dq d “ ă κ´1pYmaxq ą` (3.4a) (3.4b) (3.4a) (3.4b) The square and column matrices rMs and tRu are, respectively, computed at each time step by using 6 Hammer points on each element (Hammer et al. 1956). The new value of the damage variable is obtained from eq. (3.4b), solved locally at each integration point while computing tRu. Knowing the deformation, the variable d is computed explicitly as Y max depends only on the deformation. The computation of d ensures the admissibility condition (eq. 2.27) at each time step. Finally, q is computed by solving eq. (3.4a) with the Pardiso solver (Schenk & G¨artner 2004). Mesh A conform mesh is used, the nodes on the capsule S being shared by the ﬂuid and the solid problems. The mesh is composed of curved triangular elements containing six nodes with quadratic shape functions (P2 elements). The mesh is generated on the spherical shape corresponding to the initial conﬁguration (Figure 6). Following a previous study (Walter et al. 2010), the mesh contains NE “ 1280 P2 elements corresponding to a total of NN “ 2562 nodes. 12 N. Grandmaison, D. Brancherie and A.-V. Salsac 4. Capsule damage under isotropic inﬂation We ﬁrst analyse the damage of a spherical capsule under osmotic inﬂation. We impose radial displacements inﬂating the capsule from radius a to radius ap1 ` αptqq, where the inﬂation ratio α is such that α ě 0. We will study two cases: a monotonic increase of α and cyclic variations of α with successive increase and decrease of the capsule diameter. We compare the solution given by the solid solver to the analytical solution. The problem consists in ﬁnding the damage variable d and the external load q that satisfy the evolution law of damage (eq. 2.29) and the equilibrium of the membrane (eq. 2.7). An analytical solution of the problem exists. We look for it in the form of uniform ﬁelds that satisfy the spherical symmetry of the problem. The stretch ratio of the membrane, which is the square root of the isotropic principal value of the dilatation tensor F T ¨ F, is simply λ “ 1 ` α. The corresponding isotropic principal value T of the tension is: T “ p1 ´ dqGsp1 ´ 1 λ6 q, (4.1) (4.1) and the elastic energy release rate Y : Y “ Gs 2 p2λ2 ` 1 λ4 ´ 3q. (4.2) (4.2) As Y increases monotonically with α, the evolution law for damage (2.29) writes: As Y increases monotonically with α, the evolution law for damage (2.29) writes: d “ă κ´1pY pαmaxqq ą`, (4.3) (4.3) where αmax is deﬁned similarly to Y max in eq. (2.31). Hence, the condition for d to increase is that α is larger than any of its previous values. where αmax is deﬁned similarly to Y max in eq. (2.31). Hence, the condition for d to increase is that α is larger than any of its previous values. The external load is q “ pn, where p ě 0 is the diﬀerence between the internal and external pressures. Choosing test functions of the form ˆu “ ˆux in the equilibrium equation (2.7), we obtain the Laplace relation between T and p: T “ ap1 ` αqp 2 . (4.4) (4.4) We prescribe the radial displacements to the nodes and impose xpmq “ p1`αqXpmq, @m P r0, NNs. The pressure diﬀerence and damage variable d are obtained analytically using eq. (4.1)-(4.4), and numerically using the solid solver presented in section 3. Coupling Using a conform mesh with isoparametric elements, the loads vσw ¨ n and q are in the same space H1 h. Hence the dynamic coupling between the ﬂuid and the solid (eq. 2.6) is veriﬁed in its strong form in this space. Considering the kinematic coupling, the no-slip 13 Capsule damage in simple shear ﬂow condition (eq. 2.8) is solved at the nodes with an explicit second order Runge-Kutta scheme to ﬁnd the position of the nodes at the next temporal increment. Since the local problem of damage is solved in the solid problem with an implicit scheme, the condition of stability of the scheme of temporal integration of the ﬂuid-structure interaction problem is the same as the one initially developed by Walter et al. (2010). 4. Capsule damage under isotropic inﬂation For the numerical solution, we compute p and d as surface averages, the pressure diﬀerence p being given by q ¨ n. Between the numerical and analytical solutions, we always ﬁnd relative errors lower than 10´3% for the pressure diﬀerence p and 10´4% for damage d. We ﬁrst compare how ap{Gs (the dimensionless value of p) and d evolve with the inﬂation ratio α in the case of a monotonic inﬂation of the capsule (Figure 7). The nu- merical and analytical curves are perfectly superimposed (Figures 7 b-c) and comparison with the analytical solution of the undamaged capsule (d “ 0) shows a clear eﬀect of damage on the pressure diﬀerence (Figure 7 b). Damage is initiated at the critical value α “ αc, which corresponds to Y pαcq “ YD. The loss in elastic properties of the damaged N. Grandmaison, D. Brancherie and A.-V. Salsac 14 0 0.4 0.6 0.8 αc αℓ t α (a) 0 0.2 0.4 0.6 0.8 1 1.2 1.4 0 0.4 0.6 0.8 αc αℓ ap Gs α (b) 0 0.2 0.4 0.6 0.8 1 0 0.4 0.6 0.8 αc αℓ No damage Analytic P2 elements d α (c) Figure 7: Case of a monotonic inﬂation: for the stretch ratio α shown in (a), corresponding curves of the dimensionless pressure diﬀerence (b) and of the damage variable (c), computed for YD “ 0.2, YC “ 2.0. 0 0.2 0.4 0.6 0.8 1 1.2 1.4 0 0.4 0.6 0.8 αc αℓ ap Gs α (b) 0 0.2 0.4 0.6 0.8 1 0 0.4 0.6 0.8 αc αℓ No damage Analytic P2 elements d α (c) α Figure 7: Case of a monotonic inﬂation: for the stretch ratio α shown in (a), corresponding curves of the dimensionless pressure diﬀerence (b) and of the damage variable (c), computed for YD “ 0.2, YC “ 2.0. capsule leads to a reduction in pressure diﬀerence as compared to the undamaged case. The pressure diﬀerence returns to zero when d “ 1, which occurs when α “ αℓ. sule leads to a reduction in pressure diﬀerence as compared to the undamaged ca pressure diﬀerence returns to zero when d “ 1, which occurs when α “ αℓ. We then compare the evolution of ap{Gs and d with α in the case of a capsule subjected to cyclic inﬂations and deﬂations with increasing maximum sizes (Figure 8). 4. Capsule damage under isotropic inﬂation Capsule damage in simple shear ﬂow 15 n simple shear ﬂow 15 0 0.2 0.4 0.6 0.8 1 1.2 1.4 0 0.4 0.6 0.8 A B C α αc αℓ ap Gs (b) 0 0.4 0.6 0.8 A B C α αc αℓ t (a) (a) α 0 0.2 0.4 0.6 0.8 1 0 0.4 0.6 0.8 A B C α αc αℓ No damage Analytic P2 elements d (c) α Figure 8: Case of cycles of inﬂations and deﬂations with increasing maximum capsule size: for the stretch ratio α shown in (a), corresponding curves of the dimensionless pressure diﬀerence (b) and of the damage variable (c), computed for YD “ 0.2, YC “ 2.0. parameter values YD and YC. Following equation (4.3), the values of α initiating damage and rupture are given respectively by the equations Y pαcq “ YD and Y pαℓq “ YD ` YC, where the expression of Y pαq is obtained using equation (4.2). The critical inﬂation ratio αc thus depends solely on YD, but the limit inﬂation ratio αℓdepends on both YD and YC. Furthermore, the higher the parameter values, the higher the two threshold inﬂation ratios. 4. Capsule damage under isotropic inﬂation During the ﬁrst cycle corresponding to the inﬂation of the capsule until point A, the value of α does not exceed the critical value αc. Hence damage does not initiate and the curves of ap{Gs for the damaged and undamaged capsules coincide during inﬂation and deﬂation. For the second cycle (inﬂation until point B), the curves of d and ap{Gs coincide with the corresponding curves obtained for the monotonic size increase (Figures 7b-c). During deﬂation from point B, damage remains constant and the curve of pressure diﬀerence ap{Gs stays below the inﬂation curve when α decreases back to 0. For the third cycle, the inﬂation curves of ap{Gs and d overlap the corresponding curves of the previous deﬂation until point B. But, between points B and C, damage increases during inﬂation, and the curve of ap{Gs again coincides with the corresponding curve obtained for the monotonic size increase. The deﬂation from point C is then similar to that of the second cycle with constant damage and an ap{Gs-curve below the inﬂation one. During the last inﬂation, capsule rupture occurs, when α reaches the limit value αℓ(corresponding to d “ 1). The case of the capsule under isotropic inﬂation illustrates the eﬀects of damage on the behaviour of the capsule. For a given value of the inﬂation ratio α, the more the membrane is damaged, the lower the pressure diﬀerence (Figure 8b), in other words damage reduces the loading capacity of the membrane. For increasing d, the slope at the origin for the curve ap{Gs(α) decreases (Figure 8b), which means that damage reduces the stiﬀness of the structure. It is interesting to see how the values of αc and αℓdepend on the Capsule damage in simple shear ﬂow 15 0 0.4 0.6 0.8 A B C α αc αℓ t (a) 0 0.2 0.4 0.6 0.8 1 1.2 1.4 0 0.4 0.6 0.8 A B C α αc αℓ ap Gs (b) 0 0.2 0.4 0.6 0.8 1 0 0.4 0.6 0.8 A B C α αc αℓ No damage Analytic P2 elements d (c) Figure 8: Case of cycles of inﬂations and deﬂations with increasing maximum capsule size: for the stretch ratio α shown in (a), corresponding curves of the dimensionless pressure diﬀerence (b) and of the damage variable (c), computed for YD “ 0.2, YC “ 2.0. As reference case, we choose YD “ 0.2, YC “ 2.0 and Ca “ 0.7. The value of Ca is such that Cac ă Ca ă Caℓ. Hence the capsule is damaged but the damage stabilizes and a steady state is reached. 5.1. Coupled kinetics of motion and damage on a reference case (Ca “ 0.7) 5. Capsule damage under simple shear ﬂow We now study the damage of a capsule in simple shear ﬂow. We ﬁrst show the typical motion and evolution of damage of a capsule in a reference case and then study the inﬂuence of the capillary number on the capsule behaviour. We will see that, when the capillary number is increased, three diﬀerent regimes are found. The capsule is ﬁrst undamaged until a critical capillary number Cac is reached, corresponding to the onset of damage. Above this value, the capsule reaches a steady-state deformed shape in which it is partly damaged. When the limit capillary number Caℓis reached, rupture initiates putting a limit to the damage regime. In the last part of this section, we will ﬁnally study the inﬂuence of the material parameters YD and YC on the three identiﬁed regimes and on the values of Cac and Caℓ. N. Grandmaison, D. Brancherie and A.-V. Salsac 16 , ey z x v8 e1 e1 e2 L1 L2 L3 β O O P P 1 Figure 9: Two principal ellipses of the ellipsoid of inertia of the capsule. z x v8 e1 e2 L1 L2 β O ey e1 L3 O P P 1 Figure 9: Two principal ellipses of the ellipsoid of inertia of the capsule. Figure 9: Two principal ellipses of the ellipsoid of inertia of the capsule. Figure 9: Two principal ellipses of the ellipsoid of inertia of the capsule. t “ tc tc ă t ă t1 t “ t1 t “ t8 Current conﬁguration ez ex P ey e1 P P 1 Reference conﬁguration ez ex d 0.0 0.1 0.2 Figure 10: Map of damage at the instant of initiation of damage tc, at an intermediary instant between tc and the instant of maximum elongation t1, at time t1, and at steady state (t8). The map of damage is represented on the current and reference conﬁgurations. The current conﬁguration is observed in the shear plane pO, ex, ezq and in the plane pO, e1, eyq which is deﬁned in Figure 9. The reference conﬁguration of the capsule is observed in the shear plane pO, ex, ezq. The points P and P 1 correspond to the intersection of the membrane with the vorticity axis ey. The results are obtained for Ca “ 0.7, YD “ 0.2 and YC “ 2.0. All the pictures are at the same scale. 5. Capsule damage under simple shear ﬂow Capsule damage in simple shear ﬂow 17 t “ tc tc ă t ă t1 t “ t1 t “ t8 P Y 0.6 0 T1 1.5 0 q ¨ n 2.9 0 ´4.4 T2 T2 ě 0 ´0.24 17 Figure 11: Time evolution of diﬀerent state quantities: elastic energy release rate Y (ﬁrst row), maximum principal tension T1 (second row), normal load q¨n to visualize wrinkling (third row) and negative part of principal tension T2 (fourth row). The results are shown in the shear plane pO, ex, ezq at the same instants as in Figure 10, for Ca “ 0.7, YD “ 0.2 and YC “ 2.0. distribution at diﬀerent stages: at the onset of damage (t “ tc), at an intermediate instant while damage develops, at maximum elongation (t “ t1) and at steady state (t “ t8). The capsule states are shown in the current conﬁguration from two view points in the shear plane and in the transverse inclined plane containing the maximum principal direction e1 (Figure 9). Damage is initiated, at time tc, at the points P and P 1 which are on the vorticity axis pO, eyq. As the capsule elongates, two symmetric disjoint damaged areas form around points P and P 1, which correspond to the locations of maximum damage dmax at each instant. Due to the irreversibility of damage, the maximum values d8 max are found at P and P 1 at steady state (t “ t8). In order to see whether preferential direction of damage exists, we plotted the damage distributions on the initial capsule conﬁguration (last row of Figure 10). Damage initially develops preferentially along the direction of maximum elongation e1 but the anisotropy decreases after time t1 to reach a quasi- isotropic damage distribution at steady state. This may be induced by the tank-treading of the capsule membrane around the vorticity axis. Figure 11 gives complementary information on the evolution of the state of the capsule over time until the steady damaged state. The localisation of the energy release rate Y , and hence of damage, in the regions around the points P and P 1 (see Figure 10) is correlated with the maximum of the principal tension T1 (ﬁrst and second rows of Figure 11). 5. Capsule damage under simple shear ﬂow The capsule is delimited by a black line. Figure 10: Map of damage at the instant of initiation of damage tc, at an intermediary instant between tc and the instant of maximum elongation t1, at time t1, and at steady state (t8). The map of damage is represented on the current and reference conﬁgurations. The current conﬁguration is observed in the shear plane pO, ex, ezq and in the plane pO, e1, eyq which is deﬁned in Figure 9. The reference conﬁguration of the capsule is observed in the shear plane pO, ex, ezq. The points P and P 1 correspond to the intersection of the membrane with the vorticity axis ey. The results are obtained for Ca “ 0.7, YD “ 0.2 and YC “ 2.0. All the pictures are at the same scale. The capsule is delimited by a black line. Upon the start of the shear ﬂow at t “ 0, the initially spherical capsule rotates and takes an ellipsoidal deformed shape. It gets ﬂattened while inclining towards the direction of the ﬂow ex (Figure 9). Figure 10 shows the evolution of the capsule state over time until steady state. Note that the membrane rotates around the vorticity axis ey and has a so-called tank-treading motion. We choose to show the capsule shape and damage Capsule damage in simple shear ﬂow 17 t “ tc tc ă t ă t1 t “ t1 t “ t8 P Y 0.6 0 T1 1.5 0 q ¨ n 2.9 0 ´4.4 T2 T2 ě 0 ´0.24 Figure 11: Time evolution of diﬀerent state quantities: elastic energy release rate Y (ﬁrst row), maximum principal tension T1 (second row), normal load q¨n to visualize wrinkling (third row) and negative part of principal tension T2 (fourth row). The results are shown in the shear plane pO, ex, ezq at the same instants as in Figure 10, for Ca “ 0.7, YD “ 0.2 and YC “ 2.0. 5. Capsule damage under simple shear ﬂow Damage has no visible consequences on membrane wrinkling: the wrinkles visible on the normal load maps in the third row of Figure 11 are the same as in Walter et al. (2010) in the case without damage. They are induced by the presence of negative T2 tensions transverse to the direction of the wrinkles (fourth row of Figure 11). The capsule wall being presently modelled as a membrane devoid of bending stiﬀness, the wrinkle amplitude and wavelength are purely numerical. But the small amplitude of the negative part of T2 tensions indicates that they hardly contribute to the energy release N. Grandmaison, D. Brancherie and A.-V. Salsac 18 18 N. Grandmaison, D. Brancherie and A. V. Salsac 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 0 5 10 15 18 9γt 9γtc 9γt1 Li 2a (a) L1 L2 L3 0 0.1 0.2 0.3 0.4 0.5 0.6 0 5 10 15 18 9γt 9γtc 9γt1 D12 (b) 0.53 0.55 0.57 12 16 0 0.05 0.1 0.15 0.2 0.25 0 5 10 15 18 9γt 9γtc 9γt1 β π (c) 0.09 0.1 0.11 12 16 0 0.05 0.1 0.15 0.2 0.25 0.3 0 5 10 15 18 9γt 9γtc 9γt1 No damage Damaged λS (d) Figure 12: Temporal evolution of (a) the lengths of the axes of the ellipsoid of inertia, (b) the Taylor parameter D12, (c) the inclination angle β, and (d) the global surface expansion λS. Computed for Ca “ 0.7, YD “ 0.2 and YC “ 2.0. 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 0 5 10 15 18 9γt 9γtc 9γt1 Li 2a (a) L1 L2 L3 D 0 0.1 0.2 0.3 0.4 0.5 0.6 0 5 10 15 18 9γt 9γtc 9γt1 D12 (b) 0.53 0.55 0.57 12 16 0 0.05 0.1 0.15 0.2 0.25 0 5 10 15 18 9γt 9γtc 9γt1 β π (c) 0.09 0.1 0.11 12 16 0 0.05 0.1 0.15 0.2 0.25 0.3 0 5 10 15 18 9γt 9γtc 9γt1 No damage Damaged λS (d) Figure 12: Temporal evolution of (a) the lengths of the axes of the ellipsoid of inertia, (b) the Taylor parameter D12, (c) the inclination angle β, and (d) the global surface expansion λS. Computed for Ca “ 0.7, YD “ 0.2 and YC “ 2.0. rate Y and thus to damage. 5. Capsule damage under simple shear ﬂow Consequently, they do not lead to any numerical artefact and damage is well predicted by the present model. We now investigate how the capsule shape and deformation is inﬂuenced by damage. In Figure 12, we compare the time evolution of geometric parameters to the case of a capsule without damage. Since the shape of the capsule can be approximated by an ellipsoid of inertia, we deﬁne the principal lengths L1 and L2 of the major and minor axes (directions e1 and e2) in the shear plane pO, ex, ezq and L3, the length along the vorticity axis ey (see Figure 9). The capsule indeed elongates along the directions e1 and ey (L1 ą L3 ą 2a) and shrinks along the direction of the minor axis (L2 ă 2a) (Figure 12a). We quantify the deformation of the capsule with the Taylor parameter D12 “ pL1 ´ L2q{pL1 ` L2q which measures the distortion of the proﬁle of the ellipsoid in the shear plane (Figure 12b). The inclination of the capsule is measured by the angle β between the ﬂow direction ex and the direction of the major axis e1. Figure 12c represents the temporal evolution of β showing that the inclination angle decreases from the ﬁrst measurable value near π{4. Figure 12d shows the evolution of the global surface expansion ratio λS “ pS ´ S0q{S0. Figure 12 globally shows that a steady deformed shape is reached. All the quantities tend towards a plateau value which will be denoted with the symbol 8 hereafter. It is interesting to notice in Figure 12a that the onset of damage (t “ tc) is not visible on the Li curves. It is only close to t “ t1 that the curves slightly diverge from the case without Capsule damage in simple shear ﬂow 19 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 Ca Cac Caℓ d8 max ex ez (a) (b) Ca “ 0.6, t “ t8 Ca “ 0.7, t “ t8 (c) d “ 1 Ca “ 0.8, t “ tℓ 0 0.1 d ě 0.2 d Figure 13: Maximum damage value at steady state d8 max with respect to Ca for YD “ 0.2 and YC “ 2. 5. Capsule damage under simple shear ﬂow The inserted images represent the map of damage in the shear plane at steady state for Ca “ 0.6 (a), for Ca “ 0.7 (b) and at the instant of initiation of rupture t “ tℓfor Ca “ 0.8 (c). The colormap for d is saturated for values of d larger than 0.2. 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 Ca Cac Caℓ d8 max ex ez (a) (b) Ca “ 0.6, t “ t8 Ca “ 0.7, t “ t8 (c) d “ 1 Ca “ 0.8, t “ tℓ 0 0.1 d ě 0.2 d Ca Figure 13: Maximum damage value at steady state d8 max with respect to Ca for YD “ 0.2 and YC “ 2. The inserted images represent the map of damage in the shear plane at steady state for Ca “ 0.6 (a), for Ca “ 0.7 (b) and at the instant of initiation of rupture t “ tℓfor Ca “ 0.8 (c). The colormap for d is saturated for values of d larger than 0.2. damage. But only small diﬀerences are observed on the principal lengths Li (Figure 12a), D12 (Figure 12b) and β (Figure 12c) hereafter. In this reference case, we ﬁnd that damage has no signiﬁcant eﬀects on the motion and deformation of the capsule, suggesting that damage will be very diﬃcult to detect experimentally. The geometrical parameter that is the most aﬀected by damage ends up being the global surface expansion ratio λS (Figure 12d). Nevertheless, the diﬀerence at steady state is only of a few percent. 5.2. Eﬀect of Ca We now study the eﬀect of Ca for the same values of parameters (YD “ 0.2, YC “ 2.0) as in the reference case. The corresponding critical and limit capillary numbers are Cac “ 0.37 and Caℓ“ 0.73. The maximum value of damage at steady state d8 max is shown as a function of the capillary number Ca in Figure 13. For Ca ą Cac, it increases almost linearly with Ca until Ca „ 0.6. Above, d8 max increases more rapidly with Ca until d8 max “ 0.4. Around Ca “ Caℓ, it ﬁnally reaches the value of 1 at points P and P 1 very sharply, with a slope close to inﬁnity. It is for this reason that it is classical in damage mechanics to relax the criterion for rupture to d “ 0.9 or even d “ 0.8. Figure 13 indeed shows that they provide the same value for Ca “ Caℓ. The inserted images in Figure 13 show that the damage maxima always lie at points P and P 1. They also provide an indication of the extent of the damaged zone for increasing values of Ca. Note that for Ca “ 0.8 the damage distribution is given at the instant of initiation of rupture t “ tℓand not at steady state, as it no longer exists. In these cases, the capillary number inﬂuences mainly the values of damage in the vicinity of points P and P 1 and marginally the damaged surface. The capsule deformation and inclination at steady state are compared in Figure 14 with the non-damaged case for Ca ď Caℓ. No results are shown above Caℓ, since no steady deformed shape exists any longer (D12 diverges to inﬁnity). Despite the large eﬀect of N. Grandmaison, D. Brancherie and A.-V. Salsac 20 20 N. Grandmaison, D. Brancherie and A.-V. Salsac 0 0.1 0.2 0.3 0.4 0.5 0.6 0 0.1 0.2 0.3 0.5 0.6 Ca Cac Caℓ D8 12 (a) 0.48 0.54 0.58 0.5 0.6 0.7 0 0.05 0.1 0.15 0.2 0.25 0 0.1 0.2 0.3 0.5 0.6 Ca Cac Caℓ No damage Damaged β8 π (b) 0.09 0.1 0.12 0.5 0.6 0.7 Figure 14: Evolution of the values (a) D8 12 and (b) β8, respectively the values of D12 and β at steady state, in relation to Ca. Computed for YD “ 0.2 and YC “ 2.0. 5.2. Eﬀect of Ca 0 0.1 0.2 0.3 0.4 0.5 0.6 0 0.1 0.2 0.3 0.5 0.6 Ca Cac Caℓ D8 12 (a) 0.48 0.54 0.58 0.5 0.6 0.7 0 0.05 0.1 0.15 0.2 0.25 0 0.1 0.2 0.3 0.5 0.6 Ca Cac Caℓ No damage Damaged β8 π (b) 0.09 0.1 0.12 0.5 0.6 0.7 Figure 14: Evolution of the values (a) D8 12 and (b) β8, respectively the values of D12 and β at steady state, in relation to Ca. Computed for YD “ 0.2 and YC “ 2.0. 0 0.1 0.2 0.3 0.4 0.5 0 0.2 0.4 0.6 0.8 Ca d 8 max YC “ 2.0 YD “ 0.1 0.2 0.3 (a) 0 0.1 0.2 0.3 0.4 0.5 0 0.2 0.4 0.6 0.8 1 Ca d 8 max YD “ 0.2 YC “ 1.0 2.0 3.0 (b) Figure 15: Inﬂuence of the parameters YC and YD on the evolution of the damage value d8 max at points P and P 1 at steady state with Ca: (a) YC “ 2.0 and YD “ 0.1, 0.2, 0.3, (b) YC “ 1.0, 2.0, 3.0 and YD “ 0.2. 0 0.1 0.2 0.3 0.4 0.5 0 0.2 0.4 0.6 0.8 1 Ca max YD “ 0.2 YC “ 1.0 2.0 3.0 (b) 0 0.1 0.2 0.3 0.4 0.5 0 0.2 0.4 0.6 0.8 Ca d 8 max YC “ 2.0 YD “ 0.1 0.2 0.3 (a) d8 Figure 15: Inﬂuence of the parameters YC and YD on the evolution of the damage value d8 max at points P and P 1 at steady state with Ca: (a) YC “ 2.0 and YD “ 0.1, 0.2, 0.3, (b) YC “ 1.0, 2.0, 3.0 and YD “ 0.2. Ca on d8 max for Cac ă Ca ă Caℓ, the D8 12 and β8 curves initially remain superimposed to the non-damaged case, and it is only close to Caℓthat small diﬀerences become visible. No signiﬁcant inﬂuence of damage is, thus, found on these global quantities. It is a consequence of the localisation of damage around points P and P 1 that occurs in the case of a shear ﬂow. Although the evolution curve of D12 with Ca does not provide information on when damage is initiated (i.e. on the value of Cac), it directly provides the value of Caℓ, which corresponding to when D12 diverges to inﬁnity (initiation of breakup). 5.3. Eﬀect of YD and YC The only eﬀect of YC is to shift the Caℓdelimiting curve to higher Ca values as the capsule is then more resistant. This is what is shown by the dotted lines in Figure 16, which complete the base case (YC “ 2.0). criterion of initiation of rupture is controlled by YD ` YC, as already shown at the end of section 4. The results are synthesized in Figure 16, which provides a phase-diagram of the capsule states for a range of values of YD and YC. For a given YC, the curves CacpYDq and CaℓpYD; YCq delimit three domains in the parametric space pCa, YDq: undamaged for Ca ă Cac, damaged for Cac ă Ca ă Caℓ, ruptured for Ca ą Caℓ. The only eﬀect of YC is to shift the Caℓdelimiting curve to higher Ca values as the capsule is then more resistant. This is what is shown by the dotted lines in Figure 16, which complete the base case (YC “ 2.0). 5.3. Eﬀect of YD and YC We ﬁnally study the inﬂuence of the material parameters YD and YC on the damage of the capsule. The evolution of d8 maxpCaq is represented for diﬀerent values of YD and YC in Figure 15. We observe the same trend as in the reference case (Figure 13). For a ﬁxed value of YC, Cac and Caℓincrease with YD (Figure 15a). However, when YD is ﬁxed (Figure 15b), increasing YC does not impact when damage initiates (constant Cac) but delays when the capsule breaks up (increasing value of Caℓ). This relates to the facts that the criterion of initiation of damage is only a function of YD, whereas the Capsule damage in simple shear ﬂow 21 0 0.2 0.4 0.6 0.8 1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 Ca YD Cac CaℓpYC “ 1.0q CaℓpYC “ 2.0q CaℓpYC “ 3.0q Undamaged Damaged Ruptured Figure 16: Evolution of the critical and limit capillary numbers Cac and Caℓwith YD. The solid lines represent the limit curves of Cac and Caℓfor YC “ 0.2. They delimit three domains corresponding to three states of the capsule: undamaged, damaged and ruptured. We also show the limit curves of Caℓfor YC “ 1.0, 3.0 as dotted lines to show how the three domains evolve with the parameters. 0 0.2 0.4 0.6 0.8 1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 Ca YD Cac CaℓpYC “ 1.0q CaℓpYC “ 2.0q CaℓpYC “ 3.0q Undamaged Damaged Ruptured Figure 16: Evolution of the critical and limit capillary numbers Cac and Caℓwith YD. The solid lines represent the limit curves of Cac and Caℓfor YC “ 0.2. They delimit three domains corresponding to three states of the capsule: undamaged, damaged and ruptured. We also show the limit curves of Caℓfor YC “ 1.0, 3.0 as dotted lines to show how the three domains evolve with the parameters. criterion of initiation of rupture is controlled by YD ` YC, as already shown at the end of section 4. The results are synthesized in Figure 16, which provides a phase-diagram of the capsule states for a range of values of YD and YC. For a given YC, the curves CacpYDq and CaℓpYD; YCq delimit three domains in the parametric space pCa, YDq: undamaged for Ca ă Cac, damaged for Cac ă Ca ă Caℓ, ruptured for Ca ą Caℓ. 6. Discussion and conclusion In response to the current need for a damage model of microcapsules in ﬂow, we have developed the ﬁrst ﬂuid-structure interaction (FSI) numerical model accounting for the degradation of the capsule membrane till the onset of rupture, when it is deformed by hydrodynamic forces. We have placed ourselves within the framework of continuum damage mechanics, and simulated microdefect development by degrading the elastic material parameters through the introduction of a damage variable d. We have used an isotropic brittle damage model, in which the damage evolution of the membrane depends on the history of loading. We have integrated it in a Finite Element method that solves for the membrane deformation, which we have coupled to a Boundary Integral method to solve for the Stokes ﬂows inside and outside the capsule. 22 N. Grandmaison, D. Brancherie and A.-V. Salsac N sound ﬁbres nb broken ﬁbres λ 2 λ 2 φNH, Pf nb “ δS ´ δ ˜S δS N “ PfpλmaxqN δS δ ˜S N sound ﬁbres nb broken ﬁbres N sound ﬁbres Figure 17: The RVE consists of a bundle of elastic initially unbroken ﬁbres of speciﬁc elastic energy φNH and probability of rupture Pf (eq. (6.3)). It is subjected to an elongation λ up to the maximum elongation ratio λmax. The zone where the microdefects have appeared upon the rupture of the ﬁbres are represented in grey. Figure 17: The RVE consists of a bundle of elastic initially unbroken ﬁbres of speciﬁc elastic energy φNH and probability of rupture Pf (eq. (6.3)). It is subjected to an elongation λ up to the maximum elongation ratio λmax. The zone where the microdefects have appeared upon the rupture of the ﬁbres are represented in grey. 6.2. Capsule inﬂation test We have ﬁrst applied the model to a capsule under isotropic inﬂation, case for which we derived an analytical solution. This has allowed us to validate the numerical simulations and to show the consequences of damage on the pressure diﬀerence p between the internal and external ﬂuids. The main ﬁndings are that a given capsule remains sound up to a critical value of the inﬂation ratio αc, at which damage initiates. As the capsule further inﬂates above this critical value, the isotropic tension ﬁrst increases with the isotropic strain, reaches a maximum and then decreases: it corresponds to what is generally deﬁned as a softening behaviour. As damage builds up, the pressure diﬀerence decreases, as the global stiﬀness of the capsule is proportional to the local eﬀective surface shear modulus p1´dqGs. The pressure diﬀerence ﬁnally returns to p “ 0, which occurs when the damage variable reaches d “ 1: it corresponds to the moment when the membrane ruptures. A given capsule is thus characterized by a limit inﬂation ration αℓat which it breaks up. The inﬂation capsule test has shown how excellent the agreement is between the theoretical solution and the one obtained with the FSI damage model. If the problem had been solved in displacement (imposed pressure) as classically done in ﬁnite element numerical codes, the material softening behaviour resulting from damage would have induced a loss of stability of the uniform solution at the beginning of the regime of strain localization (Rice 1976; Benallal et al. 1993). In this regime, a small perturbation from the uniform solution would have localized damage and strain in a band of width of one element: the solution would have been strongly mesh-dependent. To solve this issue, classical solid solvers require additional methods, called localization limiters, to obtain more objective solutions (Baˇzant & Pijaudier-Cabot 1988; Simo et al. 1993). However, it is interesting to notice that even for the case of a capsule in simple shear ﬂow discussed below, where the solution is non-uniform, we did not observe the eﬀect of strain localization by changing the mesh size (results not shown). This shows how advantageous it is to implement the damage model within an explicit FSI solver, where the node displacements are imposed by the ﬂuid and the corresponding external loads exerted by the ﬂuids on the membrane are solved for in the solid problem. 6.1. Interpretation of the damage evolution law We have explained the physical meaning of the damage model in section 2, but propose to further detail the interpretation of the damage evolution law eq. (2.29). The capsule membrane being assumed to have a quasi-brittle behaviour, damage evolution is driven by Y max. As an illustration, we propose to introduce a toy model (Figure 17), consisting of a bundle of parallel elastic ﬁbres under uniaxial traction (Krajcinovic 1989; Lemaitre & Desmorat 2005). The RVE consists of N parallel elastic ﬁbres initially unbroken and subjected to an elongation ratio λ. Each ﬁbre is associated to the speciﬁc elastic energy φNH and has a brittle behaviour given by the classical energetic criterion of rupture # φNHpλmaxq ă φu ñ sound ﬁbre φNHpλmaxq “ φu ñ broken ﬁbre, (6.1) (6.1) where φu is a speciﬁc energy at rupture and λmax is deﬁned similarly to Y max in eq. (2.31). The key ingredient of this model is to consider φu as a random variable with probability density ppφuq given by the following band-limited and uniform probability density where φu is a speciﬁc energy at rupture and λmax is deﬁned similarly to Y max in eq. (2.31). The key ingredient of this model is to consider φu as a random variable with probability density ppφuq given by the following band-limited and uniform probability density ppφuq “ $ & % 1 YC @φu P rYD, YD ` YCs 0 @φu R rYD, YD ` YCs. (6.2) (6.2) where YD and YC are the parameters of the damage model introduced in eq. (2.28). Hence, from eqs. (6.1) and (6.2), the probability of rupture of a ﬁbre is given by Pfpλmaxq “ ż φNHpλmaxq 0 ppφuqdφu (6.3) (6.3) Consistent with eq. (2.9), the damage variable d corresponds to the ratio nb{N, where nb is the number of broken ﬁbres. For a large number of ﬁbres, we can postulate nb “ Consistent with eq. (2.9), the damage variable d corresponds to the ratio nb{N, where nb is the number of broken ﬁbres. For a large number of ﬁbres, we can postulate nb “ Capsule damage in simple shear ﬂow 23 Capsule damage in simple shear ﬂow 23 Capsule damage in simple shear ﬂow 23 Capsule damage in simple shear ﬂow 23 23 PfpλmaxqN, and thus d “ Pfpλmaxq. From eq. 6.1. Interpretation of the damage evolution law (6.3), we obtain d “ $ ’ ’ & ’ ’ % 0 if φNHpλmaxq ď YD φNHpλmaxq ´ YD YC if YD ď φNHpλmaxq ď YD ` YC 1 if YD ` YC ď φNHpλmaxq (6.4) (6.4) where φNHpλmaxq “ Y max (see eq. (2.19)2). We thus retrieve the damage evolution law (2.29). This toy model thus shows that the damage evolution law (2.29) is dictated by local phenomena: each ﬁbre has a binary state broken/unbroken (eq. (6.1)), for which the transition is randomly triggered. By integrating the function of rupture probability over all the ﬁbres, we obtain a deterministic damage model for the RVE, where d ranges from 0 (all the ﬁbres are unbroken) to 1 (all the ﬁbres are broken). Eq. (6.2) shows that the model parameters YD and YC delimit the range of dispersion of the speciﬁc energy at rupture in the microstructure. 6.2. Capsule inﬂation test Furthermore, the present FSI scheme is particularly robust and stable, thanks to the fact that the quantities are integrated over the surface in both the ﬂuid and solid solvers. 24 N. Grandmaison, D. Brancherie and A.-V. Salsac 6.3. Capsule under simple shear ﬂow We have then considered a capsule under simple shear ﬂow and similarly seen that there exist a critical value of the capillary number Cac, at which damage initiates, and a limit capillary number Caℓ, at which capsule rupture occurs. In the model, we have chosen to base the criterion for damage on the elastic energy release rate Y of the membrane and to use the evolution law developed by Marigo (1981) for quasi-brittle materials, in which damage evolves when Y “ YD ` YCd. The initiation of damage is then solely dictated by the threshold modulus YD, to which Cac is proportional. As for the hardening modulus YC, it governs the rate at which damage occurs: the lower YC, the faster rupture occurs. The initiation of rupture (d “ 1) and the corresponding limit capillary number Caℓare thus controlled by YC ` YD. For Cac ă Ca ă Caℓ, irreversible damage appears on the ﬂanks of the capsule at the points P and P 1 located on the ﬂow vorticity axis: it is at these locations that the internal membrane tension is the highest. As the capsule tank-treads, the two damaged zones grow around these points, but remain conﬁned in their vicinity, the maximum values remaining at P and P 1. The most striking results in this range of capillary numbers are that the capsule still reaches a steady deformed shape like in the case without damage, and that the eﬀect of damage remains non-visible on the capsule deformed shape, inclination and dynamics. Indeed, damage concentrates around the capsule poles P and P 1 in the case of a simple shear ﬂow, without propagating to the entire capsule. Note that such would not be the case under other ﬂows conditions with three-dimensional vorticity eﬀects, as the capsule rotation would lead to an isotropic distribution of damage all over the capsule membrane. Still, at present, diﬀerences in shape and inclination with the no-damage case start to be visible, when Ca gets close to Caℓ. At Ca “ Caℓ, rupture ﬁnally occurs at points P and P 1, and no steady deformed shape exists thereafter for the capsule. 6.4. Comparison with experiments of capsule damage Damage models are phenomenological and require confrontation with experimental data to assess the relevance of choice of damage evolution law. It is interesting to observe that the present ﬁndings corroborate well the results of the few experimental studies present in the literature, which showed that rupture is initiated at the points of maximum elastic tension (Husmann et al. 2005; Abkarian et al. 2008; Koleva & Rehage 2012; Unverfehrt et al. 2015; Le Goﬀet al. 2017). The damage model assumptions are thus relevant to study the dynamics of microcapsules in ﬂow. Comparing the results of the model with experiments also serves the purpose of identifying the values of the model parameters, namely YD and YC in the present model. We propose to look more closely at the results obtained by the group of Prof. H. Rehage on thin polysiloxane microcapsules subjected to a simple shear ﬂow until breakup in a counter-rotating rheometer cell (Koleva & Rehage 2012; Unverfehrt et al. 2015). They followed a given capsule under increasing values of shear rate and found that wrinkles form on the capsule membrane (Figure 18b) similarly to what was predicted by numerical models (Lac et al. 2004; Walter et al. 2010). Polysiloxane being very brittle and resistant to deformation, only a small increase in capsule deformation was observed as Ca increased (Figure 18a), and rupture occurred at only 3% of deformation. The crack formed in the region near the vorticity axis (Figure 18c), in agreement with the prediction given by our model. Similarly to what we have shown in section 5.2, no inﬂuence of damage eﬀects could be observed on the Taylor parameter curve (Figure 18a). But, even though simple shear ﬂow experiments do not allow to identify the value of Cac (and thus YD), Caℓis easily identiﬁed from the point of divergence of the Taylor parameter curve. 6.4. Comparison with experiments of capsule damage Note that 25 Capsule damage in simple shear ﬂow (a) ex ez (b) (c) (d) (e) d “ 1 (f ) onset rupture (g) Ca “ 0.005, t “ t8 Ca “ 0.01, t “ tℓ Ca “ 0.012, t ą tℓ d ě 0.3 0 d 1 0 Figure 18: Experimental results obtained by Koleva & Rehage (2012) on polysiloxane microcapsules: (a)Evolution of the Taylor parameter D12 with the capillary number Ca, (b) Formation of wrinkles at Ca “ 0.0042 , (c) Formation of a crack at Ca “ 0.01 , (d) Divergence of the capsule shape for Ca larger than Ca “ 0.01 . Numerical predictions given by the present damage FSI model for YD{Gs “ 5 ˆ 10´4 and YC{Gs “ 3.1 ˆ 10´3: (e) At Ca “ 0.005, 3D rendering showing the presence of wrinkles. (f ) At Ca “ 0.01, map of damage at t “ tℓwhen rupture initiates (d “ 1). (g) At Ca “ 0.012, map of damage at an instant of time after t “ tℓwhile the capsule shape diverges due to inﬁnite elongation, this is a case where damage initiates at the points on the vorticity axis but not rupture, which occurs in the nearby region. Pictures (a-d) are reproduced from (Koleva & Rehage 2012) with permission of The Royal Society of Chemistry. (a) (c) (d) (c) (d) onset r (g) Ca “ 0.012, t ą tℓ d 1 0 onset (g) Ca “ 0.012, t ą tℓ (e) Ca “ 0.005, t “ t8 d “ 1 (f ) Ca “ 0.01, t “ tℓ d ě 0.3 0 (f ) (e) Ca “ 0.005, t “ t8 Ca “ 0.012, t ą tℓ Ca “ 0.01, t “ tℓ d 1 0 d d Figure 18: Experimental results obtained by Koleva & Rehage (2012) on polysiloxane microcapsules: (a)Evolution of the Taylor parameter D12 with the capillary number Ca, (b) Formation of wrinkles at Ca “ 0.0042 , (c) Formation of a crack at Ca “ 0.01 , (d) Divergence of the capsule shape for Ca larger than Ca “ 0.01 . Numerical predictions given by the present damage FSI model for YD{Gs “ 5 ˆ 10´4 and YC{Gs “ 3.1 ˆ 10´3: (e) At Ca “ 0.005, 3D rendering showing the presence of wrinkles. 6.4. Comparison with experiments of capsule damage (f ) At Ca “ 0.01, map of damage at t “ tℓwhen rupture initiates (d “ 1). (g) At Ca “ 0.012, map of damage at an instant of time after t “ tℓwhile the capsule shape diverges due to inﬁnite elongation, this is a case where damage initiates at the points on the vorticity axis but not rupture, which occurs in the nearby region. Pictures (a-d) are reproduced from (Koleva & Rehage 2012) with permission of The Royal Society of Chemistry. in Koleva & Rehage (2012) the capillary number is based on the surface Young modulus instead of the surface shear modulus as in the present study. However, for the capsules of Figure 18, the authors estimated that the two moduli had practically the same values, indicating that the Poisson ratio of the membrane was negative. The polysiloxane capsules of Figure 18 are thus found to have a limit capillary number CaℓpYD, YCq “ 0.01, which provides an implicit relationship between YD and YC. Since we know from Figure 16 that the damaged domain is delimited by the curves Ca “ Cac and Ca “ 0.01, we deduce that YD P r0; 1 ˆ 10´3s and YC P r0; 4.6 ˆ 10´3s. We have run simulations assuming YD{Gs “ 5 ˆ 10´4 and YC{Gs “ 3.1 ˆ 10´3, for which CaℓpYD, YCq “ 0.01, and found a good ﬁt between the numerical predictions (Figure 18e-g) and the experimental results (Figure 18b-d). For Ca ě Caℓ, we have continued the simulations after the critical instant 26 N. Grandmaison, D. Brancherie and A.-V. Salsac t “ tℓwhere rupture initiates, and found that the totally damaged state d “ 1 of the membrane propagates in the plane perpendicular to the major axis pO, e1q and that the capsule elongates indeﬁnitely along its major axis (Figure 18g). The divergence of the capsule shape in the simulations (Figure 18g) is similar to what is observed experimentally (Figure 18d). In retrospect, it is surprising that the experiments by Chang & Olbricht (1993) did not ﬁt those by the group of Prof. Rehage. Chang & Olbricht (1993), who were the ﬁrst to study the rupture of polyamide capsules using a counter-rotating rheometer cell, found that rupture initiated at the apex of the major axis, where the capsule is the thinnest. Declaration of Interests The authors report no conﬂict of interest. 6.4. Comparison with experiments of capsule damage Although these results contradict what all the other studies of the literature have found, it could be interesting to use the damage FSI model to investigate for which damage threshold function (eq. (2.27)) the model would predict an initiation of rupture at that location. This study, based on an associated damage model with three ingredients (Table 1), could be generalized to include other dissipative phenomena, such as irreversible strains. These have for instance been taken into account by Ghaemi et al. (2016), in the case of a capsule under compression. The model, however, does not include the gradual degradation of the membrane and information on rupture is obtained by post-processing the stress-strain results. The modularity of the framework that we are proposing rep- resents a real advantage if one wants to generalize the use of the damage FSI model for crack nucleation prediction and damage property identiﬁcation. Predicting crack propagation is, however, outside the scope of the model, as it would require the use of another approach. The eXtended Finite Element Method (Sukumar et al. 2000; Mo¨es & Belytschko 2002) could then be one option among others to provide answers on the subsequent events following crack nucleation. Acknowledgements This work was supported by the European Research Council (ERC) under the Euro- pean Union’s Horizon 2020 research and innovation programme (MultiphysMicroCaps, No. ERC-2017-COG 772191) and by the French Ministry of Higher Education and Research (fellowship of Nicolas Grandmaison). 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https://openalex.org/W2991275983	https://europepmc.org/articles/pmc6877472?pdf=render	English	null	Anti-quorum Sensing and Antimicrobial Effect of Mediterranean Plant Essential Oils Against Phytopathogenic Bacteria	Frontiers in microbiology	2,019	cc-by	4,931	Anti-quorum Sensing and Antimicrobial Effect of Mediterranean Plant Essential Oils Against Phytopathogenic Bacteria Ippolito Camele1, Hazem S. Elshaﬁe1, Lucia Caputo2 and Vincenzo De Feo2 1 School of Agricultural, Forestry, Food and Environmental Sciences, University of Basilicata, Potenza, Italy, 2 Department of Pharmacy, University of Salerno, Salerno, Italy Essential oils (EOs) are one of the most interesting natural products extracted from different aromatic plants. For centuries, EOs have been considered an essential part of the traditional pharmacopeia. Many plant EOs have been reported as possible effective alternatives for commercial pesticides, and their single constituents have been used efﬁciently in food preservation for their promising anti-QS activity against several food pathogenic microorganisms. The current mini review gives a general overview over the microbicide effect as well as anti-quorum sensing and the anti-bioﬁlm formation of some common plant EOs, especially those of Lamiaceae and Verbanaceae families; these are commonly grown in the Mediterranean region and are effective against some serious food phytopathogenic bacteria. Edited by: Rosanna Tofalo, University of Teramo, Italy Reviewed by: Filippo Maggi, University of Camerino, Italy Miklós Takó, University of Szeged, Hungary Correspondence: Hazem S. Elshaﬁe hazem.elshaﬁe@unibas.it Edited by: Rosanna Tofalo, University of Teramo, Italy Reviewed by: Filippo Maggi, University of Camerino, Italy Miklós Takó, University of Szeged, Hungary *Correspondence: Hazem S. Elshaﬁe hazem.elshaﬁe@unibas.it Keywords: natural products, postharvest disease, secondary metabolites, quorum sensing, bioﬁlm Specialty section: This article was submitted to Food Microbiology, a section of the journal Frontiers in Microbiology INTRODUCTION Essential oils (EOs) are considered important natural products extracted from aromatic plants and have been used for centuries in traditional pharmacopeia (Elshaﬁe and Camele, 2017). EOs can be identiﬁed as concentrated hydrophobic liquids containing volatile aromatic compounds (Camele et al., 2012; Elshaﬁe et al., 2015b). They have several biological, nutritional, and pharmaceutical properties. Historically, they represent an important part of the traditional pharmacopeia (Elshaﬁe et al., 2015a). In addition, several EOs have demonstrated interesting antimicrobial eﬀects against many serious phytopathogenic fungi and bacteria, both in vitro and in vivo, as well as an eﬀective use in the production of pharmaceutical drugs for plant and human diseases (Mancini et al., 2014; Elshaﬁe and Camele, 2017). Bacterial bioﬁlm is considered a severe hygiene problem in the environment, plant and human health, and in the food industry. Bioﬁlms make bacteria more resistant to disinfectants and diﬀerent antimicrobial agents (Jamal et al., 2018). Many plant EOs have showed promising anti-bioﬁlm formation and quorum sensing (QS) eﬀects (Poli et al., 2018). Received: 06 August 2019 Accepted: 28 October 2019 Published: 19 November 2019 MINI REVIEW published: 19 November 2019 doi: 10.3389/fmicb.2019.02619 SIGNAL MOLECULES MEDIATED QUORUM SENSING The QS system is based on diﬀerent key elements, such as autoinducers, signal synthase, autoinducers receptors, and regulated genes (Figure 1). In general, G-ve bacteria use the Lux- R/I-type and gram-positive (G + ve) bacteria use the peptide signaling system (Nazzaro et al., 2019). Signals molecules that mediate QS are oligopeptides in G + ve, N-Acyl-homoserine Lactones (N-AHLs) in G-ve, and a family of autoinducers known as autoinducer-2 (AI-2) in both G-ve and G + ve (Miller and Bassler, 2001). In particular, the formation and the activation of N-AHLs are directly proportional to the bacterial density that enables them to act as a multicellular organism and become ready to make behavioral decisions (Withers et al., 2001). The most common bacterial food pathogens produce bioﬁlms such as the Pseudomonas species, which are able to survive at high temperatures and reduce the shelf-life of foods and ﬁsh processing. In addition, Bacillus cereus, Escherichia coli, and Staphylococcus aureus were isolated from dairy processing lines, as reported by Kerekes et al. (2013). N-AHLs are considered the main player in the bacterial pathogenesis (Miller and Bassler, 2001) as well as disease suppression by certain plant beneﬁcial bacteria (Zhou et al., 2003). When the production of N-AHLs reaches a speciﬁc threshold concentration, corresponding to a critical population density, these signals serve as co-inducers to regulate the transcription of bacterial target genes that are responsible for the pathogenicity and production of bioactive secondary metabolites. On the other hand, autoinducers can also regulate the transcription of some bacterial genes as much as the cell density (Smith et al., 2003). Autoinducers are able to coordinate a suite of virulence factors: antibiotic production (Stead et al., 1996), bioﬁlm formation (Van Delden and Iglewski, 1998), luminescence, and swarming motility (Rasmussen et al., 2000). Quorum sensing is a intercellular communication system that regulates microbe–microbe interactions (Nazzaro et al., 2019). The QS phenomenon regulates gene expression in response to the bacterial cell population size (Steindler and Venturi, 2007) and is expected to be the main function responsible for diﬀerent bacterial phenotypes (Kumari et al., 2006; Duerkrop et al., 2007). Furthermore, most bacterial bioactive secondary metabolites are synthesized by stimulating some signal molecules that mediate the process of QS (Withers et al., 2001). SIGNAL MOLECULES MEDIATED QUORUM SENSING Several studies have shed light on the QS phenomenon in many gram-negative (G-ve) bacteria, including those pathogenic to plants and animals as well as human, such as the genus of Agrobacterium, Aeromonas, Burkholderia, Chromobacterium, Citrobacter, Enterobacter, Erwinia, Hafnia, Nitrosomonas, Obesumbacterium, Pantoea, Pseudomonas, Rahnella, Ralstonia, Rhodobacter, Rhizobium, Serratia, and Yersinia. In pathogenic species, the system may also enable coordination against the host, as in case of Pseudomonas aeruginosa infections in cystic ﬁbrosis patients (Withers et al., 2001). Regarding P. aeruginosa, the opportunistic human pathogen, it secretes multiple extracellular virulence factors that cause extensive host tissue damage, BIOFILM FORMATION AND QUORUM SENSING Bioﬁlm formation is considered one of the most essential causes of bacterial resistance toward diﬀerent traditional chemical and physical treatments and antimicrobial agents (Ivanova et al., 2018). Several animal and human microbial infections are related to microbial bioﬁlm ability, which has recently become a real challenge (Coenye and Nelis, 2010). FIGURE 1 \| Schematic representation of quorum sensing: bacteria produce autoinducers molecules and detect the same molecules with speciﬁc receptors that coordinate their behavior. and these factors are regulated by the QS phenomenon, as reported by Gera and Srivastava (2006). Bioﬁlm formation is highly related to the density- dependent cell communication called QS that plays an essential role in the bioﬁlm development of many pathogenic microorganisms and triggers their resistance and virulence (Habeck, 2003). QS enables bacterial cells to have a multicellular behavior in prokaryotes and helps in regulating the virulence process, production of secondary metabolites, symbiosis, bioﬁlm formation, induction of stationary phase responses, and motility for colony escape (Withers et al., 2001). QS allows bacteria cells to monitor their local population densities and regulate the timing of communal activities. Citation: Camele I, Elshaﬁe HS, Caputo L and De Feo V (2019) Anti-quorum Sensing and Antimicrobial Effect of Mediterranean Plant Essential Oils Against Phytopathogenic Bacteria. Front. Microbiol. 10:2619. doi: 10.3389/fmicb.2019.02619 November 2019 \| Volume 10 \| Article 2619 Frontiers in Microbiology \| www.frontiersin.org 1 Biological Activity of Essential Oils Camele et al. FIGURE 1 \| Schematic representation of quorum sensing: bacteria produce autoinducers molecules and detect the same molecules with speciﬁc receptors that coordinate their behavior. In this review, we give some more information about bioﬁlm formation and the QS phenomenon, especially in food pathogenic bacteria. Moreover, this review illustrates the potential use of some plant EOs as anti-QS and bioﬁlm agents to prevent bacterial infection and avoid the drug-resistance ability of many pathogenic bacteria. Frontiers in Microbiology \| www.frontiersin.org ANTI-BIOFILM AND QUORUM SENSING EFFECTS OF EOs In particular, the family Lamiaceae is considered one of the most important families of medicinal and aromatic plants; it includes Origanum vulgare L., Majorana hortensis L., Thymus vulgaris L., Salvia oﬃcinalis L., Lavandula stoechas L., Hyptis suaveolens L., and Rosmarinus oﬃcinalis L. Besides that, Verbena oﬃcinalis L. (Verbenaceae), Carum carvi L. (Apiaceae), Citrus clementina Hort. ex Tan. (Rutaceae), Murraya koenigii (L.), and Sprengel (Rutaceae) are also considered important aromatic plants where their single constituents have promising anti-QS properties to combat Benzaid et al. (2019) studied the anti-bioﬁlm formation eﬀect of mint EO on Candida albicans and concluded that this EO has reduced the bioﬁlm formation of C. albicans. Marjoram EO also showed a promising anti-QS eﬀect against Chromobacterium violaceum, the positive sensor strains for AHL-mediated QS (Kerekes et al., 2013). Poli et al. (2018) reported that Mentha suaveolens ssp. insularis acts as an inhibitor of violacein production and the bioﬁlm TABLE 1 \| List of common plant EOs and their single constituents that display anti-QS activity. Plant scientiﬁc name Family Essential oils Raw materials Principal bioactive constituents References Carum carvi (L.) Sprengel Apiaceae Caraway Fruits Limonene 51%, sabinene 0.3%, β-myrcene 0.4%, β-pinene 0.2%, linalool 0.2%, Bornyl acetate Raal et al., 2012; Al-Haidari et al., 2016 Origanum vulgare L. Lamiaceae Oregano Leaves Carvacrol 75%, linalool 1.3%, limonene 1.3%, (E)-citral 2.5%, γ-terpinene 0.1%, 1,8-cineole 0.2%, eugenol 1.2% Mancini et al., 2014; Elshaﬁe et al., 2017; Asfour, 2018 Majorana hortensis L. Lamiaceae Marjoram Flowers α-pinene 9%, β-pinene 3.8%, limonene 6.4%, 1,8-cineole 33.5%, γ-terpinene 0.1%, linalol 9.8% Kerekes et al., 2013; Elshaﬁe et al., 2016a; Luciardi et al., 2016 Thymus vulgaris L. Lamiaceae Thyme Leaves Carvacrol 3.5%, p-cymene 11.2%, terpinene 4.8% Elshaﬁe et al., 2015a; Asfour, 2018 Salvia ofﬁcinalis L. Lamiaceae Sage Leaves Camphor 13.9%, limonene 1.4%, α-pinene 4.4%, 1,8-cineole 4.2% Elshaﬁe et al., 2016a; Asfour, 2018 Verbena ofﬁcinalis L. Verbenaceae Vervain Leaves Limonene 2.3%, 1.8-cineole, cis-Anethole 0.2%, linalol 0.1%, camphor 0.2% Duke, 1992; Rehecho et al., 2011; Chalchat and Garry, 1995 Lavandula stoechas L. Lamiaceae Lavender Flowers Fenchone 34.9%, camphone 28.9% Poli et al., 2018 Citrus clementina Hort. ex Tan. Rutaceae Clementina Peel Sabinene 31.4%, linalool 20.4% Kerekes et al., 2013; Luciardi et al., 2016; Poli et al., 2018 Murraya koenigii (L.) Sprengel Rutaceae Curry tree Leaves Caryophyllene 9.49%, caryophyllene oxide 1.02%, α- and β-phellandrene 0.07%, α-Terpinene 2.37%, linalool 0.19% Chowdhury et al., 2008; Bai and Vittal, 2014 Hyptis suaveolens L. ANTI-BIOFILM AND QUORUM SENSING EFFECTS OF EOs EOs are composed mainly from two groups of single substances, terpenoids (monoterpene, sesquiterpene and di-terpene) November 2019 \| Volume 10 \| Article 2619 Frontiers in Microbiology \| www.frontiersin.org 2 Biological Activity of Essential Oils Camele et al. diﬀerent food pathogenic microorganisms, as reported in Table 1. and phenylpropanoids (Nieto, 2017). The terpenoid group includes several compounds commonly present in the chemical composition of many plant EOs with diﬀerent percentages, such as p-cymene, pinene, limonene, sabinene, and terpinene (Kerekes et al., 2015), geraniol, menthol, linalool, citronellol, carvone, thymol, carvacrol, geranyl acetate, eugenyl acetate, geranial, neral, and 1,8-cineole (Ayala-Zavala et al., 2007; Nieto, 2017). In addition, phenylpropanoids include several aromatic compounds such as cinnamyl alcohol, cinnamaldehyde, eugenol, and methyl cinnamate (Hyldgaard et al., 2012). Since not all of the above compounds have anti-QS activity, an overview of the principal constituents of some common plant EOs extracted from diﬀerent aromatic plants and that display anti-QS activity are reported in Table 1. and phenylpropanoids (Nieto, 2017). The terpenoid group includes several compounds commonly present in the chemical composition of many plant EOs with diﬀerent percentages, such as p-cymene, pinene, limonene, sabinene, and terpinene (Kerekes et al., 2015), geraniol, menthol, linalool, citronellol, carvone, thymol, carvacrol, geranyl acetate, eugenyl acetate, geranial, neral, and 1,8-cineole (Ayala-Zavala et al., 2007; Nieto, 2017). In addition, phenylpropanoids include several aromatic compounds such as cinnamyl alcohol, cinnamaldehyde, eugenol, and methyl cinnamate (Hyldgaard et al., 2012). Since not all of the above compounds have anti-QS activity, an overview of the principal constituents of some common plant EOs extracted from diﬀerent aromatic plants and that display anti-QS activity are reported in Table 1. Origanum vulgare, one of the most eﬃcient plant EOs, is able to counteract bioﬁlm formation and the QS mechanism with its main bioactive constituents (carvacrol), which has explicated a promising eﬀect against diﬀerent food and human pathogenic bacteria, such as Salmonella enterica subsp. typhimurium and S. aureus (Asfour, 2018). In addition, other single constituents of oregano EO, such as linalool, limonene, (E)-citral, γ-terpinene, 1,8-cineole, and eugenol, have exhibited anti-QS eﬀects, as reported in several studies (Raal et al., 2012; Al-Haidari et al., 2016). Moreover, clary sage, juniper, lemon, and marjoram EOs have been examined in the food industry and showed an eﬀective anti-QS eﬀect by preventing bioﬁlm formation, especially against B. cereus, E. coli, and Pichia anomala (Kerekes et al., 2013; Luciardi et al., 2016). Raal et al., 2012; Al-Haidari et al., 2016 MODE OF ACTION formation of C. violaceum, and the Carum copticum EO showed anti-QS activity against C. violaceum (Snoussi et al., 2018). Szabó et al. (2010) reported that EOs extracted from lavender, citrus, and rosemary plants can also inhibit QS and concluded that these EOs can be used in the pharmaceutical industry for discovering new therapy for serious human infections. Many researchers have hypothesized that the possible mechanism behind the bioactivity of many EOs is due to their principal bioactive single molecules. In particular, the use of single components to control bioﬁlm formation could be, in some cases, suﬃcient, such as B. cereus and E. coli (Kerekes et al., 2013); however, other studies explained that the synergism between diﬀerent single components display better eﬀects, like in case of the Listeria monocytogenes bioﬁlm, where the synergism between a-pinene, limonene, and linalool substances can be more eﬀective than each single component (Sandasi et al., 2009). ANTI-BIOFILM AND QUORUM SENSING EFFECTS OF EOs Lamiaceae Pignut Leaves Trans-β-caryophyllene 11.3%, α-pinene (2.3), camphene 2.6%, β-myrcene 1.5%, p-cymene (11.2), limonene (7.2), γ-terpinene (1.5) Stashenko et al., 2013 Rosmarinus ofﬁcinalis L. Lamiaceae Rosemary Leaves α-pinene (26%), 1,8-cineole (25%), camphor 12% Alvarez et al., 2012; Melito et al., 2019 TABLE 1 \| List of common plant EOs and their single constituents that display anti-QS activity. Alvarez et al., 2012; Melito et al., 2019 November 2019 \| Volume 10 \| Article 2619 Frontiers in Microbiology \| www.frontiersin.org 3 Biological Activity of Essential Oils Camele et al. CONCLUSION The bioﬁlm formation of pathogenic bacteria is considered a big challenge for the food industry and human/animal health. The QS mechanism regulates the bacterial bioﬁlm formation; thus, destroying and/or disrupting this mechanism can help to prevent bioﬁlm formation and then solve many health problems. Many plant EOs display promising anti- QS properties by preventing bioﬁlm formation, which could be very important in reducing the virulence and pathogenicity of drug-resistant bacteria, especially for those that are food pathogenic. In fact, the use of plant EOs in food industry do not change the organoleptic properties of foods, and their use could thus be a promising natural alternative for several synthetic food preservatives. Finally, many plant EOs can represent a possible substitute for many traditional antimicrobial drugs, which have a signiﬁcant negative impact on the environment and human/animal health. Recently, there has been a great revolution in scientiﬁc research regarding the importance of using plant EOs in combating many pathogenic bacteria, especially against food spoilage and for human health (Khan et al., 2009; Olivero et al., 2010; Camele et al., 2012; Nieto, 2017). In particular, many interesting EOs and their single components, extracted from oregano, sage, marjoram, and vervain in particular, have been used eﬀectively against some post-harvest diseases (Mancini et al., 2014; Elshaﬁe et al., 2015a, 2016a, 2017). The three EOs extracted from V. oﬃcinalis, M. hortensis, and S. oﬃcinalis (Elshaﬁe et al., 2016a) and those extracted from leaves and fruits of Schinus terebinthifolius (Elshaﬁe et al., 2016b) showed promising antimicrobial activity against some serious phytopathogens such as Colletotrichum acutatum, Botrytis cinerea, Clavibacter michiganensis, Xanthomonas campestris, and Pseudomonas syringae pv. phaseolicola. Other plant Eos, such as O. vulgare, O. heracleoticum, and O. majorana, showed eﬀective microbicide eﬀects against some post-harvest pathogenic fungi (Aspergillus sp., Penicillium sp., Monilinia sp., and B. cinerea) and some phytopathogenic bacteria (Bacillus megaterium, C. michiganensis, X. campestris, and P. syringae pv. phaseolicola) (Della Pepa et al., 2019). MICROBICIDE EFFECT OF PLANT ESSENTIAL OILS Many foodborn pathogenic bacteria (FBPB) produce serious toxins that lead to food spoilage and human infection. Some of FBPB are characterized by the abovementioned phenomena of QS, such as E. coli, Listeria monocytogenes, Clostridium spp., S. enterica, and S. aureus (Martinoviæ et al., 2016). In addition, the synergic eﬀect between diﬀerent single constituents could trigger the antimicrobial eﬀectiveness of EOs and may reduce the resistance of many pathogenic microorganisms (Elshaﬁe et al., 2015b). Some single constituents can damage the cell walls and plasma membranes of microbial cells, alter morphology, and increase cell permeability (Elshaﬁe et al., 2019). Adebayo et al. (2012) reported that carvacrol, γ -terpinene, and p-cymene could be eﬀective on their own and also have a synergic eﬀect when they are combined. This synergistic eﬀect is due to the action of p-cymene, which works as mediator for transportation of carvacrol and γ -terpinene across the cell wall and cytoplasmic membrane of pathogenic microorganisms. On the other hand, the lipophilic properties of many single components play a role in degrading the microbe plasma membrane and, thus, lead to the lyses of the hypha wall (Elshaﬁe and Camele, 2017). In general, the use of antibiotics are the most common substances for the direct controlling of whole bacteria (Poli et al., 2018); however its use in the food industry sector for controlling FBPB is prohibited in most developed countries to avoid creating diﬀerent resistant human strains. Furthermore, many synthetic preservatives used in the food industry with antimicrobial eﬀects may causing allergies, intoxications, cancer, and other degenerative diseases (Aminzare et al., 2016). 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https://openalex.org/W3133340570	https://ejurnal.its.ac.id/index.php/teknik/article/download/54864/6404	Indonesian	null	Evaluasi dan Desain Ulang Instalasi Pengolahan Air Limbah (IPAL) di Rumah Susun Sederhana Sewa Randu Kota Surabaya	Jurnal Teknik/Jurnal Teknik ITS	2,021	cc-by-sa	5,680	Evaluasi dan Desain Ulang Instalasi Pengolahan Air Limbah (IPAL) di Rumah Susun Sederhana Sewa Randu Kota Surabaya Fitria Indaryani dan Alfan Purnomo Departemen Teknik Lingkungan, Institut Teknologi Sepuluh Nopember e-mail: alfanpurnomo@gmail.com Fitria Indaryani dan Alfan Purnomo Departemen Teknik Lingkungan, Institut Teknologi Sepuluh Nopember e-mail: alfanpurnomo@gmail.com Menurut data dari Dinas Perumahan Rakyat dan Kawasan Permukiman, Cipta Karya dan Tata Ruang, Kota Surabaya memiliki 30 Rusunawa, di mana mayoritas Rusunawa sudah dilengkapi dengan fasilitas unit Instalasi Pengolahan Air Limbah (IPAL). Namun sebagian besar IPAL yang ada di Rusunawa memerlukan perbaikan dan tidak beroperasi dengan optimal. Abstrak—Rusunawa Randu merupakan salah satu Rusunawa di Surabaya. Rusunawa Randu menghasilkan air limbah domestik baik blackwater (kakus) maupun greywater (non kakus). Rusunawa ini sudah memiliki unit pengolahan air limbah kakus berupa tangki septik di setiap bloknya, Anaerobic Biofilter dan Anaerobic Baffled Reactor (ABR). Anaerobic Biofilter berkapasitas 12 m3 dan secara eksisting melayani efluen dari tangki septik Blok D saja, sedangkan ABR berkapasitas 45 m3. Namun ABR tersebut secara eksisting belum dioperasikan, dan direncanakan untuk mengolah air limbah non kakus dari semua blok. Evaluasi IPAL Anaerobic Biofilter dan ABR dilakukan dengan cara mengukur dimensi masing-masing unit, menganalisis sampel air mbah dan membandingkan parameter waktu detensi, OLR dan HLR eksisting dengan kriteria desain. Hasil evaluasi menunjukan bahwa IPAL tersebut tidak memenuhi kriteria desain, maka dari itu hasil efluen belum memenuhi baku mutu Permen LHK No. 68 Tahun 2016. Rekomendasi dari hasil evaluasi yaitu IPAL Anaerobic Biofilter dan ABR eksisting tetap digunakan dengan menyesuaikan debit yang masuk. Anaerobic Biofilter akan mengolah air limbah kakus dari blok B, C, D dan F, dan ABR akan mengolah air limbah non kakus dari blok A, B, D dan F. Kedua IPAL eksisting tersebut, masing-masing dilengkapi unit grease trap, untuk mengolah minyak dan lemak. Rusunawa Randu merupakan salah satu Rusunawa di Surabaya, terletak di Jalan Randu Agung, termasuk dalam wilayah Kelurahan Sidotopo Wetan, Kecamatan Kenjeran. Menurut data adiministrasi kependudukan RT/RW tahun 2020, Rusunawa Randu terdiri dari 6 blok dan dihuni sebanyak 1.094 jiwa.. Rusunawa Randu menghasilkan air limbah domestik baik blackwater (kakus) maupun greywater (non kakus) yang berasal dari aktivitas penghuninya. Di Rusunawa Randu sudah terdapat tangki septik dan IPAL lanjutan dari tangki septik dengan teknologi Anaerobik Biofilter untuk mengolah limbah kakus (efluen tangki septik). Sedangkan untuk limbah non kakus di Rusunawa Randu terdapat IPAL berupa Anaerobic Baffled Reactor (ABR), namun tidak beroperasi pengolahannya, sehingga tidak terjadi pengolahan air limbah non kakus yang ada di Rusunawa Randu. D99 D99 JURNAL TEKNIK ITS Vol. 9, No. 2, (2020) ISSN: 2337-3539 (2301-9271 Print) Evaluasi dan Desain Ulang Instalasi Pengolahan Air Limbah (IPAL) di Rumah Susun Sederhana Sewa Randu Kota Surabaya Anaerobic Biofilter tersebut berkapasitas 12 m3 dan secara eksisting hanya melayani efluen dari tangki septik Blok D saja, dan ABR berkapasitas 45 m3. Berdasarkan hasil analisis sampel air limbah, baik kakus maupun non kakus di Rusunawa Randu tidak memenuhi baku mutu Permen LHK No. 68 Tahun 2016. Kata Kunci—Air Limbah Domestik, Anaerobic Baffled Reactor (ABR), Anaerobic Biofilter, Evaluasi IPAL, Rusunawa Randu. I. PENDAHULUAN R USUNAWA (Rumah Susun Sederhana Sewa) merupakan salah satu solusi menyelesaikan permasalahan keterbatasan lahan permukiman di perkotaan, khususnya untuk masyarakat berpenghasilan rendah (MBR). Namun Rusunawa juga memiliki potensi menyebabkan pencemaran air, yang berasal dari air limbah domestik penghuninya. Limbah domestik memberikan kontribusi pencemaran sebesar 60% dalam penurunan kualitas air di Kota Surabaya dan limbah industri (non domestik) sebesar 40%. Limbah domestik dapat dibagi menjadi dua macam, limbah kakus (blackwater) merupakan limbah yang berasal dari Water Closet atau tinja manusia dan limbah non kakus (greywater) merupakan limbah yang berasal dari kegiatan mencuci, memasak, mandi, atau limbah yang berasal selain dari tinja. R Evaluasi IPAL eksisting di Rusunawa Randu diharapkan dapat menganalisis permasalahan yang menyebabkan IPAL tersebut tidak beroperasi. Perencaan ulang untuk unit Anaerobic Biofilter dan ABR eksisting di Rusunawa Randu dengan menyesuaikan debit dan beban air limbah yang diolah sesuai dengan kapasitas unit - unit tersebut. Hal tersebut diharapkan bisa menjadi solusi dalam pengolahan air limbah di Rusunawa Randu, sehingga efluen air limbah telah memenuhi baku mutu Permen LHK No. 68 Tahun 2016. JURNAL TEKNIK ITS Vol. 9, No. 2, (2020) ISSN: 2337-3539 (2301-9271 Print) D100 3. Baku mutu air limbah yang digunakan adalah Permen LHK No. 68 Tahun 2016. B. Metode Evaluasi M d l i di k d l li i i i Tabel 2. Debit Air Limbah Harian Maksimum di Rusunawa Randu Blok fp Debit Harian Maksimum Non Kakus (m3/hari) Debit Harian Maksimum Kakus (m3/hari) Debit Harian Maksimum Air Limbah (m3/hari) Blok A 1,23 19,80 1,84 21,64 Blok B 1,23 22,47 2,09 24,56 Blok C 1,23 24,21 2,25 26,46 Blok D 1,23 22,63 2,10 24,73 Blok E 1,23 28,67 2,66 31,34 Blok F 1,23 29,64 2,75 32,40 Total 147,42 13,70 161,13 Tabel 1. Kualitas Air Limbah di Rusunawa Parameter Inlet IPAL Anaerobic BIofilter Outlet IPAL Anaerobic BIofilter Air Limbah Kakus Air Limbah Non Kakus pH 7,9 7,4 7,5 6,3 TSS (mg/l) 258 174 546 424 BOD (mg/l) 160 160 508 380 COD (mg/l) 300 302 955 715 Minyak Lemak (mg/l) 14 12 88 66 Amoniak (mg/l) 296,39 250,96 496,15 62,7 Total Coliform (MPN/ 100ml) 8 × 1012 2,2 × 109 2,9 × 1011 1,7 × 107 Tabel 2. Debit Air Limbah Harian Maksimum di Rusunawa Randu Tabel 1. Kualitas Air Limbah di Rusunawa Tabel 2. Tabel 1. 3. Baku mutu air limbah yang digunakan adalah Permen LHK No. 68 Tahun 2016. B. Metode Evaluasi Metode evaluasi yang digunakan dalam penelitian ini terdiri dari beberapa tahap, yaitu melakukan pengukuran dan pengambilan sampel air limbah → menganalisis sampel air limbah di laboratorium → menganalisis sistem IPAL dan menghitung kriteria desain yang di evaluasi → memperbaiki unit yang bermasalah dan dikombinaksikan alternatif unit baru apabila efluen masih belum memenuhi baku mutu. 𝑄𝑑𝑚 = debit harian maksimum dalam 1 tahun (m3/detik) 𝑄𝑎𝑣𝑒 = debit rata-rata dalam 1 tahun (m3/detik). Faktor harian air limbah di Rusunawa Randu yang didapatkan dari perhitungan dengan persamaan (1) sebesar 1,23. Hasil perhitungan debit air limbah kakus dan non kakus harian maksimum di Rusunawa Randu tiap bloknya dapat dilihat pada Tabel 1. A. Perhitungan Debit Air Limbah Debit air limbah dihitung berdasarkan debit air minum yang digunakan tiap blok di Rusunawa Randu. Debit air limbah rata-rata sebesar 80% dari debit air minum [2]. Air limbah dibagi menjadi 2 macam, air limbah kakus dan non kakus. Menurut penelitian sebelumnya, persentase banyaknya limbah kakus dan non kakus dalam air limbah adalah 8,5% dan 91,5%. Maka dari data terssebut didapatkan debit air limbah kakus dan non kakus rata-rata di Rusunawa Randu. C. Analisis Evaluasi IPAL di Rusunawa Randu IPAL yang dianalisis evaluasi yaitu IPAL eksisting anaerobic biofilter dan anaerobic baffled reactor di Rusunawa Randu. Analisis yang dilakukan dengan cara mengecek proses pengolahan dalam sistem IPAL eksisting dengan parameter waktu detensi, HLR dan OLR, kemudian dibandingkan dengan kriteria desain dari tiap unitnya. Analisis evaluasi lainnya yang dilakukan adalah mengukur dimensi eksisting tiap unit IPAL eksisting dan menghitung performa penyisihan polutan setelah diolah didalam IPAL eksisting. Berikut adalah analisis evaluasi IPAL eksisting di Rusunawa Randu. A. Perhitungan Debit Air Limbah A. Perhitungan Debit Air Limbah B. Kualitas Air Limbah di Rusunawa Randu Data primer yang digunakan dalam evaluasi ini meliputi data kualitas air limbah kakus (blackwater), non kakus (greywater), influen dan efluen dari unit anaerobic biofilter. Parameter yang diukur dalam air limbah tersebut yaitu pH, TSS, BOD, COD minyak dan lemak, amoniak dan total coliform. Data priner lainnya yaitu dimensi dari tiap unit IPAL, anaerobic biofilter dan ABR. Kualitas air limbah yang digunakan dalam penelitian evaluasi IPAL di Rusunawa berasal dari air limbah kakus (blackwater), non kakus (greywater), influen dan efluen dari unit anaerobic biofilter. Hasil uji laboratorium sampel air limbah dapat dilihat pada Tabel 2. Berdasarkan hasil analisis air limbah pada Tabel 2, dapat disimpulkan bahwa semua parameter air limbah di Rusunawa Randu tidak memenuhi baku mutu berdasarkan Permen LHK No 68 Tahun 2016, kecuali parameter pH. Data sekunder yang digunakan adalah debit air minum, yang didapatkan dari rekening PDAM tiap bulannya dan jumlah penghuni setiap blok di Rusunawa Randu. A. Ruang Lingkup A. Ruang Lingkup Ruang lingkup evaluasi IPAL di Rusunawa Randu adalah: A. Ruang Lingkup Ruang lingkup evaluasi IPAL di Rusunawa Randu adalah: Kualitas air limbah domestik di salah satu Rusunawa di Surabaya memiliki nilai COD 329,81 mg/L, BOD 182,02 mg/L dan TSS 103,33 mg/L, di mana parameter - parameter tersebut tidak memenuhi baku mutu air limbah domestik Peraturan Menteri Lingkungan Hidup dan Kehutanan No. 68 Tahun 2016 [1]. Upaya pencegahan pencemaran kualitas air dapat dilakukan dengan menerapkan salah satu alternatif teknologi Instalasi Pengolahan Air Limbah (IPAL). 1. Unit IPAL Rusunawa Randu yang dievaluasi adalah Anaerobic Biofilter untuk limbah kakus (efluen tangki septik) dan Anaerobic Baffled Reactor untuk limbah non kakus. 1. Unit IPAL Rusunawa Randu yang dievaluasi adalah Anaerobic Biofilter untuk limbah kakus (efluen tangki septik) dan Anaerobic Baffled Reactor untuk limbah non kakus. 2. Kriteria desain yang dievaluasi adalah waktu detensi, OLR, HLR dan efisiensi penyisihan tiap polutan. 2. Kriteria desain yang dievaluasi adalah waktu detensi, OLR, HLR dan efisiensi penyisihan tiap polutan. 𝑓𝑝 = faktor harian maksimum 𝑓𝑝= 𝑄𝑑𝑚 𝑄𝑎𝑣𝑒… … … … … … … … … … … … … … … … … … … (1) Keterangan : = 𝑄𝑎𝑣𝑒… … … … … … … … … … … … … … … … … … … (1 𝑓𝑝= 𝑄𝑑𝑚 𝑄𝑎𝑣𝑒… … … … … … … … … … … … … … … … … … … (1) JURNAL TEKNIK ITS Vol. 9, No. 2, (2020) ISSN: 2337-3539 (2301-9271 Print) JURNAL TEKNIK ITS Vol. 9, No. 2, (2020) ISSN: 2337-3539 (2301-9271 Print) JURNAL TEKNIK ITS Vol. 9, No. 2, (2020) ISSN: 2337-3539 (2301-9271 Print) D101 Tabel 3. Kualitas Efluen Air Limbah Grease Trap – Anaerobic Biofilter Parameter % Penyisihan di Grease Trap % Penyisihan di Bak Pengendap Anaaerobic Biofilter % Penyisihan Kompartemen Anaaerobic Biofilter % Penyisihan Efluen % Penyisihan Efluen % Penyisihan Efluen pH 7,9 - 7,9 - 7,9 - 7,9 TSS (mg/l) 258 - 258 60,6% 101,64 70% 30,61 BOD (mg/l) 160 - 160 28% 115,1 86% 23,95 COD (mg/l) 300 - 300 26% 221,3 78% 32,03 Minyak Lemak (mg/l) 14 95% 0,7 - 0,7 - 0,7 Amoniak (mg/l) 296,39 - 296,39 - 296,39 - 296,39 Total Coliform (MPN/ 100ml) 8 × 1012 - 8 × 1012 - 8 × 1012 - 8 × 1012 Tabel 3. Kualitas Efluen Air Limbah Grease Trap – Anaerobic Biofilter Tabel 3. limbah kakus berasal dari 1 blok dan kondisi limbah kakus ketika dari semua blok. = 296,4 𝑚𝑔 𝑙 NH3N − 250,96 𝑚𝑔 𝑙 NH3N 296,4 𝑚𝑔 𝑙 𝑥 100% =15% • Total coliform Influen : 8 x 1012 MPN / 100 ml Efluen : 2,2 x 109 MPN / 100 ml Efisiensi : 𝑖𝑛𝑓𝑙𝑢𝑒𝑛−𝑒𝑓𝑓𝑙𝑢𝑒𝑛 𝑖𝑛𝑓𝑙𝑢𝑒𝑛 𝑥 100% = (8 𝑥 1012𝑚𝑔 𝑙 ) − (2,2𝑥 109𝑚𝑔 𝑙) 300𝑚𝑔 𝑙 𝑥 100% = 99,97 % W kt D t i (Td) a) Dimensi reaktor IPAL anaerobic biofilter • Panjang total reaktor anaerobic biofilter = 12,4 m • Panjang reaktor anaerobic biofilter = 9,8 m • Lebar anaerobic biofilter = 1,3 m • Tinggi anaerobic biofilter = 0,74 m • Panjang bak pengendap (kompartemen 1) = 2,6 m • Lebar bak pengendap (kompartemen 1) = 1,3 m • Tinggi bak pengendap (kompartemen 1) = 0,74 m • Volume bak pengendap = 2,5 m3 • Volume reaktor anaerobic biofilter = 9,5 m3 b) Efisiensi Penyisihan c) Waktu Detensi (Td) 1) IPAL Anaerobic Biofilter Eksisting Namun dalam evaluasi unit IPAL harus menggunakan debit air limbah harian maksimum. Debit air maksimum harian maksimum didapatkan dengan cara mengkalikan faktor harian maksimum dengan debit rata-rata air limbah, baik limbah kakus dan non kakus. Permasaan faktor harian maksimum untuk air limbah dapat dilihat pada persamaan berikut : Kapasitas IPAL anaerobic biofilter di Rusunawa Randu adalah 12 m3, dengan lima kompartemen didalamnya. Kompartemen pertama merupakan bak pengendap dan kompartemen kedua hingga kelima merupakan reaktor biofilter. Kondisi eksisting sumber air limbah kakus yang masuk ke IPAL anaerobic biofilter ini berasal dari hanya 1 blok saja, blok D. Namun dalam analisis evaluasi di unit anaerobic biofilter menggunakan kapasitas debit pengolahan IPAL Rusunawa Randu dengan kondisi eksisting dari air 𝑓𝑝 = faktor harian maksimum b) Efisiensi Penyisihan • Waktu detensi untuk debit dari 1 blok D Q = 2,102 m3/hari Volume reaktor = 9,5 m3 Waktu detensi = 𝑉𝑜𝑙𝑢𝑚𝑒 𝐷𝑒𝑏𝑖𝑡 = 9,5 𝑚3 2,102 𝑚3/ℎ𝑎𝑟𝑖 = 4,52 hari atau 109,47 jam Berdasarkan hasil analisis di laboratorium dapat diketahui kualitas inlet dan outlet IPAL anaerobic biofilter, yang telah ditampilkan pada Tabel 1. • Chemical Oxygen Demand (COD) Influen : 300 mg/l Efluen : 302 mg/l Efisiensi : 𝑖𝑛𝑓𝑙𝑢𝑒𝑛−𝑒𝑓𝑓𝑙𝑢𝑒𝑛 𝑖𝑛𝑓𝑙𝑢𝑒𝑛 𝑥 100% = 300𝑚𝑔 𝑙 − 302𝑚𝑔 𝑙 300𝑚𝑔 𝑙 𝑥 100% = -1 % • Chemical Oxygen Demand (COD) Influen : 300 mg/l Efluen : 302 mg/l Efisiensi : 𝑖𝑛𝑓𝑙𝑢𝑒𝑛−𝑒𝑓𝑓𝑙𝑢𝑒𝑛 𝑖𝑛𝑓𝑙𝑢𝑒𝑛 𝑥 100% = 300𝑚𝑔 𝑙 − 302𝑚𝑔 𝑙 300𝑚𝑔 𝑙 𝑥 100% = -1 % • Chemical Oxygen Demand (COD) Influen : 300 mg/l Efluen : 302 mg/l Efisiensi : 𝑖𝑛𝑓𝑙𝑢𝑒𝑛−𝑒𝑓𝑓𝑙𝑢𝑒𝑛 𝑖𝑛𝑓𝑙𝑢𝑒𝑛 𝑥 100% = 300𝑚𝑔 𝑙 − 302𝑚𝑔 𝑙 300𝑚𝑔 𝑙 𝑥 100% = -1 % (Tidak memenuhi Kriteria Desain) (Tidak memenuhi Kriteria Desain) • Waktu detensi untuk debit dari semua blok Q = 13,7 m3/hari Volume reaktor = 9,5 m3 Waktu detensi = 𝑉𝑜𝑙𝑢𝑚𝑒 𝐷𝑒𝑏𝑖𝑡 = 9,5 𝑚3 13,7𝑚3/ℎ𝑎𝑟𝑖 = 0,69 hari atau 16,65 jam 𝑙 • Biochemical Oxygen Demand (BOD) 𝑙 • Biochemical Oxygen Demand (BOD) Influen : 160 mg/l Efluen : 160 mg/l Efisiensi : 𝑖𝑛𝑓𝑙𝑢𝑒𝑛−𝑒𝑓𝑓𝑙𝑢𝑒𝑛 𝑖𝑛𝑓𝑙𝑢𝑒𝑛 𝑥 100% = 160 𝑚𝑔 𝑙 − 160𝑚𝑔 𝑙 160𝑚𝑔 𝑙 𝑥 100% = 0 % 2) IPAL Anaerobic Baffled Reactor Eksisting 2) IPAL Anaerobic Baffled Reactor Eksisting Anaerobic Baffled Reactor (ABR) eksisting di IPAL Rusunawa Randu yang didesain mengolah air limbah non kakus dari seluruh blok. Kapasitas IPAL ABR eksisting di Rusunawa Randu adalah 45 m3, dengan lima kompartemen didalamnya. Kompartemen pertama merupakan bak pengendap dan kompartemen kedua hingga kelima merupakan reaktor pengolahan anaerobik. Berdasarkan hasil perhitungan OLR unit anaerobik biofilter apabila menampung debit dari 1 blok D atau debit dari semua blok Keduanya menghasilkan nilai yang tidak memenuhi kriteria desain. Kriteria desain OLR untuk IPAL anaerobik biofilter adalah 0,4 – 4,7 kg/ m3.hari. Ketidaksesuaian antara hasil perhitungan IPAL eksisting dengan kriteria desain mungkin dikarenakan volume reaktor IPAL yang ada terlalu besar. Karena dengan dicoba menggunakan kapasitas debit harian maksimum dari semua blok juga ternyata belum memenuhi kriteria desain OLR anaerobic biofilter atau terjadi perubahan kualitas air limbah, yang menyebabkan beban BOD ketika perencanaan anaerobic biofilter awal berbeda dengan ketika saat ini dievaluasi. a) Dimensi reaktor IPAL ABR a) Dimensi reaktor IPAL ABR a) Dimensi reaktor IPAL ABR • Panjang total ABR = 7 m • Panjang reaktor ABR = 4,9 m • Lebar ABR = 3 m • Tinggi ABR = 2,14 m • Panjang bak pengendap (kompartemen 1)= 2,1 m • Lebar bak pengendap (kompartemen 1) = 3 m • Tinggi bak pengendap (kompartemen 1) = 2,14 m • Volume bak pengendap = 13,5 m3 • Volume reaktor ABR = 31,5 m3 b) Waktu detensi Q = 147,43 m3/hari Volume reaktor = 31,5 m3 Waktu detensi = 𝑉𝑜𝑙𝑢𝑚𝑒 𝐷𝑒𝑏𝑖𝑡 = 31,5 𝑚3 147,43 𝑚3/ℎ𝑎𝑟𝑖 = 0,214 hari atau 5,1 jam (Tidak memenuhi Kriteria Desain) e) HLR (Hydraulic Loading Rate) e) HLR (Hydraulic Loading Rate) ( y g ) • HLR untuk debit dari 1 blok D Q = 2,102 m3/hari Panjang reaktor = 9,8 m Lebar reaktor = 1,3 m Luas permukaan (As) = 12,74 m2 HLR = 𝑄 𝐴𝑠 = 2,102 𝑚3 ℎ𝑎𝑟𝑖 12,74 𝑚2 = 0,165 m3./m2.hari (Tidak memenuhi Kriteria Desain) Berdasarkan hasil perhitungan waktu detensi unit ABR apabila menampung debit dari semua blok, tidak memenuhi kriteria desain. Hal tersebut dikarenakan debit yang masuk terlalu terlalu besar. Kriteria desain waktu detensi untuk IPAL ABR adalah > 8 jam. JURNAL TEKNIK ITS Vol. 9, No. 2, (2020) ISSN: 2337-3539 (2301-9271 Print) D102 Tabel 4. Kualitas Efluen Air Limbah Grease Trap – ABR Parameter % Penyisihan di Grease Trap % Penyisihan di Bak Pengendap Anaaerobic Biofilter % Penyisihan Kompartemen Anaaerobic Biofilter % Penyisihan Efluen % Penyisihan Efluen % Penyisihan Efluen pH 6,3 - 6,3 - 6,3 - 6,3 TSS (mg/l) 424 - 424 52,6% 200,84 68% 63,72 BOD (mg/l) 380 - 380 26% 282,4 71% 82,39 COD (mg/l) 715 - 715 24% 543,4 67% 181,61 Minyak Lemak (mg/l) 66 95% 3,3 - 3,3 - 3,3 Amoniak (mg/l) 62,7 - 62,7 - 62,7 - 62,7 Total Coliform (MPN/ 100ml) 1,7 × 107 - 1,7 × 107 - 1,7 × 107 - 1,7 × 107 Tabel 4. Kualitas Efluen Air Limbah Grease Trap – ABR = 0,035 𝑘𝑔/𝑚3 . hari (Tidak memenuhi Kriteria Desain) • OLR untuk debit dari semua blok Q = 13,7 m3/hari Volume reaktor = 9,5 m3 BODin (So) = 160 mg/l = 0,16 kg/m3 OLR = 𝑄 𝑥 𝑆𝑜 𝑉𝑜𝑙𝑢𝑚𝑒 = 13,7 𝑚3 ℎ𝑎𝑟𝑖 𝑥 0,16𝑘𝑔 𝑚3 9,5 𝑚3 = 0,23 kg/m3..hari (Tidak memenuhi Kriteria Desain) menampung debit dari 1 blok D atau debit dari semua blok Keduanya menghasilkan nilai yang memenuhi kriteria desain. Kriteria desain HLR untuk IPAL anaerobik biofilter adalah < 2 m3./m2.hari. = 0,035 𝑘𝑔/𝑚3 . hari (Tidak memenuhi Kriteria Desain) , (Tidak memenuhi Kriteria Desain) , (Tidak memenuhi Kriteria Desain) Berdasarkan hasil perhitungan waktu detensi unit anaerobic biofilter apabila menampung debit dari 1 blok D saja, tidak memenuhi kriteria desain. Hal tersebut dikarenakan debit yang masuk terlalu kecil. Sedangkan apabila dirancang untuk menampung debit dari seluruh blok, IPAL tidak mampu mengolah dengan optimal. Karena debit yang masuk terlalu besar, yang dapat dilihat dari hasil perhitungan waktu detensi IPAL tersebut tidak memenuhi kriteria desain juga. Kriteria desain waktu detensi untuk IPAL anaerobik biofilter adalah 24 – 48 jam. Debit maksimal yang dibutuhkan untuk memenuhi kriteria desain waktu detensi 24 jam, yaitu 9,5 m3/hari. 𝑙 • Total Suspended Solid (TSS) Influen : 258 mg/l Efluen : 174 mg/l Efisiensi : 𝑖𝑛𝑓𝑙𝑢𝑒𝑛−𝑒𝑓𝑓𝑙𝑢𝑒𝑛 𝑖𝑛𝑓𝑙𝑢𝑒𝑛 𝑥 100% = 258 𝑚𝑔 𝑙 − 174 𝑚𝑔 𝑙 258 𝑚𝑔 𝑙 𝑥 100% = 33 % IPAL tidak mampu mengolah dengan optimal. Karena debit yang masuk terlalu besar, yang dapat dilihat dari hasil perhitungan waktu detensi IPAL tersebut tidak memenuhi kriteria desain juga. Kriteria desain waktu detensi untuk IPAL anaerobik biofilter adalah 24 – 48 jam. Debit maksimal yang dibutuhkan untuk memenuhi kriteria desain waktu detensi 24 jam, yaitu 9,5 m3/hari. d) OLR (Organic Loading Rate) d) OLR (Organic Loading Rate) • OLR untuk debit dari 1 blok D Q = 2,102 m3/hari Volume reaktor = 9,5 m3 BODin (So) = 160 mg/l = 0,16 kg/m3 OLR = 𝑄 𝑥 𝑆𝑜 𝑉𝑜𝑙𝑢𝑚𝑒 = 2,102 𝑚3 ℎ𝑎𝑟𝑖 𝑥 0,16𝑘𝑔 𝑚3 9,5 𝑚3 JURNAL TEKNIK ITS Vol. 9, No. 2, (2020) ISSN: 2337-3539 (2301-9271 Print) JURNAL TEKNIK ITS Vol. 9, No. 2, (2020) ISSN: 2337-3539 (2301-9271 Print) 2) IPAL Anaerobic Baffled Reactor Eksisting c) Organic Loading Rate (OLR) Q = 147,43 m3/hari Volume reaktor = 31,5 m3 CODin (So) = 715 mg/l = 0,715 kg/m3 OLR = 𝑄 𝑥 𝑆𝑜 𝑉𝑜𝑙𝑢𝑚𝑒 = 147,43 𝑚3 ℎ𝑎𝑟𝑖 𝑥 0,715𝑘𝑔 𝑚3 31,5 𝑚3 = 3,346 kg/m3..hari ( id k hi i i i ) (Memenuhi Kriteria Desain) (Memenuhi Kriteria Desain) c) Organic Loading Rate (OLR) Q = 147,43 m3/hari Volume reaktor = 31,5 m3 CODin (So) = 715 mg/l = 0,715 kg/m3 OLR = 𝑄 𝑥 𝑆𝑜 𝑉𝑜𝑙𝑢𝑚𝑒 = 147,43 𝑚3 ℎ𝑎𝑟𝑖 𝑥 0,715𝑘𝑔 𝑚3 31,5 𝑚3 = 3,346 kg/m3..hari • HLR untuk debit dari semua blok Q = 13,7 m3/hari Panjang reaktor = 9,8 m Lebar reaktor = 1,3 m Luas permukaan (As) = 12,74 m2 HLR = 𝑄 𝐴𝑠 = 13,7 𝑚3 ℎ𝑎𝑟𝑖 9,5 𝑚2 = 1,075 m3./m2.hari g (Tidak memenuhi Kriteria Desain) D. Rancangan Ulang IPAL Eksisting di Rusunawa Randu d) Efisiensi penyisihan polutan Efisiensi penyisihan polutan yang masuk ke anaerobic biofilter dapat dilihat pada Tabel 3 [3]: e) Perencanaan grease trap untuk unit anaerobic biofilter eksisting • Debit harian maksimum total = 9,19 m3/hari • Konsentrasi minyak dan lemak = 14 mg/l = 0,014 kg/m3 • Beban minyak dan lemak = 𝑄𝑡𝑜𝑡𝑎𝑙 × 𝑘𝑜𝑛𝑠𝑒𝑛𝑡𝑟𝑎𝑠𝑖 𝑚𝑖𝑛𝑦𝑎𝑘 = 9,19 𝑚3/ℎ𝑎𝑟𝑖 𝑥 0,014 𝑘𝑔/𝑚3 = 0,1287 kg/hari Dimensi kompartemen • Jumlah kompartemen = 1 buah • Waktu detensi (td) = 10 menit = 0,007 hari • Volume Grease Trap = 𝑄𝑡𝑜𝑡𝑎𝑙 × 𝑡𝑑 = 9,19 m3/hari x 0,007 hari = 0,064 m3 • Kedalaman efektif = 0,5 meter • Freeboard = 0,3 meter • Kedalaman total = 𝑘𝑒𝑑𝑎𝑙𝑎𝑚𝑎𝑛 𝑒𝑓𝑒𝑘𝑡𝑖𝑓 + 𝑓𝑟𝑒𝑒𝑏𝑜𝑎𝑟𝑑 = 0,5 meter + 0,3 meter = 0,8 m • Luas Permukaan (As) = 𝑉𝑜𝑙𝑢𝑚𝑒 𝐾𝑒𝑑𝑎𝑙𝑎𝑚𝑎𝑛 𝑒𝑓𝑒𝑘𝑡𝑖𝑓 = 0,064 𝑚3 0,5 𝑚𝑒𝑡𝑒𝑟 = 0,13 m2 • Rasio panjang : lebar = 2 : 1 • Lebar =√𝐿𝑢𝑎𝑠 𝑝𝑒𝑟𝑚𝑢𝑘𝑎𝑎𝑛 (𝐴𝑠) 2 = √0,13 𝑚2 2 = 0,255 meter = 0,3 meter (dibulatkan) • Panjang = 𝐿𝑢𝑎𝑠 𝑝𝑒𝑟𝑚𝑢𝑘𝑎𝑎𝑛 (𝐴𝑠) 𝐿𝑒𝑏𝑎𝑟 = 0,13 𝑚2 0,3 𝑚𝑒𝑡𝑒𝑟 = 0,43 meter d) Efisiensi penyisihan polutan Rancangan ulang IPAL eksisitng di Rusunawa Randu dilakukan untuk unit anaerobic biofilter dan ABR. Hal tersebut dikarenakan berdasarkan analisis evaluasi yang telah dilakukan, terdapat kriteria desain masing-masing unit IPAL yang belum memenuhi kriteria desainnya. Kriteria desain yang tidak terpenuhi dapat menyebabkan tidak maksimal pengolahan didalam IPAL tersebut, sehingga efluen air limbah belum memenuhi baku mutu dan polutan masih berbahaya untuk dibuang ke badan air secara langsung. Berikut adalah rancangan ulang IPAL eksisting berdasarkan hasil evaluasi di Rusunawa Randu. Efisiensi penyisihan polutan yang masuk ke anaerobic biofilter dapat dilihat pada Tabel 3 [3]: Efisiensi penyisihan polutan yang masuk ke anaerobic biofilter dapat dilihat pada Tabel 3 [3]: e) Perencanaan grease trap untuk unit anaerobic biofilter eksisting = 0,155 kg/m3..hari (Tidak memenuhi Kriteria Desain) d) Efisiensi penyisihan polutan Efisiensi penyisihan polutan yang masuk ke anaerobic biofilter dapat dilihat pada Tabel 3 [3]: e) Perencanaan grease trap untuk unit anaerobic biofilter eksisting • Debit harian maksimum total = 9,19 m3/hari • Konsentrasi minyak dan lemak = 14 mg/l = 0,014 kg/m3 • Beban minyak dan lemak = 𝑄𝑡𝑜𝑡𝑎𝑙 × 𝑘𝑜𝑛𝑠𝑒𝑛𝑡𝑟𝑎𝑠𝑖 𝑚𝑖𝑛𝑦𝑎𝑘 = 9,19 𝑚3/ℎ𝑎𝑟𝑖 𝑥 0,014 𝑘𝑔/𝑚3 = 0,1287 kg/hari Dimensi kompartemen • Jumlah kompartemen = 1 buah • Waktu detensi (td) = 10 menit = 0,007 hari • Volume Grease Trap = 𝑄𝑡𝑜𝑡𝑎𝑙 × 𝑡𝑑 = 9,19 m3/hari x 0,007 hari = 0,064 m3 • Kedalaman efektif = 0,5 meter • Freeboard = 0,3 meter • Kedalaman total = 𝑘𝑒𝑑𝑎𝑙𝑎𝑚𝑎𝑛 𝑒𝑓𝑒𝑘𝑡𝑖𝑓 + 𝑓𝑟𝑒𝑒𝑏𝑜𝑎𝑟𝑑 = 0,5 meter + 0,3 meter = 0,8 m • Luas Permukaan (As) = 𝑉𝑜𝑙𝑢𝑚𝑒 𝐾𝑒𝑑𝑎𝑙𝑎𝑚𝑎𝑛 𝑒𝑓𝑒𝑘𝑡𝑖𝑓 = 0,064 𝑚3 0,5 𝑚𝑒𝑡𝑒𝑟 = 0,13 m2 • Rasio panjang : lebar = 2 : 1 • Lebar =√𝐿𝑢𝑎𝑠 𝑝𝑒𝑟𝑚𝑢𝑘𝑎𝑎𝑛 (𝐴𝑠) 2 = √0,13 𝑚2 2 = 0,255 meter = 0,3 meter (dibulatkan) • Panjang = 𝐿𝑢𝑎𝑠 𝑝𝑒𝑟𝑚𝑢𝑘𝑎𝑎𝑛 (𝐴𝑠) 𝐿𝑒𝑏𝑎𝑟 = 0,13 𝑚2 0,3 𝑚𝑒𝑡𝑒𝑟 = 0,43 meter g (Tidak memenuhi Kriteria Desain) Berdasarkan hasil perhitungan OLR unit ABR apabila menampung debit dari semua blok Keduanya menghasilkan nilai yang tidak memenuhi kriteria desain. Kriteria desain (Memenuhi Kriteria Desain). Berdasarkan hasil perhitungan HLR unit anaerobic biofilter apabila JURNAL TEKNIK ITS Vol. 9, No. 2, (2020) ISSN: 2337-3539 (2301-9271 Print) D103 = 9,19 𝑚3 ℎ𝑎𝑟𝑖 𝑥 0,16𝑘𝑔 𝑚3 9,5 𝑚3 OLR untuk IPAL ABR adalah <3 kg/ m3..hari. Ketidaksesuaian antara hasil perhitungan IPAL eksisting dengan kriteria desain mungkin dikarenakan volume reaktor IPAL yang ada terlalu besar atau debit yang masuk terlalu besar juga. = 0,155 kg/m3..hari (Tidak memenuhi Kriteria Desain) = 0,155 kg/m3..hari (Tidak memenuhi Kriteria Desain) g (Tidak memenuhi Kriteria Desain) Kriteria desain OLR untuk IPAL anaerobic biofilter adalah 0,4 – 4,7 kg/ m3..hari. Parameter OLR tidak dapat terpenuhi karena beban yang masuk ke anaerobic biofilter terlalu kecil atau volume reaktor yang terlalu besar. Karena volume reaktor tidak dapat diubah, maka dari itu beban yang masuk harus diperbesar atau diberi nutrisi lebih sebelum masuk ke anaerobic biofilter. j g d) Hydraulic Loading Rate (HLR) d) Hydraulic Loading Rate (HLR) Hydraulic Loading Rate (HLR) Q = 147,43 m3/hari Panjang reaktor = 4,9 m Lebar reaktor = 3 m Luas permukaan (As) = 14,7 m2 HLR = 𝑄 𝐴𝑠 = 147,43 𝑚3 ℎ𝑎𝑟𝑖 14,7 𝑚2 = 10,03 m3./m2.hari (Tid k hi K it i D i ) ) Hydraulic Loading Rate (HLR) Q = 147,43 m3/hari Panjang reaktor = 4,9 m Lebar reaktor = 3 m Luas permukaan (As) = 14,7 m2 HLR = 𝑄 𝐴𝑠 = 147,43 𝑚3 ℎ𝑎𝑟𝑖 14,7 𝑚2 = 10,03 m3./m2.hari (Tid k hi K it i D i ) f c) Hydraulic Loading Rate (HLR) f c) Hydraulic Loading Rate (HLR) f c) Hydraulic Loading Rate (HLR) c) Hydraulic Loading Rate (HLR) Q = 9,19 m3/hari Panjang reaktor = 9,8 m Lebar reaktor = 1,3 m Luas permukaan (As) = 12,74 m2 HLR = 𝑄 𝐴𝑠 = 9,19 𝑚3 ℎ𝑎𝑟𝑖 12,74 𝑚2 = 0,72 m3./m2.hari (Tidak memenuhi Kriteria Desain) Berdasarkan hasil perhitungan HLR unit ABR apabila menampung debit dari semua blok, menghasilkan nilai yang memenuhi kriteria desain. Kriteria desain HLR untuk IPAL ABR adalah 16,8 – 38,4 m3./m2.hari. Ketidaksesuaian antara hasil perhitungan HLR IPAL eksisting dengan HLR kriteria desain mungkin dikarenakan luas penampang (As) reaktor IPAL yang ada terlalu besar atau debit yang masuk terlalu kecil juga. , (Memenuhi kriteria desain. kriteria desain HLR untuk IPAL anaerobic biofilter adalah < 2 m3./m2.hari). • Beban minyak dan lemak • Beban minyak dan lemak Efluen • Konsentrasi minyak efluen = 𝐾𝑜𝑛𝑠𝑒𝑛𝑡𝑟𝑎𝑠𝑖 ( 𝑖𝑛𝑓𝑙𝑢𝑒𝑛𝑡 – 𝑡𝑒𝑟𝑠 2) Rancangan Ulang ABR Eksisting Rancangan ulang unit ABR eksisting debit yang masuk direncanakan mengolah air limbah non kakus dari empat blok, blok A, B, D dan F. Sehingga debit harian maksimum air limbah non kakus yang diolah ABR sebesar 94,54 m3/hari. Maka dengan debit 94,54 m3/hari, akan dicek kembali apakah sudah memenuhi kriteria desain untuk parameter waktu detensi, OLR dan HLR atau belum. Waktu detensi Q = 94,54 m3/hari Volume reaktor = 31,5 m3 Waktu detensi = 𝑉𝑜𝑙𝑢𝑚𝑒 𝐷𝑒𝑏𝑖𝑡 = 31,5 𝑚3 94,54 𝑚3/ℎ𝑎𝑟𝑖 = 0,33 hari atau 8 jam = 94,54 m3/hari Volume reaktor = 31,5 m3 𝑉𝑜𝑙𝑢𝑚𝑒 Waktu detensi = 𝑉𝑜𝑙𝑢𝑚𝑒 𝐷𝑒𝑏𝑖𝑡 31,5 𝑚 / = 0,33 hari atau 8 jam j (Memenuhi kriteria desain, kriteria desain waktu detensi untuk IPAL ABR adalah > 8 jam). a) Organic Loading Rate (OLR) Q = 94,54 m3/hari Volume reaktor = 31,5 m3 CODin (So) = 715 mg/l = 0,715 kg/m3 OLR = 𝑄 𝑥 𝑆𝑜 𝑉𝑜𝑙𝑢𝑚𝑒 = 94,54 𝑚3 ℎ𝑎𝑟𝑖 𝑥 0,715𝑘𝑔 𝑚3 31,5 𝑚3 =2,146kg/m3..hari a) Organic Loading Rate (OLR) Q = 94,54 m3/hari Volume reaktor = 31,5 m3 CODin (So) = 715 mg/l = 0,715 kg/m3 OLR = 𝑄 𝑥 𝑆𝑜 𝑉𝑜𝑙𝑢𝑚𝑒 = 94,54 𝑚3 ℎ𝑎𝑟𝑖 𝑥 0,715𝑘𝑔 𝑚3 31,5 𝑚3 =2,146kg/m3..hari a) Organic Loading Rate (OLR) a) Organic Loading Rate (OLR) Q = 94,54 m3/hari Volume reaktor = 31,5 m3 CODin (So) = 715 mg/l = 0,715 kg/m3 OLR = 𝑄 𝑥 𝑆𝑜 𝑉𝑜𝑙𝑢𝑚𝑒 = 94,54 𝑚3 ℎ𝑎𝑟𝑖 𝑥 0,715𝑘𝑔 𝑚3 31,5 𝑚3 =2,146kg/m3..hari = 𝐾𝑜𝑛𝑠𝑒𝑛𝑡𝑟𝑎𝑠𝑖 ( 𝑖𝑛𝑓𝑙𝑢𝑒𝑛𝑡 – 𝑡𝑒𝑟𝑠𝑖 3 3 g (Memenuhi kriteria desain, kriteria desain OLR untuk IPAL ABR adalah <3 kg/ m3..hari). (Memenuhi kriteria desain, kriteria desain OLR untuk IPAL ABR d l h 3 k / 3 h i) (Memenuhi kriteria desain, kriteria desain OLR untuk IPAL ABR adalah <3 kg/ m3..hari). ABR adalah <3 kg/ m3..hari). g ) ) Hydraulic Loading Rate (HLR) Q = 94,54 m3/hari Panjang reaktor = 4,9 m Lebar reaktor = 3 m Luas permukaan (As) = 14,7 m2 HLR = 𝑄 𝐴𝑠= 94,54 𝑚3 ℎ𝑎𝑟𝑖 14,7 𝑚2 g ) ) Hydraulic Loading Rate (HLR) Q = 94,54 m3/hari Panjang reaktor = 4,9 m Lebar reaktor = 3 m Luas permukaan (As) = 14,7 m2 HLR = 𝑄 𝐴𝑠= 94,54 𝑚3 ℎ𝑎𝑟𝑖 14,7 𝑚2 JURNAL TEKNIK ITS Vol. 9, No. 2, (2020) ISSN: 2337-3539 (2301-9271 Print) JURNAL TEKNIK ITS Vol. 9, No. 2, (2020) ISSN: 2337-3539 (2301-9271 Print) D104 kesalahan perencanaan ketika merencanakan IPAL ABR tersebut. kesalahan perencanaan ketika merencanakan IPAL ABR tersebut. 1) Rancangan Ulang Anaerobic Biofilter Eksisting Dalam rancanagan ulang ini, debit yang masuk IPAL anaerobic biofilter eksisting direncanakan mengolah air limbah kakus dari empat blok, blok B, C, D dan F. Sehingga debit harian maksimum air limbah kakus yang diolah anaerobic biofilter sebesar 9,19 m3/hari. Maka dengan debit 9,19 m3/hari, akan dicek kembali apakah sudah memenuhi kriteria desain untuk parameter waktu detensi, OLR dan HLR atau belum. a) Waktu detensi a) Waktu detensi a) Waktu detensi Q = 9,19 m3/hari Volume reaktor = 9,5 m3 Waktu detensi = 𝑉𝑜𝑙𝑢𝑚𝑒 𝐷𝑒𝑏𝑖𝑡 = 9,5 𝑚3 9,19 𝑚3/ℎ𝑎𝑟𝑖 = 1,033 hari atau 24,8 jam = 1,033 hari atau 24,8 jam (Memenuhi kriteria desain. Kriteria desain waktu detensi k IPAL bi bi fil d l h 24 48 j ) (Memenuhi kriteria desain. Kriteria desain waktu detensi untuk IPAL anaerobic biofilter adalah 24 – 48 jam). (Memenuhi kriteria desain. Kriteria desain waktu detensi untuk IPAL anaerobic biofilter adalah 24 – 48 jam). (Memenuhi kriteria desain. Kriteria desain waktu untuk IPAL anaerobic biofilter adalah 24 – 48 jam). b) Organic Loading Rate (OLR) Q = 9,19 m3/hari Volume reaktor = 9,5 m3 BODin (So) = 160 mg/l = 0,16 kg/m3 OLR = 𝑄 𝑥 𝑆𝑜 𝑉𝑜𝑙𝑢𝑚𝑒 b) Organic Loading Rate (OLR) JURNAL TEKNIK ITS Vol. 9, No. 2, (2020) ISSN: 2337-3539 (2301-9271 Print) = 0,45 meter (dibulatkan) • Tebal dinding = 0,1 m • Panjang total grease trap = 0,65 meter • Lebar total grease trap = 0,5 meter • Kedalaman total = 1 meter Efisiensi Penyisihan = 0,45 meter (dibulatkan) • Tebal dinding = 0,1 m • Panjang total grease trap = 0,65 meter • Lebar total grease trap = 0,5 meter • Kedalaman total = 1 meter Efisiensi Penyisihan • Persen penyisihan lemak dan minyak di grease tra = 95% [4] • Konsentrasi minyak tersisihkan = %𝑅 𝑥 𝑘𝑜𝑛𝑠𝑒𝑛𝑡𝑟𝑎𝑠𝑖 𝑚𝑖𝑛𝑦𝑎𝑘 = 95% x 0,014 kg/m3 = 0,0133 kg/m3 Efluen • Konsentrasi minyak efluen = 𝐾𝑜𝑛𝑠𝑒𝑛𝑡𝑟𝑎𝑠𝑖 ( 𝑖𝑛𝑓𝑙𝑢𝑒𝑛𝑡 – 𝑡𝑒𝑟𝑠𝑖𝑠𝑖ℎ𝑘𝑎𝑛) = 0,014 kg/m3 - 0,0133 kg/m3 = 0,0007 kg/m3 = 0,7 mg/l c) Efisiensi penyisihan polutan c) Efisiensi penyisihan polutan c) Efisiensi penyisihan polutan Efisiensi penyisihan polutan yang masuk ke ABR dapat dilihat pada Tabel 4 [5] : Efisiensi penyisihan polutan yang masuk ke ABR dapat dilihat pada Tabel 4 [5] : d) Perencanaan grease trap untuk unit ABR eksisting • Debit harian maksimum total • Persen penyisihan lemak dan minyak di grease trap = 95% [4] • Persen penyisihan lemak dan minyak di grease trap = 95% [4] • Konsentrasi minyak dan lemak = [ ] • Konsentrasi minyak tersisihkan = %𝑅 𝑥 𝑘𝑜𝑛𝑠𝑒𝑛𝑡𝑟𝑎𝑠𝑖 𝑚𝑖𝑛𝑦𝑎𝑘 = 95% x 0,014 kg/m3 = 0,0133 kg/m3 Efluen • Konsentrasi minyak efluen = 𝐾𝑜𝑛𝑠𝑒𝑛𝑡𝑟𝑎𝑠𝑖 ( 𝑖𝑛𝑓𝑙𝑢𝑒𝑛𝑡 – 𝑡𝑒𝑟𝑠𝑖𝑠𝑖ℎ𝑘𝑎𝑛) = 0,014 kg/m3 - 0,0133 kg/m3 = 0,0007 kg/m3 = 0,7 mg/l • Konsentrasi minyak tersisihkan = %𝑅 𝑥 𝑘𝑜𝑛𝑠𝑒𝑛𝑡𝑟𝑎𝑠𝑖 𝑚𝑖𝑛𝑦𝑎𝑘 = 95% x 0,014 kg/m3 = 0,0133 kg/m3 • Konsentrasi minyak tersisihkan = 0,066 kg/m3 • Beban minyak dan lemak = 6,24 kg/hari Dimensi kompartemen • Jumlah kompartemen = 1 buah • Waktu detensi (td) = 10 menit = 0,007 hari • Volume Grease Trap = 0,656 m3 • Kedalaman total = 𝑘𝑒𝑑𝑎𝑙𝑎𝑚𝑎𝑛 𝑒𝑓𝑒𝑘𝑡𝑖𝑓 + 𝑓𝑟𝑒𝑒𝑏𝑜𝑎𝑟𝑑 = 1 meter + 0,3 meter = 1,3 m • Luas Permukaan (As) = 𝑉𝑜𝑙𝑢𝑚𝑒 𝐾𝑒𝑑𝑎𝑙𝑎𝑚𝑎𝑛 𝑒𝑓𝑒𝑘𝑡𝑖𝑓 = 0,656 m3 1 𝑚𝑒𝑡𝑒𝑟 = 0,656 m2 • Rasio Panjang : Lebar = 2 : 1 • Lebar =√𝐿𝑢𝑎𝑠 𝑝𝑒𝑟𝑚𝑢𝑘𝑎𝑎𝑛 (𝐴𝑠) 2 = √0,656 𝑚2 2 = 0,57 meter = 0,6 meter (dibulatkan) • Panjang = 𝐿𝑢𝑎𝑠 𝑝𝑒𝑟𝑚𝑢𝑘𝑎𝑎𝑛 (𝐴𝑠) 𝐿𝑒𝑏𝑎𝑟 = 0,656 𝑚2 0,6 𝑚𝑒𝑡𝑒𝑟 = 1,09 meter = 1,1 meter (dibulatkan) • Tebal dinding = 0,15 m • Panjang total grease trap = 1,4 meter • Lebar total grease trap = 0,9 meter • Kedalaman total = 1,6 meter Efisiensi Penyisihan • Konsentrasi minyak tersisihkan = 95% x 0,066 kg/m3 = 0,0627 kg/m3 Efluen Konsentrasi minyak efluen = 𝐾𝑜𝑛𝑠𝑒𝑛𝑡𝑟𝑎𝑠𝑖 ( 𝑖𝑛𝑓𝑙𝑢𝑒𝑛𝑡 – 𝑡𝑒𝑟𝑠𝑖𝑠𝑖ℎ𝑘𝑎𝑛) = 0,066 kg/m3 - 0,0627 kg/m3 = 0,0033 kg/m3 = 3,3 mg/l IV. KESIMPULAN Kesimpulan dari evalasi dan desain ulang di Rusunawa Randu adalah sebagai berikut : IPAL eksisting di Rusunawa Randu ada 2, dengan teknologi anaerobic biofilter untuk mengolah limbah kakus dan anaerobic baffled reactor untuk mengolah limbah non kakus. Hasil evaluasi kedua IPAL tersebut tidak mampu menampung semua air limbah yang dihasilkan di Rusunawa Randu. Maka, IPAL anaerobic biofilter akan mengolah limbah kakus dari blok B, C, D dan F, dengan kapasitas sebesar 9,19 m3/hari. Lalu IPAL anaerobic baffled reactor akan me[ngolah limbah non kakus dari blok A, B, D dan F, dengan kapasitas sebesar 94,9 m3/hari. Masing-masing IPAL eksisting dilengkapi dengan unit grease trap, untuk mengolah minyak dan lemak. Grease = 6,4 m3./m2.hari (Tidak memenuhi Kriteria Desain) = 6,4 m3./m2.hari , (Tidak memenuhi Kriteria Desain) Kriteria desain HLR untuk IPAL ABR adalah 16,8 – 38,4 m3./m2.hari. Parameter HLR tidak dapat terpenuhi karena debit yang masuk ke ABR terlalu kecil. Karena dimensi panjang dan lebar reaktor tidak dapat diubah, maka dari itu debit yang masuk harus diperbesar. Namun apabila debit diperbesar maka parameter waktu detensi tidak dapat memenuhi kriteria desain, sehingga dapat disimpulkan ada D105 JURNAL TEKNIK ITS Vol. 9, No. 2, (2020) ISSN: 2337-3539 (2301-9271 Print) water dengan teknologi subsurface flow constructed wetland di rusunawa grudo surabaya,” J. Tek. ITS, vol. 5, no. 2, 2016. water dengan teknologi subsurface flow constructed wetland di rusunawa grudo surabaya,” J. Tek. ITS, vol. 5, no. 2, 2016. trap untuk anaerobic biofilter berkapasitas 0,064 m3, sedangkan grease trap untuk ABR berkapasitas 0,656 m3. [2] S. Iskandar, I. Fransisca, E. Arianto, and A. Ruslan, Buku 3 Sistem Pengelolaan Air Limbah Domestik – Terpusat Skala Permukiman. Jakarta: Direktorat Jenderal Cipta Karya, 2016. UCAPAN TERIMA KASIH [3] B. Gutterer, L. Sasse, T. Panzerbieter, and R. Thorsten, Decentralised Wastewater Treatment Systems (DEWATS) and Sanitation in Developing Countries, vol. 49. German: Bremen Overseas Research and Development Association (BORDA), 2009. Penulis mengucapkan terima kasih kepada Dinas Pengelolaan Bangunan dan Tanah, Dinas Kebersihan dan Ruang Terbuka Hijau dan Dinas Perumahan Rakyat dan Kawasan Permukiman, Cipta Karya dan Tata Ruang Kota Surabaya yang telah memberikan ijin dan data-data yang diperlukan dalam penelitian ini. [4] J. Wongthanate, N. Mapracha, and P. Bengjaphorn, “Efficiency of modified grease trap for domestic wastewater treatment,” J. Ind. Technol., vol. 10, no. 2, 2014. [5] A. Mahatyanta, “Perencanaan desain alternatif IPAL dengan Teknologi Anaerobic Baffled Reactor dan Anaerobic Filter untuk Rumah Susun Romokalisari Surabaya,” Institut Teknologi Sepuluh Nopember, Surabaya, 2016. DAFTAR PUSTAKA [1] A. Safrodin and S. Mangkoedirdjo, “Desain IPAL pengolahan grey
https://openalex.org/W2008646506	https://figshare.com/ndownloader/files/38736078	Turkish	null	Does rumination mediate the relationship between emotion regulation ability and posttraumatic stress disorder?	European journal of psychotraumatology	2,014	cc-by	203	Citation: European Journal of Psychotraumatology 2014, 5: 23547 - http://dx.doi.org/10.3402/ejpt.v5.23547 Ruminasyon Duygusal Düzenleme Becerisi ve Travma Sonrası Stres Bozukluğu arasındaki ilişkiye aracılık eder mi? Thomas Ehring, Anke Ehlers Thomas Ehring, Anke Ehlers Arkaplan ve amaçlar: Travma ile ilgili ruminasyonun travma sonrası stres bozukluğunun (TSSB) sürmesi ile ilgili olduğu ortaya konmuştur. Bu görüş, enlemsel, ileriye dönük ve deneysel desenlerin kullanıldığı geniş çaplı kanıtlarla bilimsel olarak da desteklenmiştir. Ancak, olumsuz sonuçlarına rağmen, travma yaşamış kişilerin neden ruminasyon yaptıkları hala belirsizdir. Mevcut araştırma düşük duygu düzenleme becerisinin travma ile ilgili ruminasyonun temelini oluşturduğu savını test etmeyi amaçlamaktadır. Yöntem: Duygu düzenleme becerisi ve travma ile ilgili ruminasyon, trafik kazası geçirmiş 93 kişide travmadan 2 hafta sonra değerlendirilmiştir. Ayrıca, TSSB semptom seviyeleri 2 haftalık bir sürecin yanı sıra, 1, 3 ve 6 aylık takiplerde de değerlendirilmiştir. Sonuçlar: Duygu düzenleme becerisinin travma ile ilgili ruminasyon ve TSSB belirtileri ile anlamlı bir ilişkisi vardır. Ayrıca, ruminasyon düşük duygu düzenleme becerisi ve TSSB arasındaki ilişkiye aracılık etmektedir Tartışma: Bulgular, travma yasayanlarda ruminasyonun işlevsel olmayan bir duygu düzenleme stratejisi olduğu görüşünü desteklemektedir. Anahtar kelimeler: Travma; TSSB; Ruminasyon; Duygu Düzenlemesi Anahtar kelimeler: Travma; TSSB; Ruminasyon; Duygu Düzenlemesi Name of translator: Emek Yuce Zeyrek-Rios n: European Journal of Psychotraumatology 2014, 5: 23547 - http://dx.doi.org/10.3402/ejpt.v5.2354
W2980780454.txt	https://zenodo.org/records/3388022/files/10140__1_216496_LE_331412.pdf	nl		Bedrijf en onderneming voor een nieuwe taak	MAB	1,937	cc-by	2,209	10 MAANDBLAD VOOR ACCOUNTANCY EN BEDRIJFSHUISHOUDKUNDE vennootschappelijke kas. Op het eerste gezicht zou hiertegen volgens voormeld principe niet het geringste bezwaar bestaan ; immers de overeenkomst van geldleening is eenzijdig en alleen de bestuurder gaat een verplichting tegenover de N.V. aan, namelijk die om het geleende geld terug te betalen. Ons rechts gevoel tornt er echter tegenop, dat een bestuurder op eigen hoertje bevoegdelijk tot deze kasplundering zou kunnen over gaan. Juridisch acht ik zulks ook niet mogelijk. Aan het con tract van geldleening ligt namelijk een ander contract ten grondslag en wel i.c. de overeenkomst, waarbij de vennootschap zich verplicht aan den directeur een geldleening te verstrekken. Het behoeft na het bovenstaande geen betoog, dat het aangaan dezer zoogenaamde voorovereenkomst den directeur niet ge oorloofd is. Uit dit voorbeeld moge tevens blijken, dat de rechts handelingen tusschen N.V. en directeur steeds aan een nauw keurige analyse belmoren te worden onderworpen, om te kun nen vaststellen, of voor bepaalde onderdeden ervan vertegen woordiging door directeuren, dan wel door commissarissen noodzakelijk is. Op gevaar af teveel in casuïstiek te vervallen, wil ik toch gaarne nog een enkele opmerking maken over die overeenkom sten, waarbij geen der partijen een verplichting op zich neemt, omdat zij slechts bestaan in het voldoen aan een verplichting. De rechtswetenschap kent als zoodanig de zoogenaamde zake lijke overeenkomsten als levering, inpandgeving en hypotheekstelling. In de boven door mij verdedigde opvatting moet het aangaan dezer overeenkomsten door een directeur in dubbele qualiteit volkomen geoorloofd worden geacht, zoowel indien deze levering enz. geschiedt aan als door de N.V. Ik mag echter niet verzwegen, dat voor wat betreft de vestiging van bestuurspand ten behoeve der N.V. op aandeelen in de betrok ken vennootschap zelf, de wet blijkens art. 48d. Wetb. van Kooph. de medewerking van commissarissen eiseht, en dus blijkbaar van de gedachte uitgaat dat i.c. tusschen N.V. en directeur tegenstrijdig belang bestaat. Ik geloof echter niet, dat uit deze bijzondere regeling een argument tegen mijn op vatting is te putten. Indien deze zoo juist bedoelde overeenkomsten de bezits overdracht van roerende lichamelijke zaken betreffen, schuilt er nog een addertje onder het gras. Het is namelijk de vraag of de bestuurder in privé deze zaken aan den bestuurder als zoodanig, dus aan zichzelf, kan overhandigen, met als resultaat dat de N.V. bezitster wordt. Op zijn zachtst gezegd maakt zulk een daad een toch wel ietwat eigenaardigen indruk! De Ilooge Raad schijnt het evenwel blijkens zijn gemeld arrest mogelijk te achten. Toch zal de directeur goed doen, het feit dat de N.V. bezitster is geworden, duidelijk aan de buitenwereld ken baar te maken. Bij effekten aan toonder b.v. door de stukken bij een bank te deponeeren ten name der vennootschap. Dat dit feit uit de boekhouding blijkt, moet niet voldoende worden geacht. In dezen zin besliste althans de Hooge Raad bij zijn arrest van 7 Maart 1934 (Nederlandsche Jurisprudentie blz. 1517), handelende over dezelfde materie als de beslissing van 31 October van dat jaar. Dat de Hooge Raad met zijn arrest aan de rechtszekerheid een grooten dienst heeft bewezen, zal na lezing van het boven staande wel niemand beweren. Een verdienste ervan is in ieder geval het feit, dat het aanleiding is geweest en nog kan zijn. zich eens wat nader te verdiepen in het begrip „tegenstrijdig belang” in den zin van artikel 51 van het Wetboek van Koop handel. Dat door de hierboven door mij gegeven nadere begrips bepaling de vennootschappelijke praktijk bijzonder gebaat zal zijn, kan ook niet Worden gezegd. De praktijk heeft weinig tijd voor en lust in rechtskundige analyse. Zonder me nog aan profetieën te buiten te willen gaan, lijkt het me niet onmoge lijk, dat de Hooge Raad ten aanzien van artikel 51 W. v. Kh. een andere meening zou blijken voor te staan, dan ten aanzien van artikel 365 B.W. Niettemin acht ik voor de contracten tusschen N.V. en directeur gevaar aanwezig. Indien ik daarom eindig met een praktische wenk, dan is het deze: de directeur eener N.V., die in de noodzakelijkheid komt te verkeeren met zich zelf overeenkomsten te gaan, mag zich hiervan door art. 51 niet laten weerhouden, doch hij moet ervoor zorgen, dat steeds de commissarissen medewerken. Alleen dit gezamenlijk optre den schept de grootste zekerheid, dat de botsing der belan gen — en ook het ontbreken daarvan — geen averij ten ge volge zal hebben. Veenendaal. J. D. LE GRAND, cand.-not. UIT HET BUITENLAND Red.: F. HAARBOSCH, CH. HAGEMAN, Drs A. TH. DE LANGE en Drs W . P. DEN TURK (Bijdragen en mededeelingen zende men aan den Secretaris der Redactie) Bedrijf en onderneming voor een nieuwe taak De September-aflevering van het als steeds lezenswaardige Zeitschrift für Handelswissenschaftliche Forschung bevat twee artikelen over nauw verband houdende onderwerpen, nl.: Die Bedeutung der betriebswirtschaftlichen Organisation für die wirtschaftliche Mobilmachung en Senkung der indu striellen Kriegskosten als gemeinwirtschaftliche Aufgabe. Prof. Ilintner als schrijver van het eerste artikel gaat er van uit, dat de fabriek het slagveld is, waar uiteindelijk de beslissing van een oorlog valt. Daarom moeten bedrijf en on derneming object zijn van krijgskundig onderzoek (Wehr- und kriegswirtschaftlicher Forschung). Daar in tijd van oorlog de behoefte aan bepaalde artikelen vele malen zoo groot is als in vredestijd (b.v. munitie) en an derzijds aan andere artikelen vele malen kleiner (te ontberen consumptiegoederen, waarvan de vijand b.v. invloed op dc voorziening heeft) zal het bedrijfsleven reeds in vredestijd voorbereid moeten worden op de veranderingen, die men er in oorlogstijd van zal verlangen, want juist in de eerste dagen en weken van een oorlog zou een falen van de industrie in dit opzicht de noodlottigste militaire gevolgen kunnen hebben. Dit mag dus niet aan de private ondernemingen, elk voor zichzelf, overgelaten worden. Er zijn uiteraard oorlogsindustrieën, die in geval van oorlog enkel haar productie te vergrooten hebben; de meerdere be hoefte (voor verschillende soorten munitie te stellen op 470001PO.OOO %) is echter zoo groot, dat men er hiermede niet komt en ook op bedrijven moet terugvallen, die slechts in oorlogstijd bepaalde voor den oorlog noodzakelijke artikelen fabriceeren; deze bedrijven moeten dus hun productie kwalitatief verande ren ; de voorbereiding hiervan eiseht o.a. een grondig onder zoek naar de samenstelling van het personeel, b.v. t.a.v. den dienstplicht. Beide soorten van bedrijven, dus zoowel zulke, die hun productie kwalitatief, als zulke, die deze kwantitatief moe ten kunnen veranderen, belmoren daarvoor de noodige gebou wen en machines reeds in vredestijd aan te schaffen en de noo dige grondstoffen en werktuigen steeds zoo rijkelijk mogelijk in voorraad te houden. Om dan te maken, dat men eenigermate met de oorlogsproductie vertrouwd raakt, moet de regeering af en toe „Lehraufträge” geven, die dan voor de regeering wel duur uitkomen, maar het nut hebben, dat te allen tijde dc oor- MAANDBLAD VOOR ACCOUNTANCY EN BEDRIJFSHUISHOUDKUNDE logsproduetie snel en met ervaring opgevat ban worden . Aan alles moet gedacht worden; in oorlogstijd zal b.v. geen mais voor de varkens beschikbaar zijn; het wordt dan nood zakelijk direct na het uitbreken van een oorlog op groote schaal varkens af te slachten en ze in te blikken. Daartoe zullen de conservenfabrieken voor deze plotselinge afslachtig op groote schaal reservevoorraden machines en koelruimte moeten hebben en de arbeiders zullen beschikbaar moeten zijn. Van alle belangrijke grondstoffen moet niet alleen de Staat een flinken voorraad aanleggen, maar alle ondernemingen, die deze grondstoffen verhandelen of verwerken, moeten hiervan steeds „besonders reichliche” voorraden aanhouden. De ondernemingen kunnen in vredestijd schadeloos gesteld worden door reducties op bepaalde belastingen, credieten tegen lage rente of proefopdrachten tegen goede prijzen. De Staat zal hebben toe te zien, dat de tijd, dien de bedrij ven noodig hebben om zich om te, schakelen op de oorlogsproduetie zoo kort mogelijk zij, opdat de Staat zelf zoo laag moge lijke voorraden behoeft aan te houden. De schrijver van het tweede artikel (Ing. Wille) onderzoekt nu hoe de Staat alle vorengenoemde kosten zoo laag mogelijk kan houden en oorlogswinst verhinderen. Hiervoor bestaan drie systemen: 1. Prijsstelling door de Overheid; onmogelijk, daar men ook de dure grensbedrij ven in de productie betrekken moet, dus aan de goede ondernemingen een groote winstmarge zou geven; bovendien zou hier veelal een bevoordeeling van groote boven kleine ondernemingen uit voortvloeien en ten slotte leidt deze methode tot de productie van zoo slecht mogelijke artikelen (die dus nog net goedgekeurd zullen worden). 2. kostengelijkmaking; de Regeering kan b.v. de, voordeelen. die bepaalde ondernemingen hebben uit een gunstige, vesti gingsplaats en de nadeelen voor andere uit een ongunstige ligging, opheffen door vrij vervoer van grondstoffen over de Spoorwegen toe te staan. Met de Spoorwegen kan de Staat dat dan wel weer regelen. 3. vergoeding aan de bedrijven van hun kosten plus een rede lijke winst. Hiervoor bestaan dan weer drie vormen, n.1.: a. de Staat neemt liet bedrijf over voor den duur van den oorlog. Dit systeem heeft het nadeel van de uitschake ling van den ondernemer, met als gevolg geen deskun dige bedrijfsleiding, geen zuinigheid, enz. 1). boekhoudkundige overname van liet bedrijf door den Staat. Bij het uitbreken van den oorlog wordt een balans gemaakt en voor het daaruit blijkende beginkapitaal is de Staat garant. Gedurende den oorlog krijgen de be drijven dan hun kosten bij wijze van voorschot op de eindafrekening vergo-ed. Deze eindafrekening met vast stelling van de winst vindt plaats na het opmaken van een balans aan het einde van den oorlog. Hier zijn wel de oorlogswinsten uitgeschakeld, maar een drang tot zuinigheid is er niet. c. vergoeding aan de bedrijven van normale kostprijzen voor alle bewerkingen, vast te stellen op grond van bedrijfsvergelijkingen, met een extra winsttoeslag voor de bedrijven, die dalende kosten toonen. De kosten van de omstelling op oorlogsproductie belmoren afzonderlijk vergoed te worden; in de calculatie worden zij opge nomen, rekening houdende met den langst mogelijken 11 oorlogsduur; bij het sluiten wan den vrede kan het saldo dan met den Staat verrekend worden. Tenslotte wijst de schrijver op het groote belang voor de bedrijven zelf van een nauwkeurige planning reeds in vredes tijd van de kosten van de omstelling op oorlogsproductie en van de oorlogsproductie zelf en werkt deze zeer gedetailleerd uit. F. H. NEDERLANDSCHE HANDELS HOOGESCHOOL TENTAMEN REKEN1NGWETENSCHAP INRICHTINGSLEER Maandag 7 September 1936 van 9—12 uur Een fabriek van stalen kantoor- en magazijnmeubelen vervaardigt haar product bestaande uit archiefkasten, kleerkasten, lessenaars, papierbakken e.d., zowel voor voorraad ter latere aflevering als op bestelling. Op bestelling te vervaardigen product kan zijn normaal product volgens de prijscouranten met geringe afwijkingen en kan ook wor den vervaardigd naar bijzondere ontwerpen. De fabricage heeft in het kort het volgende verloop. Stalen platen worden machinaal op maat gesneden, gebogen, ge stanst en gelast tot de vereiste vorm. Bij deze bewerkingen ontstaat afval, dat ten dele nog bruikbaar, ten deel onbruikbaar is. Onderdelen, die niet zelf worden vervaardigd, als kogellagers, grepen, sloten e.d. worden aangebracht, waarna het geheel met lak wordt bespoten om daarna in een gasoven te worden gedroogd. De aandrijving van alle machines geschiedt electrisch. Vrij regelmatig moet de fabricage van meubelen in voorraad wor den onderbroken om bestellingen te doen voorgaan. Hierdoor zijn steeds hoeveelheden halffabrikaat in elk stadium van bewerking aan wezig. Vaak worden aan deze voorraden stukken ontleend ter be spoediging van het gereedkomen van bestellingen. In de fabriek zijn 100 werklieden werkzaam, die allen in uurloon werken, met uitzondering van de schilders, die in stukloon arbeiden. Gevraagd wordt een beschrijving van de administratie. Maandag 7 September 1936 van 2—5 uur De A. Bank, gevestigd in een der hoofdsteden, met vele bijkantoren over het gehele land, wenst over te gaan tot centralisatie van haar incasso-bedrijf. Het is daarbij de bedoeling, dat de centrale alle incasso-wissels ontvangt, zowel van de kantoren, als van cliënten en van correspondenten en dat de afrekening c.q. de retourgaade wissels zo spoedig mogelijk de cliënt (correspondent) bereiken. De kantoren dienen snel geïnformeerd te worden betreffende creditering en retourzending met het oog op de positie van de cliënten, waarbij ook gedacht moet worden aan de mogelijkheid, dat incassopapier als onderpand dient door middel van cessie. De kantoren moe ten vooral voor beoordeling van de waarde van dit onderpand op de hoogte zijn van de retouj'-frequentie. De centrale verdtel de haar gezonden incassowissels over de kan toren en over de correspondenten ter plaatse, waar geen kantoren gevestigd zijn en laat de incassering in de hoofdplaats door eigen lopers geschieden. Er wordt incasso-provisie volgens een vastgesteld tarief berekend, benevens nota-zegel, en voorts bij retournering en retourprovisie even tueel extra-kosten, als protestkosten e.d. Men beschikt aan het hoofdkantoor over een Hollerith-installatie en denkt er over deze voor de incassocentrale te gebruiken. Evenwel wordt ook de toepassing van het z.g. „slip-systeem” overwogen. Alvorens een besluit te nemen legt men U de vraag voor aan te geven of U een van beide systemen de voorkeur zou geven. U gelieve een beschrijving te geven van de toepassing van beide systemen en daarbij te vermelden of U bij het een speciale voordelen resp. bezwaren ziet tegenover het andere, en zo ja, welke. Daarbij worden schema’s verlangd, waaruit duidelijk de verschil lende handelinge blijken, evenals de interne controle. Dinsdag 8 September 1936 van 9—12 uur Het bedrijf van de N.V. Transport- en Verhuisonderneming v/h Jos. Sterk & Co. bestaat uit het verzorgen van verhuizingen in binnenen buitenland, het in bewaring nemen van inboedels en het vervoeren van zware voorwerpen als machines, brandkasten e.d.
https://openalex.org/W1848754142	https://lipidworld.biomedcentral.com/counter/pdf/10.1186/s12944-015-0061-y	English	null	Importance of lipid accumulation product index as a marker of CVD risk in PCOS women	Lipids in health and disease	2,015	cc-by	7,117	* Correspondence: joelmaximenes@yahoo.com.br 1Universidade Federal do Maranhão, São Luís, Maranhão, Brazil 3Rua Boa Esperança, Cond. Bosque dos Pinheiros Qd. 03, C. 02 - Bairro: Turú, São Luís, Maranhão, Brazil Full list of author information is available at the end of the article © 2015 Nascimento et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background: The polycystic ovary syndrome (PCOS) is considered the most common endocrine disease during the woman's reproductive life, with prevalence ranging from 5 to 10 % of women of reproductive age. There is a paucity of studies regarding the use of the lipid accumulation product (LAP) as a risk marker for the development of cardiovascular disease (CVD). Methods: A cross-sectional study was conducted on 78 women aged 18 to 42 years seen at University Hospital of Maranhão, with a diagnosis of polycystic ovary syndrome according to the Rotterdam criteria. The following variables of interest were recorded on a protocol form: sociodemographic and behavioral data, body mass index, waist circumference, fasting glucose, total cholesterol, triglycerides, low density lipoprotein cholesterol, high-density lipoprotein cholesterol, and systolic and diastolic blood pressure. Results: Logistic regression showed that, except for HDL, all cardiovascular risk markers presented a higher chance of being altered when the lipid accumulation product was above the cut off value of 37.9 cm.mmol/L. Conclusion: The lipid accumulation product seems to be sufficient to indicate a risk of cardiovascular diseases Conclusion: The lipid accumulation product seems to be sufficient to indicate a risk of cardiovascular diseases in women with polycystic ovary syndrome. Keywords: Polycystic ovary syndrome, Cardiovascular diseases, Lipid accumulation product Importance of lipid accumulation product index as a marker of CVD risk in PCOS women Joelma Ximenes Prado Teixeira Nascimento1,3, Maria Bethânia da Costa Chein1,3, Rosângela Maria Lopes de Sousa1, Alexsandro dos Santos Ferreira1, Paula Andrea Navarro2 and Luciane Maria Oliveira Brito1 RESEARCH Open Access Importance of lipid accumulation product index as a marker of CVD risk in PCOS women Joelma Ximenes Prado Teixeira Nascimento1,3, Maria Bethânia da Costa Chein1,3, Rosângela Maria Lopes de Sousa1, Alexsandro dos Santos Ferreira1, Paula Andrea Navarro2 and Luciane Maria Oliveira Brito1 Nascimento et al. Lipids in Health and Disease (2015) 14:62 DOI 10.1186/s12944-015-0061-y RESEARCH Open Access Importance of lipid accumulation product index as a marker of CVD risk in PCOS women Joelma Ximenes Prado Teixeira Nascimento1,3, Maria Bethânia da Costa Chein1,3, Rosângela Maria Lopes de Sousa1, Alexsandro dos Santos Ferreira1, Paula Andrea Navarro2 and Luciane Maria Oliveira Brito1 Nascimento et al. Lipids in Health and Disease (2015) 14:62 DOI 10.1186/s12944-015-0061-y Nascimento et al. Lipids in Health and Disease (2015) 14:62 DOI 10.1186/s12944-015-0061-y Introduction In addition, BMI is a limited tool for the differentiation between body fat and lean mass and for the identification of their anatomical location or func- tion of different fat deposits [15]. In this respect, a new index, called the lipid accumulation product (LAP), has been proposed. The LAP index was determined using the following equation: (WC [cm] - 58) x (TG [mmol/L]). The con- centration of triglycerides in mmol/L used in the equa- tion was obtained by multiplying their concentration in mg/dL by 0.0113 [15]. The following variables of interest were recorded on a protocol form: sociodemographic and behavioral data, BMI, WC, fasting glucose, total cholesterol (TC), triglycerides (TG), low density lipopro- tein cholesterol (LDL-c), high-density lipoprotein choles- terol (HDL-c), and systolic (SBP) and diastolic blood pressure (DBP). Women with PCOS have been shown to be at an in- creased risk of developing CVD. This risk is even higher in the presence of obesity when the chances of an al- tered LAP are high [16]. Thus, the use of this index may render the evaluation of CVD risk more feasible, practical and less costly, especially at primary healthcare services in Brazil. However, few studies have investigated the applica- tion of this index to the population attending the Brazilian primary healthcare service the single health system (SUS). Other risk markers such as the Framingham Score [17] and the Reynold Risk Score [18] require a larger num- ber of variables to assess cardiovascular risk, with a consequent increase in costs and the need for additional technologies. The more rigorous guidelines of the International Dia- betes Federation (IDF) were used to establish cut off values of CVD risk, which is defined by the presence of three or more of the following variables: WC ≥80 cm con- sidering ethnic background (since no specific data for South American women are available, cutoff values for European women were used); fasting glucose ≥5.6 mmol/L or a previous diagnosis of diabetes; TG ≥1.6 mmol/L or treatment of dyslipidemia; HDL-c < 1.2 mmol/L or treat- ment of dyslipidemia; SBP ≥130 mmHg or treatment, or DBP ≥85 mmHg. In addition, the Fourth Brazilian Guide- lines for Dyslipidemias and Prevention of Atherosclerosis, which consider total cholesterol ≥5.1 mmol/L and LDL- c ≥4.1 mmol/L, were used [20, 21]. Introduction anovulation, clinical and/or biochemical signs of hyperan- drogenism, and polycystic ovaries on ultrasound (presence of 12 or more follicles measuring 2 to 9 mm in diameter or with a total volume > 10 cm3 in at least one ovary). The diagnosis is confirmed after the exclusion of other condi- tions that can cause chronic anovulation and androgen excess. Polycystic ovary syndrome (PCOS) is an endocrine dis- order which is characterized mainly by anovulation and hyperandrogenism. Clinical manifestations range from irregular menstrual cycles to the absence of menstruation associated with variable degrees of overweight and an in- creased risk of developing cardiovascular diseases, among others [1, 2]. PCOS is the most frequent gynecological endocrinopathy, affecting 5 to 10 % of premenopausal women [3]. In Brazil, Melo et al. [4] reported a prevalence of PCOS of 13.9 % in AGA women at birth and prevalence in SGA women at birth (30.2 %). Cardiovascular disease (CVD) is one of the main pub- lic health problems, accounting for almost half of deaths in Europe and North America [6] and for 32 % of deaths in Brazil [7]. Obesity is a condition seen in 40 to 50 % of women with PCOS and the intensity of its symptoms is related to the degree of obesity [8]. Obesity alone contrib- utes to the physiopathology of PCOS and is frequently as- sociated with hyperinsulinemia [9], hypertriglyceridemia and reduced high-density lipoprotein cholesterol (HDL-c) [10], arterial hypertension, and type 2 diabetes mellitus [11]. These conditions are characterized by the redistribu- tion of body fat mass, i.e., excess fat deposition in the ab- dominal region (androgenic obesity), which is associated The criteria currently used for the diagnosis of PCOS are those established by the Rotterdam consensus [5], which defines the disease as the presence of two of the three following criteria: oligomenorrhea and/or Page 2 of 8 Nascimento et al. Lipids in Health and Disease (2015) 14:62 obesity: 30.0 – 34.5 kg/m2; grade II obesity: 35.0 – 39.9 kg/m2, and grade III obesity ≥40.0 kg/m2[19]. with a higher CVD risk, particularly arterial hypertension and diabetes mellitus [12, 13]. The body mass index (BMI) is the most commonly used indicator to assess obesity. This indicator evaluates generalized obesity, but does not measure body fat dis- tribution [12, 14]. Introduction As for physical activity, inactivity was considered for those who did not perform any type of physical exercise on a regular basis for at least 3 times per week with at least 30 min. Smoking was confirmed when the habit was present - regardless of amount [21]. The use of more objective diagnostic procedures at SUS is of particular importance when considering the in- crease in the incidence of CVD related morbidity and mortality observed during the nutrition transition, espe- cially in women with PCOS. In view of the importance of the LAP index, the objective of the present study was to evaluate the association between this indicator and cardiovascular risk factors in women with PCOS, iden- tify a cutoff point and compare it with changes in other cardiovascular markers. Our hypothesis is that LAP may be uncertain in this syndrome. The patients were submitted to blood collection for biochemical determination of diagnosis of PCOS at the Centre for Clinical Research (CEPEC), University Hospital, UFMA, in the morning between 7 to 9 h, after fasting for 12 h, in the follicular phase (third the seventh day of the cycle) in those with regular menstrual cycles and any day in those without regularity. Subjects and methods The Shapiro-Wilk test was used to determine whether the quantitative variables showed a normal distribution. The frequency distribution (absolute and relative) was performed for categorical variables. Quantitative CVD risk variables are expressed as the mean and standard devi- ation. A receiver operating characteristic (ROC) curve was used to identify the LAP index as a predictor of CVD risk as well as the cutoff point, estimating the highest sensitiv- ity and specificity of this index. Logistic regression analysis was used to evaluate the association between cardiovascu- lar risk markers (BMI, fasting glucose, total cholesterol, LDL-C, HDL-C, SBP, DBP, physical activity, and smoking habits) and the LAP index. The adjusted logistic regres- sion was used to further control potential bias. It is note- worthy that there were no losses in the sample. The Stata® 12.0 program was used for statistical analysis and the Microsoft Office 2010® software for data tabulation. A level of significance of 5 % was adopted for all tests. The study was approved by the Ethics Committee of HU-UFMA (Permit No. 349/11) and all participants signed a free informed consent form. aAs for physical activity, inactivity was considered for those who did not perform any type of physical exercise on a regular basis for at least 3 times per week with at least 30 min Results Th The adjusted logistic regression was used to further control pontenciais bias. The test showed that, except for HDL-c, all cardiovascular risk markers presented a higher chance of being altered when the LAP was above the cutoff value of 37.9 cm.mmol/L. The other variables significantly increased (p <0.05) the LAP at least 8.4 times (CI > 1.0), except for HDL-c that was associated with a re- duction in LAP (IC: 0.008 to 0.56), according to Table 4. The sociodemographic and behavioral characteristics of the sample (n = 78). It is noteworthy that there was no loss in the sample (all participants contemplated the in- clusion criteria). Noted a high frequency of age group 18–26 years (50.0 %), unmarried (65.4 %), unpaid (51.3 %), non-smokers (93.6 %) and physically active (79.5 %) (Table 1). Were not included in the logistic regression model the variables waist circumference and triglycerides by these directly influence the value of the LAP. Considering the trade-off between specificity and sen- sitivity, the cut off point for the LAP index was ≥ 37.9 cm.mmol/L (sensitivity: 85.19 %, specificity: 81.35 %), corresponding to the value closer to 1.0 (best cutoff point), Fig. 1. The variables: BMI, fasting glucose, total cholesterol, LDL-C, HDL-C, SBP, DBP, physical activity and smoking habits were analyzed both in logistic regression models unadjusted, as in adjusted. All cardiovascular risk parameters were associated with statistically the LAP index (p <0.05). Among women, 41 % had changes in LAP. Among obese 17.7 % had changes in the parameter, among those with high CC, 78.9 % among those with hyperglycemia, 83.3 %. Regarding women with lipid abnormalities, there was an association with changes in LAP index by more than 60 % in all analyzed fractions, Table 2. Before of adjust, only BMI and HDL-C showed statisti- cally significant association with the LAP index, after ad- justment, BMI and DBP were associated with a higher risk of changes in LAP and HDL-C to a reduction of changes in marker. Subjects and methods An analytic cross-sectional study was conducted on 78 women aged 18 to 42 years with a diagnosis of PCOS ac- cording to the Rotterdam criteria [5]. The patients were seen at University Hospital (HU) of Maranhão (UFMA), between September 2010 and February 2012. Patients who do not contemplate such criteria were not sampled. The non-probabilistic sampling was employed. All women were submitted to clinical examination, including the measurement of body weight, height, and WC. For WC, the lowest circumference between the last rib and iliac crest [19] was considered. The reference standards of the World Health Organization (1997) were used for the classification of nutritional status based on BMI: grade III thinness ≤16.0 kg/m2; grade II thinness: 16.0 – 16.9 kg/m2; grade I thinness: 17.0 – 18.4 kg/m2; eutrophy: 18.5 – 24.9 kg/m2; overweight: 25.0 – 29.9 kg/m2; grade I An analytic cross-sectional study was conducted on 78 women aged 18 to 42 years with a diagnosis of PCOS ac- cording to the Rotterdam criteria [5]. The patients were seen at University Hospital (HU) of Maranhão (UFMA), between September 2010 and February 2012. Patients who do not contemplate such criteria were not sampled. For each patient, 20 ml was collected whole blood and stored under sterile vacuum tubes containing EDTA (for blood count) and serology (separating gel) without anti- coagulant for biochemical and hormonal evaluation, using sterile equipment for such and disposable as the bio- security standards for biological material. The non-probabilistic sampling was employed. All women were submitted to clinical examination, including the measurement of body weight, height, and WC. For WC, the lowest circumference between the last rib and iliac crest [19] was considered. The reference standards of the World Health Organization (1997) were used for the classification of nutritional status based on BMI: grade III thinness ≤16.0 kg/m2; grade II thinness: 16.0 – 16.9 kg/m2; grade I thinness: 17.0 – 18.4 kg/m2; eutrophy: 18.5 – 24.9 kg/m2; overweight: 25.0 – 29.9 kg/m2; grade I The processing of blood samples was begun at most 1 h after collection. The serum samples were stored in a freezer at −80 °C. Fasting glucose and lipid profile were analyzed by enzymatic colorimetric method. LDL-c was calculated from the Friedewald formula: LDL-c = total cholesterol (HDL-c – TG / 5), as there was in the samples, we measured the upper TG 4.5 mmol/L. Subjects and methods The biochemical measurements were performed with the Cobas 6000 Nascimento et al. Lipids in Health and Disease (2015) 14:62 Page 3 of 8 Table 1 Sociodemographic and behavioral characteristics of the sample Variable n (%) Age (years) 18 to 26 39 (50.0) 27 to 34 31 (39.7) 35 to 42 8 (10.3) Marital status Single 51 (65.4) Married 27 (34.6) Occupation Paid 35 (48.7) Unpaid 43 (51.3) Smoking Yes 5 (6.4) No 73 (93.6) Physical inactivitya Yes 62 (79.5) No 16 (20.5) aAs for physical activity, inactivity was considered for those who did not perform any type of physical exercise on a regular basis for at least 3 times per week with at least 30 min Table 1 Sociodemographic and behavioral characteristics of the sample Variable n (%) Age (years) 18 to 26 39 (50.0) 27 to 34 31 (39.7) 35 to 42 8 (10.3) Marital status Single 51 (65.4) Married 27 (34.6) Occupation Paid 35 (48.7) Unpaid 43 (51.3) Smoking Yes 5 (6.4) No 73 (93.6) Physical inactivitya Yes 62 (79.5) No 16 (20.5) aAs for physical activity, inactivity was considered for those who did not perform any type of physical exercise on a regular basis for at least 3 times per week with at least 30 min equipment, Hitachi Hight-Technologies Corporation 24–14.Nish-shimbashi.1-chome.Minato-ku, Tokyo, Japan. equipment, Hitachi Hight-Technologies Corporation 24–14.Nish-shimbashi.1-chome.Minato-ku, Tokyo, Japan. The Shapiro-Wilk test was used to determine whether the quantitative variables showed a normal distribution. The frequency distribution (absolute and relative) was performed for categorical variables. Quantitative CVD risk variables are expressed as the mean and standard devi- ation. A receiver operating characteristic (ROC) curve was used to identify the LAP index as a predictor of CVD risk as well as the cutoff point, estimating the highest sensitiv- ity and specificity of this index. Logistic regression analysis was used to evaluate the association between cardiovascu- lar risk markers (BMI, fasting glucose, total cholesterol, LDL-C, HDL-C, SBP, DBP, physical activity, and smoking habits) and the LAP index. The adjusted logistic regres- sion was used to further control potential bias. It is note- worthy that there were no losses in the sample. The Stata® 12.0 program was used for statistical analysis and the Microsoft Office 2010® software for data tabulation. A level of significance of 5 % was adopted for all tests. 24–14.Nish-shimbashi.1-chome.Minato-ku, Tokyo, Japan. Discussion d l Among the normotensive, SBP and DBP as, 82.9 % was demonstrated-associated adjustments when the prod- uct of lipid accumulation, Table 2. Cardiovascular risk parameters, were associated in women with abnormal LAP index. By comparison, women with such showed parameter higher measurements of BMI, CC, fasting glucose, total coleterol, triglycerides, LDL-c, SBP and DBP, as well as lower HDL-c. All women with an LAP index above the defined cut off point presented significantly more frequent altered mean values of the cardiovascular risk markers analyzed. The prevalence of women with an LAP index ≥37.9 cm.mmol/ L was 41 % (Table 3). Obesity marked by BMI and increased of DBP were as- sociated with an increased risk of changes in LAP (above Nascimento et al. Lipids in Health and Disease (2015) 14:62 Page 4 of 8 Fig. 1 Distribution of lipid accumulation product (LAP index) values on the ROC curve of the sample. Lipid accumulation product Fig. 1 Distribution of lipid accumulation product (LAP index) values on the ROC curve of the sample. Lipid accumulatio Table 2 Cardiovascular risk factors associated with changes in LAP Index Variable LAPa p – value ≥37.9 cm.mmol/L <37.9 cm.mmol/L (n = 32) (n = 46) BMIb, n (%) <0.001 Not obese 23 (85.2) 4 (14.8) Obese 9 (17.7) 42 (82.3) WCc, n (%) <0.001 High 30 (78.9) 8 (21.1) Normal 2 (5.0) 38 (95.0) Fasting glucose, n (%) 0.028 High 5 (83.3) 1 (16.7) Normal 27 (37.5) 45 (62.5) Total cholesterol, n (%) 0.005 High 14 (66.7) 7 (33.3) Normal 18 (31.6) 39 (68.4) Triglycerides, n (%) <0.001 High 16 (94.1 1 (5.9) Normal 16 (26.2) 45 (73.8) LDL-cd, n (%) 0.005 High 11 (73.3) 4 (26.7) Normal 21 (33.3) 42 (66.7) HDL-ce, n (%) <0.001 Normal 20 (31.3) 44 (68.8) Low 12 (85.7) 2 (14.3) SBPf (mmHg) <0.001 Normotensive 6 (17.1) 29 (82.9) Not normotensive 26 (60.5) 17 (39.5) DBPg (mmHg) <0.001 Normotensive 7 (17.1) 34 (82.9) Not normotensive 25 (67.6) 12 (32.4) *Chi-square test; aLipid accumulation product (LAP index); bBody mass index; cwaist circumference; dlow density lipoprotein; ehigh density lipoprotein; fsystolic blood pressure; gdiastolic blood pressure Table 2 Cardiovascular risk factors associated with changes in LAP Index 8 times). The increase in HDL-c was associated with pre- vention for such change. The aim of this study was to evaluate the association between LAP index and cardiovascular risk factors in women with PCOS. Discussion d l The results support the idea that women with PCOS who have the LAP index above the cut off suggested in this work showed the largest changes in mean CVD risk markers analyzed. Even with a small sample in a cross-sectional study, considered a limitation in our study, the statistical ana- lysis allowed away the null hypothesis. Still, some of the highlights in the study are the making of laboratory tests and the use of both anthropometric and blood pressure direct measurement which shall not be obtained through self-reference. The adoption of low cost risk determining factors and also with fast responses are of great help towards treat- ment, tracking and early intervention which explains the reason why such marker contributes to the early diagno- sis of the main cause for women’s mortality in the world. In this study, women with PCOS, concentrated in the age group between 18 and 26 years, showing precocious- ness in the accumulation of risk factors for CVD. Revealing that this age group, an appropriate time for intervention in the development of CVD. Physical inactivity is an import- ant cardiovascular risk factor [22, 23]. The highest per- centage of physical inactivity in this study may favour the development of CVD especially when associated with smoking [21]. However, this habit often in the sample was low compared to the study of Junior Gil et al. [24]. 0.005 <0.001 <0.001 The average age in our study was 26.3 years and the BMI was 27.01 kg/m2 with LAP index cutoff rate at ≥ 39.32 cm.mmol/L (81 % sensitivity and 82 % specificity) on CVD risk with a prevalence of 38.5 %; yet Wiltgen et al. [25] who evaluated LAP index in women with PCOS with an age average of 20.6 years and BMI average of 29.5 kg/ m2 have come to a cut off rate of 37.87 cm.mmol/L (84 % sensitivity and 79 % specificity) thus, higher rates than Nascimento et al. Discussion d l Lipids in Health and Disease (2015) 14:62 Page 5 of 8 Table 3 Distribution of cardiovascular risk markers according to cut off value of the lipid accumulation product (LAP index) Variable LAPa p <37.9 cm.mmol/L ≥37.9 cm.mmol/L BMIb (kg/m2) 23.1 ± 0.6 32.6 ± 0.9 <0.001 WCc (cm) 74.6 ± 1.3 98.9 ± 1.7 <0.001 Fasting glucose (mmol/L) 4.7 ± 0.1 5.4 ± 0.3 0.006 Total cholesterol (mmol/L) 4.4 ± 0.1 5.2 ± 0.2 <0.001 Triglycerides (mmol/L) 0.9 ± 0.0 2.0 ± 0.1 <0.001 LDL-cd (mmol/L) 2.6 ± 0.0 3.2 ± 0.1 <0.001 HDL-ce (mmol/L) 1.3 ± 0.0 1.0 ± 0.0 <0.001 SBPf (mmHg) 109.5 ± 1.7 122.8 ± 2.0 <0.001 DBPg (mmHg) 70.0 ± 1.2 80.0 ± 1.4 <0.001 Total, n (%) 46 (59) 32 (41) Values are the mean ± standard deviation aLipid accumulation product (LAP index); bbody mass index; cwaist circumference; dlow density lipoprotein; ehigh density lipoprotein; fsystolic blood pressure; gdiastolic blood pressure In this line of avaliation, 167 PCOS patients compared to 102 controls were evaluated and such study aimed at identifying coronary instability and diabetes mellitus type 2 through the frequency of CD4 (+) CD28 (null) lympho- cytes that express the level of involvement by these morbidities. It has been concluded that there is a posi- tive association between the frequency of CD4 (+) CD28 (null) lymphocytes in PCOS patients when they are com- pared to the control [27] showing that PCOS is a risk fac- tor in the development of CVD. For example it has been a study conducted with 139 women with PCOS aiming at evaluating both clinical and endocrine pictures as well as cardiovascular disease risk among the different PCOS phenotypes evaluated BMI values, series levels of follicle-stimulating hormones (FSH), luteinizing hormone (LH), progesterone, estradiol, testos- terone, dehydroepiandrosteronesulphate (S-DHEA), fasting glucose, low density lipoprotein (LDL-C), total cholesterol, high density lipoprotein (HDL-C), C-reative protein, insu- lin, sensitivity to insulin and thickness of the carotid inner layer has found that the hyperandrogenic phenotype has lower risk of cardiovascular disease in comparison to other phenotypes [28]. ours. That may be explained due to the fact that a control case was carried out where the whole PCOS group had hyperandrogenism with high levels of testosterone. None- theless, it is controversial that hyperandrogenism by itself be considered a higher risk factor of CVD. Discussion d l Taking into account that the risk of cardiometabolic disorders may be partly determined by the employed def- inition of PCOS, clinical and epidemiological studies sup- port the need to identify women with PCOS to determine the risk of cardiometabolic diseases and thus prevent and/or treat its serious consequences [29]. The combination of anovulation and hyperandrogen- ism translates PCOS in its classical form which shows the adverse metabolic phenotype of the syndrome. Such phenotype includes visceral obesity and resistance to in- sulin as well as a manifold of other traditional cardiovas- cular risk factors especially inflammation, glucose and dyslipidemia metabolism disorders [26]. Women with PCOS associated with the presence of obesity, smoking habits, dyslipidemia, high blood pressure, intolerance to glucose and subclinical vascular disease are at risk whereas those with metabolic syndrome and/or dia- betes mellitus type 2 pose high risk of CVD. It is therefore recommended that those women manage their lifestyles in order to seek the primary prevention of CVD [30]. The resulting risk increase of CVD may affect both lean women and obese ones. The subjacent mechanisms to the increase in cardiovascular risk within the PCOS context may include not only metabolic alterations, but also hormonal factors, hyperandrogenism in particular. Nevertheless, the consequences in terms of cardiovascular morbidity remain questionable due to the drawbacks in carrying out long term prospective studies aiming at iden- tifying potential factors from those clinical results [26]. y Following the above findings with different design, pro- spective study, conducted at the Endocrinology, Diabetes and Metabolic Diseases Clinic at the University Center of Sarajevo, 50 patients diagnosed with PCOS in accordance with the Rotterdam criteria – ESHRE [5] were divided into two groups according to their BMI and had the following parameters evaluated: anthropometric indices (WC, height and weight), BMI, TG, resistance to insulin and the LAP index. Results showed that women with PCOS and BMI ≤ 24.9 kg/m2 were significantly different from those with BMI > 25 kg/m2 in body weight, WC and TG. Besides the LAP index having been a marker to differentiate women who were resistant to insulin from those who were not with a cut off value of 17.91 cm.mmol/L, the women group with BMI > 25 kg/m2 showed higher values of the LAP index. We may conclude that the LAP index is a marker to differentiate resistance to insulin in women with PCOS [31]. Discussion d l Table 4 Association between biases concerning cardiovascular system risk indicators and biases in the lipid accumulation product (LAP index) through adjusted logistic regression from the female population with polycystic ovary syndrome Altered Variables LAP1 Odds Confidence Interval Z P value BMI2 ≥30 kg / m2 32.6 6.4 – 163.8 4.23 <0.0001 HDL-c3 1.2 mmol/L 0.06 0.008 – 0.56 −2.50 0.012 DBP4 ≥80 mmHg 8.4 1.86 – 37.9 2.77 0.006 1Lipid accumulation product (LAP index); 2body mass index; 3low density lipoprotein; 4diastolic blood pressure. Table 4 Association between biases concerning cardiovascular system risk indicators and biases in the lipid accumulation product (LAP index) through adjusted logistic regression from the female population with polycystic ovary syndrome Page 6 of 8 Nascimento et al. Lipids in Health and Disease (2015) 14:62 Nascimento et al. Lipids in Health and Disease (2015) 14:62 as in the controls, 28.95 %. The most common dyslipid- emia components in PCOS patients were lowered levels of HDL-c (41.13 %) and an increase in TG (24.14 %). The frequency of metabolic syndrome in PCOS patients was 25.62 % which is more than five times higher when compared to the controls. The two main risk factors were the increase in waist circumference and low levels of HDL-c [41]. It was observed that prevalence of obesity and resist- ance to insulin in women with PCOS is significantly higher when compared to the population in general mak- ing the LAP index a new, cheap and accessible predictor of the metabolic syndrome both to the general population and women with PCOS [31]. In relation the area found under curve ROC (0.8845) shows that the LAP index presents satisfactory perform- ance in those circumstances as its area above 0.70 trans- lates a test with adequate diagnosis [32]. Obese women with PCOS present high SBP and DBP, suggesting that arterial hypertension is a late sequel of the stimulatory effects of hyperinsulinemia on the sym- pathetic nervous system and vascular smooth muscle [42]. In our study, the frequency of women with altered SBP and DBP was 14.1 %. In a case–control results with 60 women aged between 18 and 45 where 42 women with PCOS and 18 controls, observed no differences be- tween the metabolic parameters and resistance to insulin in both groups. Discussion d l Comparing those parameters (metabolic and resistance to insulin) with 24-h blood pressure mon- itoring in the ambulatory (MAPA) it was shown that the only correlating parameter was the BMI, regardless of PCOS diagnosis. Such study observed that PCOS women did not show higher levels of blood pressure, glycemia, HDL-c, TG, resistance to insulin and BMI in comparison to the control group. However, women presented correl- ation among PCOS, blood pressure and BMI, suggesting that obesity is a main factor involved in blood pressure changes in those women [43]. When screening patients under risk to develop CVD it is important that diagnostic tests present optimum sensi- tivity to detect disease or alterations in apparently healthy people (asymptomatic patients) [33]. Different studies indicated that the LAP index may be used as a screening marker when identifying patients who are likely to be characterized with the atherogenic metabolic triad: fasting hyperinsulinemia, hyperapolipo protein B and high pro- portion of small LDL-c particles [15, 16, 25, 34]. Notwith- standing, the LAP index discriminatory reach to identify patients with cardiometabolic risk profile may be com- pared to those of IDF and National Cholesterol Education Program – Adult Treatment Panel III (NCEP-ATP III) [20, 35] as it shows sensitivity, specificity [25] and low cost to identify people under higher cardiovascular risk. Indeed, it is important to determine such index to evalu- ate women with PCOS once BMI alone is not capable of marking the fat deposition characteristic in the body [15]. It is doubtless that women with PCOS show high preva- lence of central obesity [36, 37]. Studies demonstrate that 50 to 60 % of those patients present abdominal obesity re- gardless of their BMI [36–39]. Therefore, the use of anthropometric indices for the diagnostic evaluation of central obesity seems to be more adequate in this subgroup of women [13]. Using the cut off identified here, the LAP index may be a more access- ible and more accurate tool to identify cardiovascular risk in women with PCOS and can be adopted and included in clinical practice by SUS on a regular basis since it is easy to be obtained and represents lower screening costs con- cerning those women under metabolic risk. Clinical practice and literature data have shown a high prevalence of cardiovascular risk indicators in women with PCOS [36, 37]. Authors’ contribution P C ll l Authors contribution JXPTN: Collection, analysis and interpretation of the data. MBCC: Coordination of the research; conception and design of the study, and collection, analysis and interpretation of the data. RMLS: Data collection. AFS: Interpretation of the data and statistical analysis. PAN: Coordination of the research; conception and design of the study, and collection, analysis and interpretation of the data. LMOB: Coordination of the research; conception and design of the study, and collection, analysis and interpretation of the data. All authors read and approved the final manuscript. Another risk factor for CVD already established is HDL-c levels below the recommended limit, irrespective of LDL-c values [10]. The percentage of women with re- duced HDL-c observed in the present study was higher than that reported by Apridonidze et al. [40] who de- tected low HDL-c levels in 49 % of women with PCOS. A recent study including 406 patients with PCOS and 342 control women aged between 17 and 40 within a certain population in Southeast Asia between 2006 and 2011, a prevalence of 52.96 % of PCOS patients with dyslipidemia was found, which was around twice as much Discussion d l Since obesity is an independent risk factor of CVD [39], in our study the prevalence of obes- ity was 34.6 % with PCOS and its control is fundamental to improve clinical and biochemical alterations [13, 38]. Another variable used was WC, which is an indicator of abdominal adiposity and is widely used as a predictor of cardiovascular risk [39]. The frequency of increased WC was 41 %, a finding indicating its association with this syndrome [37]. Competing interest The authors declare no competing interest. References Kablak-Ziembicka A, Przewlocki T, Pieniazek P, et al. The role of carotid intima-media thickness assessment in cardiovascular risk evaluation in patients with polyvascular atherosclerosis. Atherosclerosis. 2010;209:125–30. 28. Dilbaz B, Ozkava E, Cinar M, Cakir E, Dilbaz S. Cardiovascular disease risk characteristic of the main polycystic ovary syndrome phenotypes. Endocrine. 2011;39(3):272–7. doi:10.1007/s12020-011-9437-6. y 7. Ministério da Saúde. Datasus [internet]. Informações de Saúde. Estatísticas vitais – Mortalidade e nascidos vivos. [2007]. Disponível em: <http://tabnet. datasus.gov.br. 29. Cobin RH. Cardiovascular and metabolic risks associated with PCOS. Intern Emerg Med. 2013;8 Suppl 1:61–4. doi:10.1007/s11739-013-0924-z. 8. Barcellos CR, Lage SH, Rocha MP, Hayashida AS, Baracat EC, Romano A, et al. Polycystic ovary syndrome and obesity do not affect vascular parameters related to early atherosclerosis in young women without glucose metabolism disturbances, arterial hypertension and severe abnormalities of lipid profile. Gynecol Endocrinol. 2013;29(4):370–4. doi:10.3109/09513590.2012.743009. 30. Morgan CL. Jenkins_jones S, Currie CJ Rees DA. 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Receiver Operating Characteristic Analysis: A Tool for the Quantitative Evaluation of Observer Performance and Imaging Systems. J Am Col Rad. 2006;3(6):413–22. doi:10.1016/j.jacr.2006.02.021. 33. Griner PF, Mayewski RJ, Mushlin AI, Greenland P. Selection and interpretation of diagnostic tests and procedures. Ann Intern Med. 1981;94:555–600. 11. Yarak S, Bagatin E, Hassun KM, Parada MOAB, Talarico FS. Hiperandrogenismo e pele: síndrome do ovário policístico e resistência periférica à insulina. An Bras Dermatol [serial on the Internet]. 2005; 80(4): 395–410. doi:10.1590/S0365- 05962005000400011 34. Ioachimescu AG, Brennan DM, Hoar BM, Hoogwerf BJ. The lipid accumulation product and all-cause mortality in patients at high cardiovascular risk: a PreCIS database study. Obesity (Silver Spring). 2010;18(9):1836–44. PubMed. 12. Lee CMY, Huxley RR, Wildman RP, Woodward M. Received: 11 December 2014 Accepted: 15 June 2015 Received: 11 December 2014 Accepted: 15 June 2015 22. 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CD4 (+) CD 28 (null) T lymphocyte frequency, a new marker of cardiovascular risk: relationship with polycystic ovary syndrome phenotypes. Fertil Steril. 2012. 98 (6); 1609–15. doi:10.1016/j.fertnstert.2012;08.015. 6. Kablak-Ziembicka A, Przewlocki T, Pieniazek P, et al. The role of carotid intima-media thickness assessment in cardiovascular risk evaluation in patients with polyvascular atherosclerosis. Atherosclerosis. 2010;209:125–30. 6. Author details 1 21. Sociedade Brasileira de Cardiologia. IV Diretriz brasileira sobre dislipidemias e prevenção da aterosclerose. Departamento de Aterosclerose da Sociedade Brasileira de Cardiologia. Arq Bras Cardiol. 2007; 88 (supl 1): 2–19. Disponível: <http://publicacoes.cardiol.br/consenso/2013/V_Diretriz_Brasileira_de_ Dislipidemias.pdf>. Author details 1Universidade Federal do Maranhão, São Luís, Maranhão, Brazil. 2USP/Ribeirão Preto, São Paulo, São Paulo, Brazil. 3Rua Boa Esperança, Cond. Bosque dos Pinheiros Qd. 03, C. 02 - Bairro: Turú, São Luís, Maranhão, Brazil. Author details 1Universidade Federal do Maranhão, São Luís, Maranhão, Brazil. 2USP/Ribeirão Preto, São Paulo, São Paulo, Brazil. 3Rua Boa Esperança, Cond. Bosque dos Pinheiros Qd. 03, C. 02 - Bairro: Turú, São Luís, Maranhão, Brazil. 41. Zhang J, Fan P, Liu H, Bai H, Wang Y, Zhang F. Apolipoprotein A-I and B levels, dyslipidemia and metabolic syndrome in south-west Chinese women with PCOS. Hum Reprod. 2012;27(8):2484–93. doi:10.1093/humrep/des191. Acknowledgements This study was supported by the state funding agencies FAPEMA (Fundo de Amparo à Pesquisa do Maranhão) and Centro de Pesquisas Clínicas (CEPEC) da Universidade Federal do Maranhão, andthe government funding agency CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior). Page 7 of 8 Page 7 of 8 Nascimento et al. 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Page 8 of 8 Page 8 of 8 Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit
https://openalex.org/W4367172582	https://umu.diva-portal.org/smash/get/diva2:1753787/FULLTEXT01	Swedish	null	Poeter versus skådespelare i Dagens dikt 2020–2021	Tidskrift för litteraturvetenskap	2,023	cc-by	9,342	JIMMIE SVENSSON JIMMIE SVENSSON Copyright © 2023 Author(s). This is an open access article distributed under the terms of the Creative Commons CC-BY 4.0 License (https://creativecommons.org/licenses/by/4.0/) Copyright © 2023 Author(s). This is an open access article distributed under the terms of the Creative Commons CC-BY 4.0 License (https://creativecommons.org/licenses/by/4.0/) https://doi.org/10.54797/tfl.v52i2-3.6973 Forskningsartikel https://doi.org/10.54797/tfl.v52i2-3.6973 Forskningsartikel https://doi.org/10.54797/tfl.v52i2-3.6973 Forskningsartikel Inledning Dagens dikt sänds varje dag utom söndag och når då, enligt programmets hemsida, 100 000 lyssnare. Numera kan man även prenumerera på diktuppläsningarna som poddavsnitt, utan inledande klockringning och efterföljande konstmusikstycke. Dagens dikt kan därmed sägas vara den samtida poesiuppläsningens i särklass största distributionskanal. Inspelningarna är dessutom av studiomässig kvalitet, vilket är nog så viktigt när intresset som i denna studie gäller uppläsningarnas prosodiska drag, mätta med hjälp av ett digitalt verktyg. Dagens dikt förvaltar samtidigt ett arv som gör att programmet kan uppfattas som otidsenligt. Bland annat läses fortfarande flertalet dikter av skådespelare, och inte av poeterna själva. Sedan starten 1937 har förvisso en ständig utveckling skett: repertoaren, redaktionella överväganden och debatten kring programmet fram till 1955 har studerats av Solveig Lundgren, och under perioden därefter och fram till 1989 av Lars Hänström.1 Hur fördelningen mellan skådespelare och poeter sett ut har mig veterligen inte under sökts, men det framgår att skådespelarna dominerat stort. Inslaget av poetuppläsningar sägs öka 1975 i samband med att ”Månadens diktare” införs – sex dikter av en och samma poet under månadens första vecka.2 Det är också ungefär då som genombrottet för poeten som uppläsare av sin egen dikt sker i Sverige, några decennier senare än i USA,3 medan poeten dessförinnan sällan ansågs eller ens ansåg sig själv vara den bäst lämpade framföraren.4 Det finns förstås många undantag från en sådan generaliserande historieskrivning, exempelvis den redan under sin livstid erkänt skickliga Karin Boye, som gjorde flera uppläsningar för Dagens dikt i samband med en skådespelarstrejk 1939.5 Så sent som 1987, i skrivande stund det första år för vilket uppgifter om uppläsarna finns tillgängliga i Svensk mediedatabas (SMDB), lästes emellertid endast 24 dikter av poeter, alla i egenskap av månadens diktare, medan skådespelare framförde resterande 278. I det årslånga material som används i denna studie, från 2020 och 2021, är poeter nas andel betydlig högre, omkring 40 procent. 108 TFL 2022:2–3 FORSKNINGSARTIKEL Bortsett från Dagens dikt är skådespelarna satta på undantag i den samtida poesin. Skådespelaruppläsningar dominerar visserligen den starkt växande ljudboksmarkna den6 – men där spelar poesin en obetydlig roll, och ytterst lite samtidspoesi ges ens ut i ljudboksformat.7 Poetens nu självklara och ohotade auktoritet som uppläsare kommer kanske allra tydligast till uttryck på större poesifestivaler med internationella gäster, vilka publiken andäktigt lyssnar till – ibland på språk som få eller ingen förstår. Inledning Ulf Karl Olov Nilsson, mer känd som UKON, hävdar att de flesta poeter liksom han själv hyser en motvilja mot skådespelare som diktuppläsare; de läser oftast mycket bra tekniskt, men det är just det som är problemet – ”att det rör sig om någon som kallats in i egenskap av att vara bra på sitt jobb”.8 Malte Perssons diktrader kan kanske tolkas i samma riktning: ”Dikten blir ibland uppläst av Stina Ekblad. / Det borde den inte bli.”9 Skådespelaren Ekblad håller inte med. Hon betonar dessutom vikten av att vid läsningen ”[k]änna orden man framför från insidan” och av att läsa dikten ”som om den kom direkt ur mig”10 – och kanske är det just denna inlevelse som poeten vänder sig emot. Enligt en annan poet, Magnus William-Olsson, skulle nämligen schismen mellan skådespelare och poeter handla om de förstnämndas tendens och långa tradi tion av att spela en roll när de läser, av att vara någon, på motsvarande sätt som när de uppträder på scen. De skulle så att säga främst gestalta diktjaget. Poeter ser hellre att uppläsningen förmedlar texten och är diktens röst.11 pp g Möjligen skulle denna skillnad också kunna knytas till förändringar inom poesin under de senaste decennierna: ett uppbrott från den centrallyriska traditionen med dess starka och fokuserande diktjag, tänkt som en verklig person, mot en syn på texten som text, där jaget bara är en textuell funktion och subjektet inte är en instans som kan garantera dess mening.12 Skådespelares mer dramatiserande och levandegörande uppläsningar – om det nu verkligen förhåller sig så – skulle alltså kunna sägas vara förankrade i en äldre poesi och poetik.i Syftet med den här studien är att undersöka vilka prosodiska skillnader som finns mellan poeter och skådespelares diktuppläsningar, samt att kontextualisera dessa skill nader. Vilken roll kön och ålder spelar för uppläsning kommer att diskuteras, liksom i vilken mån det finns skillnader som kan spåras till textens innehåll eller form, samt huruvida uppläsningar av samma individ skiljer sig åt. Utgångspunkten för studien är ett dataset med 12 prosodiska mått för varje inspelning. Metoden i ett första och pri märt steg kan kallas fjärrlyssning: ett lyssnande som inte involverar mänskliga öron utan sker genom datorn, som först tar fram de prosodiska måtten och sedan jämför dem genom statistiska beräkningsmetoder. Fördelen är att man då kan hitta mönster, och därmed empiriskt starka belägg, i ett annars svåröverskådligt stort material. Inledning I ett andra steg, men också parallellt med det första, kompletteras fjärrlyssningen med kvalitativ närlyssning av ett mindre antal poeters och skådespelares uppläsningar. Material och statistiska förutsättningar Dagens dikts uppläsningar är i regel tillgängliga för avlyssning och som nedladd ningsbara filer – vilket väsentligt underlättar bearbetningen – i omkring ett år från sändningstillfället. Materialet i denna studie omfattar därför ett års sändningar, när JIMMIE SVENSSON TFL 109 mare bestämt från juli 2020 till och med juni 2021, tillgängliga då arbetet påbörjades. Digitaliserade program från Sveriges Radio är tillgängliga för forskare via SMDB, men diktläsningarna kommer då inte som separata ljudfiler utan som delar i längre sjok. Att själv klippa ur sådana längre filer hade varit mycket tidsödande – och kanske även upphovsrättsligt tveksamt. Uppläsningar från perioden som av för mig okänd anledning inte funnits tillgängliga för nedladdning har inte tagits med. Några mycket gamla poetuppläsningar har också uteslutits; inspelningar som inte är helt nygjorda, men som gäller poeter som levt in på 2000-talet och där ljudkvaliteten inte är påtagligt sämre har dock inkluderats. Också uppläsningar som redan sänts en gång tidigare under perioden har uteslutits – detta sker några få gånger med anledning av att poeten i fråga aktualiserats genom att ha mottagit eller nominerats till något pris. Sammanlagt har 28 diktuppläsningar uteslutits.13 För de återstående 276 läsningarna har jag tagit fram de 12 prosodiska mått som presenteras av Marit J. MacArthur och hennes team i en artikel från 2018, med hjälp av deras fritt tillgängliga programvara Voxit.14 Vad programmet mäter är pauser och talade partier, tonade och tonlösa partier, samt grundtonsfrekvensen, det som vi upp fattar som tonhöjd. Därefter utför programmet beräkningarna som resulterar i de olika måtten, till exempel den genomsnittliga pauslängden och avståndet i oktaver mellan högsta och lägsta tonen i inspelningen. Tanken är att till exempel relativt korta pauser i kombination med en på ett mer oförutsägbart sätt varierande tonhöjd kan indikera en mer vardaglig, samtalsnära stil, medan motsatsen indikerar en mer formell eller rent av stel. Kompletterad med uppgifter om uppläsarens namn, kön, födelseår, antal ord och inspelningens längd bearbetar jag denna genom programmet erhållna data med datorstödda statistiska metoder för att eventuella skillnader och mönster i det mänskligt svåröverskådliga materialet ska kunna konstateras. Material och statistiska förutsättningar Att skådespelarna således företrädesvis läser poeter av samma kön skulle kunna underlätta en nära representation eller imitation av diktens jag, när ett sådant finns – om man nu vågar utgå från att poeten och diktjaget oftast är av samma kön. Poeterna har emellertid ännu bättre förutsättningar, om de skulle eftersträva en sådan skådespelarmässig identifikation, då de i de allra flesta fall läser egenskriven dikt. Till poetgruppen har jag valt att också räkna 9 läsningar under rubriken ”Diktare läser (andras) dikt”. Antalet läsningar per individ varierar, från en enda upp till 7 för poeterna och 21 för skådespelarna. De multipla läsningarna gör utgångsläget mer komplicerat än om det bara varit fråga om en läsning per individ, som i de båda tidigare nämnda studierna. Jag har därför konsulterat en medarbetare på Statistikenheten vid Umeå universitet, Xijia Liu. Han har hjälpt mig göra bedömningen att risken för att de multipla läsningarna skulle snedvrida resultatet är liten, varför hela materialet har använts vid de kvantita tiva jämförelserna mellan grupperna. Värdena varierar så mycket mellan läsningar av samma individ, och skiljer sig inte så mycket åt mellan individer, vilket innebär att individen så att säga inte sätter något tydligt avtryck. Det hade varit problematiskt om exempelvis Josefin Iziamo, med störst andel av skådespelargruppens läsningar (12 procent), hade haft en extrem stil med genomgående avvikande värden jämfört med övriga, men så är alltså inte fallet. Alternativet, att endast välja en läsning per individ, skulle ha fört med sig den stora nackdelen att urvalet då hade blivit så mycket mindre att det blivit väsentligt svårare att hitta statistiskt signifikanta skillnader – det vill säga skillnader som inte beräknas kunna bero på slumpen eller som med hög grad av sanno likhet skulle bekräftas i ett annat, likvärdigt urval. g Xijia Liu har också utfört de inledande, mer avancerade statistiska testerna, vilka hade till syfte att pröva metodens tillförlitlighet på materialet. Först prövades huruvida det faktiskt finns någon statistiskt signifikant skillnad mellan poeter och skådespelare utifrån de 12 prosodiska måtten sammantagna. De visade sig att så är fallet, men också att variablerna kön och födelseår ger signifikanta skillnader, varför det också är meningsfullt att gå vidare och studera skillnaderna i detalj, mått för mått. Material och statistiska förutsättningar g g Här väljer jag att presentera måtten löpande, i samband med resultaten; för en mer utförlig, samlad beskrivning hänvisas till artikeln av MacArthur et al., kring läsningar av 100 poeter från USA, och till min egen studie från 2022 av ett motsvarande svensk språkigt material.15 Dessa studier skiljer sig för övrigt ifrån den föreliggande genom att de fokuserar på skillnader inom poetgruppen, och i någon mån också undersöker skillnaden mellan poesiuppläsning och andra typer av tal eller uppläsning. I min artikel diskuteras även potentiella problem med metoden, och den främsta invändningen är att väsentliga prosodiska komponenter som intensitet (ljudstyrka) och duration (vokal längd) inte ingår. Intensiteten mäts dock av programmet, men då tillförlitligheten här ställer större krav på likvärdig och hög inspelningskvalitet rekommenderar man i nuläget inte användning av dessa värden. Metoden, som alltså alltjämt är under utveckling, är så vitt jag känner till den enda i sitt slag i bemärkelserna att den utarbetats med fokus på poesi och att den, med undantag av ett mått, möjliggör helt automatiserad mätning och därmed väsentligt underlättar insamlandet av större mängder data. Att den har använts i tidigare studier innebär också att det finns något att jämföra med – här kommer jag främst referera till min egen, då den dels rör ett svenskt material och dels har ett tidsspann (2010–2020) som ligger närmare det aktuella materialet än vad som är fallet hos MacArthur et al. Fördelningen mellan skådespelare och poeter i urvalet, också uppdelade efter kön, framgår av följande tabell: 110 TFL 2022:2–3 FORSKNINGSARTIKEL Individer Läsningar Skådespelare 25 167 Kvinnor Män 14 11 94 73 Poeter 48 109 Kvinnor Män Icke-binär 20 27 1 41 67 1 Bland skådespelarna läser männen 6 dikter av kvinnliga poeter, kvinnorna 21 dikter av manliga poeter. Att skådespelarna således företrädesvis läser poeter av samma kön skulle kunna underlätta en nära representation eller imitation av diktens jag, när ett sådant finns – om man nu vågar utgå från att poeten och diktjaget oftast är av samma kön. Poeterna har emellertid ännu bättre förutsättningar, om de skulle eftersträva en sådan skådespelarmässig identifikation, då de i de allra flesta fall läser egenskriven dikt. Till poetgruppen har jag valt att också räkna 9 läsningar under rubriken ”Diktare läser (andras) dikt”. Bland skådespelarna läser männen 6 dikter av kvinnliga poeter, kvinnorna 21 dikter av manliga poeter. Material och statistiska förutsättningar Därefter beräknades med hur stor säkerhet man utifrån mätvärdena korrekt kan avgöra om det är en poet eller en skådespelare som läser: den bästa kombinationen av variabler (mått 3, 4, 7, 8, 10 och 11), med kön och födelseår givna (utifrån en uppdelning i tre åldersgrupper) ger en säkerhet på 82 procent (76 procent utan uppgifter om kön och födelseår). Det skulle alltså gå att skriva ett program, eller ett tillägg till det befintliga programmet Voxit, som 8 av 10 gånger ”gissar rätt”.16 JIMMIE SVENSSON TFL 111 En statistiskt signifikant skillnad mellan poeter och skådespelare finns för 8 av de 12 måtten, vilka i det följande behandlas i tur och ordning. En statistiskt signifikant skillnad mellan poeter och skådespelare finns för 8 av de 12 måtten, vilka i det följande behandlas i tur och ordning. Tempo, pauser och rytmisk komplexitet (mått 1–6) Att skillnaden är statistiskt signifikant behöver dock inte betyda att den är särskilt stor, och detta gäller när poeter och skådespelare jämförs utifrån de fyra första måtten. Ett mycket enkelt mått på läshastigheten, ord per minut (mått 1), erhålls genom att antalet ord i dikten divideras med inspelningens längd (i sekunder) och multipliceras med 60. Detta måste i nuläget göras med ett betydande mått av mänsklig inbland ning (i Google Cloud finns ett verktyg för att omvandla tal till text, men resultatet måste kontrolleras och ofta korrigeras). I min tidigare studie visade sig detta mått vara det som ger de mest markanta skillnaderna för poesiuppläsning jämfört med uppläst romanprosa och med fritt tal.17 Föga förvånande är skillnaden mellan poesiläsande poeter och skådespelare däremot ganska liten. Av följande låddiagram framgår att poeterna läser något snabbare, men att skillnaden mellan medianvärdena, det vill säga det mellersta värdet, markerat av strecket i lådan, endast är omkring 5 ord per minut. Medelvärdet, markerat med kryss, ligger här och genomgående i denna studie mycket nära medianvärdet. Ett låddiagram visar spridningen, med värdena uppdelade i fyra kvartiler; i första respektive fjärde kvartilen, ”svansen” under respektive över lådan, finns 25 procent av värdena (små cirklar markerar statistiskt beräknade extremvärden), medan andra och tredje kvartilen markeras av lådan. Diagram 1. Läshastighet (ord per minut) för poeter och skådespelare. Diagram 1. Läshastighet (ord per minut) för poeter och skådespelare. En ytterligare gruppering efter kön visar att det är de manliga poeterna som står för nästan hela skillnaden – men inte heller mellan dem och de långsammaste läsarna, de kvinnliga skådespelarna, är skillnaden större än 8 ord, en skillnad som hade varit svår att uppfatta om det gällt två i övrigt någorlunda likvärdiga uppläsningar.i Lådan, som alltså markerar de 50 procent av värdena som befinner sig närmast medianen, ligger för poeternas del mellan 64 och 85 ord och för skådespelarnas mel lan 61 och 79 ord. En stor del av värdena ligger därmed samlade inom ett ganska snävt intervall. Tempo, pauser och rytmisk komplexitet (mått 1–6) Läshastighet (ord per minut) grupperat efter diktens totala antal ord. Diagram 3. Läshastighet (ord per minut) grupperat efter diktens totala antal ord. En möjlig förklaring till detta kan vara att längre dikter är mer berättande, och att läshastigheten och pausmönstren i motsvarande grad närmar sig normen för prosa eller fritt tal. Skådespelarnas dikter är i genomsnitt betydligt kortare än poeternas, 105 mot 134 ord. En hypotes kunde vara att skådespelardikterna är – eller därför uppfattas som – mindre berättande och mer traditionellt lyriska. De skillnader som konstaterats så här långt skulle då inte enbart ha att göra med om uppläsaren är poet eller skåde spelare, utan även kunna bero på att grupperna till viss del läser kvalitativt olikartade dikter. Skådespelarnas dikter är oftast äldre: drygt hälften är av 1900-talspoeter, och många är äldre än så, medan poetdikterna med få undantag är från 2000-talet. Det uppbrott från centrallyriken med dess samlande diktjag som på flera håll noterats omkring millennieskiftet har också kopplats till ett uppsving för mer expansiva former, som ett uppror mot modernismens koncentrerade ideal.23 Detta återspeglas möjligen i materialet. Det ska dock betonas att programformatet också kan medföra ett slags ”centrallyrikisering” av postmodernistisk dikt; när Johan Jönson läser ett kortare stycke ur en av sina vindlande, tusensidiga böcker, för Dagens dikt försett med den idylliska titeln ”Junikväll”, riskerar det att framstå som ett fristående, traditionellt antologistycke.24 Att kategorisera dikterna kvalitativt, för att fullt ut kunna pröva hypoteser av det här slaget, om att skillnader i läsningarna skulle bero på olikartad repertoar, vore dock ytterst vanskligt. Exempelvis Elis Monteverde Burraus dikter har å ena sidan ett starkt diktjag, men å andra sidan föga av centrallyrikens typiska koncentration, utan påminner med ett slags slarvig pratighet snarare om språket i sociala medier. Fyra av Burraus läsningar återfinns för övrigt bland de tio allra snabbaste, och hos Burrau kombineras det höga tempot också med relativt korta, täta och oregelbundna pauser (värdena återfinns i undre eller övre kvartilen).25 En motsatt läsning utifrån dessa fyra mått kunde vara skådespelaren Peter Anderssons av en kort översatt dikt.26 Medan måttet för pausernas komplexitet (4) beräknas med en algoritm som dels komprimerar, dels jämför talade och tysta partier, erhålls de två följande, rytmisk kom plexitet för stavelser (5) och rytmisk komplexitet för fraser (6) baserat på växlingen mellan tonande och tonlöst. Tempo, pauser och rytmisk komplexitet (mått 1–6) Anmärkningsvärt är också att de allra snabbaste läsningarna ligger 112 TFL 2022:2–3 FORSKNINGSARTIKEL så lågt som omkring 110 ord per minut – att jämföra med min tidigare studie, där flera poetuppläsningar har betydligt högre värden och där medianvärdet för skön litterär prosauppläsning är strax över 120 ord.18 Att inte en enda läsning för Dagens dikt bryter mot vad som av hävd och konvention kan sägas vara normen för poesi, relativ långsamhet, skulle kunna bero på en mer eller mindre medveten anpassning till det ärevördiga programmets högtidliga idiom, till dess åtminstone tidigare uttalade karaktär av ”profan andaktsstund”.19 Dikterna har dessutom spelats in under ledning av en producent, i de flesta fall Lena Birgersdotter, som varit ansvarig för programmet sedan 2017. Birgersdotter uppger att uppläsarna regisseras i högre eller lägre grad, men att det inte finns några krav på en viss läshastighet.20 För min tidigare studie användes däremot enklare, ofta heminspelade läsningar från numera nedlagda Podpoesi.nu. I Dagens dikt-materialet saknas dessutom utpräglade scenpoeter – vilka möjligen brukar läsa snabbare och med mer markanta tempoväxlingar än bokpoeter.21 p g p De tre följande måtten gäller pauserna: genomsnittlig pauslängd (2) och pausfrekvens (3), samt rytmisk komplexitet för pauser (4). Även för dessa är skillnaderna mellan grup perna små: Sammantaget kan dock en tendens urskiljas. Skådespelarna har längre men glesare pauser, och pauserna infaller också på ett mer regelbundet eller förutsägbart sätt, vilket i kombination med den lägre läshastigheten gör att de kan sägas läsa på ett mer dramatiskt men också mer formellt eller stiliserat sätt än poeterna. Poeternas kortare, tätare och mer oregelbundna pauser indikerar en mer samtalsnära stil, utan att för den skull vara i närheten av median- och medelvärdena för fritt tal, vilka för exempelvis pauslängden ligger runt 0,3 sekunder.22 Som framgår av följande diagram har de flesta skådespelare och poeter betydligt längre pauser än så (skillnad och sprid ning är snarlik för de andra två pausmåtten): Diagram 2. Pauslängd (i sekunder) för poeter och skådespelare. Diagram 2. Pauslängd (i sekunder) för poeter och skådespelare. Något som kan bidra till den tidigare påtalade, relativt begränsade spridningen i läshastighet är att programformatet inte kan rymma särskilt långa dikter eller uppläs ningar – omkring 75 procent är kortare än 2 minuter. Ett tydligt samband föreligger nämligen mellan diktens längd och läshastigheten, vilket framgår när uppläsningarna delas upp i ungefär lika stora grupper på grundval av totalt antal ord: JIMMIE SVENSSON TFL 113 Diagram 3. Tempo, pauser och rytmisk komplexitet (mått 1–6) Ett mer regelbundet temporalt mönster ger ett lägre värde, vilket i sin tur kan betyda att läsningen ger ett stelare eller mer stiliserat intryck. För de båda senare måtten har ingen signifikant skillnad konstaterats mellan skådespelare och poeter, däremot märks en svag tendens till lägre värden för män jämfört med kvinnor, 114 TFL 2022:2–3 FORSKNINGSARTIKEL vilket kan verka vara i linje med den vanliga men möjligen stereotypa uppfattningen att kvinnor är mer livliga och uttrycksfulla i sitt tal.27 För alla tre måtten på rytmisk komplexitet varierar värdena ganska mycket mellan läsningar av samma individ, vilket kan tyda på att de i högre grad än övriga mått återspeglar hur den enskilda dikten ser ut. Jag återkommer till detta i studiens avslutande, kvalitativa avsnitt. Tonhöjd och dynamik (mått 7–12) Nästa serie gäller grundtonsfrekvensen, det vill säga tonhöjden. Vad gäller genomsnittlig tonhöjd (7) beror värdet i så hög grad på individens rent fysiologiska förutsättningar att det är föga förvånande att ingen signifikant skillnad finns mellan skådespelare och poeter (kvinnor och män har naturligtvis jämförts var för sig). Därmed inte sagt att det inte är möjligt för individen att mer eller mindre medvetet manipulera och variera den genomsnittliga tonhöjden efter sammanhang, på ett högst hörbart sätt. Detsamma gäller för tonalt omfång (8), och här är det möjligen så att kvinnorna, både poeterna och skådespelarna, snävar av sitt register något. Deras värden ligger nämligen väl i linje med vad jag tidigare tagit fram för kvinnliga poeter, vilka då visade sig ligga betydligt lägre än en grupp fritt talande kvinnor.28 MacArthur et al. noterar detta som ett fenomen, att kvinnliga poeter ofta läser poesi i ett betydligt lägre tonläge och mer monotont jämfört med det register de annars använder – möjligen för att vinna i auktoritet, genom att mer eller mindre medvetet imitera en manlig norm.29 Studier har också visat att kvinnor tenderar att ha ett större omfång, vilket i sin tur förknippas med ett mer uttrycksfullt eller känslobetonat tal.30 I Dagens dikt-materialet har emellertid inga skillnader konstaterats mellan kvinnor och män vad gäller omfånget, något som alltså möjligen talar för att kvin norna begränsat sitt omfång just vid poesiuppläsning. Utan att inhämta och jämföra med icke-poetiska röstprov från de diktläsande individerna kan dock inga säkra slutsatser dras. Upplevd monotoni har inte heller enbart med omfånget att göra. Tonhöjdshastig heten (9) och tonhöjdsaccelerationen (10) är nära relaterade mått och visar hur snabbt tonhöjden förändras (mätt i oktaver per sekund) respektive hur snabbt förändring arna i hastighet sker (i oktaver per sekundkvadrat). Poeterna har här signifikant lägre värden, och skillnaderna är mer markanta än vad som noterats för mått 1–4. Som Diagram 4. Tonhöjdsacceleration (i oktaver per sekundkvadrat) för poeter och skådespelare. Diagram 4. Tonhöjdsacceleration (i oktaver per sekundkvadrat) för poeter och skådespelare. JIMMIE SVENSSON TFL 115 framgår av diagrammet för accelerationen (och det ser likadant ur för hastigheten) har mer än 75 procent av poeterna värden som ligger under median- och medelvärdet för skådespelarna, och i poetgruppen finns också de individer som har allra lägst värden, i skådespelartruppen de med högst. Tonhöjd och dynamik (mått 7–12) g Detta betyder att poeternas tonhöjd inte förändras lika mycket eller lika snabbt, och att de således är mer monotona, vilket i sin tur innebär att de låter mindre livliga eller dramatiska än skådespelarna. Stor tonal variation brukar också förknippas med en mer uttrycksfull eller rent av känslosam stil, men man bör då inflika att passiva känslor så som sorg typiskt hänger samman med låga värden för bland annat den tonala variationen.31 Skillnaden och fördelningen är likartad för tonhöjdsentropin (11), och även här kan ett lågt värde indikera monotoni, nämligen för en uppläsning som genomgående håller sig nära en genomsnittlig tonhöjd. Måttet har dock snarare med förutsägbar het att göra, och även en uppläsning som på ett regelbundet sätt växlar mellan få punkter eller områden i registret, till exempel mellan de allra högsta och de allra lägsta tonerna, har låg entropi. Låg entropi motsvarar ofta ett stiliserat intryck, medan hög, då alltså nästan alla delar av registret används på ett mer oförutsägbart sätt, verkar mer naturligt och torde fungera bättre om avsikten är att gestalta diktens talare, diktjaget. Det kunde ligga nära till hands att anta att skillnaderna beror på att skådespelarna helt enkelt är skickligare rent tekniskt. De är mästare på att levandegöra, och att poeter i jämförelse med dem många gånger framstår som stela och tråkigt enahanda vore föga förvånande. Skådespelarna är säkerligen bättre på att vara just skådespelare – men mycket tyder på att många poeter inte ens försöker att tävla med dem. MacArthur uppmärksammar i en artikel från 2016 en gängse poetisk röstkliché, ”monotonous incantation”, vilken under det populära namnet Poet Voice blir utgångspunkt för den senare, kvantitativa studien.32 UKON menar i en inledningsvis refererad bok från 2019 att Poet Voice, ”som kännetecknas av en långsam, stadig rytm och fallande intonation, som i ett slags monoton besvärjelse”, blivit en dominerande praktik även bland svenska poeter.33 Det handlar alltså om ett högst medvetet sätt att läsa på: Också de mer anti-performativa läsningarna är genomtänkta, avsiktligt ickeuppträ dande, performativt antiperformativa således. Tonhöjd och dynamik (mått 7–12) Skrivarutbildningarnas samtal om, och övningar i, uppläsningssituationen kan förmodligen tillskrivas del i detta men framför allt beror det nog på en bredare samhällsutveckling där poeter liksom andra kulturarbetare alltmer har blivit entreprenörer som måste lägga ned tid och omsorg på bilden av sig själv.34 Poet Voice innebär också att den konventionella prosodin undertrycks genom ett ständigt upprepat och av kontexten eller innehållet mer eller mindre oberoende tonalt mönster. Genom att avlägsna sig från det konventionella kan poeten stilisera sitt uttryck så långt att det blir ett slags varumärke. Något tillspetsat skulle man kunna säga att rösten då blir poetens signum, snarare än diktjagets.35 Att kvantifiera Poet Voice låter sig knap past göras, åtminstone inte utifrån de mått som används här; huruvida detta med ett återkommande tonalt mönster undertrycker det prosodiskt mer konventionella och efter kontexten anpassade, framgår inte direkt av några värden. MacArthur et al., liksom min 116 TFL 2022:2–3 FORSKNINGSARTIKEL egen tidigare studie, visar dock att drag som är förknippade med Poet Voice, monotoni och regelbundenhet eller förutsägbarhet, i stor utsträckning präglar poetuppläsningar, och att dessa drag är mer framträdande hos något yngre respektive hos kvinnliga poeter. Som framgår av följande diagram över tonhöjdsentropin (mönstret är likartat för tonhöjdshastighet och -acceleration) är könsskillnaden betydande också i Dagens dikt- materialet. Poeter läser mer tonalt förutsägbart än skådespelare, och kvinnliga poeter är mer förutsägbara än manliga. Dessutom återfinns de allra lägsta noteringarna hos de kvinnliga poeterna. egen tidigare studie, visar dock att drag som är förknippade med Poet Voice, monotoni och regelbundenhet eller förutsägbarhet, i stor utsträckning präglar poetuppläsningar, och att dessa drag är mer framträdande hos något yngre respektive hos kvinnliga poeter. Som framgår av följande diagram över tonhöjdsentropin (mönstret är likartat för tonhöjdshastighet och -acceleration) är könsskillnaden betydande också i Dagens dikt- materialet. Poeter läser mer tonalt förutsägbart än skådespelare, och kvinnliga poeter är mer förutsägbara än manliga. Dessutom återfinns de allra lägsta noteringarna hos de kvinnliga poeterna. Diagram 5. Tonhöjdsentropi (enhetslöst mått) för poeter och skådespelare, uppdelade i kvinnor (K) respek tive män (M). Diagram 5. Tonhöjdsentropi (enhetslöst mått) för poeter och skådespelare, uppdelade i kvinnor (K) respek tive män (M). Tonhöjd och dynamik (mått 7–12) Som en förklaring till kvinnliga poeters och då särskilt yngre kvinnliga poeters lägre värden och därmed mindre uttrycksfulla lässtil föreslår MacArthur et al., som jag varit inne på tidigare, en koppling till sociala, kulturella och språkliga tendenser utanför poesin, så som ökande jämställdhet och ”the apparent efforts of women to imiatate male authority through creaky voice”.36 Möjligen skulle en sådan mer allmän hypotes kunna förklara varför skillnaden också finns (och till och med är större) mellan kvinnliga och manliga skådespelare; att kvinnorna inte enbart tangerar utan också passerar männens låga värden för tonal variation och därmed uttrycksfullhet skulle kunna förstås som ett slags kompensation för mäns naturligt betydligt lägre genomsnittliga tonhöjd. Det kan förstås också vara så att de kvinnliga skådespelarna influerats av den monotona trenden bland poeter. Själv har jag i tidigare artiklar lyft en rad förklaringar vilka snarare innebär poeto logisk motivering, och vilka snarare är kopplade till födelseår än till kön. Ett genombrott för en ny typ av poesi omkring millennieskiftet, och därmed hos något yngre poeter, med ett frånvarande eller, jämfört med ett romantiskt-modernistiskt paradigm, an norlunda diktjag, har redan nämnts. Vidare skulle ett poststrukturalistiskt inspirerat fokus på texten som text, parallellt med att uppläsningen blivit en allt viktigare distri butionsform under senare år, kunna ligga till grund för en förståelse av den monotona lässtilen som ett försök att representera textualiteten. Det kan i det sammanhanget vara på sin plats att påminna om William-Olssons inledningsvis refererade ståndpunkt om att uppläsningen ska vara diktens eller textens röst. Genom att konventionell prosodi – orienterad efter och ett med innehållet – undertrycks imiteras den tryckta textens JIMMIE SVENSSON TFL 117 neutralitet. Den spänning som då uppstår mellan form och innehåll och mellan faktisk läsning och en tänkt, mer konventionell läsart skulle i sin tur i högsta grad vara poetiskt produktiv, med såväl estetiska som retoriska funktioner.37 Födelseår visar sig dock vara en viktig faktor också bland skådespelarna. Att skill naden är så stor mellan könen där, större än hos poeterna, torde nämligen ha med en skev fördelning efter födelseår att göra. Jag har valt att jämföra uppläsningar av individer födda före 1970 respektive födda 1970 och senare, då detta ger den jämnast möjliga grupperingen av hela materialet (och då detta också är den gruppering jag använder i min tidigare studie). Flertalet skådespelaruppläsningar av män tillhör den äldre gruppen, medan flertalet uppläsningar av kvinnor tillhör den yngre. 118 TFL 2022:2–3 FORSKNINGSARTIKEL Kvalitativ avslutning Genom den kvantitativa, datorstödda analysen har mönster hittats i ett för mänsklig perception svåröverskådligt material. Materialet hade gärna kunnat vara ännu större, och med hjälp av en välorganiserad databas, med samtliga bevarade Dagens dikt- uppläsningar sedan 1937 som separata filer, hade det gått snabbt och enkelt att med de metoder som jag använt mig av i denna artikel undersöka till exempel hur skåde spelarnas idiom förändrats över tid och huruvida de följt samma trender som poeterna. I nuläget är det svårt att ens grovt uppskatta detta totala antal unika uppläsningar, då många återutsänds. g Av praktiska skäl stannade det alltså vid 276 läsningar i denna studie. Detta mer beskedliga material har dock den fördelen att jag har kunnat lyssna på alla uppläs ningar, åtminstone en gång. Att göra så kan också vara ett viktigt komplement, då de 12 prosodiska mått som använts är långt ifrån heltäckande, och då viktiga frågor kring användningen av dem ännu är obesvarade, till exempel i vilken mån anpassningar bör göras när de används på andra språk än engelska. De kan inte direkt spegla huruvida en läsning är prosodiskt konventionell eller ej. Att ingen av skådespelarna läser med Poet Voice har jag bara kunnat konstatera genom egen avlyssning; lässtilen kännetecknas av att monotonin och förutsägbarheten delvis sätter en mer konventionell läsart ur spel, det vill säga en som är betonad och fraserad utifrån innehållet. Lyssningen kan dessutom tjäna som ett slags prövning av huruvida det som framkommit i den datorstödda analysen är mänskligt relevant. Det är dock på sin plats att framhålla att datorn och människan ”hör” delvis olika saker, och att människan har tillgång inte bara till fler prosodiska komponenter, utan också har kontextuell kunskap och till exempel kan känna igen en äldre dikt eller dikttyp och sluta sig till att det rör sig om en skådespelaruppläsning, eller helt enkelt känna igen en välkänd poets eller en för Dagens dikt ofta anlitad skådespelares röst. Jag är övertygad om att jag åtminstone skulle klara datorns 8 av 10 rätt (se ovan). Vidare, om nu den kvantitativa analysen närmast kräver kvalitativ komplettering, kan man också ställa sig frågan om resultaten som följer av digital metodanvänd ning verkligen motiverar det trots allt ansenliga arbete som applicerandet av metoden innebär. Tonhöjd och dynamik (mått 7–12) l En gruppering efter födelseår följer här för det avslutande måttet, dynamism (12), vilket räknas fram genom att värdena för mått 5, 6, 9 och 11 viktas mot varandra och vilket är tänkt att ge ett mer överskådligt eller sammanfattande mått på hur regel bunden eller förutsägbar uppläsningen är.38 Medianvärdet för hela poetgruppen ligger för övrigt nära det för poeterna i min tidigare studie, medan skådespelargruppens ligger mycket nära de betydligt högre värdena för de två jämförelsegrupperna fritt tal respektive romanprosa.39 Diagram 6. Dynamism för poeter och skådespelare, födda 1969 och tidigare (–1969) respektive 1970 och senare (1970–). Diagram 6. Dynamism för poeter och skådespelare, födda 1969 och tidigare (–1969) respektive 1970 och senare (1970–). Som framgår är skillnaden större mellan grupperna hos poeterna än hos skådespe larna. Hos de äldre poeterna dominerar dock männen, hos de yngre skådespelarna kvinnorna. Att isolera de båda faktorerna från varandra låter sig inte fullt ut göras så som materialet ser ut (vid en gruppering efter både födelseår och kön riskerar grup perna att bli för små, eller att bestå av läsningar av ett för litet antal individer). Hos poeterna kan skillnaden mellan åldersgrupperna alltså vara poetologiskt för ankrad. Men varför märks tendensen, att de yngre är mer monotona och regelbundna, också hos skådespelarna? Återigen kan de yngre skådespelarna helt enkelt ha påverkats av hur yngre poeter läser. Men kanske finns det samtidigt, i linje med förslaget från MacArthur et al. ovan, också kopplingar till större tendenser utanför såväl poesin som scenkonsten. Här hade det naturligtvis varit intressant att undersöka om och i så fall hur skådespelarkonventionerna har förändrats över tid, också i andra typer av uppläsning, och, inte minst, vid scenframträdanden.40 118 TFL 2022:2–3 FORSKNINGSARTIKEL Kvalitativ avslutning De båda delarna av analysen bör dock ses som ömsesidigt värdefulla: i det kvantitativa och datorstödda kan mönster identifieras som sedan kan undersökas närmare kvalitativt, medan den kvalitativa analysen och kritiska reflektionen över resultaten kan bidra till att identifiera vidare frågor och önskvärda eller nödvändiga förbättringar av den digitala metoden. Att metoden som används i denna studie är prövad och eventuellt utvecklas vidare är förstås ännu viktigare den dag det blir möjligt att applicera den på ett oöverskådligt stort ljudmaterial.41 Metodutveckling kan också innebära att man hittar sätt att använda mätresultaten i kvalitativ analys, som här när en mindre del av materialet har underkastats egent lig, noggrann och systematisk närlyssning, med de aktuella texterna för ögonen. De prosodiska måtten är nämligen fortsatt användbara, främst som ett verktyg för att upptäcka skillnader mellan läsningar av en och samma individ. Sådana skillnader är intressanta, eftersom de kan säga något om i vilken grad den lästa textens form eller innehåll påverkar uppläsningen. För ett fylligt urval har jag valt ut alla läsningar av individer med 3 eller fler dikter, men, för att begränsa urvalet, färre än 13. Detta ger 65 inspelningar för poeterna och 76 för skådespelarna. JIMMIE SVENSSON TFL 119 Det främsta resultatet av den kvantitativa analysen var att poeterna framför allt är mer monotona eller tonalt förutsägbara, något som också speglas i detta mindre urval. För det sammanvägande dynamiska måttet (12) har 10 av 13 poeter värden som för samtliga läsningar är relativt låga – i första eller andra kvartilen, det vill säga under medianvärdet, för studiens samtliga läsningar. Motsvarande gäller bara för 2 av de 11 skådespelarna. Skådespelarna har större variationer mellan sina respektive läsningar än poeterna. Variationer finns särskilt vad gäller måtten för tempo, pauser och rytmisk komplexitet (1–6); exempelvis har 5 skådespelare, men ingen poet, noteringar i både i första och fjärde kvartilen för läshastigheten (det vill säga att individen finns represen terade i såväl den långsammaste som i den snabbaste fjärdedelen av hela materialet). Skälet till detta kan vara att den enskilda diktens text, dess form och/eller innehåll, har stor inverkan på hur texten läses. Så som antagits tidigare visar sig skådespelarna i de allra flesta fall ha en betydligt mer heterogen samling dikter att traktera.42 l y g g g Ordknappa dikter läses oftast långsammare än längre, vilket tidigare konstaterats. Kvalitativ avslutning Därtill tenderar dikter med kortare versrader att läsas långsammare än de med längre, och likaså i förhållande till dikter som är på prosa eller prosaliknande. Tydligast är detta hos poeterna, där det visserligen finns ganska få formmässiga skillnader mel lan de egna dikter som poeten läser, men där man i gengäld, då dikterna i övrigt är relativt likartade, kan tänka sig att formfaktorn i någon mån kan isoleras. När Kristofer Folkhammar läser en prosadikt är tempot högre, jämfört med i en dikt med relativt korta versrader, och medan prosan läses på ett relativt konventionellt, flödande sätt, dröjer han i kortversdikten längre vid ord och stavelser, och versradsindelningen görs hörbar.43 Särskilt anmärkningsvärd är Folkhammars mycket taktfasta, närmast skanderande läsning av en metricerande dikt.44 Inget motsvarande hörs hos någon skådespelare. Visst kan man hos dem ofta ana metern (och fastställa vilken meter det handlar om utan att ha dikten framför sig), men i linje med den konvention som länge varit rådande prioriteras en mer naturlig rytm framför metern.45 För måtten som gäller tonhöjden (7–11) varierar värdena i mindre utsträckning. Detta gäller i synnerhet för poeterna, och de variationer som finns kan oftast tillskrivas stora skillnader i dikternas stämning eller innehåll. Ett exempel är skådespelaren Sofia Berg-Böhms läsning av en dikt av Wislawa Szymborska, med höga värden, i kontrast till alla hennes läsningar av jiddischpoeten Anna Margolin, med närmast genomsnitt liga värden.46 Mörkret och allvaret hos Margolin gestaltas således genom en prosodiskt mindre livlig läsning. Szymborskadikten låter istället milt ironisk, lekfull eller naiv, och att den återger en katts perspektiv hörs bland annat i en betydligt högre genomsnittlig tonhöjd.47 Värdena för Margolindikterna är alltså mycket homogena, vilket också genomgående är fallet när skådespelare läser en och samma poet. g g Hos skådespelarna förekommer andra typer av gestaltningar – nog så hörbara, men utan att de ger direkt avtryck i några mätvärden. Etienne Glasers andfådda eller Christian Fex liksom ålderdomssvaga läsning är exempel på utpräglat skådespeleri, an passat efter diktens innehåll eller motiv.48 Skiftningar i tonfall, för att markera nya eller kontrasterande stämningslägen, tankar, infall eller röster i diktens värld, förekommer genomgående och mer påtagligt hos skådespelarna än hos poeterna, något som också ligger bakom de högre respektive lägre värden för de tonala måtten som konstaterats i den kvantitativa jämförelsen. Kvalitativ avslutning 120 TFL 2022:2–3 FORSKNINGSARTIKEL Men skiftningar behöver inte enbart vara tonala, utan kan parallellt eller istäl let innebära ändringar i tempo, intensitet (ljudstyrka) och vokallängd. Saga Gärde är ensam bland skådespelarna om att ha genomgående låga tonala värden (i första kvartilen), men låter trots detta nyanserad och skiftningsrik på ett för skådespelarna typiskt sätt.49 Omvänt kunde den höga dynamiken och de stora tonala variationerna i poeten Marie Lundquists läsningar indikera en närhet till de mest dynamiska eller uttrycksfulla skådespelarna: Amanda Ooms, Anna Azcárate och Emil Almén. Lund quist skiljer sig dock från dessa genom ett relativt lågt värde för tonhöjdsentropin, vilket indikerar att hennes tonala mönster är mer förutsägbart, och vid en avlyssning förstärks intrycket av regelbundenhet genom andra prosodiska faktorer. De betonade stavelserna (vilka framträder i samverkan mellan ton, intensitet och längd) förefaller att falla mycket tätt och regelbundet, och även på stavelser som normalt inte skulle vara betonade av semantiska skäl, och även de tonala mönstren upprepas på ett påfallande sätt. Lundquist tycks således följa ett slags inre melodi, som till stora delar ersätter en prosodiskt mer konventionell läsart.50 Den kognitivt orienterade versforskaren Reuven Tsur har visat hur poesiuppläs ningen kan överbrygga skillnaden mellan hur vi lyssnar till språkliga respektive icke- språkliga ljud, så som musik. Det sker, menar Tsur, genom att poesin lyfter fram språkets form på ett för konventionellt språkbruk avvikande och därmed påtagligt sätt. Därigenom aktiveras det som Roman Jakobson kallade den poetiska språkfunk tionen.51 Lundquists ordmusik kan rent av vara så fängslande, att man stannar vid att lyssna till hur och glömmer vad hon läser. Något liknande förekommer inte hos skådespelarna: deras läsningar är nog så ofta vackra, men alltid snarare uppmärksamma på innehållet. p I Lundquists fall handlar det utan tvekan om en högst medveten, idiosynkratisk läsart. I några fall skulle förmodligen en erfaren Dagens dikt-lyssnare kunna identifiera poeter som just poeter genom alltför avvikande röster jämfört med skådespelarna: Arne Johnsons skrovliga men intima stämma eller Birgitta Lillpers dalmål kan vara exempel här. Ofta är det emellertid vanskligt att försöka skilja mellan stilisering och karaktärsdrag som snarare beror på arv och miljö, särskilt om man inte har röstprov från något icke-poetiskt sammanhang att jämföra med. Kvalitativ avslutning Som jag har varit inne på tidigare skulle en läsart som i lägre grad än normalt påverkas av kontext och innehåll kunna tänkas imitera den neutralitet som texten har tills den möter en läsare. Det är då fråga om att gestalta en mer allmän idé om textualitet, och det kan vara svårt att se hur detta är förenligt med en ambition att förmedla en specifik text, att vara ”diktens röst”, för att ännu en gång återknyta till William-Olsson i artikelns inledning. Som MacArthur anmärker går det partikulära i den enskilda dikten förlorat vid en sådan läsning: ”We feel that we are listening to any poem, not this particular one.”56 Frågan är i sådana fall om inte den typiska skådespelaruppläsningen kommer närmare dikten: ”Dikten är det viktiga, inte jag. Jag är den som dikten går igenom.”57 Mot denna Stina Ekblads föresats kan man dock invända att just hennes röst är alltför välkänd och igenkännlig för att utan vidare kunna lånas ut till ett diktjag. Poeterna Freke Räihä och Jörgen Lind sluter sig delvis till den monotona upp läsningsstilen, genom lätt mässande eller sjungande, och förlängda vokaler. Men de skiljer också ut sig genom större hörbar variation mellan dikterna och, i synnerhet hos Lind, inom den enskilda dikten. Det är dock inte som hos Folkhammar uppenbart vad som föranleder dessa variationer. Mitt intryck är att de i högre grad är intuitivt uppmärksamma på mönster de hittar under läsningens gång – eller som de möjligen redan hade i huvudet när de skrev. Linds sonora stämma dröjer och hänger sig närmast lustfyllt vid språkljuden, vilka i kombination med innehållsmässiga skiftningar kan verka motivera ett ändrat tonfall.58 Vid andra tillfällen tar han tillvara på en möjlighet till upprepning av rytmiska figurer, som när han läser ”en stretande björk / vid en grönskande brant” (oOooO / ooOooO).59 Med dessa variationer låter Lind livlig och inlevelsefull, och det mekaniska och för texten likgiltiga intryck som kan inträda vid den typiska Poet Voice-läsningen uteblir. Därmed inte sagt att han ger avkall på detta att göra rösten till personligt varumärke. Ja, han kan nog sägas ligga nära den enskilda dikten till både form och innehåll. Frågan är emellertid om en läsning som Linds hade varit möjlig för någon annan än poeten själv. Kvalitativ avslutning Denna osäkerhet kan gälla Johnsons tydliga och täta inandningar – att jämföra med skådespelares inandningar, vilka tenderar att ha en mer uppenbar dramatisk-retorisk funktion än Johnsons.52 Johnson är dock skådespelarlik i den meningen att hans läsning tjänar bilden av diktjaget väl – den förefaller vara nära anpassad efter det personliga och vardagliga berättande som utmärker hans 6 dikter. Till skillnad från Lundquist låter han inte någon stiliserad prosodi bryta denna typ av autenticitet. Lundquist är alltså relativt dynamisk. Vanligare, vilket också framgått i den kvanti tativa delen av studien, är att poeternas prosodiska stilisering går mot det monotona. Helena Boberg, Elis Monteverde Burrau och Johan Jönson är de klaraste exemplen i det mindre urvalet (i en läsning varierar dock Jönson genom att markera de återkom mande inskotten av typen ”pengarna räcker inte” genom ett viskande, lägre tonfall, och långa pauser).53 Burraus läsning är mässande eller lätt sjungande med ett ständigt åter kommande tonalt mönster, särskilt påfallande i intonationsfrasens slut, på ett sätt som delvis sätter konventionell prosodi ur spel. Han kan därmed exemplifiera röstklichén Poet Voice (det visar sig alltså att han läser högst stiliserat, trots vad jag antytt tidigare, JIMMIE SVENSSON TFL 121 utifrån de fyra första måtten, om en mer vardaglig stil). Också Elisabeth Berchthold har en tendens att undertrycka den mer normala prosodin, dock inte genom monotoni eller tonal regelbundenhet, utan snarare på ett så dröjande, närmast överartikulerat sätt, att det stundom kan låta som om hon läser en lista med ord som inte hänger sam man i meningar – vilket också har att göra med den ibland fragmenterade syntaxen.54 g g g y Det torde ha framgått att poeterna i hög grad är orienterade mot formen. Det kan gälla visuella drag i den tryckta texten, och ytterligare ett exempel på detta kan vara Lillpers tendens att pausa vid versradsslut, också vid överklivning – hörbart åt minstone när man lyssnar med texten för ögonen.55 Lundquists, Burraus och andras tonalt regelbundna eller förutsägbara läsningar har däremot ingen för mig uppenbar förankring i deras texter. En uppläsning uppstår naturligtvis alltid i mötet mellan texten, med dess olika egenskaper, och uppläsarens vilja och förmåga till gestaltning. I dessa och andra fall tycks emellertid det sistnämnda vara överordnat – med andra ord tror jag att Lundquist och Burrau skulle kunna läsa nästan vilken text som helst på samma säregna vis. Noter 1 Solveig Lundgren, Dikten i etern. Radion och skönlitteraturen 1925–1955 [diss.] (Uppsala: Avd. för litteratursociologi vid Litteraturvetenskapliga institutionen, 1994), 68–103; Lars Hänström, Lär mig att mäta tiden. Dagens dikt 1955–1989 (Stockholm: Skrifter utgivna av Stiftelsen Etermedia i Sverige, 1997). 2 Hänström. Lär mig att mäta tiden, 93f. 2 Hänström. Lär mig att mäta tiden, 93f. 3 Fredrik Nyberg, Hur låter dikten? Att bli ved II [diss.] (Göteborg: Autor, 2013), 53; Peter Middleton, Distant Reading. Performance, Readership, and Consumption in Contemporary Poetry (Tuscaloosa: University of Alabama Press, 2005), 61ff. 3 Fredrik Nyberg, Hur låter dikten? Att bli ved II [diss.] (Göteborg: Autor, 2013), 53; Peter Middleton, Distant Reading. Performance, Readership, and Consumption in Contemporary Poetry (Tuscaloosa: University of Alabama Press, 2005), 61ff. yf 4 Lundgren, Dikten i etern, 62; Lesley Wheeler, Voicing American Poetry. Sound and Perfor mance from the 1920s to the Present (Ithaca: Cornell University Press, 2008), 4. f 4 Lundgren, Dikten i etern, 62; Lesley Wheeler, Voicing American Poetry. Sound and Perfor mance from the 1920s to the Present (Ithaca: Cornell University Press, 2008), 4. 5 Lundgren, Dikten i etern, 76, 235 (not 24). Vad som sannolikt är en av dessa inspelningar, av dikten ”Evighet”, återutsändes för övrigt under den period från vilken materialet till denna studie hämtats; den har liksom övriga inspelningar fått ett ID-nummer motsva rande sändningens år, månad och dag: 210604. Fortsättningsvis används dessa nummer vid referens till enskilda inspelningar. 5 Lundgren, Dikten i etern, 76, 235 (not 24). Vad som sannolikt är en av dessa inspelningar, av dikten ”Evighet”, återutsändes för övrigt under den period från vilken materialet till denna studie hämtats; den har liksom övriga inspelningar fått ett ID-nummer motsva rande sändningens år, månad och dag: 210604. Fortsättningsvis används dessa nummer vid referens till enskilda inspelningar. 6 Karl Berglund, ”Strömmade bästsäljare. Litteraturkonsumtion i digitala prenumerations tjänster utifrån Storytels användardata”, i Från Strindberg till Storytel. Korskopplingar mellan ljud och litteratur, Julia Pennlert & Lars Ilshammar red. (Göteborg: Daidalos, 2021), 327–359 (352); i Berglunds material läses endast 4 procent av deckarna av förfat taren, medan andelen för kvalitetslitteratur är 22 procent. 6 Karl Berglund, ”Strömmade bästsäljare. Litteraturkonsumtion i digitala prenumerations tjänster utifrån Storytels användardata”, i Från Strindberg till Storytel. Korskopplingar mellan ljud och litteratur, Julia Pennlert & Lars Ilshammar red. (Göteborg: Daidalos, 2021), 327–359 (352); i Berglunds material läses endast 4 procent av deckarna av förfat taren, medan andelen för kvalitetslitteratur är 22 procent. Kvalitativ avslutning När han läser en dikt av Anne Sexton är han faktiskt mer konventionell och lik en skådespelare.60 Vad som hos Lind uppfattas som idiosynkratiskt, för att inte säga excentriskt, och därmed autentiskt, hade kanske bara uppfattats som konstigt om någon annan läst. Om så är fallet är kanske problemet med skådespelare inte bara sättet 122 TFL 2022:2–3 FORSKNINGSARTIKEL de läser på. Även UKONs invändning, som det nu är dags att återvända till, behöver då kompletteras. Problemet är inte primärt att skådespelarna är just skådespelare, utan att de inte själva skrivit texten de läser. de läser på. Även UKONs invändning, som det nu är dags att återvända till, behöver då kompletteras. Problemet är inte primärt att skådespelarna är just skådespelare, utan att de inte själva skrivit texten de läser. Noter 7 Karl Berglund, ”Kvalitetslitteratur och medieformat”, i Skilda världar. Kvalitetslitteraturens villkor i Sverige idag, Jerry Määttä, Ann Steiner & Karl Berglund (Stockholm: Svenska Förläggareföreningen, 2022), särskilt 70ff. gg gf 8 Ulf Karl Olov Nilsson, Röstautograferna (Stockholm: Norstedts, 2019), 66f. 9 Malte Persson, Till dikten (Stockholm: Bonniers, 2018), 31. 10 Stina Ekblad, Här brusar strömmen förbi (Stockholm: Weyler, 2020), 92f. Också poeten Börje Lindström (med tre läsningar under den undersökta perioden) menar att skåde spelarna generellt är att föredra numera, och att de poeter som nedvärderar skådespelare talar om hur det var på 1980-talet och tidigare, då skådespelare ofta läste som om de stod på scen (mejl till artikelförfattaren 2022-09-01). 11 ”Läsa poesi – högläsningens konst”, Örnen och Kråkans poesipodd: https://soundcloud. com/user-107040597/lasa-poesi-hoglasningens-konst [08:55–11:00]. William-Olsson samtalar här med skådespelaren Paula Brandt. 12 Marjorie Perloff, ”Language Poetry and the Lyric Subject. Ron Silliman’s Albany, Susan Howe’s Buffalo”, Critical Inquiry vol. 25 (1999:3), 405–438, https://doi.org/10.1086/448929; Peter Stein Larsen, Drømme og dialoger. To poetiska traditioner omkring 2000 (Odense: Syd dansk Universitetsforlag, 2009); Ian Patterson, ”No man is an I. Recent developments in the lyric”, i The Lyric Poem. Formations and Transformations, Marion Thain red. (Cambrid ge: Cambridge University Press, 2013), 217–236; Jimmie Svensson, ”Poet Voice på svenska. Poetisk tradition i uppläsningar av Ida Börjel, Athena Farrokhzad och Jenny Wrangborg”, Nordisk poesi vol. 5 (2020:2), 92–110, https://doi.org/10.18261/issn.2464-4137-2020-02-04. p p g 13 200701–3, 200717, 200722, 200730, 200928–30, 201001, 201027, 201031, 201214, 201220, 201224–5, 210311, 210313, 210316, 210324, 210326, 210424, 210510–12, 210514, 210604, 210615. JIMMIE SVENSSON TFL 123 14 Marit J. MacArthur, Georgia Zellou & Lee M. Miller, ”Beyond Poet Voice. Sampling the (Non-) Performance Styles of 100 American Poets”, Journal of Cultural Analytics (2018:1), 1–72, https://doi.org/10.31235/osf.io/5vazx. Programvaran finns tillgänglig här: https:// github.com/MillerLab-UCDavis/Voxit/tree/master/Voxit.f 15 MacArthur et al., ”Beyond Poet Voice”, 29ff.; Jimmie Svensson, ”Fjärrlyssning. En kvanti tativ studie av uppläst svensk samtidspoesi”, Edda vol. 109 (2022:3), 184–205, https://doi. org/10.18261/edda.109.3.4. 16 Testerna/metoderna som jag här redovisar resultaten för (enligt Xijia Lius rapport) är inom statistiken kända som MANOVA respektive logistic regression. 17 Svensson, ”Fjärrlysning”, 190. 18 Ibid. 19 Hänström, Lär mig att mäta tiden, 41. 20 Mejl till artikelförfattaren 2022-01-16; under fem veckor i september och början av okto ber var en semestervikarie producent, och under den undersökta perioden återutsändes även flera äldre uppläsningar. l 21 Svensson, ”Fjärrlyssning”, 191f. 22 MacArthur et al., ”Beyond Poet Voice”, 30; Svensson, ”Fjärrlyssning”, 192. Noter 23 Eva Lilja, Bergur Djuurhus & Rasmus Dahl Vest red., ”Inledning”, i Långa dikter. Berät telse, experiment, politik (Bergen: Alvheim & Eide, 2016), 7–15. 24 210617. I Johan Jönson, ProponeisiS (Stockholm: Bonniers, 2021), opag., inleds stycket med det ord som för utsändningen gjorts till titel; jfr Harry Martinsons ”Juninatten” som sänds någon vecka senare i juni (210626). 25 210608–10 och 210612. 26 210414. 26 210414. 27 Svensson, ”Fjärrlyssning”, 195. 28 Svensson, ”Fjärrlyssning”, 195f. 29 MacArthur et al., ”Beyond Poet Voice”, 46.f 30 I. R. Titze, ”Physiologic and accoustic difference between male and female voices”, h 30 I. R. Titze, ”Physiologic and accoustic difference between male and female voices”, The Journal of the Accoustical Society of America vol. 85 (1989:4), 1699–1707, https://doi org/10.1121/1.397959; jfr Svensson, ”Fjärrlyssning”, 195f. S M i K b Wi f i d M i h A R h & M hi S h i 3 , y gf , The Journal of the Accoustical Society of America vol. 85 (1989:4), 1699–1707, https://doi. / / jf S ”Fjä l i ” f 31 Se t.ex. Maria Kraxenberger, Winfried Menninghaus, Anna Roth & Mathias Scharinger, ”Prosody-Based Sound-Emotion Associations in Poetry”, Frontiers in Psychology vol. 9 (2018), 1–10, https://doi.org/10.3389/fpsyg.2018.01284. 32 Marit J. MacArthur, ”Monotony, the Churches of Poetry Reading and Sound Studies”, PMLA vol. 131 (2016:1), 38–63, https://doi.org/10.1632/pmla.2016.131.1.38. 33 Nilsson, Röstautograferna, 45. 34 Nilsson, Röstautograferna, 51. 35 Se vidare Svensson, ”Poet Voice på svenska”, för tre exempel på sådana individuella tillämpningar. Jfr även beskrivningen av Marie Silkeberg av Hans Kristian S. Rustad, ”Nordisk poesifestival \| Rolf Jacobsen-dagerne”, i Digtoplæsning. Former og fællesskaber, Louise Mønster, Hans Kristian S. Rustad & Michael Kallesøe Schmidt (Bergen: Fagbokforlaget, 2022), https://doi.org/10.55669/oa1103: ”Silkeberg har en karakteristisk opplesningsstemme. Det er først og fremst denne som sitter igjen når man har hørt henne lese opp, og som gjør det nærmest umuligt å lese hennes dikt uten å bli påminnet hennes særegne, distanserte og nærmest mekaniske stemme samt bestemte og selvsikre framtoning.” (50). 36 MacArthur et al., ”Beyond Poet Voice”, 69f. 37 Svensson, ”Poet Voice på svenska”, 97, 107f.; Svensson, ”Fjärrlyssning”, 197ff. 38 I studien används följande formel (se Svensson, ”Fjärrlyssning”, 187): Dynamism = Pitch speed/1.092050992 + Pitch entropy/3.331034878 + (Complexity of syllables/13.18735087+C omplexity of Phrases/3.022951534)/2. Noter 124 TFL 2022:2–3 FORSKNINGSARTIKEL 39 Svensson, ”Fjärrlyssning”, 193; medianvärdet är för poeterna 2,96, för fritt tal 3,56 och för uppläst romanprosa 3,66 – att jämföra med 3,14 för poeterna och 3,53 för skådespelarna i föreliggande studie (ett mindre antal individer förekommer i båda materialen). 39 Svensson, ”Fjärrlyssning”, 193; medianvärdet är för poeterna 2,96, för fritt tal 3,56 och för uppläst romanprosa 3,66 – att jämföra med 3,14 för poeterna och 3,53 för skådespelarna i föreliggande studie (ett mindre antal individer förekommer i båda materialen). 40 Jfr Hänström, Lär mig att mäta tiden, 20f., om hur uppläsningarna i Dagens dikts barndom kritiserades för att vara alltför präglade av den tidens teaterkonventioner 40 Jfr Hänström, Lär mig att mäta tiden, 20f., om hur uppläsningarna i Dagens dikts barndom kritiserades för att vara alltför präglade av den tidens teaterkonventioner. 41 För en introduktion till metoddiskussioner inom fältet digital humaniora, se Johan Jarlbrink & Fredrik Norén red., ”Inledning”, i Digitala metoder i humaniora och samhälls vetenskap (Lund: Studentlitteratur, 2021), 11–26. 41 För en introduktion till metoddiskussioner inom fältet digital humaniora, se Johan Jarlbrink & Fredrik Norén red., ”Inledning”, i Digitala metoder i humaniora och samhälls vetenskap (Lund: Studentlitteratur, 2021), 11–26. 42 Poeten Erik Bergqvist, som bara läser en egen dikt (210622) och därutöver en av Eva- Liisa Manner (200728), en av Gunnar Harding (200921) och två av Jacques Ancet i egen översättning (201117 och 201205) har också relativt stora variationer i värden. 43 200707 och 200711; ett annat exempel är Elisabeth Berchtholds prosaliknande 210505 och 210506 jämfört med den mer fragmentariska 210521. 44 200706.f 43 200707 och 200711; ett annat exempel är Elisabeth Berchtholds prosaliknande 210505 och 210506 jämfört med den mer fragmentariska 210521. 6 43 200707 och 200711; ett annat exempel är Elisabeth Berchtholds prosaliknande 210505 och 210506 jämfört med den mer fragmentariska 210521. 44 200706. 45 Se vidare Eva Lilja, Svensk metrik (Stockholm: Norstedts, 2006), 173ff. Sammanlagt 36 schematiskt metriska dikter finns i materialet, alla utom en (210112) lästa av skådespelare, exempelvis Rilke av Anna Pettersson (201020) och Tranströmer av Christian Fex (201109). Utifrån de 12 prosodiska måtten skiljer sig inte läsningarna av metriska dikter från övriga; här mäts dock inte intensitet och längd, vilka tillsammans med tonhöjden kan utgöra den upplevda betoningens fonetiska korrelat. 46 210122 (Szymborska) och 210201–6 (Margolin). 46 210122 (Szymborska) och 210201–6 (Margolin). 46 210122 (Szymborska) och 210201–6 (Margolin). Noter 47 Att relativt hög tonhöjd förbinds med mindre varelser och låg med större följer en univer sell biologisk kod, se vidare Nina El Zarka, ”Pragmatic functions and the biological codes. Evidence from the prosody of sentence topic and focus in Egyptian Arabic declaratives”, i Prosody and Iconicity, Sylvie Hancil & Daniel Hirst red. (Amsterdam & Philadelphia: John Benjamins, 2013), 109–126, https://doi.org/10.1075/ill.13.06zar. 47 Att relativt hög tonhöjd förbinds med mindre varelser och låg med större följer en univer sell biologisk kod, se vidare Nina El Zarka, ”Pragmatic functions and the biological codes. Evidence from the prosody of sentence topic and focus in Egyptian Arabic declaratives”, i Prosody and Iconicity, Sylvie Hancil & Daniel Hirst red. (Amsterdam & Philadelphia: John Benjamins, 2013), 109–126, https://doi.org/10.1075/ill.13.06zar. 48 200914 respektive 210416. 49 Exempelvis i 201024. 50 200716, 201215 och 210118.h 51 Reuven Tsur, Toward a Theory of Cognitive Poetics (Brighton & Portland: Sussex Academic Press, 2008), 216–234. 51 Reuven Tsur, Toward a Theory of Cognitive Poetics (Brighton & Portland: Sussex Academic Press, 2008), 216–234. 52 Johnson (exempelvis 210405) kan här jämföras med skådespelarna Almén (201022) och Ooms (210315). 53 210423. 54 Särskilt 210505. 55 201102 och 201107. 56 MacArthur, ”Monotony, the Churches of Poetry Reading and Sound Studies”, 46. 57 Ekblad, Här brusar strömmen förbi, 93. 58 Hör exempelvis 210419, från ”Den mörka tonen…” [0:46] och några versrader framåt. 58 Hör exempelvis 210419, från ”Den mörka tonen…” [0:46] och några versrader framå 59 Jörgen Lind, Mott (Stockholm: Bonniers, 2021), 122. JIMMIE SVENSSON TFL 125
https://openalex.org/W2899934935	https://periodicos.ufsc.br/index.php/textodigital/article/download/1807-9288.2018v14n1p126/37110	Portuguese	null	Intertextualidade e estranhamento em "Amor de Clarice", de Rui Torres	Texto digital	2,018	cc-by	5,630	Intertextuality and estrangement in “Amor de Clarice”, by Rui Torres Vinícius Carvalho Pereiraa Intertextuality and estrangement in “Amor de Clarice”, by Rui Torres Vinícius Carvalho Pereiraa a Departamento de Letras, Universidade Federal de Mato Grosso, Cuiabá, Brasil - viniciuscarpe@gmail.com Palavras-chave: Rui Torres. Poesia digital. Releitura. Intertextualidade. Estranhamento. Resumo: No âmbito da literatura de vanguarda, o lugar-comum “toda obra enseja múltiplas leituras” corre um duplo risco: o de implodir, sob o peso de textos que não permitem leitura alguma (ou que a tanto se propõem), dado o hermetismo formal; e o de explodir, mediante a pressão centrífuga de obras que não são as mesmas a cada leitura sequer na materialidade discursiva. É dessa categoria de obras movediças que se fala aqui, na condição de aporias da leitura literária no século XXI, em que inovações estruturais do texto vão com frequência a reboque de inovações tecnológicas, as quais permitem transformações materiais na obra. No caso da ciberliteratura, tal afirmação torna-se ainda mais pertinente, uma vez que os recursos computacionais, cada vez mais sofisticados, vêm ensejando uma poética antes inimaginável, sobretudo em sua multiplicidade de figurações e leituras, dada a condição de devir em que habita sua substância. Nesse contexto, optou-se neste artigo pela análise da obra Amor de Clarice, do poeta português Rui Torres. Esta, valendo-se de distintos recursos tecnoestéticos digitais, potencializa a máxima “Toda obra enseja múltiplas leituras”, uma vez que relê o conto “Amor”, de Clarice Lispector, por meio de uma materialidade discursiva informática que se dissolve e transforma a cada clique no mouse. O adágio perde, assim, ares de lugar-comum desgastado pelo uso na academia e retoma, por vias algorítmicas, uma potência que só a arte enquanto estranhamento estético – proposta, em última medida, de toda releitura – pode deter. Keywords: Rui Torres. Digital poetry. Re-reading. Intertextuality. Estrangement. Abstract: In avant-garde literature, the commonplace “all works engender many readings” is twice at risk: the risk of imploding, overwhelmed by texts that do not allow any reading (or that are not supposed to do it), due to their formal hermeticism; or the risk of exploding, owing to the centrifugal pressure of works that do not remain the same even in material terms. We speak here of these ever- shifting works, as aporias of literary readings in the 21st century, where structural innovations in texts go hand in hand with technical innovations that permit material changes in texts. Keywords: Rui Torres. Digital poetry. Re-reading. Intertextuality. Estrangement. Texto Digital, Florianópolis, v. 14, n. 1, p. 126-138, jan./jun. 2018. http://dx.doi.org/10.5007/1807-9288.2018v14n1p126 Texto Digital, Florianópolis, v. 14, n. 1, p. 126-138, jan./jun. 2018. http://dx.doi.org/10.5007/1807-9288.2018v14n1p126 Esta obra foi licenciada com uma Licença Creative Commons Atribuição 4.0 Internacional SOBRENOME AUTOR, SOBRENOME AUTOR, SOBRENOME AUTOR story “Amor”, by Clarice Lispector, through a computational discursive materiality that dissolves and changes after every click on the mouse. Thereby, this dictum overcomes academia commonsenses and, through algorithmic means, recaptures a potential that lies only in art as an aesthetical estrangement – the proposal of any re-reading after all. story “Amor”, by Clarice Lispector, through a computational discursive materiality that dissolves and changes after every click on the mouse. Thereby, this dictum overcomes academia commonsenses and, through algorithmic means, recaptures a potential that lies only in art as an aesthetical estrangement – the proposal of any re-reading after all. INTRODUÇÃO Toda obra enseja múltiplas leituras: primeira lição de qualquer curso de Literatura, seja no ensino básico ou superior, variando apenas o grau de elaboração da paráfrase e os argumentos mobilizados. No entanto, a ideia geral permanece a mesma, especialmente quando se consideram obras produzidas em suportes semelhantes, para regimes de leitura análogos, com estatuto literário igualmente chancelado pela academia. Tomando, porém, obras da literatura de vanguarda, o lugar-comum “toda obra enseja múltiplas leituras” corre um duplo risco: o de implodir, sob o peso de textos que não permitem leitura alguma (ou que a tanto se propõem), dado o hermetismo formal; e o de explodir, mediante a pressão centrífuga de obras que, sequer na materialidade discursiva (FOUCAULT, 1986), permanecem as mesmas a cada leitura. É dessa categoria de obras movediças que se fala aqui, na condição de aporias da leitura literária no século XXI, em que inovações estruturais do texto vão com frequência a reboque de inovações tecnológicas, as quais permitem transformações materiais na obra. No caso da ciberliteratura, tal afirmação torna-se ainda mais pertinente, uma vez que os recursos computacionais, cada vez mais sofisticados, vêm ensejando uma poética antes inimaginável, sobretudo em sua multiplicidade de figurações e leituras, dada a condição de devir em que habita sua substância. Diante da miríade de textos com que a Internet embaralha nossos olhos – variedade vertiginosa acessível a um clique –, opta-se neste artigo por uma análise mais detida da obra digital Amor de Clarice, do poeta português Rui Torres, professor e pesquisador da Universidade Fernando Pessoa, bem como membro de uma série de grupos de pesquisa e projetos internacionais na área de Estéticas Digitais. Tal autor vem se dedicando há quase duas décadas ao labor de crítico, pesquisador e poeta das literaturas digitais, tendo criado textos antológicos nessa vertente estética, tais como Tema procura-se (2004). Nessa obra, um poema é formado aleatoriamente por um motor textual, o qual recombina substantivos e adjetivos de seu banco de dados, automaticamente ou mediante cliques do É dessa categoria de obras movediças que se fala aqui, na condição de aporias da leitura literária no século XXI, em que inovações estruturais do texto vão com frequência a reboque de inovações tecnológicas, as quais permitem transformações materiais na obra. Intertextuality and estrangement in “Amor de Clarice”, by Rui Torres Vinícius Carvalho Pereiraa In the case of cyberliterature, this utterance is even more relevant, since computer resources, increasingly sophisticated, claim for an unthought-of poetic, multiple in figurations and readings, due to the ever-shifting condition of its substance. In this article, we analyze Amor de Clarice, by the Portuguese poet Rui Torres. By means of varied digital technoaesthetic resources, this work potencializes the adage “all works engender many readings”, as it re-reads the short Esta obra foi licenciada com uma Licença Creative Commons Atribuição 4.0 Internacional Esta obra foi licenciada com uma Licença Creative Commons Atribuição 4.0 Internacional SOBRENOME AUTOR, SOBRENOME AUTOR, SOBRENOME AUTOR 1 Um primeiro dilema, de ordem comezinha, imposto pela ciberliteratura, coloca-se aqui: tratando-se de obra de autoria coletiva (ou difusa, ou compartilhada, como queira o referencial teórico adotado), a quem se atribui a obra para fins de registro (em citações na academia, patentes na indústria, ou contratos de direitos autorais no mercado editorial)? Apenas ao autor do texto verbal? Inclui-se o autor ou performer de componentes multimodais, como imagens e som? Considera-se o programador, que escreve algoritmos que constituem o estrato formal da obra? E o leitor, que acaba co-criando na medida em que seus cliques desencadeiam mudanças na materialidade do texto? Essa questão será tangenciada mais à frente no texto, mas, por ora, para fins de citação nos padrões acadêmicos, optou-se por atribuir a autoria de Amor de Clarice a Rui Torres, (co?)criador do texto verbal. INTRODUÇÃO No caso da ciberliteratura, tal afirmação torna-se ainda mais pertinente, uma vez que os recursos computacionais, cada vez mais sofisticados, vêm ensejando uma poética antes inimaginável, sobretudo em sua multiplicidade de figurações e leituras, dada a condição de devir em que habita sua substância. Diante da miríade de textos com que a Internet embaralha nossos olhos – variedade vertiginosa acessível a um clique –, opta-se neste artigo por uma análise mais detida da obra digital Amor de Clarice, do poeta português Rui Torres, professor e pesquisador da Universidade Fernando Pessoa, bem como membro de uma série de grupos de pesquisa e projetos internacionais na área de Estéticas Digitais. Tal autor vem se dedicando há quase duas décadas ao labor de crítico, pesquisador e poeta das literaturas digitais, tendo criado textos antológicos nessa vertente estética, tais como Tema procura-se (2004). Nessa obra, um poema é formado aleatoriamente por um motor textual, o qual recombina substantivos e adjetivos de seu banco de dados, automaticamente ou mediante cliques do 127 SOBRENOME AUTOR, SOBRENOME AUTOR, SOBRENOME AUTOR leitor, em ritmo articulado ao de vozes que soam ao fundo, lendo um texto outro que nunca aparece na tela. Também com base em diferentes algoritmos de combinatória e recursos multimodais, Rui Torres produziu obras em Flash que põem em releitura e fluxos de hibridação clássicos da literatura portuguesa, como 8 brincadeiras para Salette Tavares (2010), relendo textos da poetisa experimentalista; Baladas eletrônicas para AlletSator (2009), com base em Álea e Vazio (1971), de E. M. de Melo e Castro, e Aforismos (1988), de Pedro Barbosa; Húmus poema contínuo (2006), jogando com Húmus (1967), de Herberto Helder, e Húmus (1917), de Raul Brandão; e Mar de Sophia (2004), a partir de poemas de Sophia de Mello Breyner Andresen e Lewis Carroll. No conjunto da produção torreana, explicitamente intertextual e digital, optou-se neste artigo pela análise da obra Amor de Clarice, porque esta, valendo-se de distintos recursos tecnoestéticos, enseja um número ainda maior de efeitos de sentido – e portanto de leituras –, de modo a explodir a máxima “Toda obra enseja múltiplas leituras”. Tal aforismo perde assim ares de lugar-comum desgastado pelo uso na academia e retoma, por vias algorítmicas, uma potência que só a arte enquanto estranhamento estético – proposta, em última medida, de toda releitura – pode deter. 2 Portal de literatura portuguesa experimental, sob curadoria de diversos pesquisadores, coordenados pelo próprio Rui Torres. “AMOR DE CLARICE”: RELEITURA E ESTRANHAMENTO Amor de Clarice (TORRES1, 2005) é um ciberpoema produzido no bojo dos experimentalismos literários digitais em Portugal, composto a partir do conto “Amor” (1998), de Clarice Lispector. Segundo a taxonomia adotada pelo Arquivo Digital da PO.EX2 (2015), referência nos estudos portugueses de ciberliteratura, Amor de Clarice é uma obra metatextual alógrafa, pois se enquadra entre “textos acerca de obras e práticas artísticas produzidos por agentes que não são autores das obras” (PO.EX, 2015, n.p.). 2 Portal de literatura portuguesa experimental, sob curadoria de diversos pesquisadores, coordenados pelo próprio Rui Torres. 128 SOBRENOME AUTOR, SOBRENOME AUTOR, SOBRENOME AUTOR Tal categorização segue princípios estabelecidos por Genette (1982), que cunhou o conceito de metatextualidade para se referir a relações transtextuais do tipo comentário, frequentemente englobando gêneros como o ensaio, a dissertação ou o prefácio. A partir daí, percebe-se o entendimento de Rui Torres diante de seu poema como crítica literária ao conto da modernista brasileira, e não apenas como uma releitura artística. Tal perspectiva é corroborada por Torres em recensão crítica sobre Amor de Clarice, na qual o autor afirma que concebeu a releitura com vistas a “ampliar o lado libertador da personagem [...] no sentido de fazer uma re-escrita poética de certas frases, metáforas e imagens que Lispector explora ao longo do seu texto e que consider[a] características duma ilegibilidade fundamental” (TORRES, 2003, p.1, grifo nosso). Ao longo da resenha, o autor sublinha ainda a ideologia feminista subjacente ao conto clariceano, na medida em que a personagem principal, por um momento de epifania, liberta-se da rotina esvaziada e insossa que a sociedade lhe impunha na condição de mulher3. Nesse diapasão, os procedimentos de desnaturação da percepção e da significação que a releitura ciberpoética opera nos planos da expressão e do conteúdo “ampliariam” a transgressão da personagem. Amor de Clarice é composto de interfaces multimodais que transcriam o conto “Amor” ao recortar e recombinar elementos de sua superfície textual (palavras, lexias, orações ou frases). Estes, rearranjados sintaticamente em unidades novas, piscam, deslizam e se sobrepõem na interface, em direções e tempos distintos, como se vê na Figura 1, que representa uma captura da segunda tela do poema. Por sua vez, o leitor, enquanto assiste à deriva das palavras, pode também perpetrar desconstruções da materialidade discursiva do poema, tanto no estrato visual quanto no fônico. 3 De maneira bastante sucinta, pode-se dizer que o enredo do conto se centra no processo epifânico por que passa a protagonista Ana, ao ver, do assento do bonde onde estava, um cego que mascava chicletes na calçada. Tal evento dispara-lhe um processo de ressignificação de si e do mundo a seu redor, bem como uma desnaturação da percepção, que culminam em uma desconstrução de suas certezas mais íntimas acerca de sua vida então tomada pela rotina de mulher casada, mãe, dona de casa, onde não havia espaço para o imprevisto, a desordem, o caos. “AMOR DE CLARICE”: RELEITURA E ESTRANHAMENTO Para tal, pode arrastar suas frases para a direita ou para a esquerda com o cursor, criando novas associações ou sobreposições vocabulares – deslocamento ou abjuração de sentidos? –, ou clicar sobre as palavras para que estas sejam pronunciadas novamente na leitura em off – replicação ou refração da voz? Perguntas, enfim, que toda a crítica da desconstrução (DERRIDA, 2008), em sua empreitada contra discursos monolíticos e canônicos – inclusive os literários –, bem sabe fazer. 129 3 De maneira bastante sucinta, pode-se dizer que o enredo do conto se centra no processo epifânico por que passa a protagonista Ana, ao ver, do assento do bonde onde estava, um cego que mascava chicletes na calçada. Tal evento dispara-lhe um processo de ressignificação de si e do mundo a seu redor, bem como uma desnaturação da percepção, que culminam em uma desconstrução de suas certezas mais íntimas acerca de sua vida então tomada pela rotina de mulher casada, mãe, dona de casa, onde não havia espaço para o imprevisto, a desordem, o caos. 129 SOBRENOME AUTOR, SOBRENOME AUTOR, SOBRENOME AUTOR Fig. 1 – Segunda tela de Amor de Clarice. Fig. 1 – Segunda tela de Amor de Clarice. Fonte: TORRES, 2005. Fonte: TORRES, 2005. Tal releitura se complica ainda mais quando o leitor percebe que a sintaxe de desmontes e rearranjos não se restringe a cada cena da obra individualmente, com suas frases formadas por partes do conto. Em vez disso, os jogos de combinatória percorrem todo o Amor de Clarice, como um leitmotiv de poética de fragmentos. Tal releitura se complica ainda mais quando o leitor percebe que a sintaxe de desmontes e rearranjos não se restringe a cada cena da obra individualmente, com suas frases formadas por partes do conto. Em vez disso, os jogos de combinatória percorrem todo o Amor de Clarice, como um leitmotiv de poética de fragmentos. Como regra desse ludo literário-computacional, o poema de Torres é composto por duas séries de interfaces, cada uma segmentada em 26 unidades, entre as quais se distribuem fragmentos e transcriações de cenas do conto, como se nos informa na tela inicial de Amor de Clarice. Da primeira tela (paratexto anterior ao poema, descrevendo sua macroestrutura) à segunda, exibida acima, a passagem é unidirecional: ao clicar em “Iniciar poema”, o leitor é invariavelmente remetido à mesma tela (Figura 1), que apresenta um mosaico lexical da cena mais emblemática do conto, quando Ana, no clímax da narrativa, sofre uma experiência drástica, passando a ver o mundo de outro modo. No texto de Clarice, essa mudança de perspectiva é metaforizada pela alteração dos sentidos da protagonista, especialmente do tato: “A rede de tricô era áspera entre os dedos, não íntima como quando a tricotara. A rede perdera o sentido e estar num bonde era um fio partido” (LISPECTOR, 1998, p.22). A malha que até então atava docilmente os ovos carregados por Ana no bonde torna-se aí um objeto estranho, incômodo à mão que testemunha o quebrar dos ovos – o irromper de um novo mundo. Já no ciberpoema, mais que casca, clara ou gema, rompem-se também as frases do conto para, recombinadas as palavras, gerar inauditas imagens da transformação por que passa a personagem, surgidas como dos ovos que rebentam e escorrem na tela revelada na Figura 1: “quebram-se os ovos/e a rede”, “mas os dias forjados 130 SOBRENOME AUTOR, SOBRENOME AUTOR, SOBRENOME AUTOR para sempre se rompem”, “ana prende os instantes entre os dedos” (TORRES, 2005). Fig. 1 – Segunda tela de Amor de Clarice. Junto à protagonista, o leitor do ciberpoema alucina a visão diante da tela movediça (qual o mundo que Ana via do bonde) e o tato angustiado do mouse ou do teclado (rede de tricô tão áspera), a partir de um simples evento que desencadeia todo o torvelinho estético seu e de Ana. Na obra de Clarice, uma visão no bonde; na de Rui Torres, um clique na tela. Lançado, então, o leitor do ciberpoema em vertigem análoga à da personagem, ao questionar sua própria percepção do mundo, resta-lhe apenas adentrar a também vertiginosa obra digital, no que a passagem da segunda tela à próxima se dá necessariamente de modo aleatório. Um clique em qualquer parte da tela na Figura 1 lança-o randomicamente para dentro ou da primeira ou da segunda série de interfaces que recontam a história da protagonista, como lance de dados caro aos jogos de azar e combinatória. Se lançado na primeira série, o leitor se defrontará com uma sucessão de interfaces que têm, como plano de fundo, sobreposições de excertos do conto clariceano, possivelmente sugerindo os vários estratos de significação de uma obra literária – suas “múltiplas leituras” –, as quais explodem exponencialmente no jogo da transcriação digital (Figura 2). No primeiro plano de cada uma dessas telas, mais sintagmas que recombinam trechos da obra original, ora subvertendo, ora amplificando seus sentidos. Em off, ouve-se a leitura dos sintagmas tremeluzindo na tela. Já se lançado na segunda série, o leitor encontrará segmentos que têm como plano de fundo vídeos de objetos turvos e pulsantes em loop, os quais remetem, como índices, à narrativa de “Amor” (Figura 3), tais quais uma xícara, um relógio, ou um vaso de flor. No primeiro plano, novamente sintagmas recombinando o léxico do conto grassam à deriva, enquanto no fundo se escuta a leitura de trechos do conto de Clarice. 131 SOBRENOME AUTOR, SOBRENOME AUTOR, SOBRENOME AUTOR Fig. 2 e 3 – Respectivamente, telas aleatórias da primeira e da segunda série de vídeos de Amor de Clarice. Fonte: TORRES, 2005. g. 2 e 3 – Respectivamente, telas aleatórias da primeira e da segunda série de vídeos de Amor de Clarice. Fonte: TORRES, 2005. Fig. 1 – Segunda tela de Amor de Clarice. Para navegar hipertextualmente ao longo de cada série ou “zapear” entre elas, dispõe-se de um menu no canto superior direito da tela, o qual permite avançar ou recuar entre as interfaces de uma mesma série; voltar ao menu principal (e talvez mudar de série, a depender da aleatória saída da segunda tela do poema); ou navegar de modo randômico, saltando entre as interfaces de qualquer uma das séries. Para ensejar todos esses procedimentos estéticos no plano da expressão, a equipe que compôs Amor de Clarice teve de lançar mão de uma série de recursos algorítmicos, que desencadeiam a combinatória dos elementos. Nessa versão do ciberpoema, Rui Torres fez a programação em Flash/Actionscript; Ana Carvalho foi responsável pelos vídeos da segunda série; Carlos Morgado e Luis Aly desenvolveram os sons musicais executados ao fundo; e Nuno M. Cardoso emprestou a voz que lê trechos recombinados por Torres na primeira série e trechos do conto original na segunda (respectivamente, o hipotexto e o hipertexto, conforme nomenclatura adotada por Genette (1982) e retomada por Torres (n.d.) na apresentação de Amor de Clarice). Diante dessa empreitada arquitetônica em equipe, pode-se entender a releitura do conto clariceano como procedimento engenhoso, de trapaça e subversão de uma obra do cânone brasileiro em um jogo a várias mãos, bem ao gosto do que Barthes definiu como sendo a Literatura: “Essa trapaça salutar, essa esquiva, esse logro magnífico que permite ouvir a língua fora de seu poder, no esplendor de uma revolução permanente da linguagem, eu a chamo, quanto a mim: literatura” (BARTHES, 1992, p. 16). 132 SOBRENOME AUTOR, SOBRENOME AUTOR, SOBRENOME AUTOR A ideia de trapaça aqui constela uma série de sentidos, uma vez que o logro só se dá dentro do jogo, subvertendo suas regras sem romper com ele, tal como Barthes viu no funcionamento da Literatura dentro da língua, ludibriando o sistema sem estourar o código. Ademais, a ideia de subversão que a trapaça veicula está intimamente relacionada à tônica feminista do conto “Amor” e à releitura crítica feita por Torres em seu ciberpoema, conforme o autor português ressalta em resenha sobre a obra (TORRES, 2003). SOBRENOME AUTOR, SOBRENOME AUTOR, SOBRENOME AUTOR Clarice reforçam a literariedade do poema, se considerarmos a perspectiva formalista de Chklovsky (2013a), pois complexificam a materialidade da obra e a percepção que ela conclama, chocando-se trapaceiramente contra o horizonte de expectativas dos leitores conhecedores (ou não) do conto original. No que tange a esse logro, que refrata os sentidos que do texto se esperavam, tornando opaca sua superfície, também no plano do conteúdo de Amor de Clarice comparece explicitamente o tema da desautomatização dos sentidos, “aumenta[ndo] a dificuldade da forma e a duração da percepção” (CHKLOVSKY, 2013a, p.91). Conforme comentado anteriormente, o poema se inicia com um mosaico verbal da cena em que Ana passa a ver o mundo de outra forma, metaforizada pelo estranhamento tátil ante a rede de tricô que sustinha os ovos, então rompidos. Curiosamente, na tela do ciberpoema que narra essa cena (Figura 1), o texto verbal que aparece como plano de fundo não é um trecho do conto de Clarice; trata-se, pois, de trapaça à trapaça, subvertendo a sobreposição de hipotexto e hipertexto anteriormente apresentada na primeira série de Amor de Clarice. Em vez de frases de “Amor”, o plano de fundo dessa tela é excepcionalmente composto de fragmentos de texto teórico sobre o Formalismo Russo, em que a primeira palavra que se lê, no canto superior à esquerda, é o próprio nome de Chklovsky, além de termos caros à discussão dos formalistas sobre o estranhamento perceptivo, como “automatizada” e “habitual”. Tal referência direta a Chklovsky no corpo do videopoema clama metatextualmente, portanto, por uma atenção mais detida para os procedimentos tecnoestéticos desnaturalizadores da percepção que figuram como linha de força na obra. Em “Amor”, toda a perspectiva de Ana diante de si e do mundo é desnaturalizada quando, do bonde, a protagonista vê um cego no ponto a mascar chicletes, o que desencadeia, entre outros estranhamentos, o choque sensorial com a cesta de ovos. Tal visão do homem cego e sua goma de mascar, supostamente banal, causa reverberações epifânicas na moça – como na cacofonia de textos sobrepostos em Amor de Clarice –, conforme se lê no excerto a seguir, retirado do conto original: 134 (...) o coração batia-lhe violento, espaçado. Inclinada, olhava o cego profundamente, como se olha o que não nos vê. Ele mascava goma na escuridão. Sem sofrimento, com os olhos abertos. Fig. 1 – Segunda tela de Amor de Clarice. Por fim, um sistema que atinja efeitos expressivos inventando novas regras dentro daquelas que já existem é a definição mais simples, mas mais honesta, que se pode dar de uma mensagem poética computacional, em que o desvio, a literariedade, a trapaça, ou o que o valha, é sempre programado, ao menos enquanto potência, nas regras do sistema. Neste ponto da discussão, toma-se como argumento para a leitura aqui proposta uma similaridade entre procedimentos poéticos e computacionais, a qual por vezes surpreende a academia na área de Letras por analisar recursos, em tese, estranhos ao campo literário, como as tecnologias das hipermídias digitais. Diz-se aqui “em tese” porque, desde Aristóteles (1973), as fronteiras entre tecnologia e arte são tênues (senão inexistentes em determinados contextos culturais), haja vista o caráter de artifício, maquinaria e técnica, em oposição à natureza e à espontaneidade, que marca o termo grego tekhné, em sua ambiguidade entre o técnico e o artístico. Sob semelhante perspectiva, mais especificamente aplicada ao campo literário, Chklovsky (2013b, p. 213) afirma que, “para o escritor, respeitar a tradição [é] depender de um conjunto de normas literárias, conjunto que, como a tradição das invenções, é formado pelas possibilidades técnicas do momento”. No caso de Amor de Clarice, releia-se essa máxima acrescentando o prefixo “des-” ao verbo “respeitar”, uma vez que a desconstrução intertextual do conto clariceano se dá justamente nas possibilidades técnicas (ou tecnoestéticas) de nosso momento presente, isto é, no bojo das tecnologias digitais. Nesse sentido, adota-se aqui o termo “tecnoestético” para sublinhar que cada procedimento técnico adotado pela equipe de desenvolvimento do ciberpoema é também um recurso expressional, com vistas a atingir a percepção do leitor por meio do estranhamento estético (ou desnaturalização dos sentidos), de modo algo análogo ao que se propunham os tropos catalogados à exaustão pelos retóricos e gramáticos da Renascença. Assim, enquanto mecanismos retóricos, os algoritmos que compõem a programação subjacente a Amor de 133 133 SOBRENOME AUTOR, SOBRENOME AUTOR, SOBRENOME AUTOR SOBRENOME AUTOR, SOBRENOME AUTOR, SOBRENOME AUTOR Ana olhava-o. E quem a visse teria a impressão de uma mulher com ódio. Mas continuava a olhá-lo, cada vez mais inclinada — o bonde deu uma arrancada súbita jogando-a desprevenida para trás, o pesado saco de tricô despencou-se do colo, ruiu no chão — Ana deu um grito, o condutor deu ordem de parada antes de saber do que se tratava — o bonde estacou, os passageiros olharam assustados. Incapaz de se mover para apanhar suas compras, Ana se aprumava pálida. Uma expressão de rosto, há muito não usada, ressurgia-lhe com dificuldade, ainda incerta, incompreensível. O moleque dos jornais ria entregando-lhe o volume. Mas os ovos se haviam quebrado no embrulho de jornal. Gemas amarelas e viscosas pingavam entre os fios da rede. O cego interrompera a mastigação e avançava as mãos inseguras, tentando inutilmente pegar o que acontecia. O embrulho dos ovos foi jogado fora da rede e, entre os sorrisos dos passageiros e o sinal do condutor, o bonde deu a nova arrancada de partida. (LISPECTOR, 1998, p. 21-22). Vendo o cego, ou aquele que não a vê, Ana acaba por enxergar aquilo que não se vê, senão quando de um choque que desloca os procedimentos perceptivos e os agudiza, em episódio que Torres (2003) classifica como epifânico, muito próximo da singularização da imagem prevista no pensamento chklovskyiano. Diante do comezinho mascar de chicletes pelo cego, Ana desperta para uma sensibilidade exacerbada, doída diante do mundo que não pode mais tolerar: “A piedade a sufocava, Ana respirava pesadamente. Mesmo as coisas que existiam antes do acontecimento estavam agora de sobreaviso, tinham um ar mais hostil, perecível... O mundo se tornara de novo um mal-estar” (LISPECTOR, 1998, p.22). Vendo o cego, ou aquele que não a vê, Ana acaba por enxergar aquilo que não se vê, senão quando de um choque que desloca os procedimentos perceptivos e os agudiza, em episódio que Torres (2003) classifica como epifânico, muito próximo da singularização da imagem prevista no pensamento chklovskyiano. Diante do comezinho mascar de chicletes pelo cego, Ana desperta para uma sensibilidade exacerbada, doída diante do mundo que não pode mais tolerar: “A piedade a sufocava, Ana respirava pesadamente. Mesmo as coisas que existiam antes do acontecimento estavam agora de sobreaviso, tinham um ar mais hostil, perecível... O mundo se tornara de novo um mal-estar” (LISPECTOR, 1998, p.22). SOBRENOME AUTOR, SOBRENOME AUTOR, SOBRENOME AUTOR O movimento da mastigação fazia-o parecer sorrir e de repente deixar de sorrir, sorrir e deixar de sorrir — como se ele a tivesse insultado, (...) o coração batia-lhe violento, espaçado. Inclinada, olhava o cego profundamente, como se olha o que não nos vê. Ele mascava goma na escuridão. Sem sofrimento, com os olhos abertos. O movimento da mastigação fazia-o parecer sorrir e de repente deixar de sorrir, sorrir e deixar de sorrir — como se ele a tivesse insultado, 134 SOBRENOME AUTOR, SOBRENOME AUTOR, SOBRENOME AUTOR Tal temática do despertar sensorial, dominante em “Amor” e aludida já nas referências a Chklovsky no início de Amor de Clarice, é potencializada pelos próprios procedimentos de composição do ciberpoema, por meio de recursos tecnoestéticos ensejados por sua textualidade digital movediça. Assim, se toda releitura nos convida a olhar de forma diferente uma obra já conhecida – por contingência do que seja a intertextualidade –, Amor de Clarice o faz enquanto desnaturação de segundo grau, pois nos adensa a visão de um texto que já tratava, enquanto matéria narrada, de um adensamento perceptivo. Na Figura 4, pode-se observar um quadro em que a cena da epifania no bonde é retomada na segunda série de interfaces do poema de Rui Torres. 135 SOBRENOME AUTOR, SOBRENOME AUTOR, SOBRENOME AUTOR Fig. 4 – Tela de Amor de Clarice, relendo a cena da epifania no bonde. Fonte: TORRES, 2005. Fonte: TORRES, 2005. À primeira vista, ressalta no vídeo a permuta do meio de transporte em que se passa a cena: em vez do bonde, com seus solavancos que marcam o novo rumo na vida de Ana, aparece em Amor de Clarice um vagão de metrô, que sacoleja ininterruptamente no vídeo, turvo como a percepção amplificada de Ana. Tal troca do veículo sobre trilhos amplia seus efeitos de sentido se pensarmos que a passagem do bonde ao metrô é uma mudança tecnológica que impacta na forma como as pessoas se deslocam sobre o mundo (ou na forma como elas veem o mundo que se desloca da janela do carro). Do mesmo modo, a mudança tecnológica do suporte impresso para o digital, que permite a releitura de “Amor” no ciberpoema de Torres, desloca a percepção do leitor, confrontando-o com um texto que se escreve como multimodalidade impermanente, em que vídeos, sons e textos escritos cintilam por apenas alguns instantes na tela – tal qual o mundo que Ana vira por um instante da janela do bonde. Ainda no que tange ao componente audiovisual desse trecho do ciberpoema, é interessante pensar que, enquanto no conto vemos a cena pelos olhos de um narrador onisciente, que de um não lugar enxerga Ana no bonde e o cego na rua, na releitura de Torres a focalização é interna ao metrô, deslocando o ponto zero da enunciação que constitui a narrativa e o que dela se nos dá a ver. Da visão externa à interna, do bonde ao metrô, opera-se uma transmutação semelhante à que vive a personagem que antes via o “de fora” do mundo e agora se defronta com o “de dentro” das coisas que rebenta a seus olhos: choque dos sentidos mobilizado por toda releitura – do mundo como um texto ou do texto como um mundo. No jogo dos vários planos simultâneos que o texto de Torres enseja, sobrepondo som, imagem e texto verbal com ajuda de recursos tecnoestéticos digitais, os excertos do conto, 136 136 SOBRENOME AUTOR, SOBRENOME AUTOR, SOBRENOME AUTOR recombinados no primeiro plano do quadro, ressignificam o vídeo e toda a releitura dessa cena. Fig. 4 – Tela de Amor de Clarice, relendo a cena da epifania no bonde. Assim, as frases “olha ana profundamente como se olha o que nos vê”, “e o bonde/súbito, como quem não vê ana” e “gritam/como quem grita a quem não nos vê” (TORRES, 2005) retomam a questão da alternância de perspectivas – o narrador ou Ana, o bonde ou o metrô, o ver e o ser visto – que a epifania mobiliza. Estando essas frases constantemente sumindo e aparecendo na tela, a visão cambaleante de Ana passa aos olhos do leitor, incapaz de apreender o todo da cena. CONSIDERAÇÕES FINAIS Voltamos, pois, às múltiplas leituras que todo texto enseja, mas aqui múltiplas até enquanto camadas textuais simultâneas na mesma visada da obra. Se ver duas coisas ao mesmo tempo é não ver nenhuma das duas, na verdade, mas sim uma terceira que as relaciona, o jogo de sobreposições de Amor de Clarice, em que deslizam constantemente o conto de Clarice, sua releitura por Torres e as teorias de Chklovsky, desnaturalizam nossos modos de ler a página ou a tela, causando ao leitor o estranhamento de quem vê um cego mascar chicletes. Ou o estranhamento de quem vê um poema incrustado em código computacional, instâncias a priori tão díspares no entendimento tradicional do que seja o literário. Tal estranhamento, no caso de Amor de Clarice, constrói-se a partir de engenhosas remissões intertextuais, regra básica do ludo aqui analisado. Esta se instala por meio de procedimentos de escrita ainda pouco explorados na área dos Estudos Literários: a escrita em códigos de programação, que permite uma poética algorítmica e, ao mesmo tempo, desconstrutora, na medida em que postula obstáculos constantes a qualquer estabilidade semiótica, seja na interpretação ou mesmo na materialidade discursiva do poema. Como produto de tal jogo, lê- se um texto movente, em que a cada leitura do móbile, um outro texto dá-se a ver: fragmentos recombinados na interação entre leitor e letra, mediada por uma combinatória codificada em poéticos algoritmos. REFERÊNCIAS ARISTÓTELES. Ética a Nicômaco. In: Os Pensadores. v. 4. São Paulo: Nova Cultural, 1973. ARISTÓTELES. Ética a Nicômaco. In: Os Pensadores. v. 4. São Paulo: Nova Cultural, 1973. 137 ARISTÓTELES. Ética a Nicômaco. In: Os Pensadores. v. 4. São Paulo: Nova Cultural, 1973. 137 137 SOBRENOME AUTOR, SOBRENOME AUTOR, SOBRENOME AUTOR ARQUIVO Digital da PO.EX. 2015. Disponível em: <http://www.po-ex.net>. Acesso em: 15 dez. 2017. ARQUIVO Digital da PO.EX. 2015. Disponível em: <http://www.po-ex.net>. Acesso em: 15 dez. 2017. BARTHES, Roland. Aula. São Paulo: Cultrix, 1992. CHKLOVSKY, Victor. A arte como procedimento. In: TODOROV, Tzvetan. Teoria da literatura: textos dos formalistas russos. São Paulo: Unesp, 2013a. ______. A construção da novela e do romance. In: TODOROV, Tzvetan. Teoria da literatura: textos dos formalistas russos. São Paulo: Unesp, 2013b. DERRIDA, Jacques. Gramatologia. São Paulo: Perspectiva, 2008. FOUCAULT, Michel. A arqueologia do saber. Rio de Janeiro: Forense, 1986. GENETTE, Gérard. Palimpsestes: la littérature au second degré. Paris: Seuil, 1982. LISPECTOR, Clarice. Amor. Laços de Família. Rio de Janeiro: Rocco, 1998. TORRES, Rui. Amor de Clarice. In: Electronic Literature Collection. 2005. Disponível em: <http://collection.eliterature.org/2/works/torres_amordeclarice.html>. Acesso em: 29 nov. 2017. ______. Ler Clarice Lispector, re-escrevendo Amor. 2003. Disponível em :<http://www.ufp.pt/~rtorres/ens.htm>. Acesso em: 30 nov. 2017. Recebido: 16 de janeiro de 2018 Aceito: 21 de maio de 2018 Recebido: 16 de janeiro de 2018 Aceito: 21 de maio de 2018 138
https://openalex.org/W4372328822	https://www.qeios.com/read/8APEYH/pdf	English	null	Review of: "A Methods Note on Remote Sensing Platforms and Large-Scale Archeological Impact Assessments (AIA) in the Philippines"	null	2,023	cc-by	155	Qeios, CC-BY 4.0 · Review, May 6, 2023 Qeios ID: 8APEYH · https://doi.org/10.32388/8APEYH Chong Xu Chong Xu Potential competing interests: No potential competing interests to declare. This manuscript introduces how various aerial remote sensing platforms can conduct archaeological impact assessments on scattered or large-scale project sites, providing archaeologists with efficient and targeted utilization of limited time and resources, following simplified workflow. The specific suggestions are as follows: (1) The background should be introduction. In this section, there is a lack of clarification on the review of the study direction. It is incomprehensible that there were no references in the introduction. (2) Discussion should actually be a conclusion. (3) The real discussion part seems to be missing. The authors should discuss the progressiveness and limitations of this work from the aspects of methods and results in combination with previous work. In addition, it is also necessary to provide some prospects. Qeios ID: 8APEYH · https://doi.org/10.32388/8APEYH 1/1
https://openalex.org/W4361807363	https://zenodo.org/records/7787253/files/16.pdf	English	null	Interactive Materials And Their Impact On The Relationship Of The Sculptural Form To The Environment, The Recipient And The Work Parts	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	9,551	Thus, the research problem is summarized in the following question: Is it possible to enrich the interactive relationship of the sculptural form with its environment, the recipient and the work parts among themselves through the implementation of light-reflecting surfaces as one of the interactive materials? R h Obj ti Is it possible to enrich the interactive relationship of the sculptural form with its environment, the recipient and the work parts among themselves through the implementation of light-reflecting surfaces as one of the interactive materials? Abstract Art has become more accommodating of the human experience in the various fields of objective sciences, meanwhile it preserves the subjective aspects of the artist. The works ofartdo not include pictures, paintings and sculptures only, but also contain devices, machines and raw materials with interactive properties that provide the viewer with greater opportunities to participate and gain theoretical and non- theoretical experiences. In modern times, theartwork has become an active cell rather than a passive space that conveys and represents something, whether itis real or imaginary. This is to claim that what is going on the painting or in the statue does not represent animage or a form only, but rather an eventor an occurrence or a happening. Therefore, most artistic trends have often deviated from the principle of representation, and focused more on exploring abstract relationships, with the aim of widening the visual experience via novel progressivethoughts and visions, rather than arousing the imagination in a specific limited topic. Thus, it is possible for art to enrich and expand the borders and scope of the artistic work to extend the limits of the form or the shape on which the artwork is implemented. This is achieved by creating interactive relationships in more than one way. On one hand, the relations between the work of art and its environmental location, with its different characteristics and components. On the other hand, the relation between the work of art and its viewer or perceiver to go beyond the limits of the simple emotional interpretation of the work, and the interactive relations of the parts of the stereoscopic work with each other and the instability of those relationships. Such interactive relations are regularly changing and creating new relationships among the parts of the artworks. Thus, making the work more dynamic and varying. To do this, the artist must use materials that have distinctive characteristics in building and forming the sculptural work, which the researcher has called (interactive materials). Research problem: p The research focuses on using one of the materials that have such interactive properties, of which the researcher has chosen the materials that interact effectively with light, which are the light-reflecting materials, thus reflecting everything around them in the environment as well as the recipient. The three axes (environment, recipient, and form of art) Introduction hi h In this research, “Interactive materials” are referred to as those materials that have distinctive properties which, when used in the construction of artwork, allow variable and unstable relationships, whether by dynamic or mechanical movement, or changing light or color relationships, which allows the sculptural work to interact more with the environment, the recipient and the parts of the work of art among themselves. Research problem: Multicultural Education Multicultural Education Multicultural Education Volume 9, Issue 3, 2023 Interactive Materials And Their Impact On The Relationship Of The Sculptural Form To The Environment, The Recipient And The Work Parts Wael Fathy Ibrahim Mohamed Interactive Materials And Their Impact On The Relationship Of The Sculptural Form To The Environment, The Recipient And The Work Parts Wael Fathy Ibrahim Mohamed Article Info Abstract Article History Received: December 29, 2022 Art has become more accommodating of the human experience in the various fields of objective sciences, meanwhile it preserves the subjective aspects of the artist. The works ofartdo not include pictures, paintings and sculptures only, but also contain devices, machines and raw materials with interactive properties that provide the viewer with greater opportunities to participate and gain theoretical and non- theoretical experiences. In modern times, theartwork has become an active cell rather than a passive space that conveys and represents something, whether itis real or imaginary. This is to claim that what is going on the painting or in the statue does not represent animage or a form only, but rather an eventor an occurrence or a happening. Therefore, most artistic trends have often deviated from the principle of representation, and focused more on exploring abstract relationships, with the aim of widening the visual experience via novel progressivethoughts and visions, rather than arousing the imagination in a specific limited topic. Thus, it is possible for art to enrich and expand the borders and scope of the artistic work to extend the limits of the form or the shape on which the artwork is implemented. This is achieved by creating interactive relationships in more than one way. On one hand, the relations between the work of art and its environmental location, with its different characteristics and components. On the other hand, the relation between the work of art and its viewer or perceiver to go beyond the limits of the simple emotional interpretation of the work, and the interactive relations of the parts of the stereoscopic work with each other and the instability of those relationships. Such interactive relations are regularly changing and creating new relationships among the parts of the artworks. Thus, making the work more dynamic and varying. To do this, the artist must use materials that have distinctive characteristics in building and forming the sculptural work, which the researcher has called (interactive materials). Accepted: March 31, 2023 Keywords : Art, The Human Experience,Sciences, Relationship DOI: 10.5281/zenodo.7787253 Introduction Research Objectives: j an interactive relationship between the sculptural form and its environment. j - Finding an interactive relationship between the sculptural form and its environment. - Enriching the interactive relationship between the sculptural work and the recipient through the use of reflective surfaces in its construction. - Enriching the relationships of work parts and changing them by using reflective surfaces in building the sculptural form. • Research Hypotheses: 124 125 The researcher assumes that: - The use of reflective surfaces in building the sculptural form enriches its interactive relationship with its environment. - The use of reflective surfaces in building the sculptural form enriches its interactive relationship with its environment. - The use of reflective surfaces in building the sculptural form enriches its interactive relationship with its environment. - The use of reflective surfaces in building the sculptural form enriches its interactive relationship with the recipient. - The use of reflective surfaces in building the sculptural form enriches its interactive relationship with the recipient. - The use of reflective surfaces in building the sculptural form enriches the interactive relationships bet parts. Theoretical framework Light as a natural phenomenon: Perhaps natural phenomena are one of the most important inspirations for the artist to implement the idea of a work of art, and therefore the artist’s observation and contemplation of natural phenomena deepens his sensory experience and enables him to benefit from its data and its visible and mysterious aesthetic and structural features. Consequently, this is considered an enrichment in dealing with these suggestive phenomena and transcending the limits of their tangible physical structural form. Artist, Adolph Luther, stats that, "A fundamental shift has occurred in the artist's vision of nature. He has moved from looking at its apparent elements to contemplating the hidden forces that hide behind phenomena. With this shift in outlook, the artist is in agreement with both the scientist and engineer in their quest to understand and contemplate natural phenomena. Light is one of the most important natural phenomena that help us perceive things around us generally and plastic art particularly. In fact, without light, we cannot see things and therefore we cannot perceive them. Light as a phenomenon has several characteristics and laws. Among those characteristics is the phenomenon of light reflection, which has been of great benefit to the researcher in achieving the research aims. Reflective materials: Some light-reflecting materials have a very high degree of light reflection on them. It is worthy of noting, in this regard thatthe technological awareness of the nature and treatment of different raw materials had had a direct impact on the implementation and progression of many reflective materials.” In the nineteenth century, scientific theories had proved the benefit of chemistry and awareness of changes in the internal composition of materials, in reaching more effective treatments for materials, and this indicated a fundamental change in the degree to which the understanding of the basic nature of materials stands. Furthermore,at the end of the century, scientists had developed new methods for producing aluminum, carbon, silicon and stainless-steel metals, so that these metals became of a high degree of quality and brilliance. In the twentieth century, the discovery of the internal structure of the materials resulted in the development of polymeric fibers, plastics, plastic mirrors, high-quality metal alloys, glass fibers, and other reflective materials, whichshow the most accurate reflection on their surfaces. The types of materials and reflective surfaces and their characteristics vary, including glass mirrors, plastic mirrors, and metals with reflective surfaces. Moreover, each of these types differs in the images formed on their (reflected) surfaces depending on the shape of the surface, whether it is flat, convex, or concave, and these differences are due to the different angles of light reflection from the reflective surface. These are in turn subject to mathematical regulation according to the laws of light reflection. This is to argue that building stereoscopic sculptural forms using reflective surfaces requires a special mathematical organization • Reflection It means a change in the direction of the light waves falling on a reflective surface, so when the surface of the material receives some energy waves and rejects others, it radiates on what surrounds it. However, this does not mean that the light energy is divided into two parts so that one part is retained bythe material composition, while the other part is radiated. On the contrary, it rather means that the amount of energy input is great whereas the output is small, and the difference between them varies in capacity from one reflective surface to another according to the degree of polishing and color and the degree of transparency and opacity. Reflective materials: Abstraction And Emancipation From The Subject Scientific discoveries related to the laws of light affected some artists’ tendency towards liberation. This trend is apparently clear among the impressionist artists who are interested in the language of form and the process of vision, and liberated from the literary subject that preoccupied feelings, from contemplating the aesthetic and structural relations in the artwork. Thus,the historian of the movement,Franka Steal, states: What distinguishes the impressionists from other artists is their treatment of the subject for its tonal differences, not for the subject itself. From the point of view of the modern artist, the subject of the artwork is the work itself because of its constructive style, pure artistic values of lines, colors and shapes, and the relationships it contains arising from the distribution of lights and shadows and the elements of the artwork. Such artistic construction, whichis achieved through reflective surfaces, as well as the concepts of time and space, has changed dramatically because of their influential role in interacting with the sculptural work. Time and place have their role in perceiving the work of art. In this sense, “Philosophy at the beginning of the twentieth century emphasized the supremacy of the values and arts of temporal movement, not the arts of spatial stability… Modern science changed the concepts of time and space, especially with the emergence of the theory of relativity at the beginning of the twentieth century. Absolute place or absolute time are relative, where Einstein made it clear that any calculations of a world in which everything is in motion must depend on the position of the observer, which is a variable position. At a certain distance, the world thus appeared to be a continuous spatio-temporal entity. Time reveals similarity (or resemblance) through continuity (or permanency) and reveals change (or difference) through succession (or sequence). Likewise, space reveals unity, as it is (connected) and reveals multiplicity(or plurality) in that it is a complex of parts. Relationship to the environment: Relationship to the environment: As the works of art that use reflective surfaces in their creation are more compatible with their internal and external environment, as their surfaces acquire the distinctive character of that environment, whether the color, formal, or kinetic rhythm of them. y Furthermore, it is possible for the artwork to interact with any environment in which it is clarified, which results in dynamisms in the workplace, and makes it valid to be located in specific environments rather than others This is because it will reflect images of that environment and thus the interdependence will be achieved through it as it makes its environment richer through the modification of the reality of the environment which gives other forms of the environment. Mathematical Organization: Mathematical Organization: g The mathematical organization is an organization prepared with calculated mathematical relations, to which all elements of work are subject, with the aim of creating specific effects, and not just an organization through which the work is only balanced. Therefore, when investing light-reflecting surfaces, whether in the fields of public, scientific or artistic life, it is necessary to rely on mathematical calculations and geometric angles in assembling the component parts of the work. This is because the general character that can be realized through the geometric organization behind which lies a mathematical system is represented in the fall of light rays in straight lineswhose reflection at the angle of incidence determines the direction of the new reflection, and so on. Therefore, the artistic works formed by reflective surfaces require when organizing a conscious understanding of the laws of reflection and the changes arising from the diversity of different surfaces as well as the engineering construction of the relations of 126 juxtaposition, contrast and contact of those units, and the angles of their openness to the internal and external void of the artwork, as well as the impact and influence of the units established with what this void contains of variables Light, movement, color and shape, as factors, are subject to construction processes to mathematical systems that include the laws of reflection and calculations of emerging variables, which is a necessity to control the final form of the artwork and the variables expected to be added to it. Therefore, these works tend to be abstract and liberal from the subject, and the main goal is to reach calculated Therefore, these works tend to be abstract and liberal from the subject, and the main goal is to reach c plastic formulations that contain many variables and shapes resulting from light reflections. Abstraction And Emancipation From The Subject It is the connection of the spatio-temporal reality of these two characters that made it impossible to establish any separation between “space and time.” For this reason, “Alexander” decides that what replaces time with its unity is place, and that what deprives space of its abundance is time, and if it were not for that, time would be just a succession, and space would be just an empty unit.Furthermore, the use of such interactive materials in building the stereoscopic sculptural form, among which are the reflective surfaces, emphasizes this concept of time and place effectively. In fact, it demonstrates that the presence of the work in a place that reflects its elements and characteristics makes it a part of that place, and is affected by the movement and scope of the viewer from different angles of reflection, resulting in new formations and relationships. Thus, allowing the viewer himself to become part of the work, as he is regarded among theessential factors of the work environment when he is next to the artwork. Thus, the light-reflecting material can be used as an interactive material to enrich the relation stereoscopic work with the environment, scenes, light and work system: Relationship to the viewer: The viewer of works of art created with materials with high light reflection, is not subject to a specific vision, but always sets out to discover new horizons in which he is liberated from the constraints of rigid vision. Thus, with the new configurations, the vision is extended for the viewer. It also arouses the viewer’s imagination, as the images on reflective surfaces do not represent a material reality, but rather it is a subjective combination between the existential and the non-existential, or the present and the absent at the same time. It enriches the viewer’s imagination, by the interaction between the internal and external image as derived from the viewer’s personal experience. Reflective surfaces also contribute to the viewer’s awareness of the environment in various and novel ways, where the shapes appear sometimes larger and sometimes smaller than their original size, compressed, 127 elongated, or repeated by meeting the reflective surfaces at certain angles, which multiplies the reflected elements and increases the sense of depths and voids. In fact, it may be claimed that the attractive sensory appearance of the lights emanating from the reflective surfaces provokes visual perception of the viewer, and gives a new and attractive appearance to the viewed objects. Application framework: In light of this theoretical framework, the researcher attempted to create stereoscopic sculptural formulations based on reflective surfaces as one of the interactive materials, using mathematical systems in the construction of these stereoscopic bodies to achieve research objectives and verify the research hypotheses. The following is a presentation and analysis of the above mentioned works. Relationship to light: p g Reflections change according to the amount of the incident light and the angle of its incidence, which makes the dynamics of time also in the realization of the stereoscopic work formed by the reflective surfaces. For example, we can imagine a field work made of reflective surfaces. Its appearance in the morning light is different from at noon time, as the intensity and angle of the light are also different from its appearance in the evening and its relationship to artificial lighting and its different colors, which makes the work different in its appearance at each time from the other. Thus,dynamics and deviations are achieved in the visual perception of the artwork. Relationship to the system at work: This is the system to which the reflective shapes are subject within the artwork, and it affects the ways in which these shapes interact with each other, and the ways in which those shapes interact with external influences in the environment. Through the system, surfaces do not become merely reflectionsof objects, but science interacts dynamically with place and time, thus completing the excitement of the recipient as he views the stereoscopic shapes, especially those in the external environment (Outdoor) at any moment throughout the day. Application framework:  First Work  First Work Pic. (A) Pic. (B) Materials: Reflective balls + glass mirrors Dimensions: 52 x 48 x 60 cm Thi k i d f l i d il b ll d i i d f id i h h id Pic. (A) Pic. (A) Pic. (B) Pic. (B) Materials: Reflective balls + glass mirrors Dimensions: 52 x 48 x 60 cm This work is made fromglass mirrors and silver balls, and it is composed of a pyramid with three sides and equal angles of 120between the three parts. Furthermore, it consists also of two silver-colored balls, one of them, namely, the largest, is in the lower spatial part, between the parts constituting the pyramid shape. The other one is situated at the top of the pyramid shape. The base is in the form of a rectangle at the top and its sides open at an angle of inclination to the outside, and the shape is perpendicular to the base. It is obviously, noticeable in picture (A) that the parts of the figure are reflected in the environment surrounding the work in different ways and angles, giving different lighting and shades. Thus, the work gains its colors in relation to the structural organization of the work. It is alsonotable that the base reflects part of the surrounding environment, which gives the work unreal extra dimensions in the vision. As for the picture (B), with the change in the angle of vision of the artwork, we find that the environmental reflection on the work has varied in terms of its colors and shades, and the parts of the work have been reflected on each other, especially on the left part of the work in the picture. Thus, it resulted in an overlap and repetition 128 of the image of the parts and the environment. In addition to this, it also gave depth inside the surface, and made the reflection of the great ball on the work more noticeable, which gave it an impression of both repetition and extension within the base. Thus, it gave rise to the intensification of the elements of the work and other new formative relationships within the work. Therein, we can observe in the same process the presence of a miniature reflection in the confined part between the other two representing the two sides of the pyramid, which gave new perceptual images of the work.  Second Work: p g p p p q It is noticeable that in picture (A) parts of the environment are reflected on parts of the work, and the change of color in the upper part is the result of the reflection of colored objects in the environment. We can also notice the reflective variations on the ball, which are in miniature because of the convexity of the surface of the ball. As for the picture (B), with different viewing angles, there are some deviations in the form of the work, because f th fl ti f th k t th b hi h it t i i id th b (i i ) Th l It is noticeable that in picture (A) parts of the environment are reflected on parts of the work, and the change of color in the upper part is the result of the reflection of colored objects in the environment. We can also notice the reflective variations on the ball, which are in miniature because of the convexity of the surface of the ball. As for the picture (B), with different viewing angles, there are some deviations in the form of the work, because of the reflection of the work parts on the base, which gave it an extension inside the base (imaginary). The color of the artwork changed with the changing reflections from the environment on it. This confirms that in picture ( c) , the shape of the extension of the shape inside the base has changed, and this resulted in new relationships between the parts of the work themselves. Third Work:  First Work g p p g Finally, we can realize that with the different angles of vision of the artwork, the perceptual picture changes as a result of the change in the light reflections of the work parts, and the diversity and difference of the reflected images, whether they are the product of an interaction between thework and the environment, or between each of the artwork parts among themselves.  Second Work:  Second Work:  Second Work: Pic. (A) Pic. (C) Pic. (B) Materials: stainless steel + glass mirrors Dimensions 45 x 25 x 58 cm Pic. (B) Materials: stainless steel + glass mirrors Dimensions 45 x 25 x 58 cm Pic. (C) Pic. (A) Pic. (A) This work is made fromstainless steel, but its base is of square, flat glass mirrors. It consists of three main units, each of which represents a right triangle, part of which has been removed at a straight angle in the form of a curve, which gave a variety between straight and curved lines. Its three parts are of different sizes arranged from the largest to the smallest. Two parts were installed consecutively at a cropped angle in-betweenof 90 degrees. the third and smaller part is installed upside down on the other side, in such a way so that the curved part faces the larger part of the shape. Finally, a silver reflective ball is placed over the smaller part, and the base is in the form of a rectangular parallelepiped of flat square mirrors. angle in-betweenof 90 degrees. the third and smaller part is installed upside down on the other side, in such a way so that the curved part faces the larger part of the shape. Finally, a silver reflective ball is placed over the smaller part, and the base is in the form of a rectangular parallelepiped of flat square mirrors. It is noticeable that in picture (A) parts of the environment are reflected on parts of the work, and the change of color in the upper part is the result of the reflection of colored objects in the environment. We can also notice the reflective variations on the ball, which are in miniature because of the convexity of the surface of the ball. As for the picture (B), with different viewing angles, there are some deviations in the form of the work, because of the reflection of the work parts on the base, which gave it an extension inside the base (imaginary). The color of the artwork changed with the changing reflections from the environment on it. This confirms that in picture ( c) , the shape of the extension of the shape inside the base has changed, and this resulted in new relationships between the parts of the work themselves. Pic. (B) Materials: copper foil + glass mirrors Dimensions: 25 x 25 x 43 cm Materials: copper foil + glass mirrors Dimensions: 25 x 25 x 43 cm Materials: copper foil + glass mirrors Di i 25 25 43 Dimensions: 25 x 25 x 43 cm This work is made of brass and the base is made of squared mirrors. The work consists of a basic unit, namely, the cube, and on top of it is another relatively small unit in the form of a triangular prism. The base cube has been placed in such a way so that one of the corners is the base and the other is the top. The corner of the top bears the small prism, so that the angle of the prism is the fulcrum point on the corner of the cube. The researcher made formations on the surfaces of the cube to enrich them. We note in picture (A)that the reflection of the environment on the shape and the base, and the reflection of the base on the shape, have enriched the value of the artistic work with a diversity of shadows and lighting for different reflections. In picture (B), we notice a change in the perceptual image of the shape of the work, because of the difference in the scope of vision. The extension of the lines of the cube inside the base is also obviously noticeable. The surface of the prism also reflected part of the surface of the cube, which supported the formal interaction between them. The different angles of the constructive formation and reflection, which were made by the researcher on the surface of the cube , gave the shape a diversity and richness.  Fourth Work: Pic. (A) Pic. (B) Pic. (C) Materials: glass mirrors + reflective balls. Dimensions: 58 x 42 x 60 cm This work is made from several materials, namely, glass mirrors, plastic mirrors, stainless steel, and transparent glass.  Fourth Work: Pic. Pic. Pic. Materials: glass mirrors + reflective balls. Dimensions: 58 x 42 x 60 cm Materials: glass mirrors + reflective balls. 5 Dimensions: 58 x 42 x 60 cm This work is made from several materials, namely, glass mirrors, plastic mirrors, stainless steel, and transparent glass. Dimensions: 58 x 42 x 60 cm k is made from several materials, namely, glass mirrors, plastic mirrors, stainless steel, and transparent g It consists of several parts. Third Work: Third Work: Third Work: 129 Pic. (A) Pic. (B) Pic. (B) Fifth Work:  Pic. (B) Materials: copper foil + glass mirrors Dimensions: 25 x 25 x 43 cm Each unit cuts one of its corners with a curved e all is placed under it. The whole work represents a semi-pyramidal structure he environment is reflected on the three parts of the work, and the parts of the work are reflected on hese reflections are noticeable in the places where the component parts meet with each other. It is oting that there is also a reflection of the balls on the triangular parts, and the work is reflected which is formed by the glass mirrors. Thus, a sense of replication and repetition of balls and shapes which results in an extension of the work forms in the imaginary depth inside the base. In fact, th nteractive relationships that have arisen between the work parts themselves, and between the artwo  Fifth Work: Materials: glass mi Dimensions: Materials: glass mirrors + stainless steel. Dimensions: 40 x 31 x 58 cm This work is made of stainless steel, with a base of glass mirrors. It consists of three triangular units graded from largest to smallest, in the form of a regular repetition. Each unit cuts one of its corners with a curved error, and a ball is placed under it. The whole work represents a semi-pyramidal structure The environment is reflected on the three parts of the work, and the parts of the work are reflected on each other. These reflections are noticeable in the places where the component parts meet with each other. It is worthy of noting that there is also a reflection of the balls on the triangular parts, and the work is reflected on its base which is formed by the glass mirrors. Thus, a sense of replication and repetition of balls and shapes is created, which results in an extension of the work forms in the imaginary depth inside the base. In fact, the new and interactive relationships that have arisen between the work parts themselves, and between the artwork and the environment could be obviously observedthrough the optical reflections of the work surfaces. Thus, the dynamic vision of the form is obtained in agreement with the environmental variables, and changes in the angles of vision.  Sixth Work:  Sixth Work: Pic. (B) Materials: copper foil + glass mirrors Dimensions: 25 x 25 x 43 cm Firstly, there is a cube of transparent glass, the inner surface of which is glass mirrors. Secondly, inside this cube another small cube of stainless steel is leaning its weight on one of its corners and carrying a small silvery ball. Thirdly, on the sides inside this stainless cube, there are two relatively larger silvered balls. In addition to this, around the work itself, there are slices of plastic mirrors connected with different angles of reflection and of different lengths. Furthermore, directly behind the work, there are three strips of plastic mirrors at different angles. Finally, at the bottom of the glass cube there are parallel rectangles of mirrors divided into squares. This artwork is placed on a plate of glass mirrors. In picture (A), the extent to which images from the environment are reflected in the work in different ways and at different angles as well as their reflected colors are noticeable. This is explained in such a way that the balls and the small cube have been reflected on the inner surface of the big square and in the plastic, mirror slices on the sides of the cube. Furthermore,it is worthy of noting that the reflection of the balls and slices on the lower mirrors gave the work an imaginary depth inside the base. This is confirmed by image (B) where the angle of vision has differed, leading to different angles of reflection, and seeing different repetitions of the hierarchical part in the back slices. That is to say that as the repetitions of balls in different shapes in the side slices is observed, the cube’s reflection on the floor of the large cube becomes more noticeable, whose mirrors are divided into squares, thus, creating new relationships between the parts of the work, the diversity of the perceptual image, and the intensification of units more, as it is typically noticed in picture (C). This leads to the diversity of forms of work with different viewing angles and interaction with the surrounding environment, including the viewer as well, thus achieving a dynamism of vision in the realization of the stereoscopic artwork. 130  Fifth Work: Materials: glass mirrors + stainless steel. Dimensions: 40 x 31 x 58 cm his work is made of stainless steel, with a base of glass mirrors. It consists of three triangular units g argest to smallest, in the form of a regular repetition. Sixth Work: 131  Seventh Work: Pic. (A) Pic. (B) Materials: glass mirrors + stainless steel. Dimensions: 40 x 35 x 59 cm This figure consists of two parts. The first represents the main part, which is in the form of a rectangular parallelepiped on the vertical axis to the left in picture (A). The curved part has been deleted from the top and bottom, each of which is in the opposite direction of the other. An inward displacement has been made in the upper part of the cuboid so that it results in an angle of 90 between it and its complement part. Furthermore, there is another part that represents a small triangle. Recurrently, the same curved part has been removed, and installed in a reverse axis (in terms of direction) of the cuboid, to achieve equilibrium in the mass. As a result, a lower void is located, and in the upper part of the curve there is another relatively larger silver ball. The figure is constructed in accordance to a geometrical mathematical system in such a way that achieves both symmetry and equilibrium between its components. In picture (A), the reflections of environmental shapes and colors on the Pic. (A) Pic. (B) Materials: glass mirrors + stainless steel. Dimensions: 40 x 35 x 59 cm This figure consists of two parts. The first represents the main part, which is in the form of a rectang Pic. (B) Materials: glass mirrors + stainless steel. Dimensions: 40 x 35 x 59 cm Pic. (A) Pic. (A) Pic. (B) This figure consists of two parts. The first represents the main part, which is in the form of a rectangular parallelepiped on the vertical axis to the left in picture (A). The curved part has been deleted from the top and bottom, each of which is in the opposite direction of the other. An inward displacement has been made in the upper part of the cuboid so that it results in an angle of 90 between it and its complement part. Furthermore, there is another part that represents a small triangle. Recurrently, the same curved part has been removed, and installed in a reverse axis (in terms of direction) of the cuboid, to achieve equilibrium in the mass. As a result, a lower void is located, and in the upper part of the curve there is another relatively larger silver ball. Materials: glass mirrors + reflective balls. Dimensions: 40 x 41 x 60 cm This work is made of strips of plastic mirrors, silver balls and a base of glass mirrors. This work is made of strips of plastic mirrors, silver balls and a base of glass mirrors. It consists of a base of glass mirrors in the shape of the letter (L) and on top of it there are several reflective balls. Behind them are placed longitudinal strips of plastic mirrors, three of them are greater in length than the other two. As shown in the picture of the artwork, the reflection of the external environment on the parts of the work, as well as the reflection of its colors are clearly observed. Furthermore, the reflection of part of the scenes on the work in the middle background slide of the pink-colored work is also perceptible.Through the reflection,the viewer can easily notice that the balls are repeated inside the slides, which gave a sense of condensation and depth. Thus, new relationships have emerged between each of theballs, as well as between the balls and back slides, andin relation to the base as well. The lines of the slides are extended on the base, and the silver balls are repeated, which gave them different images from their reality. Thus, the diversity of the perceptual images of the work is achieved, which in turn vary according to different viewing angles. As it is shown in the work,it represents the extent of the interaction that is achieved through the reflective surfaces between the work and its environment, and between the work and the scenes, and the interaction of the parts of the work with each other, which led to the realization of the dynamic vision of the form.  Eighth Work: Furthermore,as it is clear in picture (B),a reflection related to its appearance in the structural organization of the work is observed on the hierarchical shape inside the cube, and the upper shape with the presence of repetitions of those images on the inner side surfaces of the cube. In terms of the interaction of the parts with each other, this isnoticeable in both pictures. In picture (A), there is a repetition of the pyramid inside the cube inside, as well as a repetition in the upper pyramidal shape, especially at the line of their convergence. In picture (A), the reflection of the environment surrounding the work on the lower cube and the upper hierarchical shape are remarkably noticeable. Furthermore,as it is clear in picture (B),a reflection related to its appearance in the structural organization of the work is observed on the hierarchical shape inside the cube, and the upper shape with the presence of repetitions of those images on the inner side surfaces of the cube. In terms of the interaction of the parts with each other, this isnoticeable in both pictures. In picture (A), there is a repetition of the pyramid inside the cube inside, as well as a repetition in the upper pyramidal shape, especially at the line of their convergence. g This is intensely apparent in picture (B) inside the cube, where the distortions and hesitations in the images of the environment are noticed. This is the result of the interaction of the surfaces of the upper hierarchical shape with the surfaces of the pyramid inside the cube, due to the transparency of the separating part between them, which gave an opportunity for interaction and the emergence of repetitions and distortions in the perceived shape images. This is obviously perceived in the clear difference between the two images (A) and (B), which results from changing the perceptual angle. It could be concluded and deduced that the difference in the images that perceive the shape with different angles contributes to the achievement of the dynamic shape in its vision.  Seventh Work: The figure is constructed in accordance to a geometrical mathematical system in such a way that achieves both symmetry and equilibrium between its components. In picture (A), the reflections of environmental shapes and colors on the 132 figure are represented in the green color in itsvariant degrees. The reflections are also evident in the base, which is in the form of a rectangle, composed of mirrors divided into square parts. Picture (A) represents the reflections of the work parts on each other in their meeting places, which gives it an extension and an illusion of depth and repetition. This is noticed in the reflection of the base on the bottom of the figure, which removes the separation between them and makes it an integrated unit. This is further achieved by the extension of the lines of the figure and by their reflection on the base.As the angles of vision varied,the angles of reflection willconsequently change, causing a change in the relationships between the elements, in terms of their perception of the viewer, thus achieving the interaction and dynamism of the figure.  Eighth Work:  Eighth Work: Pic. (A) Pic. (B) Materials: Reflective ball + glass mirrors + glass cube. Dimensions: 30 x 30 x 57 cm The work consists of a cube of glass mirrors open from the front and the upper face of it is transparent gla  Eighth Work: Pic. (A) Pic. (B) Materials: Reflective ball + glass mirrors + glass cube. Dimensions: 30 x 30 x 57 cm The work consists of a cube of glass mirrors open from the front, and the upper face of it is transparent glass. A pyramidal shape of glass mirrors has been placed inside it. The second part of the work is a pyramidal shape of glass mirrors interspersed with a lower void with a reflecting ball. In picture (A), the reflection of the environment surrounding the work on the lower cube and the upper hierarchical shape are remarkably noticeable. Furthermore,as it is clear in picture (B),a reflection related to its appearance in the structural organization of the work is observed on the hierarchical shape inside the cube, and the upper shape with the presence of repetitions of those images on the inner side surfaces of the cube. In terms of the interaction of the parts with each other, this isnoticeable in both pictures. In picture (A), there is a repetition of the pyramid inside the cube inside, as well as a repetition in the upper pyramidal shape, especially Pic. (B) Materials: Reflective ball + glass mirrors + glass cube. Pic. (A) Pic. (B) Materials: Reflective ball + glass mirrors + glass cube. The work consists of a cube of glass mirrors open from the front, and the upper face of it is transparent glass. A pyramidal shape of glass mirrors has been placed inside it. The second part of the work is a pyramidal shape of glass mirrors interspersed with a lower void with a reflecting ball. The work consists of a cube of glass mirrors open from the front, and the upper face of it is transparent glass. A pyramidal shape of glass mirrors has been placed inside it. The second part of the work is a pyramidal shape of glass mirrors interspersed with a lower void with a reflecting ball. In picture (A), the reflection of the environment surrounding the work on the lower cube and the upper hierarchical shape are remarkably noticeable.  Ninth Work:  Ninth Work: 133 Pic. (A) Pic. (B) Pic. (B) The work consists of several parallelograms of rectangles, covered with a material (with a reflective surface, and the other surface is a “reflective sticker”), and they were assembled in an aesthetic form, according to an appropriate mathematical organization. A furthertriangle wrapped with the same material was added to the figure, and the figure was placed on a base of glass mirrors resting on a silver ball, so that it is given an aesthetic composition resulting from the reflection on the base. p g Picture (A)was taken from above, which means that it has a horizontal projection and the base, and the shape reflect the color of the sky. Furthermore, parts of the figure (according to the angles of their reflections) have reflected parts of the environment that formed pictures of different appearances, characteristics and colors on the parts of the figure. It is noticeablethat the triangular part has the effect of the soft texture on the reflection, and the clarity of the image reflected on it. In the other image (B), the reflection of the shape on the base has led to the realization of a new composition that differs in its appearance and aesthetic value from image (A). This is seen in an extension and repetition of the components of the shape in the illusory spatial depth in the base. It is also noticeable the reflection of the environment on the base and the merging of its images with the form, which leads to the realization of the type of interaction between the elements of the artwork itself and the perceiver.  Tenth Work: Pic. (A) Pic. (B) Materials: stainless steel + reflective balls + glass mirrors. Dimensions: 50 x 50 x 52 cm Pic. (B) Pic. (A) Materials: stainless steel + reflective balls + glass mirrors. Dimensions: 50 x 50 x 52 cm The work consists of several parts. The main part is in the form of an inverted pyramidal formation, permeated from the top by a void in the middle, in which a cube of coated plastic (with a reflective sticker) is placed, and it is based on one of its corners, and the upper corner holds a small silver ball.  Ninth Work: As for the lower part, with comparison to the upper balls, relatively larger silver balls were placed, and the work was fixed on a base of glass mirrors divided into four parts. As for picture (A), the reflection of the environment on the upper part of the figure, and on parts of the base are observed.As part of the base is reflected on the figure, the appearance of the surface in its perceived imagehas varied, which is also clearly realizedseen in picture (B). In picture (B), the reflective surfaces of the main part of the shape are filled with green, which is the color of the surrounding environment. It is also noticed that the reflection of the parts on each other in the places where they meet, and the reflection of the shape with its reflective balls on the base, have contributed to more depth, repetition, intensification and overlap in the images perceived by the figure.Changes in some parts of the base in terms of color, due to the reflection of other forms of the environment on it other than the green color are also noticeable. Thus, by comparing the two pictures (A) and (B), and analyzing them technically, it can be noted the extent of the change in the form, and the extent of its interaction with the environment. Likewise, the viewer who is part of the environment, and the extent to which the parts of the work interact with each other, and the existence of new relationships, all of which arise through changing the viewing angles, and depending on the reflective materials, thus, achieving the dynamic vision of the form.  Eleventh Work : Twelfth Work:  Eleventh Work : Twelfth Work: 134  Thirteenth Work:  Thirteenth Work: Research Results • The mathematical organization is an important factor that must be taken into consideration when performing stereoscopic works of art based on scientific mathematical laws. • The stereoscopic artwork executed with reflective surfaces does not end once its implementation is completed, as it is prepared to be completed in a changing manner in all its scenes, or when the place (environment) changes, or any stimulus appears in the environment that contributes novelty to the work through its reflection. • The mathematical organization is an important factor that must be taken into consideration when performing stereoscopic works of art based on scientific mathematical laws. p p Research problem: The problem is summarized in the following question: - Is it possible to enrich the interactive relationship of the sculptural form with each of its environment, the recipient, and the work parts among themselves through the use of light-reflecting surfaces as one of the interactive materials? Research Results Through the above presentation and analysis of the artworks, the findings could be concluded in thefollowing: • The surfaces of the stereoscopic figure executed with reflective surfaces interact with the environment through the reflection of images from the environment, and the presence of interference and repetition of these images, as well as the presence of distortion in the images. Through the above presentation and analysis of the artworks, the findings could be concluded in thefollowing: • The surfaces of the stereoscopic figure executed with reflective surfaces interact with the environment through the reflection of images from the environment, and the presence of interference and repetition of these images, as well as the presence of distortion in the images. p g • The surfaces of the stereoscopic figure executed with reflective surfaces interact with the viewer in being part of the environment surrounding the work, when it is in the environmental space reflected on the figure. p g • The surfaces of the stereoscopic figure executed with reflective surfaces interact with the viewer in being part of the environment surrounding the work, when it is in the environmental space reflected on the figure. g p g • The parts of the stereoscopic figure executed on the reflective surfaces interact with each other, which gives a diversity in the perceptual images of the figure, and new relationships between those parts also arise. • The parts of the stereoscopic figure executed on the reflective surfaces interact with each other, which gives a diversity in the perceptual images of the figure, and new relationships between those parts also arise. y p p g g p p  Achieving perceptual paradoxes among the viewer, because of the differences in perceptual images of the form executed on reflective surfaces, and their diversity in different viewing angles.  Achieving perceptual paradoxes among the viewer, because of the differences in perceptual images of the form executed on reflective surfaces, and their diversity in different viewing angles. • The stereoscopic artwork executed with reflective surfaces does not end once its implementation is completed, as it is prepared to be completed in a changing manner in all its scenes, or when the place (environment) changes, or any stimulus appears in the environment that contributes novelty to the work through its reflection. Refrences Refrences Adel, Rahman: "A View in the Light" Published Researd The Artist union Gallery, Cairo, Opera House.2005. Ehab Ahmed Abdel Reda: “Artistic trends in the formation of postmodernism”, published research, Academic Adel, Rahman: "A View in the Light" Published Researd The Artist union Gallery, Cairo, Opera House.2005. Ehab Ahmed Abdel Reda: “Artistic trends in the formation of postmodernism”, published research, Academic Rahman: "A View in the Light" Published Researd The Artist union Gallery, Cairo, Opera House.2005. Ahmed Abdel Reda: “Artistic trends in the formation of postmodernism” published research Academ Adel, Rahman: A View in the Light Published Researd The Artist union Gallery, Cairo, Opera House.2005. Ehab Ahmed Abdel Reda: “Artistic trends in the formation of postmodernism”, published research, Academic J l C ll f Fi A U i i f B hd d ISSUE 72 2015 g y p Ehab Ahmed Abdel Reda: “Artistic trends in the formation of postmodernism”, published research, Academic Journal, College of Fine Arts, University of Baghdad, ISSUE 72, 2015. Ezz El-Din Ismail: "Art and Man", Dar Al-Qalam, Beirut, 1974. Ezz El-Din Ismail: "Art and Man", Dar Al-Qalam, Beirut, 1974. Ezz El-Din Ismail: "Art and Man", Dar Al-Qalam, Beirut, 1974. Kelly James: " the sculptural idea" , Burgess Publishing Company ,2004. Kelly James: " the sculptural idea" , Burgess Publishing Company ,2004. Naim Attia: "Modern Art is an attempt towards Comprehension", Iqra series, Dar Al Maaref, Cairo, 199 Shaker Abdel Hamid: " Visual Arts and the Genius of Perception", Egyptian General Authority for Book - Cairo, 2002. Zakaria Ibrahim: "Studies in Contemporary Philosophy", Part 1, Nasr Library, Cairo, 1998. G. L. Lidl: "Material Science", Journal of Science, June, Volume 4, Number 6, Kuwait. -Abdel Fattah Riad: "Training in Plastic Arts", A Study in the Psychology of Vision and its Role in Arousing Aesthetic Feelings, Dar Al-Nahda Al-Arabia, Cairo 2000. -Abdul Mohsen Saleh: "Energy: its nature, forms, sources", the world of thought, Part 5, No. 2, Ministry of Information, Kuwait 1974. ra Hami Matar: "On Values and Reason in Philosophy and Civilization", Ain for Human and Soci Studies and Research, Cairo-2016. Studies and Research, Cairo-2016. -Amy Dempsey , stles , schools , and movement (an encyclopedic guide to modern art ) ,Thames & Hudson , London 2002 y Dempsey , stles , schools , and movement (an encyclopedic guide to modern art ) ,Thames & Hudson London , 2002.  Research aims : - Finding an interactive relationship between the sculptural form and its environment. - Enriching the interactive relationship between the sculptural work and the recipient using reflective surfaces in its construction. - Enriching the interactive relationship between the sculptural work and the recipient using reflective surfaces in its construction. 135 - Enriching the relationships of work parts and changing them using reflective surfaces in building the sculptural figure. - Enriching the relationships of work parts and changing them using reflective surfaces in building the sculptural figure. The researcher assumes that: The researcher assumes that: The researcher assumes that: - The use of reflective surfaces in building the sculptural figure enriches its interactive relationship with its environment. - The use of reflective surfaces in building the sculptural figure enriches its interactive relationship with its environment. - The use of reflective surfaces in building the sculptural figure enriches its interactive relationship with the recipient. p - The use of reflective surfaces in building the sculptural figure enriches the interactive relationships between its parts. The following results were also obtained:  The surfaces of the stereoscopic figure executed with reflective surfaces interact with the environment through the reflection of images from the environment, and the presence of interference and repetition of these images, as well as the presence of distortion in the images. • The surfaces of the stereoscopic figure executed with reflective surfaces interact with the viewer in being part of the environment surrounding the work, when it is in the environmental space reflected on the figure. • The parts of the stereoscopic figure executed on the reflective surfaces interact with each other, which gives a diversity in the perceptual images of the figure, and new relationships between those parts also arise. p p g diversity in the perceptual images of the figure, and new relationships between those parts also arise. y p p g g , p p • Achieving perceptual paradoxes among the viewer, because of the differences in perceptual images of the form executed on reflective surfaces, and their diversity in different viewing angles.  The stereoscopic artwork executed with reflective surfaces does not end once its implementation is completed, as it is prepared to be completed in a changing manner in all its scenes, or when the place (environment) changes, or any stimulus appears in the environment that contributes novelty to the work through its reflection. g • The mathematical organization is an important factor that must be taken into consideration when performing stereoscopic works of art based on scientific mathematical laws. "The authors would like to thank the ‎Deanship of Scientific Research at Umm Al-Qura University ‎for supporting this work by Grant Code:(22UQU4330151DSR01)"  Research Hypotheses:  Research Hypotheses: Refrences -Anwar Ali Alwan Abbas, Diaa Hammoud Muhammad Al-Aaraji: "The Aesthetics of Color and Movement in Visual Art", University of Babylon - Faculty of Fine Arts, University of Babylon Journal of Human Sciences Volume 20, Issue 9, 2013 -Dalia Fouad Mahmoud ATTIA: "The Role Of Design in Maximizing the Benefit Of the Optical Illusion Art In the Field Of InteriorDesign", Internation Journal Of Designand Fashion Studies, Volume 3, Issue 2, 2020, 6 – 8 -Ghada Mahmoud Ibrahim Auf, "The Evolution of Visual Deception to Keep Up with the Global Development", Journal of Architecture and Arts, Eleventh Issue, Part Two, 2012. -Irwin : " Perceiving on integrated visual word": In D.E. meyer& S. Kornblum (Eds.)Attention and performance XIV: Synergies in experimental psychology, artificial intelligence, and cognitive neuroscience Cambridge, MA: MIT Press.1993. 136 -Naglaa Saad Zaghloul Jaber:"The Importance of Perception in the Art of Visual Deception and Its Causes", The Fourth International Scientific Conference of the Faculty of Arts, Zaitouna University, Jordan, 2014. -Naglaa Saad Zaghloul Jaber:"The Importance of Perception in the Art of Visual Deception and Its Causes", The Fourth International Scientific Conference of the Faculty of Arts, Zaitouna University, Jordan, 2014. -Najm Eid Haidar: "Aesthetics, its Prospects and Development", Second Edition, University of Baghdad 2001. -Steven M.Lavalle: "Virtual Reality",University of Illinois ,Cambridge university press ,2017. -Subhi Ahmed Al-Dulaimi, Mona Ali Duaij Al-Kinani: "Renewable Energy: Concept - Sources - Importance" Ibsar Publishers and Distributors, Amman, Jordan, 2022. -Yang, Yee-soo; Kim, Sung-yoen:"The Formativeness Expression of Space Expansion using Optical Illusion on Optical Fiber", Journal of Korea Design Forum, P: 91-104, 2017. Author Information Prof. Wael Fathy Ibrahim Mohamed Advisor to the Deanship of Development and Quality at Umm Al-Qura University AndProfessor of Sculpture, Faculty of Specific Education - Fayoum University -Najm Eid Haidar: "Aesthetics, its Prospects and Development", Second Edition, University of Baghdad 2001. y y g y p -Subhi Ahmed Al-Dulaimi, Mona Ali Duaij Al-Kinani: "Renewable Energy: Concept - Sources - Importance" Ibsar Publishers and Distributors, Amman, Jordan, 2022. g, Yee-soo; Kim, Sung-yoen:"The Formativeness Expression of Space Expansion using Optical Illusion o Optical Fiber", Journal of Korea Design Forum, P: 91-104, 2017. Author Information Prof. Wael Fathy Ibrahim Mohamed Advisor to the Deanship of Development and Quality at Umm Al-Qura University AndProfessor of Sculpture, Faculty of Specific Education - Fayoum University
https://openalex.org/W4321476687	https://zenodo.org/records/7664659/files/Postolachi.pdf	English	null	Strengthening the Professional Identity of Primary School Teachers through Effective Time Management	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	3,010	EX CATHEDRA EX CATHEDRA Strengthening the Professional Identity of Primary School Teachers through Effective Time Management Iulia POSTOLACHI Iulia POSTOLACHI asist. univ., Universitatea Alecu Russo din Bălți Abstract: The article outlines the professional identity of primary school teachers, which is a component of artistic excellence that involves performance in the career field of pro fessionalization. Values of the axiological system of professional identity are addressed and emphasis is placed on the examination of social awareness and time management – a factor that contributes to the personal development of teachers, high achievement, personal satisfaction, and fulfilment, implying compliance with methodological principles in the valorisation of time. The concept of time management comprises a number of definitions that describe a system of activities, processes, and resources that we use to make effective use of our time. Time management is a way of living and working, in which we manage our own time efficiently. Keywords: professional identity, artistic excellence, teachers, primary education, artistic transposition, social resonance, time management. Rezumat: Articolul prezintă descrierea identității profesionale a cadrelor didactice din învățământul primar, aceasta fiind o componentă a excelenței artistice ce presupune performarea în cariera domeniului de profesionalizare. Textul vizează unele valori ale sistemului axiologic al identității profesionale. Accentul se pune pe examinarea rezonanței sociale și gesti onarea corectă a timpului, factor ce contribuie la dezvoltarea personală a cadrelor didactice, la atingerea celor mai înalte scopuri, la satisfacția și împlinirea personală și presupune respectarea principiilor metodologice în valorificarea timpului. Conceptul de gestionare a timpului cuprinde o serie de definiții care descriu un sistem de activități, procese şi resurse pe care le folosim pentru a utiliza în mod eficient timpul propriu. Managementul timpului este un mod de viață și de muncă, prin care ne folosim propriul timp într-un mod eficient. Cuvinte-cheie: identitate profesională, excelență artistică, cadre didactice, învățământ primar, transpunere artistică, rezonanță socială, gestionarea timpului. Professional identification in primary education has an emotional impact on teachers, signifying a more effec tive approach to the educational process. The term identity encompasses principles, values, knowledge and skills woven into integrity [12, p. Iulia POSTOLACHI 194].f Therefore, professional identity is all about the effective conduct of the teaching process, namely: • use of the curriculum and the methodological guidelines to implement the modular curriculum; Therefore, professional identity is all about the effective conduct of the teaching process, name Therefore, professional identity is all about the effective conduct of the teaching process, namely: • use of the curriculum and the methodological guidelines to implement the modular curriculum; th d l t f l t j t d l t d t hi t i l Therefore, professional identity is all about the effective conduct of the teaching process, namely: • use of the curriculum and the methodological guidelines to implement the modular curriculum; • the development of long-term projects and related teaching materials; • the development of long-term projects and related teaching materials; ABORDAREA CRITICĂ A LIBERTĂȚII DE EXPRIMARE A PROFESORILOR ÎN CLASĂ 11 EX CATHEDRA • active-participatory, learner-centered learning; i • active-participatory, learner-centered learning; i • active-participatory, learner-centered learning; i or friends. In this example, resonance is determined by both similarity and complementarity, with causes on both the conscious and unconscious levels [2]. • identification of opportunities to make the edu cational process more efficient; [ ] Time constitutes a very valuable resource in edu cational context. Its limited, irreversible, irrevocable, non-transferable nature gives it uniqueness and value. Time, in turn, gives value to activity, i.e. good management of this resource enables efficient activity. The author who laid the foundations of scientific management, F. Taylor, postulated the principle of strict time recording and work norming, taking a huge step towards increasing efficiency in the organization [3]. At present, management means the efficient and effective running of an activity. From this perspective, the teacher, as a classroom manager, must know how to manage time resources efficiently in relation to the present needs. Performance-oriented teachers know that time is measurable and continuous, and time manage ment is a priority for the success of an organization. Time, as a key resource, is extremely important not only at an individual level but also at an organizational level. Iulia POSTOLACHI Modern teachers, as a rule, depend directly on the efficiency of their use of time, which has also led to the specification of management on this dimension – time management, which, in current management thinking, is precisely about optimizing the design and planning of teaching activity, focusing on setting objectives, planning, and setting edu cational priorities. Identifying this dimensionality of time in classroom managerial activity is very important, as it highlights the notion of rational and efficient use of time to achieve the desired results [4, p. 286]. • constructive communication and networking in the social and teaching environment; the social and teaching environment; • monitoring one’s own learning and training process [8, p. 78]. Figure 1 renders the values of the professional iden tity of artistic excellence of primary school teachers: Figure 1 renders the values of the professional iden tity of artistic excellence of primary school teachers: Figure 1. A graphic representation of professional identity Social resonance and time management Professional inte grity and efficiency Artistic transposition and sense of humor Professional identity Social resonance and time management Professional inte grity and efficiency Artistic transposition and sense of humor Professional identity Figure 1. A graphic representation of professional identity Scientific concerns about artistic transposition and sense of humor are gaining momentum in the system of language and literature education objectives, resulting in the development of strategies in this direction. They are aimed at: Time management is a personal process of planning, anticipating, and reacting in a planned, predictive, effecti ve, and efficient manner (as defined by P. R. Godin), based on three pillars: planning, organizing, and controlling time [1]. The concept of educational/teaching planning may be delimited according to the temporal and spatial resources specifically provided for the achievement of socially and individually determined pedagogical goals. Organization is a prerequisite for time efficiency. Well-planned and we ll-organized activities are enhanced by positive thinking/a positive attitude and communication. • establishing the values of the mother tongue, national literature, universal literature, and the ethnic groups concerned; f • attitudinal, affective integration into national and European value systems; • the development of personal values; • training in the ability to receive and produce values; • education in the spirit of national pride and fundamental civic obligations [7, p. 4]. The examination of social resonance and time management is felt differently by each individual. STRENGTHENING THE PROFESSIONAL IDENTITY OF PRIMARY SCHOOL TEACHERS THROUGH EFFECTIVE TIME MANAGEMENT Iulia POSTOLACHI For example, a song may give pleasure to one person and displeasure to another. Control is a key pillar in time management. Therefore, the basic paradigm of the third generation is about control based on the principles we establish. This is why institu tionalized time management is dependent on the educa tional curriculum, especially a curriculum that propels the school into the future and is based on the conceptual component, which generalizes between the coordinates of (1) a new educational ideal – the formation of a free, creative, autonomous and open personality, (2) changes in society – the transition to a democratic, computerized society, and (3) new pedagogical trends – the transition to a competence-centered design. On the other hand, this didactic design must aim to place education in the perspec The phenomenon of resonance is much more com plex when it occurs in the process of social interaction, and its explanation becomes much more elusive. As an example, the following situation may be envisaged: subject A interacts with subject B; both subjects were frustrated in their childhood by the joy of receiving gifts; nowadays, subject A feels great pleasure when he has the opportunity and the possibility to bring joy to his loved ones by giving them gifts, and subject B feels fulfilled and at peace when he receives gifts from family STRENGTHENING THE PROFESSIONAL IDENTITY OF PRIMARY SCHOOL TEACHERS THROUGH EFFECTIVE TIME MANAGEMENT 12 EX CATHEDRA tive of (1) lifelong learning, (2) interdisciplinarity and (3) competency-based curriculum as a concept and model for designing and implementing learning [4, p. 290]. from the term ‘control’. We should admit that the need to control time depends on the perception of how each individual uses their time. In general, rigorous time control allows for highlighting: Time is a resource that cannot be stored, and how we use it influences our success, health, and happiness. The more organized we are, the less stressed we are, and the better our health will be. At the same time, not managing our time properly puts our health at risk. • the proportion of time allocated/used for each task; • satisfactory actions within a given time frame; • problems encountered; • knowledge acquired; Correct time management will contribute to the per sonal development of teachers, the achievement of their highest goals, personal satisfaction, and fulfillment [10, p. 64]. • results obtained etc. • results obtained etc. Time management problems can be solved by imple menting timing techniques. The classical time management techniques are: management by objectives, spreadsheets, SWOT analysis, Gantt charts, mind maps, and evaluation of meetings. Much more up-to-date and efficient, but requiring investment, is specific software that allows to adapt the working style to Western standards [5, p. 189]. At academic year level, time management highli ghts the distribution and sequencing of time devoted to strictly didactic activities, assessment and examination, holidays etc. The valorization of pedagogical time implies compliance with the following methodological principles applicable to all the variables involved: We need to plan very carefully and rigorously: • which tasks we want to accomplish; • what is the order of priorities; • how long it is going to take us to achieve them; • how our short-term goals relate to our medium- and long-term objectives and planned outcomes; p p g g principles applicable to all the variables involved: • the distribution of the time reserved for each subject, according to the competences adopted in the school curriculum; and long-term objectives and planned outcomes; • resistance to disruptive events and time-wasting tendencies; • the means by which we monitor the achievement of tasks and how we have planned our work. • breaking down the subject matter into teaching units that can be related to time units; • alternating school activities with predominantly intellectual objectives with activities with mainly moral, technological, aesthetic, and psychophy sical objectives, and formal and non-formal training activities; Time management is actually self-management. Thus, employing the skills one possesses in order to better plan tasks at work and to delegate responsibilities more effectively is the key to optimal time management [5, p. 186]. Time management/timing has many advan tages for teacher managers. It encourages them: • the school timetable should be tailored to the pupils’ real interests and possibilities, based on the specific circumstances of the school and the subjects studied: faculty council, extracurricular activities, peer lessons etc. Iulia POSTOLACHI Of course, for efficient time management, one must take into account the set priorities, objectives, and plans, how to implement and assess them etc. Efficient time management is achieved not by solving tasks ran domly or in the order they appear, but by relating them to objectives and priorities. • results obtained etc. • to achieve the desired goal; • to set priorities; • to obtain an overview of the tasks to be accom plished; The concept of time management has been imposed as a process of planning and controlling the time used for specific activities, in particular in order to increase its effectiveness, efficiency, and productivity, which may only be achieved through proper organization of educational time – a sequence of social time specialized in preparing persons to enter learning or dedicated to their training in a profession, the formation of socially prescribed behaviors, or the acquisition of specific infor mation. Educational time is the period during which the subjects undergo a training program (directly or indirectly, explicitly or implicitly) at the level of such institutions as family, school, or cultural institutions [9, p. 46]. • to communicate better; • to achieve more results per unit of time; REFERINȚE BIBLIOGRAFICE: 1. Baciu S. Paradigma managementului calității în instituțiile de învățământ superior. Chișinău: ASEM, 2014. 1. Baciu S. Paradigma managementului calității în instituțiile de învățământ superior. Chișinău: ASEM, 2014. 2. Birău C. Mecanisme psihologice implicate în re zonanţa afectivă interindividuală. Pe: https://www. armyacademy.ro/reviste/1_2005/a11.pdf (Accesat la 15.01.2023). 3. Hawking S. W. Scurtă istorie a timpului. Bucureşti: Humanitas, 2015. 4. Melnic N. Timpul – resursă-cheie în educaţie. În: In stitutul de Ştiințe ale Educației: ascensiune, perfor manțe, personalități. 10 decembrie 2021. Chişinău: Print Caro, 2021, pp. 286-292. The ability to organize and manage time influences a person’s professional success by highlighting their time management pillars: planning, organizing, and controlling time. Identifying time management processes involves:i time. Identifying time management processes involves:i • defining project activities and determining their sequence, their technological and organizational constraints; 5. Melnic N. Principii, metode şi tehnici de manage ment eficient al timpului educaţional. În: Dezvol tarea Armatei Naţionale în contextul aprofundării reformelor democratice. Materialele conferinţei cu participare internaţională, 1 martie 2017. Chişinău: Notograf Prim SRL, 2017, pp. 185-193i • estimating the duration of the activities, which can be done in a deterministic or probabilistic way; • the drawing up of the project execution schedule, using classical or modern scheduling methods, depending on complexity; 6. Melnic N. Semnificaţiile timpului în educaţie şi în dezvoltarea umană. În: Univers Pedagogic, nr. 3 (51), 2016, pp. 33-37.i • monitoring and updating the schedule as it is being fulfilled, which includes measuring and reporting progress in schedule execution and re source use, taking corrective action, and updating and adjusting the schedule [11, p. 277]. 7. Pogolșa L. Eficientizarea învățământului – vector al politicilor educaționale moderne. În: Efici entizarea învăţământului – vector al politicilor educaţionale moderne. Conferinţă ştiinţifică in ternaţională, partea I, 11-12 decembrie 2014. Chişinău: Cavaioli, 2014, pp. 3-15. There are no “recipes” for time management, but there are methods (prioritization, organization, and planning), techniques (setting targets/objectives); set ting organizational/institutional goals; identifying key areas; formulating tasks; setting priorities and tools (SMART model, benchmarking, ABC schemes, diaries, calendars, organizers etc.) for personalizing one’s own time management [11, p. 278]. In order to acquire time management skills, teachers must adopt tools and tech niques that characterize them and suit their personality, lifestyle and professional activity [11, p. 280]. 8. Proțiuc S. Rolul proiectelor educaționale în dezvoltarea instituției de învăţământ profesional tehnic. În: Univers Pedagogic, nr. 1 (57), 2018, pp. 76-84. 9. Reaboi-Petrachi V. Didactica Pro..., nr.1 (137) anul 2023 • to delegate tasks; • to maintain a work-life balance; • to develop their creativity; fi • to overcome difficulties and to better adapt to change; • to contribute to an improvement in the quality of life of all students in the classroom [5, p. 188]. In this respect, time control and planning are cru cial. In the field of time management, control consists of measuring the progress or results of tasks carried out, in a given period, in order to identify dysfunctions and correct them. Time control has two purposes: to measure the activities being carried out and to adjust them in line with fluctuations in the variables involved, a psychological purpose linked to the fear that stems When referring to time management, we mostly think about professional activity and less about what we do with the rest of our time. But some techniques that are learned for work can also be successfully applied to private life. STRENGTHENING THE PROFESSIONAL IDENTITY OF PRIMARY SCHOOL TEACHERS THROUGH EFFECTIVE TIME MANAGEMENT 13 EX CATHEDRA There are strategies we can learn in order to manage our time more successfully. Although the idea of time management has been around for more than 100 years, the term time management did not come into general use until the 1970s, and it was not until the early 1980s that people began to think of time as a means (resource) that can be managed. Time management is the ability to decide what is important in our lives. A large number of speci alists (business and personal coaches) are increasingly promoting and studying the issue of time management as an essential resource in increasing productivity and success in both work and private life. A person with good time management is able to accomplish more tasks in less time, while being more relaxed and able to strike a balance between personal and professional life. l time into an ally, is to learn to manage it properly, to learn to set our priorities, to plan carefully the things we have to accomplish and to try and seize opportu nities when they arise [6, p. 36]. REFERINȚE BIBLIOGRAFICE: Managementul timpului în teoria şi practica educaţională. În: Integrare prin cercetare și inovare. Ştiinţe socioumanistice. Vol. 2, septem brie 2016. Chișinău: CEP USM, 2016, pp. 46-49. 10. Ștefăneț D. Managementul timpului – instrument de organizare și eficientizare. Pe: https://ibn.idsi. md/sites/default/files/imag_file/64-69_13.pdf. In conclusion: We should point out that today, when there is a lot of talk of educational reform, the time dimension can be an essential benchmark for achieving quality education, in line with the requi rements of the present day, but above all with those of the future and in light of the past. Identifying the dimension of time is highly commendable, as it hi ghlights the notion of efficiency, so as to answer the question of what is being offered to education in the past, the present and the future, and what is the value of this offer. The only thing we can do, in order to turn 11. Tarnovschi A. Gestionarea timpului – resursă psi hologică în activitatea profesională a cadrelor di dactice. În: Profesionalizarea cadrelor didactice: tendințe și realități sociopsihopedagogice, 14 mai 2021. Chişinău, 2021, pp. 277-280. 12. Tolstaia S. Identitatea profesională a psihologu lui narativ. În: Studia Universitatis Moldaviae, nr. 5 (85), Seria Științe ale educației, 2015, pp. 194-196. STRENGTHENING THE PROFESSIONAL IDENTITY OF PRIMARY SCHOOL TEACHERS THROUGH EFFECTIVE TIME MANAGEMENT 14
https://openalex.org/W2531572830	https://europepmc.org/articles/pmc5062870?pdf=render	English	null	Designer Leptin Receptor Antagonist Allo-aca Inhibits VEGF Effects in Ophthalmic Neoangiogenesis Models	Frontiers in molecular biosciences	2,016	cc-by	8,354	ORIGINAL RESEARCH published: 13 October 2016 doi: 10.3389/fmolb.2016.00067 Designer Leptin Receptor Antagonist Allo-aca Inhibits VEGF Effects in Ophthalmic Neoangiogenesis Models Roberta Coroniti 1, Rafal Farjo 2, Didier J. Nuno 2, Laszlo Otvos 3, Laura Scolaro 1 and Eva Surmacz 1* 1 Sbarro Institute for Cancer Research and Molecular Medicine, Temple University, Philadelphia, PA, USA, 2 Department of Biology, Temple University, Philadelphia, PA, USA, 3 EyeCRO, Oklahoma, OK, USA Experimental and clinical data suggest that pro-angiogenic, pro-inﬂammatory and mitogenic cytokine leptin can be implicated in ocular neovascularization and other eye pathologies. At least in part, leptin action appears to be mediated through functional interplay with vascular endothelial growth factor (VEGF). VEGF is a potent regulator of neoangiogenesis and vascular leakage with a proven role in conditions such as proliferative diabetic retinopathy, age-related macular degeneration and diabetic macular edema. Accordingly, drugs targeting VEGF are becoming mainstream treatments for these diseases. The crosstalk between leptin and VEGF has been noted in different tissues, but its involvement in the development of eye pathologies is unclear. Leptin is coexpressed with VEGF during ocular neovascularization and can potentiate VEGF synthesis and angiogenic function. However, whether or not VEGF regulates leptin expression or signaling has never been studied. Consequently, we addressed this aspect of leptin/VEGF crosstalk in ocular models, focusing on therapeutic exploration of underlying mechanisms. Here we show, for the ﬁrst time, that in retinal (RF/6A) and corneal (BCE) endothelial cells, VEGF (100 ng/mL, 24 h) stimulated leptin mRNA synthesis by 70 and 30%, respectively, and protein expression by 56 and 28%, respectively. In parallel, VEGF induced RF/6A and BCE cell growth by 33 and 20%, respectively. In addition, VEGF upregulated chemotaxis and chemokinesis in retinal cells by ∼40%. VEGF-dependent proliferation and migration were signiﬁcantly reduced in the presence of the leptin receptor antagonist, Allo-aca, at 100–250 nmol/L concentrations. Furthermore, Allo-aca suppressed VEGF-dependent long-term (24 h), but not acute (15 min) stimulation of the Akt and ERK1/2 signaling pathways. The efﬁcacy of Allo-aca was validated in the rat laser-induced choroidal neovascularization model where the compound (5 µg/eye) signiﬁcantly reduced pathological vascularization with the efﬁcacy similar to that of a standard treatment (anti-VEGF antibody, 1 µg/eye). Cumulatively, our results suggest that chronic exposure to VEGF upregulates leptin expression and function. As leptin can in turn activate VEGF, the increased abundance of both cytokines could amplify pro-angiogenic and pro-inﬂammatory environement in the eye. Thus, combined therapies targeting ObR and VEGF should be considered in the treatment of ocular diseases. Edited by: Ali Tavassoli, Edited by: Ali Tavassoli, University of Southampton, UK Reviewed by: Amaia Rodríguez, University of Navarra, Spain David Stec, University of Mississippi Medical Center, USA Correspondence: Eva Surmacz surmacz@temple.edu Specialty section: This article was submitted to Chemical Biology, a section of the journal Frontiers in Molecular Biosciences Received: 15 August 2016 Accepted: 27 September 2016 Published: 13 October 2016 Citation: Coroniti R, Farjo R, Nuno DJ, Otvos L, Scolaro L and Surmacz E (2016) Designer Leptin Receptor Antagonist Specialty section: This article was submitted to Chemical Biology, a section of the journal Frontiers in Molecular Biosciences Received: 15 August 2016 Accepted: 27 September 2016 Published: 13 October 2016 Keywords: leptin, ObR antagonist, peptide drug, VEGF, ocular neoangiogenesis INTRODUCTION by ObR antagonist Allo-aca, implicating leptin signaling in the pathological processes caused by high glucose levels (Parrino et al., 2014). Leptin, a pluripotentcytokine produced in the adipose tissue, has been ﬁrst discovered as a hormone regulating energy balance and appetite via hypothalamic signals (Wauters et al., 2000; Scolaro et al., 2010). In addition to its metabolic functions in the CNS, leptin is known to regulate multiple physiological and pathological processes, e.g., immune responses, hematopoiesis, bone remodeling, cardiovascular function, normal and neoplastic cell growth (Surmacz, 2013; Surmacz and Otvos, 2015; Upadhyay et al., 2015; Meek and Morton, 2016; Naylor and Petri, 2016). Although adipocytes are the main source of leptin, the hormone can be produced by diﬀerent types of cells and in diﬀerent organs (Scolaro et al., 2010; Sweeney, 2010; Surmacz, 2013). Adding to the bulk of experimental data, some recent clinical reports suggest that leptin can be involved in eye pathologies. For instance, in patients with proliferative diabetic retinopathy (PDR) or retinal detachment (RD), intravitreous leptin levels were signiﬁcantly elevated compared with that in patients with other ocular diseases (Gariano et al., 2000; Kovacs et al., 2015). In addition, the study suggested that locally produced leptin, not simply leptin derived from circulation, could be involved in the pathogenesis of PDR and RD (Gariano et al., 2000). Similarly, a small study conﬁrmed higher vitreous leptin levels in PDR relative to other retinopathies (Maberley et al., 2006). Whether leptin is causally related to the progression of DR is still under investigation. This study focuses on the angiogenic function of leptin and its involvement in ocular neovascularization. The leptin receptor (ObR) is expressed in vascular endothelial cells and studies in vitro demonstrated that leptin can induce angiogenic diﬀerentiation as well as proliferation and migration of endothelial cells, including cells of ophthalmic origin (Bouloumié et al., 1998; Sierra-Honigmann et al., 1998; Cao et al., 2001; Park et al., 2001; Anagnostoulis et al., 2008; Ferla et al., 2011; Garonna et al., 2011; Scolaro et al., 2013; Parrino et al., 2014; Adya et al., 2015). In mouse models, transgenic overexpression of the leptin gene (ob) potentiated ischemia-induced retinal neovascularization, while leptin deﬁciency due to ob inactivation, signiﬁcantly reduced ocular angiogenesis, proving again the role of this cytokine in neovascularization (Suganami et al., 2004). INTRODUCTION Similarly, leptin was not able to induce neovascularization in corneas of fa/fa Zucker rats that lack functional ObR, underlying the importance of leptin signaling in this process (Sierra- Honigmann et al., 1998). An important aspect of leptin’s role in the regulation of key processes implicated in eye diseases is its functional connection with vascular endothelial growth factor (VEGF), a major regulator of neoangiogenesis and vascular leakage with a proven role in ocular pathologies such as PDR, age- related macular degeneration (AMD) and diabetic macular edema (DME) (Miller, 2016). Notably, experimental evidence suggests that leptin can induce and amplify VEGF expression and signaling. For instance, in tetrandrine-induced corneal neovascularization model, leptin is found coexpressed with VEGF (Sun et al., 2011). In ischemia-induced neovascularization, leptin can potentiate vessel formation through induction of VEGF expression (Suganami et al., 2004). In endothelial HUVEC cells, leptin stimulats angiogenesis simultaneously with upregulation of VEGF expression (Bouloumié et al., 1998; Sierra- Honigmann et al., 1998). Furthermore, in HUVEC cells, leptin- mediated angiogenesis and intracellular signaling through the p38 MAPK/Akt/COX-2 pathway is partially reduced with a VEGFR inhibitor, implicating this receptor in leptin response (Garonna et al., 2011). In syngeneic mammary cancer models, the inhibition of leptin signaling signiﬁcantly reduces the levels of VEGF and its receptor VEGFR2 (Newman and Gonzalez- Perez, 2014). The codependence of VEGF and leptin expression has been also noted in ob/ob mice where lack of functional leptin is associated with increased circulating concentrations of VEGF-A and leptin replacement normalizes VEGF-A levels in this model. In addition, leptin regulation of VEGF-A expression has been demonstrated in obese subjects before and after weight loss (Gómez-Ambrosi et al., 2010). We have recently demonstrated that leptin is a potent mitogenic and angiogenic factor in retinal and corneal endothelial cells (Scolaro et al., 2013; Parrino et al., 2014). We have described that these leptin functions are associated with the modulation of the activity or expression of several signaling molecules involved in proliferation, inﬂammatory activity and angiogenesis, including the transcription factor STAT3, common kinases Ras, ERK1/2 and Akt, pro-inﬂammatory mediators and regulators COX2 and NF-κB. Furthermore, we have found that leptin can upregulate its own mRNA and protein expression in retinal and corneal cells, suggesting the existence of leptin autocrine circuits in the eye. Citation: Coroniti R, Farjo R, Nuno DJ, Otvos L, Scolaro L and Surmacz E (2016) Designer Leptin Receptor Antagonist Allo-aca Inhibits VEGF Effects in Ophthalmic Neoangiogenesis Models. Front. Mol. Biosci. 3:67. doi: 10.3389/fmolb.2016.00067 October 2016 \| Volume 3 \| Article 67 Frontiers in Molecular Biosciences \| www.frontiersin.org 1 Leptin Antagonist Blocks VEGF Action Coroniti et al. Frontiers in Molecular Biosciences \| www.frontiersin.org INTRODUCTION We have validated leptin involvement in the above processes using a selective and highly eﬃcacious ObR antagonist Allo-aca, a leptin peptidomimetic that blocks ObR activation and biological activity at low-mid nmol//L concentrations in vitro in diﬀerent cell types (Scolaro et al., 2013; Parrino et al., 2014). However, whether or not leptin/VEGF crosstalk is a bilateral relationship, i.e., if VEGF can inﬂuence leptin expression and function, has never been studied. Consequently, this aspect of leptin/VEGF crosstalk and the potential of therapeutic exploration of the underlying mechanism is the subject of the present paper. The mechanisms of leptin expression in the eye are still under investigation. Previous studies have shown that eye injury, ischemic or hyperglycemic conditions can increase leptin expression (Suganami et al., 2004; Sun et al., 2010, 2011). We have recently reported that hyperglycemia can induce leptin mRNA and protein expression in retinal endothelial cells and that this process is associated with increased angiogenesis, cell growth and migration. These eﬀects can be partially reversed At present, several biologic drugs targeting VEGF and/or its receptor have been approved for ophthalmology use (van der Giet et al., 2015; Miller, 2016). However, new treatments, perhaps with broader therapeutic spectrum, are needed to decrease adverse eﬀects and/or complement anti-VEGF drugs (Tang and Kern, 2011; Truong et al., 2011; Chen et al., 2013; van der Giet et al., October 2016 \| Volume 3 \| Article 67 2 Leptin Antagonist Blocks VEGF Action Coroniti et al. β-actin using a TaqMan probe (Life Technologies). The levels of leptin mRNA relative to β-actin mRNA were determined using a comparative CT method (Life Technologies). All reactions were done in triplicate and an average CT value (±SD) for all RNAs was calculated. The individual experiments were repeated at least 3 times. 2015). In this context, targeting leptin, a known pro-angiogenic and pro-inﬂammatory factor whose function is intimately related to VEGF, could prove provide an attractive targeted therapy for pathological neovascularization in the eye (Cheung et al., 2010). Transwell Migration Assays The eﬀects of VEGF on chemotactic properties of RF/6A cells were studied using Transwell inserts (8.0 µm pore size) (Corning, Tewksbury, MA). The cells (5–6th passage) were plated at concentrations 5 × 104 cells/well and allowed to migrate through the membrane for 24 h. Then, non-migrated cells in the upper chamber were removed and the cells that migrated across the membranes were stained with Giemsa for 20 min and counted. To test chemotactic eﬀects of VEGF on RF/6A cells, 100 ng/mL VEGF was added to the lower chamber only. The involvement of VEGF-dependent chemokinesis was assessed using 100 or 250 nmol/L Allo-aca. Each migration assay was done in triplicate and repeated at least 3 times and the mean number of migrated cells ± SD was determined. Proliferation Assay The cells (5–7th passage) were plated in 24-well plates at concentrations 5–8 × 104 and 1–1.5 × 105 cells/well for RF/6A and BCE cells, respectively. At semi-conﬂuence, the cells were shifted to SFM for 24 h and then treated with 50–250 ng/mL of VEGF for 24 h, without 100–250 nmol/L Allo-aca. All assays were done in triplicate and repeated 3 times. Cell numbers were determined by direct counting as describe by us previously (Scolaro et al., 2013). The percentage decrease/increase in cell number vs. control SFM was calculated and expressed as mean ± standard deviation (SD). Cell Lines and Growth Conditions The cellular assays were performed using monkey endothelial retinal cells (RF/6A) and bovine endothelial corneal cells (BCE). The cells were purchased and cultured as recommended by the supplier (American Type Culture Collection, Rockville, MD, USA) and describe by us in detail previously (Scolaro et al., 2013). Cell culture reagents and media were purchased from Cellgro (Herndon, VA, USA). Before treatments, the cells were synchronized in serum-free medium (SFM) containing 10 µM FeSO4 0.5% bovine serum albumin, 1% FBS, 1% Pen/Strep. We have shown before that RF/6A and BCE ocular endothelial cell lines express ObR and respond to leptin with the activation of various biological functions, i.e., growth, signaling, angiogenesis, migration (Scolaro et al., 2013; Parrino et al., 2014). Wound-Healing (Scratch) Assay The ObR antagonist, Allo-aca, a short leptin-based peptidomimetic (H-alloThr-Glu-Nva-Val-Ala-Leu-Ser-Arg- Aca-NH2) was used to inhibit ObR signaling and function. The process of Allo-aca design, development and eﬃcacy in vitro, including the ophthalmic models, and in vivo has been reported by us before (Otvos et al., 2011a,b; Scolaro et al., 2013; Parrino et al., 2014). VEGF (human recombinant, VEGF 165) was purchased from Gibco Life Technologies (Grand Island, NY). Directional cell migration in vitro was assessed using a wound- healing assay. Linear scratches (3 per plate) were produced in 100% conﬂuent cultures of RF/6A cells using a 200 µL tip. The cultures were then shifted to SFM, SFM containing 100 ng/mL VEGF, or SFM with 100 ng/mL VEGF plus 100–250 nmol/L Allo- aca for 24 h. Wound dimensions (at least 6 ﬁelds/experimental condition) were recorded with Olympus 1 × 81 phase-contrast microscope at 2.0x magniﬁcation and images were acquired using Metamorph 7.5 program. The scratch areas were quantiﬁed using the Adobe Acrobat Pro program and the areas expressed in arbitrary units (AU). Immunoﬂuorescence Leptin protein was detected in RF/6A and BCE cells by immunoﬂuorescence (IF), as described by us before (Bartella et al., 2008; Cascio et al., 2008; Scolaro et al., 2013). In short, 1 × 105 cells were plated on glass coverslips in normal growth medium. After 24 h, the cells were synchronized in SFM for 24 h and then treated either with 100 ng/mL VEGF in the absence or presence of 100 or 250 nmol/L Allo-aca for 24 h. Next, the cells were washed with PBS, ﬁxed in methanol, and permeabilized in 0.2 Triton X-100%. Leptin expression was detected using pAb A-20 (1:25 dilution; 2 h) and goat anti- rabbit IgG-FITC (1:1000 plus 1.5% blocking goat serum; 1 h). In control experiments, primary Abs were replaced by non- immune serum. To visualize cell nuclei, the coverslips were mounted with UltraCruz Mounting Medium containing DAPI (5 µg/mL of 4′,6-diamidino-2-phenylindole). The expression of leptin was detected using Olympus 1 × 81 phase-contrast microscope at 3.2x magniﬁcation. The percentage of positive cells was determined in 10 visual ﬁelds. All reagents were purchased from Santa Cruz Biotechnology (Dallas, TX). Neovascularization (CNV) Assays ( ) y All animal experiments conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. A 22-day study was conducted in female 6 week old Brown Norway rats to determine the antiangiogenic/vascular disrupting eﬀects of the leptin receptor antagonist, Allo-aca, in a laser induced model of CNV. The animals were divided into 3 separate treatment groups of 6 animals per group. On day 1, laser treatments were performed on all groups using a 520 nmol/L thermal laser to generate a total of three lesions per eye. On day 3, group 1 received bilateral intravitreal (ivt) injections of the vehicle, group 2 (positive control) received bilateral ivt injections of an anti-VEGF antibody (R&D Systems, AF564) at 1 µg/eye, and group 3 (test) received bilateral intravitreal injections of Allo- aca at 5 µg/eye. Intravitreal administration of Allo-aca was well tolerated by the rats and no adverse events were observed. On Day 22 (3-weeks post-laser treatment), ﬂuorescein angiography was performed and lesion size area was determined following hand tracing of the lesions using image analysis software ImageJ. The experiments were performed under animal protocol 11- 156-H approved by the IACUC committee of the University of Oklahoma Health Sciences Center. VEGF Induces Leptin mRNA and Protein Expression in Ocular Endothelial Cells. Allo-aca Reduces These Effects The eﬀects of VEGF on leptin expression were assessed in ocular endothelial cell models RF/6A and BCE. In both cell lines, VEGF was tested at 100 ng/mL concentrations for 24 h. The treatment signiﬁcantly induced leptin mRNA expression in both cell lines (Table 1). VEGF upregulation of leptin mRNA was more pronounced in RF/6A cells (∼1.7-fold) vs. BCE cells (∼1.3-fold). Similarly, exposure to VEGF for 24 h signiﬁcantly increased the number of cells with well detectable expression of the leptin protein. In both cell lines, the level of leptin-positive cells under SFM conditions was below 1%, while upon VEGF treatment, 56 ± 5% of RF/6A cells and 28 ± 3% of BCE cells displayed leptin expression (Figure 1). The upregulation of leptin expression in both cell lines by VEGF was statistically signiﬁcant. ObR Antagonist Allo-aca Inhibits VEGF Mitogenic Effects In addition to strong pro-angiogenic activities, VEGF is known to induce mitogenesis in diﬀerent endothelial cell models (Lu et al., 2010). Similarly, leptin can increase cell growth in BCE and RF/6A cells, as previously demonstrated by us (Scolaro et al., 2013). Because VEGF increased leptin expression in these cell models, we speculated that at least part of VEGF mitogenic action is mediated through the leptin/ObR axis. To test this hypothesis, we examined if VEGF-mediated growth could be reduced in the presence of the ObR antagonist, Allo-aca. TABLE 1 \| VEGF induces leptin mRNA expression in BCE and RF6A cells. BCE Leptin mRNA levels (fold ± SD over SFM) RF/6A Leptin mRNA Expression (fold ± SD over SFM) SFM 1.0 1.0 VEGF 1.3 ± 0.2 1.7 ± 0.1* VEGF + Allo-aca 0.9 ± 0.1# 0.6 ± 0.2# Leptin mRNA levels in cells treated with 100 ng/mL VEGF for 24 h were measured by QRT- PCR as described in Materials and Methods. Statistically signiﬁcant differences (p ≤0.05) vs. SFM are marked with * and vs. VEGF with #. TABLE 1 \| VEGF induces leptin mRNA expression in BCE and RF6A cells. BCE Leptin mRNA levels (fold ± SD over SFM) RF/6A Leptin mRNA Expression (fold ± SD over SFM) SFM 1.0 1.0 VEGF 1.3 ± 0.2* 1.7 ± 0.1* VEGF + Allo-aca 0.9 ± 0.1# 0.6 ± 0.2# Leptin mRNA levels in cells treated with 100 ng/mL VEGF for 24 h were measured by QRT- PCR as described in Materials and Methods. Statistically signiﬁcant differences (p ≤0.05) vs. SFM are marked with * and vs. VEGF with #. TABLE 1 \| VEGF induces leptin mRNA expression in BCE and RF6A cells. Quantitative Real Time PCR (qRT-PCR) Qua t tat e ea e C (q C ) Leptin mRNA was detected by qRT-PCR as described in detail previously (Scolaro et al., 2013). Brieﬂy, RF/6A and BCE cells at semi-conﬂuence were placed in SFM for 24 h, pretreated or not with 250 nmol/L Allo-aca for 1 h, and then treated with 100 ng/mL VEGF for 6 and 24 h. RNA was isolated from cultures using Trizol Reagent (Life Technologies, Grand Island, NY) and 4 µg of RNA was reverse transcribed using the High-Capacity cDNA Kit (Life Technologies). The RT products were used to amplify leptin sequences using TaqMan probes Bt03211909_m1 for bovine leptin (Gene ID: 280836) and Rh02788316_m1 for monkey leptin (Gene ID: 698728) (Life Technologies). For normalization, parallel reactions were run on each sample for October 2016 \| Volume 3 \| Article 67 Frontiers in Molecular Biosciences \| www.frontiersin.org 3 Leptin Antagonist Blocks VEGF Action Coroniti et al. RESULTS g g The eﬀects of Allo-aca on VEGF-induced signaling in RF/6A cells were tested by Western immunoblotting (WB). In brief, cells were synchronized in SFM for 24 h and then treated with 100 ng/mL VEGF in the presence or absence of 250 nmol/L Allo-aca for 24 h or were left untreated. Next, total cellular proteins were obtained and the expression and activation of signaling molecules was evaluated with speciﬁc antibodies (Abs) as described previously (Scolaro et al., 2013). The following primary Abs from Cell Signaling Technology (Danvers, MA) were used: phospho-Akt, Akt Ser473 pAb, 1:500; total Akt, Akt pAb, 1:1000; phospho-STAT3, STAT3 Tyr705, D3A7 mAb, 1:500; total STAT3, STAT3 79D7 mAb, 1:500; phospho-ERK1/2, p44/42 mitogen-activated protein kinase (MAPK; ERK1/2) pAb Thr202/Tyr204, 1:1000; total ERK1/2, p44/42 MAPK pAb, 1:1000. The experiments were repeated at least 3 times. Densitometry Evaluation of Protein Expression As leptin is known to stimulate its own expression, we probed if the inhibition of leptin signaling can decrease the above VEGF eﬀects. To this end, we employed a peptide antagonist of the leptin receptor, Allo-aca, that has been shown to block leptin signaling and action in numerous in vitro and in vivo models (Otvos et al., 2011a,b; Scolaro et al., 2013; Parrino et al., 2014). In the present work, Allo-aca at 250 nmol/L reduced VEGF- dependent leptin mRNA expression in both cell lines below base levels (Table 1). Similarly, addition of Allo-aca at 250 nmol/L to VEGF treatment reduced the number of leptin-positive cells in RF/6A and BCE cultures by 75 ± 4 and 80 ± 5%, respectively (Figure 1). The intensity of bands corresponding to studied proteins was measured in all WB as described before using Image J program (National Institutes of Health; Scolaro et al., 2013). The modiﬁcations in protein expression/phosphorylation were evaluated as decrease/increase vs. SFM (% ± SD), diﬀerences with p ≤0.05 were considered signiﬁcant. Allo-aca Inhibits VEGF-Induced Chemotaxis and Chemokinesis in RF/6A Retinal Endothelial Cells TABLE 2 \| VEGF induces cell growth in BCE and RF/6A cells. Treatment BCE Growth response (% ± SD over SFM) RF/6A Growth response (% ± SD over SFM) VEGF 50 9.0 ± 0.9* 7.1 ± 0.2* VEGF 100 20.4 ± 1.7* 33.1 ± 1.9* VEGF 250 16.2 ± 1.8* 29.3 ± 1.9* VEGF 100 + Allo-aca 100 −4.0 ± 0.0* 4.0 ± 0.1* VEGF 100 + Allo-aca 250 −18.0 ± 1.2* −4.1 ± 1.3 Allo-aca inhibits VEGF mitogenic effects. Proliferation assays were carried out as described in Materials and Methods. Increase of cell number (%) over that in SFM is shown. VEGF was used at 50–250 ng/mL; Allo-aca was used at 100 and 250 nmol/L. Statistically signiﬁcant differences (p ≤0.05) vs. SFM are marked with asterisk. VEGF as well as leptin are well recognized as regulators of chemotaxis and chemokinesis–processes that are intimately involved in angiogenic diﬀerentiation. To assess if VEGF eﬀects on chemotaxis and chemokinesis are mediated indirectly through ObR, we used RF/6A cells, characterized by robust migratory abilities in vitro. The migration of RF/6A cells was measured in wound-healing and Transwell assays. TABLE 2 \| VEGF induces cell growth in BCE and RF/6A cells. In wound-healing assay, basal cell migration was observed even in SFM at 24 h after cell plating, likely due to the activity of autocrine pro-migratory factors (e.g., leptin) produced by RF/6A cells (Scolaro et al., 2013). Allo-aca inhibits VEGF mitogenic effects. Proliferation assays were carried out as described in Materials and Methods. Increase of cell number (%) over that in SFM is shown. VEGF was used at 50–250 ng/mL; Allo-aca was used at 100 and 250 nmol/L. Statistically signiﬁcant differences (p ≤0.05) vs. SFM are marked with asterisk. Allo-aca inhibits VEGF mitogenic effects. Proliferation assays were carried out as described in Materials and Methods. Increase of cell number (%) over that in SFM is shown. VEGF was used at 50–250 ng/mL; Allo-aca was used at 100 and 250 nmol/L. Statistically signiﬁcant differences (p ≤0.05) vs. SFM are marked with asterisk. Addition of VEGF at 100 ng/mL further stimulated cell motility, reﬂected by signiﬁcantly reduced (∼40%) scratch FIGURE 1 \| VEGF induces leptin protein expression in BCE and RF6A cells. Cells were synchronized in SFM and stimulated with 100 ng/mL VEGF for 24 h in the presence or absence of 250 nmol/L Allo-aca (Allo). Control cells were left untreated in SFM. Statistical Analysis The results of in vitro and CNV experiments were analyzed by a two-tailed distribution paired Student’s t-test; p ≤0.05 were considered statistically signiﬁcant. Leptin mRNA levels in cells treated with 100 ng/mL VEGF for 24 h were measured by QRT- PCR as described in Materials and Methods. Statistically signiﬁcant differences (p ≤0.05) vs. SFM are marked with * and vs. VEGF with #. October 2016 \| Volume 3 \| Article 67 Frontiers in Molecular Biosciences \| www.frontiersin.org 4 Leptin Antagonist Blocks VEGF Action Coroniti et al. area compared with that under untreated SFM conditions (p ≤0.05) (Figure 2). The eﬀects of VEGF were totally abolished (p ≤0.05) in the presence of 250 nmol/L Allo-aca (Figure 2). First, we found that VEGF at 50–250 ng/mL induced proliferation in BCE and RF/6A cells. In both cell lines, the best growth response was observed with VEGF used at 100 ng/mL (Table 2). In the presence of Allo-aca at 100 or 250 nmol/L, VEGF-induced proliferation was either reduced to base or below base levels (Table 2), suggesting that leptin pathways are implicated in VEGF response. In the Transwell assay, VEGF at 100 mg/mL induced directional migration of ∼37% of RF/6A cells. Addition of Allo- aca at 100 nmol/L partially (∼46%) reduced VEGF eﬀects, while Allo-aca at 250 nmol/L blocked VEGF-directed migration by ∼92% (Table 3). Allo-aca Inhibits VEGF-Induced Chemotaxis and Chemokinesis in RF/6A Retinal Endothelial Cells Treatment Migrating cells (% ± SD over SFM) SFM 0.0 VEGF 37.6 ± 7.0* VEGF + Allo-aca 100 20.4 ± 2.3* VEGF + Allo-aca 250 3.4 ± 0.8 Chemotaxis induced by VEGF was measured in Boyden chamber assays as described in Materials and Methods. VEGF was used at 100 ng/mL; Allo-aca was used at 100 and 250 nmol/L. The number of migrating cells in SFM is taken as 0%. Statistically signiﬁcant differences (p ≤0.05) vs. SFM are marked with asterisk. TABLE 3 \| Allo-aca inhibits VEGF-induced chemotaxis in RF/6A cells. Treatment Migrating cells (% ± SD over SFM) SFM 0.0 VEGF 37.6 ± 7.0* VEGF + Allo-aca 100 20.4 ± 2.3* VEGF + Allo-aca 250 3.4 ± 0.8 Chemotaxis induced by VEGF was measured in Boyden chamber assays as described in Materials and Methods. VEGF was used at 100 ng/mL; Allo-aca was used at 100 and 250 nmol/L. The number of migrating cells in SFM is taken as 0%. Statistically signiﬁcant differences (p ≤0.05) vs. SFM are marked with asterisk. TABLE 3 \| Allo-aca inhibits VEGF-induced chemotaxis in RF/6A cells. STAT3, ERK1/2, Akt, which are common for VEGFR and ObR were assessed (Figure 3). g At 15 min, VEGF greatly (p ≤0.05) increased the phosphorylation of ERK1/2 (130 ± 16%) and Akt (181 ± 31%), but had no signiﬁcant eﬀect on STAT3 (p ≥0.05). None of these acute VEGF responses were aﬀected by the presence of Allo- aca (p ≥0.05). In contrast, at 24 h VEGF induced robust and signiﬁcant STAT3 activation (203 ± 24%) as well as maintained increased phosphorylation of ERK1/2 (109 ± 11%) and Akt (46 ± 5%) In the presence of Allo-aca, the long-term VEGF eﬀects on ERK1/2 and Akt were well suppressed (by 80 ± 12% and 91 ± 10%, respectively), while the phosphorylation of STAT3 was only moderately reduced (by 50 ± 10%) (p ≥0.05) (Figure 3). Chemotaxis induced by VEGF was measured in Boyden chamber assays as described in Materials and Methods. VEGF was used at 100 ng/mL; Allo-aca was used at 100 and 250 nmol/L. The number of migrating cells in SFM is taken as 0%. Statistically signiﬁcant differences (p ≤0.05) vs. SFM are marked with asterisk. Allo-aca Inhibits VEGF-Induced Chemotaxis and Chemokinesis in RF/6A Retinal Endothelial Cells The expression of leptin protein (green immunoﬂuorescence) was detected with speciﬁc Abs while cell nuclei (blue ﬂuorescence) were detected with DAPI, as described in Materials and Methods. The bar represents 10 µm. FIGURE 1 \| VEGF induces leptin protein expression in BCE and RF6A cells. Cells were synchronized in SFM and stimulated with 100 ng/mL VEGF for 24 h in the presence or absence of 250 nmol/L Allo-aca (Allo). Control cells were left untreated in SFM. The expression of leptin protein (green immunoﬂuorescence) was detected with speciﬁc Abs while cell nuclei (blue ﬂuorescence) were detected with DAPI, as described in Materials and Methods. The bar represents 10 µm. October 2016 \| Volume 3 \| Article 67 5 Frontiers in Molecular Biosciences \| www.frontiersin.org Coroniti et al. Leptin Antagonist Blocks VEGF Action FIGURE 2 \| Allo-aca inhibits VEGF-induced chemokinesis in RF6A cells. The scratch assays were preformed as described in Materials and Methods. Immediately after wounding, the cells were photographed (control, C) and then placed for 24 h in either SFM or incubated with 100 ng/mL VEGF in the presence or absence of 250 nmol/L Allo-aca (Allo). The scratch areas before and after treatments were measured as described in Materials and Methods and the data are shown in the graph. Statistically signiﬁcant changes (p ≤0.05) are marked (SFM vs. VEGF ; VEGF vs. VEGF+Allo-aca #). The initial scratch boundaries (C) have been marked in the picture. The bar represents 250 µm. FIGURE 2 \| Allo-aca inhibits VEGF-induced chemokinesis in RF6A cells. The scratch assays were preformed as described in Materials and Methods. Immediately after wounding, the cells were photographed (control, C) and then placed for 24 h in either SFM or incubated with 100 ng/mL VEGF in the presence or absence of 250 nmol/L Allo-aca (Allo). The scratch areas before and after treatments were measured as described in Materials and Methods and the data are shown in the graph. Statistically signiﬁcant changes (p ≤0.05) are marked (SFM vs. VEGF ; VEGF vs. VEGF+Allo-aca #). The initial scratch boundaries (C) have been marked in the picture. The bar represents 250 µm. TABLE 3 \| Allo-aca inhibits VEGF-induced chemotaxis in RF/6A cells. Allo-aca Inhibits Ocular Neovascularization in the CNV Animal Model To examine if VEGF activates intracellular pathways indirectly through ObR, we stimulated RF/6A cells with 100 ng/mL VEGF in the presence or absence of 250 nmol/L Allo-aca. Both acute (15 min) and long-term (24 h) eﬀects on signaling molecules such as Allo-aca has previously been shown to reduce leptin- induced angiogenic eﬀects in ocular endothelial cell models in vitro (Scolaro et al., 2013; Parrino et al., 2014), however the eﬃcacy of this inhibitor in a relevant in vivo model October 2016 \| Volume 3 \| Article 67 Frontiers in Molecular Biosciences \| www.frontiersin.org 6 Leptin Antagonist Blocks VEGF Action Coroniti et al. FIGURE 3 \| Allo-aca inhibits several VEGF-induced signaling pathways. RF/6A cells were stimulated for 15 min or 24 h with 100 ng/mL VEGF in the presence or absence of 250 nmol/L Allo-aca (Allo); control cells were left untreated in SFM. The expression of phosphorylated (p) and total (Tot) proteins was assessed by WB and quantiﬁed as described in Materials and Methods. The representative WB results are shown. The numbers under WB panels represent densitometry values (%) of phosphorylated and total proteins in the blot shown, with the value in SFM taken as 100%. The average increase/decrease values from different experiments are given under Results. Statistically signiﬁcant differences vs. untreated cells are marked with * and vs. VEGF with #. FIGURE 3 \| Allo-aca inhibits several VEGF-induced signaling pathways. RF/6A cells were stimulated for 15 min or 24 h with 100 ng/mL VEGF in the presence or absence of 250 nmol/L Allo-aca (Allo); control cells were left untreated in SFM. The expression of phosphorylated (p) and total (Tot) proteins was assessed by WB and quantiﬁed as described in Materials and Methods. The representative WB results are shown. The numbers under WB panels represent densitometry values (%) of phosphorylated and total proteins in the blot shown, with the value in SFM taken as 100%. The average increase/decrease values from different experiments are given under Results. Statistically signiﬁcant differences vs. untreated cells are marked with * and vs. VEGF with #. DISCUSSION have never been tested. We used the well-recognized rat CNV model (Shah et al., 2015) to induce robust ocular neovascularization and probe the activity of Allo-aca to suppress laser-induced lesions. As described before, leptin mRNA is detectable in CNV-treated rat eyes (Scolaro et al., 2013). Several biologic drugs targeting VEGF and/or its receptor have been approved for the use in ophthalmology. At present, VEGF neutralizing drugs, ranibizumab (a humanized monoclonal antibody fragment with molecular weight of 48 kDa), and bevacizumab (a recombinant humanized monoclonal antibody with molecular weight of 149 kDa), that were engineered to bind with high aﬃnity and to neutralize all biologically active isoforms of VEGF (Ferrara et al., 2006; Scolaro et al., 2013) and aﬂibercept (a fusion protein containing the second immunoglobulin domain of VEGFR-1 and the third immunoglobulin domain of VEGFR- 2, which binds all isoforms of VEGF, VEGF-B, and placental growth factor; Holash et al., 2002; Miller et al., 2013; Scolaro et al., 2013) are approved for the treatment of wet AMD and DME, and experimentally used for other eye diseases, e.g., PDR (Willard and Herman, 2012; Scolaro et al., 2013). Most trials have shown beneﬁts with the use of intravitreal anti-VEGF agents for both DME and PDR (Cheung et al., 2010; Scolaro et al., 2013). However, adverse eﬀects (systemic and ocular) and development of resistance to the treatment have been noted with long-term Several biologic drugs targeting VEGF and/or its receptor have been approved for the use in ophthalmology. At present, VEGF neutralizing drugs, ranibizumab (a humanized monoclonal antibody fragment with molecular weight of 48 kDa), and bevacizumab (a recombinant humanized monoclonal antibody with molecular weight of 149 kDa), that were engineered to bind with high aﬃnity and to neutralize all biologically active isoforms of VEGF (Ferrara et al., 2006; Scolaro et al., 2013) and aﬂibercept (a fusion protein containing the second immunoglobulin domain of VEGFR-1 and the third immunoglobulin domain of VEGFR- 2, which binds all isoforms of VEGF, VEGF-B, and placental growth factor; Holash et al., 2002; Miller et al., 2013; Scolaro et al., 2013) are approved for the treatment of wet AMD and DME, and experimentally used for other eye diseases, e.g., PDR (Willard and Herman, 2012; Scolaro et al., 2013). Most trials have shown beneﬁts with the use of intravitreal anti-VEGF agents for both DME and PDR (Cheung et al., 2010; Scolaro et al., 2013). DISCUSSION However, adverse eﬀects (systemic and ocular) and development of resistance to the treatment have been noted with long-term The eﬃcacy of Allo-aca was tested in parallel with the “standard treatment”, i.e., the VEGF Ab. The experiment demonstrated that treatment with either 1 µg anti-VEGF Ab/eye or 5 µg/eye of Allo-aca resulted in signiﬁcant reduction of lesion size vs. the vehicle control group. When compared with CNV rats injected with NaCl, anti- VEGF intraocular injection inhibited new vessels formation by ∼35% (p < 0.01) while injections with ObR antagonist reduced neoangiogenesis by ∼30% (<0.05) at 22 days post procedure. Notably, there was no signiﬁcant diﬀerence in lesion size between the group receiving anti-VEGF Ab and Allo-aca (Figure 4). As expected, due to the multifactorial and complex nature of the CNV (Shah et al., 2015), none of the treatments resulted in complete reduction of the lesions. October 2016 \| Volume 3 \| Article 67 Frontiers in Molecular Biosciences \| www.frontiersin.org 7 Leptin Antagonist Blocks VEGF Action Coroniti et al. FIGURE 4 \| Allo-aca inhibits laser-induced choroidal neovascularization in vivo. Choroidal neovascularization (CNV) was induced by bilateral laser treatment as described in Materials and Methods. Three days post-injury, the animals received bilateral intravitreal injections of NaCl, an anti-VEGF antibody 5 µg/eye or Allo-aca 5 µg/eye. Three weeks post-laser treatment, ﬂuorescein angiography was performed and lesion size area was determined with image analysis software. The representative angiography results (fundus images of 3 lesions) in NaCl and Allo-aca treated eyes are shown. The graphs show the average area of lesion size area at the experimental end point. Statistical signiﬁcance calculated with Student’s t-test. p ≤0.05; p ≤0.01. FIGURE 4 \| Allo-aca inhibits laser-induced choroidal neovascularization in vivo. Choroidal neovascularization (CNV) was induced by bilateral laser treatment as described in Materials and Methods. Three days post-injury, the animals received bilateral intravitreal injections of NaCl, an anti-VEGF antibody 5 µg/eye or Allo-aca 5 µg/eye. Three weeks post-laser treatment, ﬂuorescein angiography was performed and lesion size area was determined with image analysis software. The representative angiography results (fundus images of 3 lesions) in NaCl and Allo-aca treated eyes are shown. The graphs show the average area of lesion size area at the experimental end point. Statistical signiﬁcance calculated with Student’s t-test. p ≤0.05; **p ≤0.01. endothelial cells. These eﬀects are accompanied by increased cell growth and cell and are abolished in the presence of ObR antagonist, Allo-aca. DISCUSSION In addition, VEGF promotes cell chemotaxis and chemokinesis in retinal endothelial cells, which at least in part is restricted by Allo-aca. Likewise, many long- term, but not acute, intracellular signaling eﬀects of VEGF are blocked in the presence of Allo-aca. These VEGF-induced intracellular signals (ERK1/2, Akt) are common for ObR and VEGFR pathways. All these observations suggest that VEGF indirectly activates leptin pathways by upregulating leptin expression, thus, potentiating leptin signaling. Increased leptin concentration, in turn, likely can activate its own expression as well as upregulate VEGF expression, thereby greatly enhancing pro-angiogenic environment in the eye. use, thus targeting pro-angiogenic factors other than VEGF could be prove to be an eﬀective alternative or complementary therapy for pathological neovascularization in the eye (Praidou et al., 2010; Tang and Kern, 2011; Truong et al., 2011; Stewart, 2012; Willard and Herman, 2012) In addition to VEGF, many other molecular players including pro-angiogenic and pro-inﬂammatory cytokines, anti-angiogenic factors, integrins and matrix proteinases have been implicated in ocular pathologies (Praidou et al., 2010; Truong et al., 2011; Wang et al., 2012). Recent data, including that from our laboratory, suggest that leptin, a cytokine implicated in neovascularization, proliferation and inﬂammation can induce growth and angiogenic diﬀerentiation of ocular endothelial cells in vitro (Cao et al., 2001; Suganami et al., 2004; Sun et al., 2011; Scolaro et al., 2013) and might be implicated in DR (Gariano et al., 2000; Parrino et al., 2014). Furthermore, targeting ObR has been shown to inhibit most of biological eﬀects of leptin in ocular disease models (Scolaro et al., 2013; Parrino et al., 2014). This new direction in evaluating leptin a target in ocular neovascularization needs further assessment in light of reports showing diﬀerential (and model speciﬁc) leptin inﬂuence on such components of vasculature as vascular smooth muscle cells (Oda et al., 2001; Bohlen et al., 2007; Rodriguez et al., 2010). We previously demonstrated that inhibition of angiogenesis in vitro by combined ObR and VEGF inhibitors is more eﬃcacious than individual treatments (Ferla et al., 2011). Our present study shows that the eﬃcacy of ObR antagonist Allo-aca in restricting experimental neovascularization in animal models is similar to that achieved with compounds targeting VEGF. This suggests that development of therapeutic approaches interfering simultaneously or sequentially with both pathways could be clinically beneﬁcial. DISCUSSION Our previous preliminary observations suggested that simultaneous inhibition of leptin and VEGF pathways was more eﬃcacious in suppressing angiogenesis than individual treatments in HUVEC cells (Ferla et al., 2011). The existence of leptin/VEGF crosstalk has been noted in diﬀerent experimental models (Sierra-Honigmann et al., 1998; Suganami et al., 2004; Gonzalez-Perez et al., 2010; Ferla et al., 2011; Garonna et al., 2011). However, the functional links between leptin and VEGF have not been suﬃciently explored in the context of eye disease and relevant therapeutic interventions. While leptin has been shown to stimulate VEGF expression and potentiate ocular neovascularization, the impact of VEGF on leptin signaling has never been tested. AUTHOR CONTRIBUTIONS Conception and design of the work (ES, RC, RF); acquisition, analysis, or interpretation of data (RC, DN, RF, LS, LO, ES); drafting and revising of the manuscript and approval of the ﬁnal version (ES, RF, LO). All authors have agreed to the content of this manuscript and are accountable for all aspects of the work described. Frontiers in Molecular Biosciences \| www.frontiersin.org REFERENCES Maberley, D., Cui, J. Z., and Matsubara, J. A. (2006). Vitreous leptin levels in retinal disease. Eye (Lond). 20, 801–804. doi: 10.1038/sj.eye.6702011 Adya, R., Tan, B. K., and Randeva, H. S. (2015). 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Development of ranibizumab, an anti-vascular endothelial growth factor antigen binding fragment, as therapy for neovascular age-related macular degeneration. Retina 26, 859–870. doi: 10.1097/01.iae.0000242842.14624.e7 Rodríguez, A., Gómez-Ambrosi, J., Catalan, V., Fortuño, A., and Frühbeck, G. (2010). Leptin inhibits the proliferation of vascular smooth muscle cells induced by angiotensin II through nitric oxide-dependent mechanisms. Mediat. Inﬂamm. 2010:105489. doi: 10.1155/2010/105489 Gariano, R. F., Nath, A. K., D’Amico, D. J., Lee, T., and Sierra-Honigmann, M. R. (2000). Elevation of vitreous leptin in diabetic retinopathy and retinal detachment. Invest. Ophthalmol. Vis. Sci. 41, 3576–3581. Scolaro, L., Cassone, M., Kolaczynski, J. REFERENCES W., Otvos, L. Jr., and Surmacz, E. (2010). Leptin-based therapeutics. Expert Rev. Endocrinol. Metab. 5, 875–889. doi: 10.1586/eem.10.61 Garonna, E., Botham, K. M., Birdsey, G. M., Randi, A. M., Gonzalez-Perez, R. R., and Wheeler-Jones, C. P. 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Leptin stimulates ischemia-induced retinal neovascularization: possible role of vascular endothelial growth factor expressed in retinal endothelial cells. Diabetes 53, 2443–2448. doi: 10.2337/diabetes.53.9.2443 Kovacs, K., Marra, K. V., Yu, G., Wagley, S., Ma, J., Teague, G. C., et al. (2015). Angiogenic and inﬂammatory vitreous biomarkers associated with increasing levels of retinal ischemia. Invest. Ophthalmol. Vis. Sci. 56, 6523–6530. doi: 10.1167/iovs.15-16793 Sun, G., Su, G., Zhang, M., Zeng, Q., and Shi, Q. (2011). Eﬀect of tetrandrine on the expression of leptin (LP) and vascular endothelial growth factor (VEGF) in corneal neovascularization of rats. Sci. Res. Essays 6, 5008–5013. Lu, J., Zhang, K., Nam, S., Anderson, R. FUNDING This study was supported in part by a research grant from Novo Nordisk Diabetes Innovation Program to ES. Here, we demonstrate for the ﬁrst time that VEGF increases leptin mRNA and protein expression in retinal and corneal October 2016 \| Volume 3 \| Article 67 Frontiers in Molecular Biosciences \| www.frontiersin.org 8 Leptin Antagonist Blocks VEGF Action Coroniti et al. REFERENCES A., Jove, R., and Wen, W. (2010). Novel angiogenesis inhibitory activity in cinnamon extract blocks VEGFR2 kinase and downstream signaling. Carcinogenesis 31, 481–488. doi: 10.1093/carcin/ bgp292 Sun, J., Xu, Y., Sun, S., Sun, Y., and Wang, X. (2010). Intermittent high glucose enhances cell proliferation and VEGF expression in retinal endothelial cells: the October 2016 \| Volume 3 \| Article 67 Frontiers in Molecular Biosciences \| www.frontiersin.org 9 Coroniti et al. Leptin Antagonist Blocks VEGF Action role of mitochondrial reactive oxygen species. Mol. Cell. Biochem. 343, 27–35. doi: 10.1007/s11010-010-0495-5 role of mitochondrial reactive oxygen species. Mol. Cell. 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The ObR antagonist described in the paper was co-invented by LO and ES and is covered by the US patent 8778890 “Leptin antagonist and methods of use,” currently licensed to Allysta, Inc. for ophthalmology development. The company did not inﬂuence or prescreen data before submission for publication. Sweeney, G. (2010). Cardiovascular eﬀects of leptin. Nat. Rev. Cardiol. 7, 22–29. doi: 10.1038/nrcardio.2009.224 Tang, J., and Kern, T. S. (2011). Inﬂammation in diabetic retinopathy. Prog. Retin. Eye Res. 30, 343–358. doi: 10.1016/j.preteyeres.2011.05.002 Truong, A., Wong, T. Y., and Khachigian, L. M. (2011). Emerging therapeutic approaches in the management of retinal angiogenesis and edema. J. Mol. Med. 89, 343–361. doi: 10.1007/s00109-010-0709-z All the other authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Upadhyay, J., Farr, O. M., and Mantzoros, C. S. (2015). The role of leptin in regulating bone metabolism. Metab. Clin. Exp. 64, 105–113. doi: 10.1016/j.metabol.2014.10.021 Copyright © 2016 Coroniti, Farjo, Nuno, Otvos, Scolaro and Surmacz. Frontiers in Molecular Biosciences \| www.frontiersin.org REFERENCES This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. van der Giet, M., Henkel, C., Schuchardt, M., and Tolle, M. (2015). Anti- VEGF drugs in eye diseases: local therapy with potential systemic eﬀects. Curr. Pharm. Des. 21, 3548–3556. doi: 10.2174/13816128216661502251 20314 Wang, S., Park, J. K., and Duh, E. J. (2012). Novel targets against retinal angiogenesis in diabetic retinopathy. Curr. Diab. Rep. 12, 355–363. doi: 10.1007/s11892-012-0289-0 October 2016 \| Volume 3 \| Article 67 Frontiers in Molecular Biosciences \| www.frontiersin.org 10
https://openalex.org/W2138537724	https://europepmc.org/articles/pmc4228825?pdf=render	English	null	Pulmonary Embolism following Cessation of Infliximab for Treatment of Miliary Tuberculosis	Case reports in pulmonology	2,014	cc-by	1,881	Hindawi Publishing Corporation Case Reports in Pulmonology Volume 2014, Article ID 479025, 3 pages http://dx.doi.org/10.1155/2014/479025 Hindawi Publishing Corporation Case Reports in Pulmonology Volume 2014, Article ID 479025, 3 pages http://dx.doi.org/10.1155/2014/479025 Hindawi Publishing Corporation Case Reports in Pulmonology Volume 2014, Article ID 479025, 3 pages http://dx.doi.org/10.1155/2014/479025 Correspondence should be addressed to Farid Moosavy; fmoosavy@christianacare.org Received 23 June 2014; Revised 15 September 2014; Accepted 18 September 2014; Published 29 October 2014 Academic Editor: Hasan Bayram Academic Editor: Hasan Bayram Copyright © 2014 B. Lee and F. Moosavy. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. We report a case of a 41-year-old male who presented with tachycardia and swelling of his left arm six weeks after he started antituberculosis treatment and stopped his rheumatoid arthritis infliximab treatment. He was diagnosed with pulmonary embolism by chest CT and initially treated with warfarin, which interacted with his antituberculosis treatment. This presentation of deep vein thrombosis and pulmonary embolism as part of immune reconstitution inflammatory syndrome has not been previously reported for infliximab treated patients. 1. Introduction anti-TNF and steroid therapy were discontinued and replaced with hydroxychloroquine and sulfasalazine. The day before his admission, the patient noticed swelling and pain in his left neck and mild dyspnea. The swelling progressed down his entire arm over the course of the day. The patient was a lifelong nonsmoker and had no prior history of deep vein thrombosis (DVT). Biologic agents are increasingly used as disease-modifying antirheumatic drugs (DMARDS) because of their ability to slow down progression of the disease. However, their use can lead to increased susceptibility to tuberculosis. Tuberculosis treatment, in turn, typically involves elimination of immuno- suppression by these biologic agents. Rapid changes in the immune system can lead to a heightened and generalized inflammatory response, usually referred to as immune recon- stitution inflammatory syndrome. On physical examination, he had a BMI of 24 and was afebrile but tachycardic to 130. He had cervical and supraclavicular lymphadenopathy. His entire left arm was erythematous, swollen, and tender to palpation. Range of motion of that extremity was reduced secondary to pain.t CT of neck revealed a thrombus within the left axillary and left subclavian veins extending to the proximal left brachiocephalic vein, in addition to enlarged right and left supraclavicular lymph nodes, without obvious vascular compression (Figure 1). CT angiography of chest showed pulmonary emboli involving the right lower lobe in addition to previously noted nodular opacities of miliary tuberculosis (Figure 2).i Brian Lee1 and Farid Moosavy2 1 Thomas Jefferson University, 1025 Walnut Street, Philadelphia, PA 19107, USA 2 1 Thomas Jefferson University, 1025 Walnut Street, Philadelphia, PA 19107, USA 2 Attending, Pulmonary and Critical Care Section, Christiana Care Health System, 4745 Ogletown-Stanton Road, Map 1, Suite 220, Newark, DE 19713, USA hf 2 Attending, Pulmonary and Critical Care Section, Christiana Care Health System, 4745 Ogletown-Stanton Road, Newark, DE 19713, USA Correspondence should be addressed to Farid Moosavy; fmoosavy@christianacare.org 2. Case Presentation A 41-year-old male, a recent immigrant, presented to the emergency room with swelling and pain in his neck and left arm. He had a history of rheumatoid arthritis and started treatment with infliximab, methotrexate, and high dose prednisone one year prior to admission. Six weeks prior to admission, he developed night sweats and weight loss, which was diagnosed as miliary tuberculosis by CT imaging and positive sputum culture. His tuberculosis was treated with rifampin, pyrazinamide, isoniazid, and ethambutol. His His hemogram was significant for normocytic anemia and neutrophilia. Electrolytes, renal and liver function tests, hepatitis panels, and blood cultures were normal and his 2 Case Reports in Pulmonology (a) (b) (c) Figure 1: CT angiography showing (a) thrombus (arrow) in the left subclavian vein extending into the proximal innominate. Bilateral necrotic supraclavicular lymphadenopathy (star) does not show compression of the subclavian vein in the (b) axial and (c) coronal views. (c) (a) (b) (a) (b) (c) Figure 1: CT angiography showing (a) thrombus (arrow) in the left subclavian vein extending into the proximal innominate. Bilateral necrotic supraclavicular lymphadenopathy (star) does not show compression of the subclavian vein in the (b) axial and (c) coronal views. Figure 2: CT angiography demonstrating lobar and segmental pulmonary emboli (arrow) in the right lower lobe. medication such as steroids and thus are more likely to have negative tuberculin skin tests [5].l Immune reconstitution inflammatory syndrome (IRIS) has been well described in HIV-positive patients upon initia- tion of antiretroviral therapy [6]. IRIS has also been reported in patients on infliximab who develop tuberculosis [7, 8]. There is a five-to-sixteen-week period between cessation of infliximab treatment and onset of symptoms, such as progression of lymph node swelling, infiltrates, or pleural effusion [9]. To our knowledge, however, the development of pul- monary thromboembolic disease has never been described in the setting of IRIS specifically occurring as a consequence of discontinuation of anti-TNF therapy. We speculate that this complication was the result of concurrent biomechanical and biochemical factors as delineated below. Figure 2: CT angiography demonstrating lobar and segmental pulmonary emboli (arrow) in the right lower lobe. acid-fast smear was now negative. His coagulation profile was normal except for an elevated D-dimer of greater than 1000 ng/mL. 2. Case Presentation An extensive work-up for thrombophilia was unremarkable with normal lupus anticoagulant, silicone- clotting test, Factor V Leiden, prothrombin 20210, Cardi- olipin IgG, IgM 5, and beta-2 glycoprotein.h Virchow’s triad has been used to explain the association of lower extremity DVT with TB in multiple reports [10]. In our case, lymph node enlargement could have mechanically contributed to this unusual case of upper extremity DVT, presumably with impeding the flow in the adjacent veins at least in certain positions. Cervical lymphadenopathy is the most common lymphadenopathy in TB [11], but any lym- phatic chain, including the supraclavicular lymph nodes, can easily be involved in the setting of generalized inflammatory resurgence as part of immune reconstitution as in this case. The patient was treated with enoxaparin and his swelling and dyspnea subsided. A warfarin regimen was attempted at 5 mg, but his INR remained 1.2. The patient was subsequently discharged being on rivaroxaban, prednisone 20 mg, and trimethoprim-sulfonamide prophylaxis, in addition to his antituberculosis regimen. g p Both RA and TB could have presumably contributed to a hypercoagulable state in this patient. Early in tuberculosis, procoagulants such as fibrin degradation products (FDP) and tissue plasminogen activator (t-PA) are increased [12]. Although their levels normalize over the course of 12 weeks of treatment, they can still be susceptible to DVT [12]. In a review of autopsies in 1948, Zahn and Peirce found that 1.5% of tuberculosis subjects had DVT and less than 0.1% had pul- monary embolism as the cause of death [13]. In a 2013 review of 30 subjects who had DVT after receiving antituberculosis therapy, Kouismi et al. documented 5 cases of pulmonary tuberculosis that resulted in pulmonary embolism [10]. While pulmonary embolism is a rare complication of tuberculosis, rheumatoid arthritis is also an autoinflammatory disorder associated with endothelial dysfunction through the rise in immune mediators and cytokines and can have similar thromboembolic complications [14]. Conflict of Interests The authors declare that there is no conflict of interests regarding the publication of this paper. Case Reports in Pulmonology pattern and concepts in management,” Postgraduate Medical Journal, vol. 77, no. 905, pp. 185–187, 2001. Overall, in our patient, the reduction in RA treatment and initiation of antituberculous treatment could have conceiv- ably created an immune reconstitution inflammatory syn- drome leading to a procoagulant milieu. To our knowledge, this is the first report of a pulmonary thromboembolic man- ifestation of immune reconstitution inflammatory syndrome as a result of withdrawal of infliximab treatment following miliary tuberculosis. [12] S. C. Robson, N. W. White, I. Aronson, R. Woollgar, H. Goodman, and P. Jacobs, “Acute-phase response and the hyper- coagulable state in pulmonary tuberculosis,” British Journal of Haematology, vol. 93, no. 4, pp. 943–949, 1996. [13] D. W. Zahn and C. T. Peirce, “Venous thrombosis and pul- monary embolism in tuberculosis,” The American Journal of Medicine, vol. 5, no. 5, pp. 716–728, 1948. [14] W.-S. Chung, C.-L. Peng, C.-L. Lin et al., “Rheumatoid arthritis increases the risk of deep vein thrombosis and pulmonary thromboembolism: a nationwide cohort study,” Annals of the Rheumatic Diseases, vol. 73, pp. 1774–1780, 2014. 3. Discussion It is well known that disease-modifying antirheumatics, such as infliximab, significantly increase the risk of tuberculo- sis [1]. Patients receiving interferon and adalimumab are more at risk compared to those receiving etanercept [2]. Recent reports indicate cumulative incidence rates as high as 499/100,000 in rheumatoid arthritis and 287/100,000 in inflammatory bowel disease [3].h l Thus, both active and latent tuberculosis are contraindi- cations to the use of anti-TNF-alpha drugs, and candidates for infliximab therapy are screened with a tuberculin skin test, which, for this group, is positive if it is greater than 6 mm [4]. However, these patients are often on anti-inflammatory 3 References [1] J. Keane, S. Gershon, R. P. Wise et al., “Tuberculosis associated with infliximab, a tumor necrosis factor 𝛼-neutralizing agent,” The New England Journal of Medicine, vol. 345, no. 15, pp. 1098– 1104, 2001. [2] W. G. Dixon, K. L. Hyrich, K. D. Watson et al., “Drug- specific risk of tuberculosis in patients with rheumatoid arthritis treated with anti-TNF therapy: results from the British Society for Rheumatology Biologics Register (BSRBR),” Annals of the Rheumatic Diseases, vol. 69, no. 3, pp. 522–528, 2010. [3] J. Cheng and Q. Cao, “P-030 infliximab and tuberculosis infection: a meta-analysis,” Inflammatory Bowel Diseases, vol. 19, supplement 1, p. S37, 2013. [4] K. G. Saag, G. T. Gim, N. M. Patkar et al., “American College of Rheumatology 2008 recommendations for the use of nonbi- ologic and biologic disease-modifying antirheumatic drugs in rheumatoid arthritis,” Arthritis Care and Research, vol. 59, no. 6, pp. 762–784, 2008. [5] S. Agarwal, S. K. Das, G. G. Agarwal, and R. Srivastava, “Steroids decrease prevalence of positive tuberculin skin test in rheumatoid arthritis: implications on anti-TNF therapies,” Interdisciplinary Perspectives on Infectious Diseases, vol. 2014, Article ID 430134, 5 pages, 2014.l [6] H.-Y. Sun and N. Singh, “Immune reconstitution inflammatory syndrome in non-HIV immunocompromised patients,” Cur- rent Opinion in Infectious Diseases, vol. 22, no. 4, pp. 394–402, 2009. [7] S. M. Arend, E. M. S. Leyten, W. P. J. Franken, E. M. Huisman, and J. T. van Dissel, “A patient with de novo tuberculosis during anti-tumor necrosis factor-𝛼therapy illustrating diagnostic pit- falls and paradoxical response to treatment,” Clinical Infectious Diseases, vol. 45, no. 11, pp. 1470–1475, 2007. [8] E. M. T. Salvana, G. S. Cooper, and R. A. Salata, “Mycobacterium other than tuberculosis (MOTT) infection: an emerging disease in infliximab-treated patients,” Journal of Infection, vol. 55, no. 6, pp. 484–487, 2007. [9] C. G. Vidal, S. R. Fern´andez, J. M. Lacasa et al., “Paradoxi- cal response to antituberculous therapy in infliximab-treated patients with disseminated tuberculosis,” Clinical Infectious Diseases, vol. 40, no. 5, pp. 756–759, 2005. [10] H. Kouismi, M. Laine, J.-E. Bourkadi, and G. Iraqi, “Association of deep venous thrombosis with pulmonary tuberculosis,” Egyptian Journal of Chest Diseases and Tuberculosis, vol. 62, no. 3, pp. 541–543, 2013. [11] B. C. Jha, A. Dass, N. M. Nagarkar, R. Gupta, and S. 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https://openalex.org/W3036330497	https://www.researchsquare.com/article/rs-23746/latest.pdf	English	null	Sex differences in Alzheimer’s-related Tau biomarkers and a mediating effect of testosterone	Biology of sex differences	2,020	cc-by	8,451	Sex differences in Alzheimer’s-related Tau biomarkers and a mediating effect of testosterone Erin Sundermann (  esundermann@ucsd.edu ) University of California San Diego https://orcid.org/0000-0001-5821-8035 Matthew S. Panizzon University of California, San Diego Xu Chen University of California San Diego Murray Andrews University of California San Diego Douglas Galasko University of California San Diego Sarah J. Banks University of California, San Diego Research Keywords: Alzheimer’s disease, phosphorylated-Tau, testosterone, APOE, sex, cerebrospinal fluid Posted Date: June 1st, 2020 DOI: https://doi.org/10.21203/rs.3.rs-23746/v2 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published on June 19th, 2020. See the published version at https://doi.org/10.1186/s13293-020-00310-x. Sex differences in Alzheimer’s-related Tau biomarkers and a mediating effect of testosterone Erin Sundermann (  esundermann@ucsd.edu ) University of California San Diego https://orcid.org/0000-0001-5821-8035 Matthew S. Panizzon University of California, San Diego Xu Chen University of California San Diego Murray Andrews University of California San Diego Douglas Galasko University of California San Diego Sarah J. Banks University of California, San Diego Research Keywords: Alzheimer’s disease, phosphorylated-Tau, testosterone, APOE, sex, cerebrospinal fluid Posted Date: June 1st, 2020 DOI: https://doi.org/10.21203/rs.3.rs-23746/v2 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published on June 19th, 2020. See the published version at https://doi.org/10.1186/s13293-020-00310-x. Research License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published on June 19th, 2020. See the published version at https://doi.org/10.1186/s13293-020-00310-x. Page 1/20 Abstract Women show greater pathological Tau biomarkers than men along the Alzheimer’s disease (AD) continuum, particularly among apolipoprotein ε-E4 (APOE4) carriers; however, the reason for this sex difference in unknown. Sex differences often indicate an underlying role of sex hormones. We examined whether testosterone levels might influence this sex difference and the modifying role of APOE4 status. Analyses included 172 participants (25 cognitively normal, 97 mild cognitive impairment, 50 AD participants) from the Alzheimer’s Disease Neuroimaging Initiative (34% female, 54% APOE4+, aged 55- 90). We examined the separate and interactive effects of plasma testosterone levels and APOE4 on cerebrospinal fluid phosphorylated-tau181 (p-Tau) levels in the overall sample, and the sex difference in p-Tau levels before and after adjusting for testosterone. A significant APOE4-by-testosterone interaction revealed that lower testosterone levels related to higher p-Tau levels among APOE4 carriers regardless of sex. As expected, women had higher p-Tau levels than men among APOE4 carriers only, yet this difference was eliminated upon adjustment for testosterone. Results suggest that testosterone is protective against p-Tau particularly among APOE4 carriers. The lower testosterone levels that typically characterize women may predispose them to pathological Tau, particularly among female APOE4 carriers. Background Furthermore, exogenous testosterone supplementation led to improved performance over time in a range of cognitive domains including global cognition [19,33], psychomotor speed [33], executive function [33], osterone on AD-related outcomey nificant contributor tonitive impairmentthey suggest that the f AD pathology and that this cogand spatial and verbal memory [34,35], although not always [36]. Animal and human studies demonstrate that the effects of testosterone may depend on APOE genotype. The APOE4 allele is associated with lower testosterone levels in men [31], and with downregulation of androgen receptors in mice, resulting in reduced binding of testosterone [37]. Experimental manipulations of testosterone levels in male and female mouse models relate to changes in cognitive function more so among APOE4 carriers than APOE3 carriers, [37,38] suggesting that APOE4 carriers are more sensitive to the effects of testosterone on the brain. In humans, the direction of the APOE4 by testosterone interaction is less consistent, whereby Panizzon et al. found that low testosterone levels in men related to smaller hippocampal volumes and poorer episodic memory among APOE4 carriers only [39,40], whereas, Hogervorst et al. found that low testosterone levels related to a greater likelihood of an AD diagnosis among APOE4 non-carriers only [31]. The APOE4 by testosterone interaction has yet to be examined either in women or in relation to hallmark AD pathologies. In the Alzheimer’s Disease Neuroimaging Initiative (ADNI), we aimed to replicate previous findings of greater pathological Tau biomarkers in women versus men at-risk for AD by way of the APOE4 allele, and to extend these findings by testing the hypothesis that testosterone may contribute to this sex difference. To this end, we examined the relationship of circulating total and free testosterone levels and their interaction with APOE4 with CSF levels of p-Tau across and within sex while adjusting for amyloid-β (Aβ) biomarkers and other covariates. Furthermore, we determined whether lower testosterone levels in women partially account for their higher p-Tau levels. Extrapolating from animal studies, we hypothesized that lower testosterone levels would relate to higher p-Tau levels across sex and more so among APOE4 carriers versus non-carriers. Furthermore, we hypothesized that the higher p-Tau levels in female versus male APOE4 carriers will diminish upon adjustment for testosterone. Background There are critical gaps in our understanding of sex differences in Alzheimer’s disease (AD) including the higher prevalence of AD [1,2], the steeper cognitive decline [3–5] and a stronger effect of the apolipoprotein E ε4 allele (APOE4) on AD risk in women versus men [6–8]. Sex differences in underlying AD pathology have also been reported, with autopsy [9], neuroimaging [10] and cerebrospinal fluid (CSF) [11,12] studies reporting higher levels of pathological tau (referred here simply as “Tau”) in women versus men who are either diagnosed with or are at-risk for AD by way of the APOE4 allele or clinically- significant beta-amyloid (Aβ) plaque deposition in the brain. . Because Tau topography is closely tethered to clinical presentation [13], the higher levels of Tau in women may be a contributing factor to the higher prevalence and more aggressive clinical profile of AD in women. The reasons for higher levels of Tau in women are unknown; however, a potential mechanism may stem from differences in sex hormones. Animal studies report a protective role of testosterone against the hyperphosphorylation of tau (p-Tau) in both male and female rats [14–16], suggesting that the typically lower testosterone levels in women may be a risk factor for pathological Tau. In women, circulating estradiol levels experience a substantial decline during menopause, whereas post-menopausal women continue to demonstrate a range of circulating testosterone levels that continue to be lower than levels in men [17]. Despite this sex difference in hormone levels, most studies examining links between testosterone and AD-related outcomes have been solely in men [18–23], and the link between testosterone and Tau has been minimally examined in humans. Page 2/20 Despite some inconsistencies [24,25], a wealth of evidence indicates an association between low testosterone levels, poorer cognitive function [26–30], and greater odds or risk for AD [18,20–23,31,32], with these associations more clearly defined in men [18,20,32,21–23,26–29,31] than in women [26,27]. Suggestive of a more causative than consequential role for testosterone on AD-related outcomes, longitudinal studies have shown that low free and/or total testosterone levels precede development of AD dementia [23] and cognitive dysfunction on measures of global cognition [26,29] and episodic memory [29] . Background Because of a previously-reported link between testosterone and Aβ pathology in a rodent model [14] and evidence of an effect of Aβ on Tau development [41], we also examined whether testosterone and Tau associations were independent of CSF Aβ levels. Methods Participants and Data Source Page 3/20 Page 3/20 Data were extracted from ADNI, a publically-accessible dataset available at adni.loni.usc.edu. ADNI is a longitudinal, multi-site, cohort study that began in 2003 as a public-private partnership. Information about ADNI can be found at www.adni-info.org. The primary goal of ADNI is to test whether neuroimaging measures and other biological and clinical markers can be combined to measure the progression of MCI and early AD. ADNI study visits involve neuroimaging, neuropsychological, clinical and biomarker assessments. The general enrollment inclusion/exclusion criteria for ADNI have been described elsewhere [42]. This specific study was limited to ADNI1 participants with CSF p-Tau levels, as determined by the Roche Elecsys assay, and plasma testosterone levels from their baseline visit. The current sample consisted of 172 participants (113 men and 59 women) aged 55-90 years including 25 (15%) cognitively normal, 97 (56%) MCI and 50 (29%) AD dementia individuals. Fluid Biomarkers Plasma levels of total testosterone and sex hormone binding globulin (SHBG) were measured on the Luminex xMAP platform by Biomarkers Consortium Plasma Proteomics Project Rules-Based Medicine multiplex (http://www.rulesbasedmedicine.com) as part of a panel of 190 analytes related to a diverse array of human disease. A Box-Cox transformation was applied to raw assay values to normalize the distribution. Detail of assay methods and normalization procedures are described in “Biomarkers Consortium Plasma Proteomics Data Primer 02Aug2013 Final.pdf” and available for download at http://adni.loni.usc.edu/data-samples/access-data/. We utilized CSF concentrations of p-Tau (pg/mL), phosphorylated at threonine 181, and Aβ as determined by the Roche Elecsys assay (Roche, Basel, Switzerland). Detailed methods and quality control procedures for p-Tau measures can be found at http://adni.loni.ucla.edu. Increased CSF p-tau181 levels occur in AD but not in other neurodegenerative disorders. SHBG is a protein that binds testosterone rendering it biologically unavailable. Thus, SHBG levels were used to measure levels of bound versus unbound or free testosterone using the following formula: total testosterone/SHBG×100. All analyses were repeated substituting free for total testosterone levels to determine whether results were driven by bioavailable testosterone. Statistical Analyses Continuous variables that were not normally distributed were transformed via log- or Box-Cox transformations to improve normality. Sample characteristics by sex and APOE4 status were assessed using independent t-tests for continuous variables and chi-square tests for categorical variables. First, we used linear regression to examine the separate and interactive effects of sex and APOE4 status on p-Tau levels while adjusting for age, education and cardiovascular risk factors available in ADNI (i.e., body mass index [BMI] and self-reported history of cardiovascular events). Men were compared to women (reference group) and APOE4 carriers to APOE4 non-carriers (reference group). Next, we used linear regression to examine the effect of testosterone and its interaction with APOE4 status on p-Tau levels in the overall sample and within sex. In addition to the previously-mentioned covariates, we adjusted for sex in analyses in the overall sample. Significant interactions were probed via analyses stratified by APOE4 status. Next, stepwise linear regressions were conducted in the overall sample to examine sex differences Page 4/20 Page 4/20 in p-Tau levels after adding testosterone (step 2) to the initial model that adjusted for age, education and cardiovascular risk factors (step 1). Analyses were compared before and after covarying for Aβ1-42 levels in order to determine the specificity of findings to p-Tau. in p-Tau levels after adding testosterone (step 2) to the initial model that adjusted for age, education and cardiovascular risk factors (step 1). Analyses were compared before and after covarying for Aβ1-42 levels in order to determine the specificity of findings to p-Tau. Table 1. Sample characteristics by APOE4 carrier status and sex. Results Among 172 participants, there were 79 APOE4 non-carriers (25 women and 54 men) and 93 APOE4 carriers (34 women and 59 men). The sample was 97% White, with a mean age of 75, and mean years of education of 15. In the overall sample, APOE4 carriers were younger, showed poorer global cognition (lower mean MMSE score), had higher p-Tau levels, were less likely to be cognitively normal and more likely to be AD dementia patients compared to non-carriers (ps<.05; Table 1). Mean testosterone level was lower in APOE4 carriers versus non-carriers, although not significantly (p=.09). When comparing men and women by APOE4 status, female APOE4 carriers were significantly younger than male APOE4 carriers (p=.002). As expected, mean total and free testosterone levels were lower in women than in men regardless of APOE4 status (ps<.001). In replication of previous findings, p-Tau levels were higher in women versus men but only among APOE4 carriers (p=.001). Table 1. Sample characteristics by APOE4 carrier status and sex. Sex differences in p-Tau by APOE4 status In line with hypotheses and our unadjusted analyses (Table 1), a significant sex by APOE4 interaction on p-Tau levels (B=-5.76, β=-0.24, standard error [SD]=2.96, p=.05) when adjusting for covariates (i.e., age, education and cardiovascular risk factors) indicated higher p-Tau levels in women versus men among APOE4 carriers only (B=-11.16, β=-0.31, SD=3.85, p=.005). Analyses stratified by APOE4 status actually revealed an opposing sex difference among non-carriers, whereby p-Tau levels were higher in men versus women, although not significantly (B=6.09, β=0.22, SD=3.22, p=.06; Figure 1). Results Page 5/20 APOE4- (n=79) APOE4+ (n=93) p-value (effect size)a APOE4- APOE4+ Women n=25 Men n=54 p-value (effect size)a Women n=34 Men n=59 p-value (effect size)a Age, Mean (SD) 76.6 (7.2) 74.0 (6.7) .02 (.37) 77.0 (6.3) 76.4 (7.7) .74 71.3 (7.4) 75.6 (5.7) .002 (.65) Years of education, Mean (SD) 15.9 (3.0) 15.3 (3.2) .20 15.7 (2.7) 16.0 (3.1) .64 14.7 (2.9) 15.6 (3.4) .17 White, n (%) 76 (96.2%) 91 (97.8%) .54 25 (100%) 51 (94.4%) .49 32 (94.1%) 59 (100%) .06 Cognitive status <.001 (.40) .26 .65 Cognitively normal, n (%) 22 (27.8%) 3 (3.2%) 10 (40.0%) 12 (22.2%) 1 (2.9%) 2 (3.4%) MCI, n (%) 45 (57.0%) 52 (55.9%) 12 (48.0%) 33 (61.1%) 17 (50.0%) 35 (59.3%) AD dementia, n (%) 12 (15.2%) 38 (40.9%) 3 (12.0%) 9 (16.7%) 16 (47.1%) 22 (37.3%) Global cognition (MMSE), Mean (SD) 27.1 (2.2) 25.8 (2.5) <.001 (.55) 27.6 (2.2) 26.9 (2.2) .15 25.6 (2.5) 25.9 (2.5) .56 BMI, Mean (SD) 26.5 (4.0) 25.8 (3.7) .28 25.7 (4.9) 26.8 (3.5) .24 25.5 (4.0) 26.0 (3.6) .55 Self-reported history of cardiovascular events, n (%) 61 (77.2%) 62 (66.7%) .13 16 (64.0%) 45 (83.3%) .06 23 (67.6%) 39 (66.1%) .88 b 61 0 59 1 41 61 9 60 8 80 58 9 59 2 93 (%) (SD) Plasma total testosterone levelc (ng/mL), Mean (SD) 0.2 (0.4) 0.1 (0.5) .09 -0.3 (0.4) 0.5 (0.1) <.001 (2.74) -0.4 (0.4) 0.4 (0.2) <.001 (2.53) Plasma free testosterone levelc (ng/mL), Mean (SD) 13.4 (24.8) 6.5 (27.7) .09 -16.9 (19.5) 27.5 (9.8) <.001 (2.9) -22.0 (22.7) 23.0 (13.0) <.001 (2.4) CSF p-Tau181 level (pg/mL), Mean (SD) 26.8 (13.1) 35.9 (17.2) <.001 (.59) 23.5 (8.6) 28.3 (14.5) .13 43.7 (22.6) 31.5 (11.1) .001 (0.68) CSF Aβ1-42 level (pg/mL), Mean (SD) 1240.4 (702.9) 639.1 (292.2) <.001 (1.12) 1286.7 (765.1) 1218.9 (678.7) .69 658.7 (627.8) 627.8 (327.0) .63 Note. aEffect sizes are provided for significant differences; Cohen’s d is provided for mean differences (0.2 = small, 0.5 = medium, 0.8 = large) and a phi coefficient is provided for differences in proportions (0.1 = small, 0.3 = medium, 0.5 = large). bPulse pressure = systolic – diastolic blood pressure. cTestosterone levels were normalized based on a Box-Cox transformation. MCI = mild cognitive impairment; AD = Alzheimer’s disease; APOE4 = apolipoprotein E ɛ4 allele; MMSE = Mini Mental Status Examination; BMI = body mass index. CSF = cerebrospinal fluid. Explanatory role of testosterone in sex difference in p-Tau In testing the mediating role of testosterone in the sex difference in p-Tau levels, we found that the significantly higher p-Tau levels in female APOE4 carriers versus male APOE4 carriers was eliminated after adjusting for testosterone levels (B=3.21, β=0.09, SE=5.78, p=.58; Figure 1). Conversely, the trend for higher p-Tau levels in men versus women among APOE4 non-carriers changed minimally after adjusting for testosterone (B=9.47, β=0.34, SE=5.84, p=.10). Again, results were unchanged when substituting free for total testosterone and when adjusting for Aβ levels. Relationship between Testosterone and p-Tau by APOE4 status In the overall sample, there was a significant relationship between lower total testosterone levels and higher CSF p-Tau levels (B=-13.26, β=-.39, p=.002; Figure 2), but, more importantly, there was a significant total testosterone X APOE4 status interaction on p-Tau levels (B=-17.78, β=-0.40, SD=4.9, p<.001). Analyses stratified by APOE4 status revealed that lower total testosterone levels were associated with higher p-Tau among APOE4 carriers (B=-17.36, β=-0.50, SE=5.41, p=.002) but not non-carriers (B=-4.45, Page 7/20 Page 7/20 Page 7/20 β=-0.15, SE=6.4, p=.49). Results in the overall and the APOE4-stratified analyses were unchanged when substituting free for total testosterone and when including Aβ levels as a covariate in the model. In sex-stratified analyses, the range of total testosterone levels were lower in women (range=-1.2-0.2, median=-0.28) versus men (range:-0.6-0.7, median=0.5) although overlapping. Within the distribution of lower testosterone levels in women (B=-13.83, β=-0.27, SE=5.88, p=.02) and the distribution of higher levels in men (B=-15.85, β=-0.24, SE=6.32, p=.01), there was a negative association between testosterone and p-Tau levels suggestive of a continuous, linear relationship (Figure 3). These associations occurred regardless of APOE4 status as indicated by non-significant testosterone by APOE4 interactions in women (B=-12.55, β=-.23, SE=13.13, p=.34) or men (B=10.19, β=0.20, SE=13.66, p=.46). However, the testosterone by APOE4 interaction on p-Tau in the overall sample appeared to be mostly driven by women in that the testosterone and p-Tau relationship was marginally significant among female APOE4 carriers (B=-18.06, β=-0.34, SE=8.89, p=.05) but not among female non-carriers (B=-0.27, β=-0.01, SE=5.1, p=.96; Figure 3). In contrast, the testosterone and p-Tau relationship was a trend in both male APOE4 carriers (B=-13.38, β=-0.26, SE=6.75, p=.053) and non-carriers (B=-24.24, β=-0.26, SE=13.78, p=.08), Despite the specificity of a testosterone and p-Tau link to female APOE4 carriers, we were likely underpowered to detect a APOE4 by testosterone interaction given the smaller sample size in female-specific analyses (n=53). Results in both men and women were unchanged when substituting free for total testosterone and when adjusting for Aβ levels. Discussion In replication of previous findings, we found higher CSF p-Tau levels in women versus men specifically among APOE4 carriers. Our novel finding was significant relationship between low testosterone levels and higher p-Tau among APOE4 carriers. Our hypothesis concerning a potential mechanistic role of testosterone in the sex difference in p-Tau was supported in that the significant sex difference in p-Tau levels among APOE4 carriers was eliminated when adjusting for testosterone levels. Findings suggest that the lower testosterone levels in women are a significant contributor to their higher levels of p-Tau compared to men. Previous animal and cell culture studies have described a protective role of testosterone against Tau pathology [15,16,43]; however, to the best of our knowledge, we are the first to report a testosterone and Tau link in a human sample. Page 8/20 Testosterone offers a number of neuroprotective effects including improvements in synaptic plasticity [44,45] and synaptic density in hippocampal nerouns [46–49], heightened cerebral blood flow and glucose metabolism [50], reductions in inflammation and oxidative stress [51,52], and prevention against Aβ plaque deposition and their neurotoxic effects [14,53,54]. Although the biological basis underlying the testosterone and tau link is unclear, most relevant to Tau pathogenesis may be testosterone’s anti- inflammatory actions [55]. A role for gliosis and neuroinflammation in Tauopathy is evidenced by greater microglial activity and altered inflammatory pathway markers (e.g., interleukin-6, tumor necrosis factor-α) correlating with Tau burden [56–59] as well as inflammation-related AD risk factors that contribute to Tauopathy such the genetic factors of TREM2 [60] and APOE4 [61] and the environmental factors of traumatic brain injury [62,63] and viral infection [64,65]. Evidence suggests bidirectional effects between neuroinflammation and Tau propagation whereby inflammation can initiate and propagate Tau pathology while Tau aggregates can directly activate microglia and secretion of pro-inflammatory cytokines [66–68]. In early AD, Aβ plaques stimulate microgliosis and release of inflammatory cytokines [69] suggesting that testosterone’s protection against Aβ plaque deposition may contribute to its anti- inflammatory properties and, in turn, decreased p-Tau. However, our results were unchanged after adjusting for Aβ suggesting that the mechanisms underlying the testosterone and p-Tau link are independent of Aβ. Research into the potential mediating role of neuroinflammation in the testosterone and Tau link is warranted. Prior studies have also reported a testosterone by APOE4 interaction on cognitive function in animal models [37,38] and on AD risk [31] and hippocampal volume [39] in humans. Discussion Similar to the majority of these studies, the pattern of interactive effects indicated an association between testosterone and p-Tau only among APOE4 carriers. In fact, the inclusion of APOE4 non-carriers, particularly women, in our analyses across APOE4 status weakened the relationship observed between testosterone and p-Tau among APOE4 carriers. In the sex-stratified analyses, the testosterone and p-Tau relationship is stronger in men versus women when combining APOE4 carriers and non-carriers; however, this relationship is stronger in female versus male APOE4 carriers. These findings underscore the importance of accounting for APOE4 status when examining testosterone and tau links. There is biological plausibility for a testosterone by APOE interaction. In the brain, the APOE protein is a key transporter of lipoproteins. Given testosterone’s role in triglyceride and high density lipoprotein cholesterol metabolism [70,71], the shared role of APOE and testosterone in this lipoprotein pathway offers possibilities for interactive. The APOE4 allele is associated with an increased susceptibility to inflammation [61]. Thus, it is possible that APOE4 carriers are the most likely to benefit from the testosterone’s protective actions against inflammation and, in turn, Tau. In animal studies, APOE4 is associated with a reduction in cytosolic androgen receptor (AR) levels in the neocortex [37] leading to the possibility that the adverse effects of low testosterone levels are further amplified in APOE4 carriers that have fewer or less efficient AR to support testosterone signaling. We extend pervious findings by demonstrating testosterone by APOE4 interactive effects on p-Tau and their potential specificity to women. Page 9/20 Our results suggest that higher levels of p-Tau in women versus men is likely capturing an association between the low testosterone levels that are commonly seen in women and higher p-Tau. In fact, we found that the higher p-Tau levels in female APOE4 carriers versus male APOE4 carriers was eliminated when adjusting for testosterone suggesting that differences in testosterone between men and women is a central mechanism underlying this sex difference. These findings may have implications for the well- evidenced higher AD risk in women considering that Tau pathology is closely tied to neurodegeneration and clinical symptomology. Our findings also challenge the concept that testosterone is a ‘male hormone’ in which the implications of low levels on AD-related outcomes are mostly circumscribed to men despite women having lower testosterone levels than men overall as well as age-related declines. Discussion Our results offer a potential mechanism for the strongly, yet not consistently [72] supported finding of a stronger effect of APOE4 in women versus men on AD risk [6–8,72]. If APOE4 has a stronger effect on AD-related outcomes in the context of low testosterone levels, as suggested by our data, then this would lead to a greater susceptibility of women to these effects. Our findings may also help to explain inconsistencies in the literature regarding an effect of APOE4 on Tau. Other biomarker [73], neuroimaging [74] and autopsy [75] studies found a more robust association between APOE4 and Tau in women versus men, whereas studies that did not compare by sex have shown inconsistent findings in the APOE4 and Tau link [73,76–80]. If the Tau and APOE4 relationship is dependent on testosterone, as our results suggest, the presence of this relationship may be related to the proportion of men versus women in a sample. In non-sex-stratified analyses, an association between APOE4 and Tau may be obscured in samples that are predominantly male and, thus, likely characterized by higher testosterone levels. This study has limitations. Our smaller sample size likely limited statistical power particularly when examining the testosterone and APOE4 interaction in sex-stratified analyses. Levels of circulating estradiol were not available in the ADNI, which precluded us from examining whether it is testosterone or the aromatization of testosterone to estradiol that is responsible for the observed association. However, previous animal work found testosterone’s neuroprotective effects against Aβ [81] and p-Tau [16] to be independent of estradiol levels suggesting that androgenic mechanisms are implicated in these effects [81]. CSF levels of testosterone may be more reflective of testosterone activity in the brain; however, only plasma-based levels were available to us. Because of our cross-sectional design, we were precluded from determining the temporal relationship between testosterone and Tau. Although previous findings suggest that testosterone’s effects predate AD outcomes, there is potential for bidirectional given evidence that AD pathology may negatively feedback on testosterone levels by hindering production of sex steroid hormones [82,83]. Lastly, ADNI is a convenience sample of mostly white and well-educated volunteers compared with the general US population, which limits generalizability of results. In conclusion, we found a relationship between lower testosterone levels and higher CSF p-Tau that was specific to APOE4 carriers. The specificity of this relationship to APOE4 carriers seemed to be driven by women. Perspectives and Significance Our findings inform a knowledge gap in our understanding of greater Tauopathy in women versus men on the AD trajectory and in the repeated demonstration of a stronger APOE4 effect in women. Our findings may also help to enlighten disparities in the literature regarding an APOE4 and Tau relationship. This study represents a call to researchers and clinicians that it is equally important to examine the effects of testosterone on AD-related outcomes in women as it is in men, if not more. Our findings stress the need to examine the effects of testosterone on AD-related outcomes in women in addition to men. Our findings have clinical relevance in that low testosterone is a potentially modifiable risk factor. Although numerous studies have investigated the effects of testosterone supplementation on cognitive function and AD risk with mixed findings (Wolf et al., 1999), very few studies have examined the effects of testosterone supplementation in women and with regard to APOE4 status. Follow-up studies should investigate (a) the association between testosterone levels and cortical Tau as measured by PET, (b) the effect of testosterone supplementation on Tau burden and (c) the mediating role of neuroinflammation in the testosterone and Tau link. Availability of Data and Materials The dataset supporting the conclusions of this article is available in the ADNI repository [adni.loni.usc.edu]. Consent for publication Not applicable Ethics approval and consent to participate This research was approved by the Institutional Review Boards of all participating ADNI sites, and written informed consent was obtained for all participants. Competing interests E. Sundermann reports no disclosures relevant to the manuscript. M. Panizzon reports no disclosures relevant to the manuscript. M. Andrews reports no disclosures relevant to the manuscript. X Chan reports no disclosures relevant to the manuscript. D Galasko reports no disclosures relevant to the manuscript. S. Banks reports no disclosures relevant to the manuscript. Discussion We replicated a consistent finding of higher p-Tau levels in women versus men at-risk for AD; Page 10/20 Page 10/20 however, this difference was eliminated after adjusting for testosterone. Results suggest that testosterone has a protective role against Tau particularly among APOE4 carriers, and that low testosterone levels that are more characteristic of women than men may predispose one to Tau. however, this difference was eliminated after adjusting for testosterone. Results suggest that testosterone has a protective role against Tau particularly among APOE4 carriers, and that low testosterone levels that are more characteristic of women than men may predispose one to Tau. Perspectives and Significance Authors’ contributions Erin E. Sundermann: Conceptualization, Methodology, Data Curation, Formal Analyses, Writing – Original Draft, Project Administration. Matthew Panizzon: Conceptualization, Methodology, Writing – Review and Editing. Xu Chen: Conceptualization, Writing – Review and Editing. Murray Andrews: Conceptualization, Visualization, Writing – Review and Editing. Douglas Galasko: Methodology, Writing – Review and Editing. Sarah J. Banks: Conceptualization, Writing – Original Draft, Methodology, Writing – Review and Editing Funding Page 11/20 This work was supported by the NIH [grant numbers AG049810, AG05131, R01 AG056410]. Data collection and sharing for this project was funded by the Alzheimer's Disease Neuroimaging Initiative (ADNI) (National Institutes of Health Grant U01 AG024904) and DOD ADNI (Department of Defense award number W81XWH-12-2-0012). ADNI is funded by the National Institute on Aging, the National Institute of Biomedical Imaging and Bioengineering, and through generous contributions from the following: AbbVie, Alzheimer’s Association; Alzheimer’s Drug Discovery Foundation; Araclon Biotech; BioClinica, Inc.; Biogen; Bristol-Myers Squibb Company; CereSpir, Inc.; Cogstate; Eisai Inc.; Elan Pharmaceuticals, Inc.; Eli Lilly and Company; EuroImmun; F. Hoffmann-La Roche Ltd and its affiliated company Genentech, Inc.; Fujirebio; GE Healthcare; IXICO Ltd.; Janssen Alzheimer Immunotherapy Research & Development, LLC.; Johnson & Johnson Pharmaceutical Research & Development LLC.; Lumosity; Lundbeck; Merck & Co., Inc.; Meso Scale Diagnostics, LLC.; NeuroRx Research; Neurotrack Technologies; Novartis Pharmaceuticals Corporation; Pfizer Inc.; Piramal Imaging; Servier; Takeda Pharmaceutical Company; and Transition Therapeutics. The Canadian Institutes of Health Research is providing funds to support ADNI clinical sites in Canada. Private sector contributions are facilitated by the Foundation for the National Institutes of Health (www.fnih.org). The grantee organization is the Northern California Institute for Research and Education, and the study is coordinated by the Alzheimer’ Therapeutic Research Institute at the University of Southern California. 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CSF = cerebrospinal fluid. p-Tau = phosphorylated Tau. APOE4 = apolipoprotein E ε4 allele. Relevant covariates were age, education, BMI and self-reported history of cardiovascular events. Testosterone represents plasma-based total testosterone levels. covariates. CSF = cerebrospinal fluid. p-Tau = phosphorylated Tau. APOE4 = apolipoprotein E ε4 allele. Relevant covariates were age, education, BMI and self-reported history of cardiovascular events. Testosterone represents plasma-based total testosterone levels. Page 19/20 Figure 2 Relationship between plasma testosterone levels and CSF p-Tau levels overall (A) and by APOE4 status (B) Using multivariable linear regression, we found a significant total testosterone X APOE4 status interaction on p-Tau levels that revealed an association between lower total testosterone levels and higher p-Tau levels among APOE4 carriers (n=93) but not among APOE4 non-carriers (n=79). CSF = Figure 2 Figure 2 Relationship between plasma testosterone levels and CSF p-Tau levels overall (A) and by APOE4 status (B) Using multivariable linear regression, we found a significant total testosterone X APOE4 status interaction on p-Tau levels that revealed an association between lower total testosterone levels and higher p-Tau levels among APOE4 carriers (n=93) but not among APOE4 non-carriers (n=79). CSF = Relationship between plasma testosterone levels and CSF p-Tau levels overall (A) and by APOE4 status (B) Using multivariable linear regression, we found a significant total testosterone X APOE4 status interaction on p-Tau levels that revealed an association between lower total testosterone levels and higher p-Tau levels among APOE4 carriers (n=93) but not among APOE4 non-carriers (n=79). CSF = Page 19/20 Page 19/20 cerebrospinal fluid. p-Tau = phosphorylated Tau. APOE4 = apolipoprotein E ε4 allele. Analyses covaried for age, sex, education, BMI and self-reported history of cardiovascular events. cerebrospinal fluid. p-Tau = phosphorylated Tau. APOE4 = apolipoprotein E ε4 allele. Analys for age, sex, education, BMI and self-reported history of cardiovascular events. Figure 3 for age, sex, education, BMI and self-reported h Figure 3 p y Figure 3 The sex-stratified relationship between plasma testosterone levels and CSF p-Tau levels overall (A,B) and by APOE4 status (C,D). Using multivariable linear regression, we found negative associations between testosterone and p-Tau levels in both women and men. Although the testosterone X APOE4 status interactions were not significant in either sex, the significant testosterone X APOE4 interaction in the overall sample appeared to be driven by women. When analyses were stratified by sex, we found that the testosterone X APOE4 status interaction on p-Tau levels was driven by women. CSF = cerebrospinal fluid. p-Tau = phosphorylated Tau. APOE4 = apolipoprotein E ε4 allele. Analyses adjusted for age, education, BMI and self-reported history of cardiovascular events. Figure 3 The sex-stratified relationship between plasma testosterone levels and CSF p-Tau levels overall (A,B) and by APOE4 status (C,D). Using multivariable linear regression, we found negative associations between testosterone and p-Tau levels in both women and men. Although the testosterone X APOE4 status interactions were not significant in either sex, the significant testosterone X APOE4 interaction in the overall sample appeared to be driven by women. When analyses were stratified by sex, we found that the testosterone X APOE4 status interaction on p-Tau levels was driven by women. CSF = cerebrospinal fluid. p-Tau = phosphorylated Tau. APOE4 = apolipoprotein E ε4 allele. Analyses adjusted for age, education, BMI and self-reported history of cardiovascular events. The sex-stratified relationship between plasma testosterone levels and CSF p-Tau levels overall (A,B) and by APOE4 status (C,D). Using multivariable linear regression, we found negative associations between testosterone and p-Tau levels in both women and men. Although the testosterone X APOE4 status interactions were not significant in either sex, the significant testosterone X APOE4 interaction in the overall sample appeared to be driven by women. When analyses were stratified by sex, we found that the testosterone X APOE4 status interaction on p-Tau levels was driven by women. CSF = cerebrospinal fluid. p-Tau = phosphorylated Tau. APOE4 = apolipoprotein E ε4 allele. Analyses adjusted for age, education, BMI and self-reported history of cardiovascular events. Page 20/20 Page 20/20
https://openalex.org/W4298282612	https://zenodo.org/record/2673067/files/How%20the%20Abstract%20Become%20Concrete.pdf	English	null	How the Abstract Becomes Concrete: Irrational Numbers are Understood Relative to Natural Numbers and Perfect Squares	Zenodo (CERN European Organization for Nuclear Research)	2,017	cc-by	16,879	Running head: UNDERSTANDING IRRATIONAL NUMBERS Running head: UNDERSTANDING IRRATIONAL NUMBERS 1 How the abstract becomes concrete: How the abstract becomes concrete: How the abstract becomes concrete: Irrational numbers are understood relative to natural numbers and perfect squares Purav Patel1, Sashank Varma2 1Department of Educational Psychology, University of Wisconsin – Madison, Madison, WI, USA 2Department of Educational Psychology, University of Minnesota – Twin Cities, Minneapolis, MN, USA Corresponding Author: Purav Patel Room 859, Educational Sciences Building Department of Educational Psychology University of Wisconsin-Madison 1025 West Johnson Street Madison, WI 53706-1796 Email: patel011393@gmail.com Keywords: irrational, perfect square, magnitude, number line, strategy, arithmetic, reference, individual differences rrational numbers are understood relative to natural numbers and perfect squares Purav Patel Purav Patel Email: patel011393@gmail.com Keywords: irrational, perfect square, magnitude, number line, strategy, arithmetic, reference, individual differences irrational, perfect square, magnitude, number line, strategy, arithmetic, reference, UNDERSTANDING IRRATIONAL NUMBERS 2 2 Abstract Mathematical cognition research has largely emphasized concepts that can be directly perceived or grounded in visuospatial referents. These include concrete number systems like natural numbers, integers, and rational numbers. Here, we investigate how a more abstract number system, the irrationals denoted by radical expressions like √2, is understood across three tasks. Performance on a magnitude comparison task suggests that people interpret irrational numbers – specifically, the radicands of radical expressions – as natural numbers. Strategy self- reports during a number line estimation task reveal that the spatial locations of irrationals are determined by referencing neighboring perfect squares. Finally, perfect squares facilitate the evaluation of arithmetic expressions. These converging results align with a constellation of related phenomena spanning tasks and number systems of varying complexity. Accordingly, we propose that the task-specific recruitment of more concrete representations to make sense of more abstract concepts (referential processing) is an important mechanism for teaching and learning mathematics. UNDERSTANDING IRRATIONAL NUMBERS 3 Introduction The historical development of number systems can be characterized as a gradual progression from the concrete to the abstract. Natural numbers, which denote the cardinalities of sets, were understood directly and formalized first. They are concrete in the sense that they map directly to quantities in the material world such as the number of berries in one’s hand. According to the mathematician Leopold Kronecker, “God created” this relatively concrete number system, whereas more abstract number systems are “the work of man” (Bell, 1986). In other words, more abstract number systems were discovered later and constructed hierarchically upon the natural numbers (Landau, 1966). These systems are still relatively concrete in the sense that they can be interpreted as corresponding to real-world quantities like basement floors for integers and pie pieces for rationals (Van de Walle, Karp, & Bay-Williams, 2010). The historical development of number systems can be characterized as a gradual progression from the concrete to the abstract. Natural numbers, which denote the cardinalities of sets, were understood directly and formalized first. They are concrete in the sense that they map directly to quantities in the material world such as the number of berries in one’s hand. It is with the irrational numbers, which include √2 and 𝜋, that mathematicians discovered a number system lacking material referents or models that build on intuition (Struik, 1987). Such abstraction is associated with many surprising properties. For instance, there are more irrational numbers than natural numbers, integers, or rational numbers. The set of all irrationals is uncountably infinite in cardinality, whereas the latter sets are each countably finite (Struik, 1987). Mathematics teachers, in an attempt to facilitate understanding, encourage students to think of irrational numbers like 𝜋 by using rational number approximations like 3.14. Unfortunately, this can lead to conceptual confusions (González-Martín, Giraldo, & Souto, 2013; Sirotic & Zazkis, 2007). Unfortunately, this can lead to conceptual confusions (González-Martín, Giraldo, & Souto, 2013; Sirotic & Zazkis, 2007). Unfortunately, this can lead to conceptual confusions (González-Martín, Giraldo, & Souto, 2013; Sirotic & Zazkis, 2007). Cognitive science research has left irrational numbers unexamined, focusing instead on more concrete number systems. For example, the natural numbers are thought to be grounded in directly perceptible objects and internally represented as continuous magnitudes on a mental UNDERSTANDING IRRATIONAL NUMBERS 4 number line (Moyer & Landauer, 1967). For multiple number systems, researchers have acknowledged the necessity of mastering associated symbol systems. However, the focus is often on how these symbol systems recruit and restructure magnitude representations. Examples include incorporating the inverse relationship between positive and negative integers into the mental number line and capturing the ratio relationship between the numerators and denominators of fractions more precisely (Matthews & Chesney, 2015; Varma & Schwartz, 2011). For more abstract number systems, do magnitude representations continue to be heavily recruited or does notation-specific strategic processing become more important (Bender, Schlimm, & Beller, 2015; Zhang & Norman, 1995)? We addressed this question using irrational numbers as a test case, specifically those denoted by radical expressions like √2 . We investigated the task demands and stimulus properties that cause adults to rely on magnitude representations versus strategic processing. Our findings reveal the vital roles that concrete natural number referents play in understanding abstract irrational number expressions. They have implications for extending theories of numerical cognition and improving instructional design in mathematics education. Our approach is grounded in prior studies of how people understand natural numbers, integers, and rational numbers. Natural numbers The natural numbers are the set of countable numbers {0, 1, 2, … }.1 Their mental representation has been studied extensively using the magnitude comparison (MC) task. The 1 Mathematicians disagree on whether the natural numbers begin with 0 or 1. The original Peano- Dedekind axioms begin with 1 (Dedekind, 1963/1888). However, more recent presentations of UNDERSTANDING IRRATIONAL NUMBERS 5 primary finding is that when comparing which number in a pair is greater or lesser, response time decreases as the difference between the numbers increases (Moyer & Landauer, 1967). For instance, people can judge the greater or lesser number in the pair (2, 9) more quickly than in the pair (5, 6). This well-replicated performance pattern is known as the distance effect. It manifests whether participants compare symbolic numbers or visuospatial numerosities like sets of dots (Buckley & Gillman, 1974). The distance effect is commonly interpreted as evidence that natural numbers are represented in part as psychophysically scaled magnitudes. It has been observed as early as infancy with numerosities (Xu & Spelke, 2000) and kindergarten with symbolic numbers (Sekuler & Mierkiewicz, 1977). Over development, children’s representation of numerical magnitude changes such that response times decrease overall and the slope of the distance effect decreases (Sekuler & Mierkiewicz, 1977). These changes indicate improved precision of magnitude representations. The magnitude representation of natural numbers and its development have also been probed using the number line estimation (NLE) task. In the bounded version of this task, participants are presented with a number line with the left and right endpoints labeled (e.g., as 0 and 100, respectively) and the middle segment left blank (Siegler & Opfer, 2003). Numbers are presented one at a time and in random order; the goal is to mark the position of each number on these axioms often begin with 0. This reflects the subsequent development of set theory and reconstruction of the natural numbers on this foundation with 0 mapped to the empty set (e.g., Halmos, 1960; Bostock, 2009). In addition, the current ISO standard for mathematical notation defines the natural numbers as beginning with 0 (ISO, 2009). Our definition of the natural numbers follows what we take to be the modern consensus. UNDERSTANDING IRRATIONAL NUMBERS 6 6 the number line. Performance is typically measured by average absolute error – the absolute difference between the correct position of the number and the position selected by the participant. Higher error values signify worse performance. Over development, children’s accuracy on the NLE task improves gradually (Siegler, Thompson, & Opfer, 2009). There is a rich debate in the literature about the representations and processes that underlie performance on this task and its change over time. Some have proposed that children initially represent natural numbers in a compressed, logarithmically spaced fashion. Over development, this incorrect representation is thought to improve by shifting to linear spacing (Ashcraft & Moore, 2012; Opfer, Thompson, & Kim, 2016; Opfer, Siegler, & Young, 2011; Siegler & Booth, 2004; Siegler & Opfer, 2003; Thompson & Opfer, 2010). Others have argued that the representation is proportional between critical landmarks such as the midpoint (Barth & Paladino, 2011; Cohen & Blanc-Goldhammer, 2011; Slusser, Santiago, & Barth, 2013). Still others have proposed that the representation is piecewise linear between critical landmarks like place-value boundaries (Ebersbach, Luwel, Frick, Onghena, & Verschaffel, 2008; Landy, Charlesworth, & Ottmar, 2016; Landy, Silber, & Goldin, 2013; Moeller, Pixner, Kaufmann, & Nuerk, 2009). We return to these proposals in the Discussion when considering the results of the current study. the number line. Performance is typically measured by average absolute error – the absolute difference between the correct position of the number and the position selected by the participant. Higher error values signify worse performance. Over development, children’s accuracy on the NLE task improves gradually (Siegler, Thompson, & Opfer, 2009). There is a rich debate in the literature about the representations and processes that underlie performance on this task and its change over time. Some have proposed that children initially represent natural numbers in a compressed, logarithmically spaced fashion. Over development, this incorrect representation is thought to improve by shifting to linear spacing (Ashcraft & Moore, 2012; Opfer, Thompson, & Kim, 2016; Opfer, Siegler, & Young, 2011; Siegler & Booth, 2004; Siegler & Opfer, 2003; Thompson & Opfer, 2010). Others have argued that the representation is proportional between critical landmarks such as the midpoint (Barth & Paladino, 2011; Cohen & Blanc-Goldhammer, 2011; Slusser, Santiago, & Barth, 2013). UNDERSTANDING IRRATIONAL NUMBERS Still others have proposed that the representation is piecewise linear between critical landmarks like place-value boundaries (Ebersbach, Luwel, Frick, Onghena, & Verschaffel, 2008; Landy, Charlesworth, & Ottmar, 2016; Landy, Silber, & Goldin, 2013; Moeller, Pixner, Kaufmann, & Nuerk, 2009). We return to these proposals in the Discussion when considering the results of the current study. Individual differences in the magnitude representations of natural numbers, as indexed by the slope of the distance effect in the MC task, predict variation in mathematical achievement for elementary (De Smedt, Verschaffel, & Ghesquière, 2009) and middle school students (Halberda, Mazzocco, & Feigenson, 2008). Likewise, NLE performance predicts mathematical achievement in elementary school students, although not older students learning more advanced topics (Sasanguie, De Smedt, Defever, & Reynvoet, 2012). Instructional studies support a causal link between numerical magnitude representations and arithmetic skills. Young children who were UNDERSTANDING IRRATIONAL NUMBERS UNDERSTANDING IRRATIONAL NUMBERS 7 tutored on the magnitudes of small natural numbers via a board game performed better on simple arithmetic problems than their peers who received a control lesson (Siegler & Ramani, 2009). Integers UNDERSTANDING IRRATIONAL NUMBERS 8 8 distance between the integers increases, so does the response time. Krajcsi and Igacs (2010) detected this effect too. As with natural numbers, patterns of estimation bias on the NLE task has been used to suggest a logarithmic-to-linear shift in the mental representation of integers. When estimating the positions of positive numbers in the range 0 – 1000 and negative numbers in the range -1000 – 0, second graders exhibit logarithmic magnitude representations. By contrast, fourth and sixth graders exhibit linear representations for both ranges (Brez, Miller, & Ramirez, 2015). This logarithmic-to-linear shift extends to larger number ranges and older people. Middle school students’ estimates of integers in the negative range -10000 – 0 and the combined range -1000 – 1000 are linear, though with lower accuracy for negative numbers (Young & Booth, 2015). Adults’ estimates of integers in the combined range -100 – 100 are linear and show comparable accuracy for positive and negative numbers (Ganor-Stern & Tzelgov, 2008). Performance on NLE tasks like these depends partly on recognizing the symmetry of the integers around the zero point (Saxe, Earnest, Sitabkhan, Haldar, Lewis, & Zheng, 2010). The inverse distance effect for the MC task and symmetry effects on the NLE task led Tsang, Blair, Bofferding, and Schwartz (2015) to design a manipulative that encourages learners to incorporate the zero point in their magnitude representation of the integers. Instruction with the manipulative improved arithmetic problem-solving for elementary school students on difficult items like 3 + 𝑥= 0. Integers The natural numbers combined with the negative numbers form the integers {…, -2, -1, 0, 1, 2, …}. Comprehending the meaning of this number system requires engaging in additional symbolic processing. For instance, when judging the greater number in the pair (-3, -9), it is necessary to inhibit the whole number interpretations of “-3” as “3” and “-9” as “9”. Children initially compare such integer pairs by using a symbolic strategy. They convert the negative numbers to natural numbers by ignoring the minus signs, comparing the numerical magnitudes, and finally reversing this judgment (Varma & Schwartz, 2011). This strategy results in a distance effect when comparing pairs of negative numbers. However, when comparing mixed integer pairs like (-4, 9), judgments can be made by noticing that positives are greater than negatives. The result is the absence of a distance effect in elementary school children. With respect to adults’ comparison of mixed integer pairs, the literature is consistent. Ganor-Stern, Tzelgov, and collaborators claimed that adults compare mixed integer pairs like children; they apply the rule that positives are greater than negatives. Their studies have largely not found distance effects under standard conditions (Ganor-Stern, Pinhas, Kallai, & Tzelgov, 2010; Tzelgov, Ganor-Stern, & Maymon-Schreiber, 2009). On the contrary, Varma and Schwartz (2011) found a developmental trend. Strategic processing of integers is eventually substituted by accessing a restructured magnitude representation that incorporates the symmetry of positives and negatives around the zero point. Adults represent the negative number line as a reflection of the positive number line, incorporating the additive inverse property 𝑥+ −𝑥= 0. This shift manifests as an inverse distance effect when comparing mixed integer pairs – as the UNDERSTANDING IRRATIONAL NUMBERS Rational numbers Rational numbers are the set of numbers that can be written as a ratio of two integers such that the denominator is non-zero. Magnitude comparison with symbolic fractions and decimals, as well as with non-symbolic ratios, produces the distance effect (DeWolf, Grounds, 2 The failure to find a logarithmic-to-linear shift for rational numbers may be an artifact of the relatively older children who have been tested to date. It is possible that 3rd and 4th graders, who have just recently learned about fractions, will exhibit logarithmic representations. Siegler et al. (2011) argued against the expectation of logarithmic representations even in younger children. UNDERSTANDING IRRATIONAL NUMBERS 10 and Thompson (2014) found that children reported using such anchors to segment the number line and estimate more accurately. and Thompson (2014) found that children reported using such anchors to segment the number line and estimate more accurately. The precision of rational number magnitude representations is also associated with higher-level mathematical skills. Symbolic fraction magnitude representations predict achievement across a range of measures – fraction arithmetic, algebra, grade school standardized exams, and high school mathematical achievement (Siegler et al., 2012). Similarly, accuracy on NLE tasks using decimals predicts mathematical achievement in elementary school students (Schneider, Grabner, Zurich, & Paetsch, 2009). Finally, nonsymbolic ratio precision predicts college students’ knowledge of fractions and algebra (Matthews, Lewis, & Hubbard, 2016). Causal support for the link between magnitude knowledge and arithmetic skills comes from interventions that devote more instructional time to mastering the magnitudes of fractions. These interventions show that emphasizing magnitude comprehension boosts arithmetic skills for high and low achievers alike (Fuchs et al., 2013). UNDERSTANDING IRRATIONAL NUMBERS 9 Bassok, & Holyoak, 2014; Matthews & Chesney, 2015; Matthews & Lewis, 2016; Siegler, Thompson, & Schneider, 2011; Varma & Karl, 2013). Thus, rational numbers are also thought to be integrated on the mental number line. As children begin to understand fraction magnitudes, the slope of the distance effect is nearly zero. Over time, fraction magnitude information becomes more accessible and the slope increases. By adulthood, the slope stabilizes (Gabriel, Szucs, & Content, 2013). These behavioral changes are paralleled by the discovery and use of strategies to facilitate performance. For instance, children and young adults often report using unit fractions like ½ as anchors to perform fraction magnitude comparison (Fazio, Dewolf, & Siegler, 2016; Siegler & Thompson, 2014) In contrast to natural numbers and integers, studies using the NLE task suggest that the representation of rational numbers may be linear early on. That is, it may not undergo the logarithmic-to-linear developmental shift that has been proposed for natural numbers and integers. Both 10-year-olds and adults exhibit highly linear estimation patterns when performing NLE with decimal proportions or fractions (Iuculano & Butterworth, 2011). However, the degree of linearity does improve with development. On average, 8th graders make more accurate estimates of fractions than 6th graders; at the individual level, a higher percentage of 8th graders exhibit linear representations than 6th graders (Siegler et al, 2011).2 These developmental gains may result in part from using unit fractions like ½ as anchors (Spinillo & Bryant, 1991). Siegler 2 The failure to find a logarithmic-to-linear shift for rational numbers may be an artifact of the relatively older children who have been tested to date. It is possible that 3rd and 4th graders, who have just recently learned about fractions, will exhibit logarithmic representations. Siegler et al. (2011) argued against the expectation of logarithmic representations even in younger children. UNDERSTANDING IRRATIONAL NUMBERS Irrational numbers Irrational numbers are incommensurable. Unlike rational numbers, they cannot be expressed as ratios of integers such that the denominator is non-zero. Their decimal expansions are infinitely long, non-repeating, and ostensibly random. When they were first proposed, the notion seemed outlandish. According to a famous anecdote, the Greek mathematician who first proved their existence was drowned at sea for challenging the ratio doctrine of numbers. It was not until the late 1800s that irrationals were formalized and properly integrated onto the real number line (Dedekind, 1963/1888). Number theorists distinguished two subclasses of irrationals – algebraic irrationals and transcendental numbers. Algebraic irrationals, like √20 2 , are the 3 For the curious, √34 10 ≈1.422 and √68 15 ≈1.325. Hence, √34 10 > √68 15 . UNDERSTANDING IRRATIONAL NUMBERS 11 solutions to polynomial equations and can be denoted by expressions of the form √𝑥 𝑦 . Transcendental numbers, like 𝜋 and 𝑒, are not the solutions to any polynomial equation. solutions to polynomial equations and can be denoted by expressions of the form √𝑥 𝑦 . Transcendental numbers, like 𝜋 and 𝑒, are not the solutions to any polynomial equation. Just one other study has explored whether the same human “number sense” that allows us to compare the magnitudes of natural numbers, integers, and rational numbers extends to algebraic irrationals (Obersteiner & Hofreiter, 2017). Participants performed magnitude comparisons with irrationals of the form √𝑥 𝑦 . Both the root y and the radicand x could vary. The researchers tested whether participants compared the magnitudes of irrationals by accessing their holistic magnitudes or by focusing on the root and radicand components. Comparisons were faster when both numbers contained a common root component, as in the pair (√12 9 , √63 9 ). Likewise, comparisons were faster when both numbers contained a common radicand component, as in the pair ( √34 10 , √34 13 ). When both the roots and radicands differed, as in the pair ( √34 10 , √68 15 ), response times were slower.3 Unexpectedly, response times for the common component pairs were not predicted by the distances between the root or radicand components. These mixed results require explanation. One reason might be that the irrational number stimuli were unusually complex. They do not occur in typical contexts like solving quadratic equations. Another possibility is the highly expert sample composed of mathematics graduate students and professors who may have used specialized strategies. It remains unclear how typical adults understand less complex irrational numbers like √2. Our stimuli are also algebraic irrationals, though we focus on square root expressions of the form √𝑥. Such expressions are often encountered when solving quadratic equations. They denote irrational numbers when the radicand x is not a perfect square (e.g., √2) and natural UNDERSTANDING IRRATIONAL NUMBERS UNDERSTANDING IRRATIONAL NUMBERS 12 numbers when the radicand is a perfect square (e.g., √9 ). In the following sections, we will collectively refer to both cases as radical expressions because both contain the radical sign. Research questions and hypotheses UNDERSTANDING IRRATIONAL NUMBERS prime factors and then shift pairs of common factors outside the radical sign – the prime factorization strategy. We exemplify it by the solution procedure: prime factors and then shift pairs of common factors outside the radical sign – the prime factorization strategy. We exemplify it by the solution procedure: √72 = √2 × 36 = √2 × 2 × 18 = √2 × 2 × 2 × 9 = √2 × 2 × 2 × 3 × 3 = 2√2 × 3 × 3 = 2 × 3√2 = 6√2 A more efficient approach is to factor the radicand into its largest perfect square factors and directly reduce these – the perfect squares factorization strategy: A more efficient approach is to factor the radicand into its largest perfect square factors and directly reduce these – the perfect squares factorization strategy: Third, we investigated whether individual differences in the mental representation and processing of irrationals explain variation in conceptual and procedural knowledge of this number system. Some have proposed that magnitude representations are the core of numerical and arithmetic performance (Link, Nuerk, & Moeller, 2014; Siegler, 2016). In contrast to this view, we hypothesize that the influence of magnitude representations on arithmetic problem- solving attenuates as the abstraction of number systems increases. Arithmetic performance with irrationals may depend less on magnitude representations and more on symbolic strategies. Research questions and hypotheses First, we asked whether radical expressions are represented as continuous magnitudes integrated on the mental number line much like natural numbers, integers, and rational numbers. We refer to this proposal as the mental number line hypothesis. An alternative proposal is that people use processes that capitalize on the discrete structure of radical expressions. For instance, when judging the greater or lesser number in the pair (√3 , √8 ), people may ignore the radical signs and only compare the radicand components (3, 8) using their magnitude representations of natural numbers. This is possible because when x and y are non-negative, judgments of (√𝑥 , √𝑦 ) and (x, y) are equivalent. We refer to this as the equivalence strategy hypothesis. Second, we investigated whether people process irrational numbers by strategically referencing more concrete concepts like natural numbers and perfect squares. Specifically, we hypothesized the possible use of a multiplication strategy on the MC task whereby perfect square pairs like (√4, √9 ) are transformed to computationally analogous “tie” multiplication problems like 2 × 2 and 3 × 3. Such a transformation might facilitate comparison because tie problems are processed more quickly than non-tie problems of comparable size (Ashcraft, 1992; Parkman, 1972). On the NLE task, irrational numbers might be estimated in relation to perfect squares – the landmark strategy. For example, people may estimate the positions of irrational numbers like √3 by referencing the positions of neighboring perfect squares like √1 and √4 . Finally, people may leverage their knowledge of perfect squares during arithmetic problem-solving. When simplifying √72 for instance an inefficient strategy would be to decompose the radicand into its simplifying √72, for instance, an inefficient strategy would be to decompose the radicand into its UNDERSTANDING IRRATIONAL NUMBERS 13 Participants Overall, 81 undergraduate students from a large Midwestern university were recruited via flyers posted on campus. We posted flyers in buildings housing classrooms and in the student union. Participants’ ages ranged from 18 to 24 years (M = 20.5, SD = 1.7). The sample consisted UNDERSTANDING IRRATIONAL NUMBERS 14 of more females than males (57 vs. 24). This is more skewed than the overall percentage of female versus male undergraduates at the university (52% vs. 48%). However, it is consistent with the percentage of females in the College of Education (61% vs. 39%), where our lab is located. On a questionnaire, 30.3% of participants self-reported that they were enrolled in a major emphasizing quantitative and analytic skills such as science, technology, engineering, mathematics, finance, or economics. This percentage is consistent with the distribution of such majors at the university. Experimental sessions spanned about 60 minutes and all participants received $12 in compensation. Our protocol was approved by the local Institutional Review Board. -------------------------------------------Insert Table 1 about here - Design and materials We employed a within-subjects experimental design whereby each participant completed all levels of the four experimental tasks. See Table 1 for an overview of the design and materials. Experimental materials and data are available via the Open Science Framework (https://osf.io/6s9pc/#). UNDERSTANDING IRRATIONAL NUMBERS 15 Table 1. Overview of the design and materials used in this study. Note. The stimuli for the strategy questionnaire and the irrationals knowledge test are examples Task Condition Stimuli Number of Items Dependent Variables Magnitude comparison (MC) natural numbers 0, 1, 2…, 9 204 accuracy response time one-digit radicals √0 , √1 , √2, …, √9 204 Number line estimation (NLE) natural numbers 1, 2, 3, …, 9 11 absolute error response time R2 value one-digit radicals √1 , √2, √3, …, √9 11 perfect squares √1 , √4, √9, …, √81 11 two-digit radicals √10 , √20, √30, … , √90 11 Strategy questionnaire natural numbers How did you place 7? 2 strategy type one-digit radicals How did you place √2? 2 perfect squares How did you place √9? 2 two-digit radicals How did you place √40? 2 Irrationals knowledge test number concepts Is √2 rational or irrational? 8 accuracy strategy type density concepts How many irrationals are between 0 – 1? 2 operation concepts The sum of a rational and an irrational is ...? 2 simple arithmetic Simplify 2√48. 10 complex arithmetic Simplify √80 + √18 − √20. 7 Table 1. Overview of the design and materials used in this study. UNDERSTANDING IRRATIONAL NUMBERS 16 Magnitude comparison task. The first task was magnitude comparison (MC) and its stimuli were defined by four variables – type, distance, size, and perfect square status. The type variable refers to whether a number pair consisted of natural numbers or radical expressions. The natural number pairs were formed from the combinations of 0, 1, 2, …, 9 with same-number pairs excluded (90 pairs). We were particularly interested in pairs where both numbers were perfect squares, like (4, 9). Hence, we included an additional copy of each of the 12 perfect square pairs in the stimulus set. In total, 102 natural number pairs were used in each of blocks 1 and 2. Participants judged the greater of two numbers in the first block and the lesser in the second block. Natural number pairs were mirrored by 102 pairs of radical expressions formed in the same way from √0 , √1 , √2, …, √9 . These pairs were used in blocks 3 and 4. Block 3 required greater judgments and block 4 required lesser judgments. Within each block, number pairs were presented in random order. Overall, there were 408 trials. With respect to the distance and size variables, we used two definitions to evaluate the equivalence strategy and mental number line hypotheses (research question 1).4 The distance 4 The distance between the numbers being compared and the average size of the numbers being compared are indices of the precision of the magnitude representations required for successful task performance. These variables, which originate from the earliest studies of the magnitude comparison task (Moyer & Landauer, 1967; Parkman, 1971), continue to dominate studies of adults. By contrast, more recent studies of children have typically used a single variable such as the ratio of the numbers being compared (Halberda et al., 2008; Xu & Spelke, 2000). Because the current study is one of the first to investigate how adults understand irrational numbers, it is valuable to document whether the distance and size effects observed for other number classes extend to the irrationals. Hence, we included the distance and size variables in our analyses. UNDERSTANDING IRRATIONAL NUMBERS 17 variable was the absolute difference between the numbers. The first definition of distance focused on the radicand components. Thus, the distance of the pair (√4 , √9 ) was \|4 −9\| = 5. The second definition focused on the holistic magnitude values of the radical expressions. Thus, the distance of the pair (√4 , √9 ) was \|√4 −√9\| = 1. The size variable was defined as the average of the two numbers. The first definition again used the radicand components. Thus, the size of the pair (√4 , √9 ) was (4 + 9) 2 ⁄ = 6.5. The second definition again used the holistic magnitude values of the radical expressions. Thus, the size of the pair (√4 , √9 ) was (√4 + √9) 2 ⁄ = 2.5. The perfect square variable was defined by whether both natural numbers or radicands were perfect squares like (4, 9) or (√4 , √9 ) or not. Two dependent variables, accuracy and response time (RT) in milliseconds, were collected. Number line estimation task. The second task was number line estimation (NLE) and it included one within-subjects variable (type) with four levels – natural numbers, one-digit radicals, perfect square radicals, and two-digit radicals. For each type, participants saw 11 stimuli. Overall, there were 44 trials. The data from the smallest stimulus and the largest stimulus were not analyzed because these mapped to one of the poles of the unmarked number line (0 and 10) in all but one case, noted below. The first block consisted of the natural numbers 0, 1, 2, …, 10. The values 0 and 10 were not analyzed. The second block consisted of the one-digit radicals √0 , √1 , √2, …, √10 . The values √0 and √10 were not analyzed, meaning all analyzed stimuli had one-digit radicands. This is the one aforementioned case where the largest stimulus (√10 ) does not map to the right pole (10). The third block included the perfect squares √0 , √1 , valuable to document whether the distance and size effects observed for other number classes extend to the irrationals. Hence, we included the distance and size variables in our analyses. UNDERSTANDING IRRATIONAL NUMBERS 18 √4, …, √100. The values √0 and √100 were not analyzed. The fourth block included the two- digit radicals √0 , √10 , √20, …, √100. The values √0 and √100 were not analyzed, meaning all analyzed stimuli had two-digit radicands. Note that a confound exists in these stimulus sets. The values √1 , √4 , and √9 are shared across two types – one-digit radicals and perfect square radicals. This was unavoidable; it was forced by the distribution of perfect squares. The 11 stimuli of each type were presented in random order within a block. Two dependent variables, average absolute error and linearity of estimates, were formed. Average absolute error was the absolute difference between a participant’s selected position and the target position5. Linearity of estimates was the relationship between participants’ selected positions and target positions as estimated by linear regression (R2). Strategy questionnaire. The third task was the strategy questionnaire for the NLE task. Participants completed a paper-and-pencil strategy questionnaire composed of two prompts for each of the four blocks – natural numbers, one-digit radicals, perfect square radicals, and two- digit radicals. Overall, there were 8 items. All prompts were in the form “How did you decide where to place x?”, where x was a stimulus from one of the four blocks. See Table 1 for examples. The independent variable was number type (natural numbers, one-digit radicals, 5 We used average absolute error (AAE) instead of percent average error (PAE). PAE is useful when participants make estimates over multiple scales (Siegler & Booth, 2004) because it allows performance to be compared between scales. Participants in the current experiment made estimates over just one scale (0-10). Therefore, we preferred the simpler AAE measure. Note that the statistical results are the same regardless of which measure is used because PAE is just a linear scaling of AAE. UNDERSTANDING IRRATIONAL NUMBERS UNDERSTANDING IRRATIONAL NUMBERS 19 perfect square radicals, and two-digit radicals) and the dependent variable was self-reported estimation strategy. Irrationals knowledge test. The fourth and final task was the irrationals knowledge test. We constructed this novel paper-and-pencil test to measure conceptual and procedural knowledge of irrational numbers. Several studies have investigated conceptual and procedural knowledge of rational numbers and arithmetic equivalence (Alibali, Knuth, Hattikudur, McNeil, & Stephens, 2007; Booth, Newton, & Twiss-Garrity, 2014; Rittle-Johnson, Siegler, & Alibali, 2001). With these studies in mind, we operationally defined conceptual knowledge of irrational numbers as the ability to categorize particular numbers as members of this system, reason about the properties of irrationals, and relate these properties to those of other number systems. We operationally defined procedural knowledge of irrational numbers as the ability to simplify radical expressions and perform arithmetic operations on them. Some test items were adapted from a study documenting students’ misconceptions about irrational numbers (Fischbein, Jehiam, & Cohen, 1995). Conceptual items were distributed across three subsections. They required participants to classify numbers like 𝜋 as either rational or irrational (8 items), answer questions about the density of rational and irrational numbers (2 items), and reason conceptually about the results of arithmetic operations on arbitrary rational and irrational numbers (2 items). Most of these items were in the selected response format. Some test items were adapted from a study documenting students’ misconceptions about irrational numbers (Fischbein, Jehiam, & Cohen, 1995). Conceptual items were distributed across three subsections. They required participants to classify numbers like 𝜋 as either rational or irrational (8 items), answer questions about the density of rational and irrational numbers (2 items), and reason conceptually about the results of arithmetic operations on arbitrary rational and irrational numbers (2 items). Most of these items were in the selected response format. Procedural items were distributed over two subsections. Subsection 4 required performing simple arithmetic operations to reduce radical expressions (10 items) and subsection 5 required evaluating complex arithmetic expressions by performing operations on multiple radical expressions (7 items). All procedural items were fill-in-the-blank. Overall, there were 29 items. Example items from all five subsections are in Table 1. For the full test, refer to th Procedural items were distributed over two subsections. Subsection 4 required performing simple arithmetic operations to reduce radical expressions (10 items) and subsection 5 required evaluating complex arithmetic expressions by performing operations on multiple radical expressions (7 items). UNDERSTANDING IRRATIONAL NUMBERS All procedural items were fill-in-the-blank. Overall, there were 29 items. Example items from all five subsections are in Table 1. For the full test, refer to th UNDERSTANDING IRRATIONAL NUMBERS 20 Supplementary Materials, section 2. Correct items earned one point each and partial credit was not awarded. Two dependent variables were constructed – accuracy on each section and arithmetic problem-solving strategies. Procedure After obtaining informed written consent, participants were seated alone in a quiet laboratory room in front of a Windows PC with an extended keyboard, a mouse, and a monitor measuring 55.6 cm diagonally. Participants completed the tasks in a fixed order. Magnitude comparison task. Participants first completed the MC task, implemented in the program OpenSesame 2.9.5 (Mathôt, Schreij, & Theeuwes, 2012). In blocks 1 and 2 (natural numbers), they made greater and lesser comparisons, respectively. In blocks 3 and 4 (one-digit radicals), they made greater and lesser comparisons, respectively. Within each block, stimuli were presented in random order against a black background. Each trial began with a blank screen for 250 ms, after which a white fixation dot was shown in the center of the screen for 1000 milliseconds. This was followed by a blank screen for 500 milliseconds. A number pair was presented next, with each number offset about 5 centimeters on either side of the screen’s center. All numbers were displayed in white and in the font Cambria Math (point 60). Participants indicated the greater or lesser number by pressing the key below the target number – “Z” for the number on the left and “M” for the number on the right. The stimuli were visible until participants made a response. Feedback was then provided to discourage participants from trading accuracy for speed. Correct responses were followed by Correct in green and incorrect responses by Incorrect in red. This feedback was displayed for 500 milliseconds. Participants required about six minutes to complete each of the four blocks. UNDERSTANDING IRRATIONAL NUMBERS 21 Number line estimation task and strategy questionnaire. Next, participants completed the NLE task written in JavaScript and hosted online (www.psycholab.org/FreeLab). They estimated the positions of natural numbers in block 1, one-digit radicals in block 2, perfect square radicals in block 3, and two-digit radicals in block 4. Within each block, trials were presented in random order. Participants were instructed to be as accurate as possible and that speed was irrelevant. On each trial, a natural number or radical expression was shown below a number line. These stimuli were shown in black against a white background. The number line was only labeled at the endpoints, zero on the left and ten on the right. The middle area was left blank. Participants estimated the number’s position on the line by left-clicking with a computer mouse. To prevent them from rushing through the task, a blank screen was shown for 1.5 seconds between trials and clicks during this interval were ignored. To avoid learning effects, feedback was not provided. After each trial, the cursor remained where the participant last clicked. Overall, participants needed about six minutes to complete this task. Afterward, they were given the strategy questionnaire to complete at their own pace. Irrationals knowledge test. Finally, participants completed the irrationals knowledge test without the use of electronic devices. They completed the five sections in order: (1) number concepts, (2) density concepts, (3) operation concepts, (4) simple arithmetic, and (5) complex arithmetic. To avoid learning effects, no feedback was provided. Approximately 25 minutes were allotted to respond. Finally, demographic information was collected, participants were debriefed, and compensated with $12 in cash. Results UNDERSTANDING IRRATIONAL NUMBERS 22 Results Results Magnitude comparison task Average accuracy across magnitude comparison blocks was high (98.2%). To determine whether there was a speed-accuracy tradeoff, we correlated the average response times and average accuracy of all participants. These variables were not significantly correlated (r = -.079, p = .486). Because of the ceiling effect in accuracy and the lack of a speed-accuracy tradeoff, subsequent analyses concentrated on the response time data by excluding all trials with incorrect responses. This meant excluding 1.8% of the overall data. Following common practice in the literature, we also excluded response times outside of the interval 200-2000 ms (Varma & Karl, 2013). This was a further 2.6% of the overall data. Recall that the first research question asks whether the magnitudes of radical expressions are compared by referencing a mental number line or by using the equivalence strategy. The second research question, concerning the use of referential strategies, is also relevant here for answering whether perfect square radicals like (√4 , √9 ) are processed more quickly via the multiplication strategy. Addressing these questions requires sensitivity to the natural confound between size and perfect square status in our stimulus set. To understand this confound, consider all natural number pairs formed by the numbers 0-9. In the range 0-9, the average is 4.5. Three of the four perfect squares in this range are less than the average (0, 1, and 4), whereas only one perfect square is greater than the average (9). As a result, the perfect square pairs were on average of smaller size than the non-perfect-square pairs. To control for this confound, an uncontrollable consequence of the non-uniform distribution of perfect squares among the natural numbers, we used a hierarchical linear regression approach. First, we estimated the effect of size. After accounting for that variance, we estimated the effect of perfect square status. After accounting for that variance, we estimated the effect of perfect square status. UNDERSTANDING IRRATIONAL NUMBERS 23 We investigated whether the canonical distance and size effects replicated for natural number comparisons and extended to radical expression comparisons. The size effect is the finding that after controlling for distance, response times are slower if the numbers being compared are larger (Parkman, 1971). For instance, the pair (7, 8) is compared more slowly than the pair (2, 3). Additionally, we investigated whether the type of a stimulus (natural numbers or radical expressions) and perfect square status (yes or no) affected RTs. Finally, we included the interaction between type and perfect square status in the hierarchical regression analysis. This is because if there is an advantage for comparing perfect squares, then it may only apply to radical expression pairs like (√1 , √4 ), but not to natural number pairs like (1, 4). The dependent variable was RT and the five predictor variables were entered sequentially: distance, size, type (natural numbers or radical expressions), perfect square status (yes or no), and the type by perfect square interaction. We addressed the first research question in two hierarchical regression analyses. The first analysis defined the distance and size variables by the radicand components of the radical expressions (e.g. (√1 , √4 ) was converted to (1, 4)), consistent with the equivalence strategy hypothesis. The second analysis defined the distance and size variables by the radical expressions’ holistic magnitude values (e.g. (√1 , √4 ) was not converted), consistent with the mental number line hypothesis. For the first analysis, the final model is shown in Table 2. Distance was entered during step 1; it was a significant predictor of RT, R2 = .278, F(1, 88) = 33.9, p < .001. Size was entered during step 2 and it accounted for significant additional variance, ∆R2 = .183, R2 = .461, F(1, 87) = 29.6, p < .001. Thus, we replicated the distance and size effects for natural numbers and extended them to radical expressions (Fig. 1). Number type was entered during step 3; it -------------------------------------------Insert Table 2 about here - -------------------------------------------Insert Table 2 about here ------------------------------------------- --Insert Table 2 about here - -------------------------------------------Insert Figure 1 about here ------------------------------------------ --------------Insert Table 2 about here ------------------------------------------- UNDERSTANDING IRRATIONAL NUMBERS 24 explained the most additional variance in RTs, ∆R2 = .471, R2 = .932, F(1, 86) = 598.5, p < .001. The positive β weight means that radical expressions required more time to compare than natural numbers. Perfect square status was entered during step 4; it did not explain significant additional variance in RTs, ∆R2 = .001, R2 = .993, F(1, 85) = 1.07, p = .303. The interaction between number type and perfect square value was entered during step 5; it was also a nonsignificant predictor, ∆R2 = .000, R2 = .993, F(1, 84) = .01, p = .935. explained the most additional variance in RTs, ∆R2 = .471, R2 = .932, F(1, 86) = 598.5, p < .001. The positive β weight means that radical expressions required more time to compare than natural numbers. Perfect square status was entered during step 4; it did not explain significant additional variance in RTs, ∆R2 = .001, R2 = .993, F(1, 85) = 1.07, p = .303. The interaction between number type and perfect square value was entered during step 5; it was also a nonsignificant predictor, ∆R2 = .000, R2 = .993, F(1, 84) = .01, p = .935. number type and perfect square value was entered during step 5; it was also a nonsignificant predictor, ∆R2 = .000, R2 = .993, F(1, 84) = .01, p = .935. -------------------------------------------Insert Figure 1 about here 25 UNDERSTANDING IRRATIONAL NUMBERS Fig. 1. Distance and size effects, defined by the radicand components and consistent with the equivalence strategy hypothesis, were found for natural number and radical expression magnitude comparisons. Shaded regions are 95% confidence envelopes. Fig. 1. Distance and size effects, defined by the radicand components and consistent with the equivalence strategy hypothesis, were found for natural number and radical expression magnitude comparisons. Shaded regions are 95% confidence envelopes. magnitude comparisons. Shaded regions are 95% confidence envelopes. UNDERSTANDING IRRATIONAL NUMBERS UNDERSTANDING IRRATIONAL NUMBERS 26 Table 2. Final hierarchical linear regression models predicting RTs on the magnitude comparison task. The models differed on whether distance and size were defined by the radicand components (top) or the holistic magnitude values of the radical expressions (bottom). Table 2. Final hierarchical linear regression models predicting RTs on the magnitude comparison task. The models differed on whether distance and size were defined by the radicand components (top) or the holistic magnitude values of the radical expressions (bottom). Model Predictor Std. β t p R2 Radicand Components / Equivalence Strategy Constant 107.49 .000 .933 Distance -.535 -18.40 .000 Size .434 15.06 .000 Type .687 22.68 .000 Perfect Square -.033 -.804 .424 Type X Perfect Square -.003 -.082 .935 Holistic Magnitudes / Mental Number Line Constant 43.03 .000 .838 Distance -.481 -8.99 .000 Size .199 -3.74 .000 Type .687 14.56 .000 Perfect Square .024 .368 .714 Type X Perfect Square -.003 -.053 .958 UNDERSTANDING IRRATIONAL NUMBERS 27 To summarize, the first regression analysis defined the distance and size variables by the radicand components of radical expressions. The second regression analysis defined distance and size by the holistic magnitude values of the radical expressions. Distance, size, and type effects were again observed (Table 2). Critically, the first analysis explained more of the variance in RTs than the second analysis (93.3% versus 83.8%). Thus, these findings favor the equivalence strategy hypothesis over the mental number line hypothesis. Radical expressions like (√8 , √3 ) appear to be converted to natural numbers like (8, 3) and then compared using the mental number line. Recall that the second research question asked whether people strategically reference the more concrete natural numbers and perfect squares to process the more abstract irrationals. The superior fit of the first model (radicand components) over the second model (holistic Recall that the second research question asked whether people strategically reference the more concrete natural numbers and perfect squares to process the more abstract irrationals. The superior fit of the first model (radicand components) over the second model (holistic magnitudes) provides evidence for the proposal that natural numbers are referenced to make sense of irrational numbers. However, the failure of both perfect square status and the type by perfect square status interaction to reach significance indicates that perfect squares are not referenced. That is, people do not appear to speed comparison of natural number or radical expression pairs composed of perfect squares by automatically activating associated “tie” multiplication problems. Note that there are two reasons to be cautious when interpreting this failed prediction. First, the distance, size, and type variables entered during the initial steps explained most of the variance in RTs. Little variability remained to be explained by the time the perfect square variables were entered. Second, although perfect squares may not be privileged on speeded tasks like magnitude comparison, they may benefit from special processing on unspeeded tasks like number line estimation and arithmetic problem-solving. For these reasons, -------------------------------------------Insert Figure 2 about here -------------------- ---------------------Insert Figure 2 about here ------------------------------------------ UNDERSTANDING IRRATIONAL NUMBERS 30 -------------------------------------------Insert Figure 3 about here --- UNDERSTANDING IRRATIONAL NUMBERS 28 we return to the second question, whether people reference perfect squares when processing irrationals, when analyzing the NLE and arithmetic problem-solving data. we return to the second question, whether people reference perfect squares when processing irrationals, when analyzing the NLE and arithmetic problem-solving data. To investigate individual differences, we separately estimated the first model (radicand components / equivalence strategy) for each participant. There was considerable variation between participants in the magnitudes of the β weights for the five predictors (Fig. 2), reflecting the complexity of understanding numerical processing. In later sections, we use these variables to predict individual differences in conceptual and procedural knowledge of irrational numbers. UNDERSTANDING IRRATIONAL NUMBERS 29 Fig. 2. Distributions of the β weights for the distance, size, type, perfect square, and type by . 2. Distributions of the β weights for the distance, size, type, perfect square, and type by fect square interaction variables in linear regressions predicting each participant’s magnitu ributions of the β weights for the distance, size, type, perfect square, and type by Fig. 2. Distributions of the β weights for the distance, size, type, perfect square, and type by perfect square interaction variables in linear regressions predicting each participant’s magnitude comparison RTs. Fig. 2. Distributions of the β weights for the distance, size, type, perfect square, and type by perfect square interaction variables in linear regressions predicting each participant’s magnitude comparison RTs. -------------------------------------------Insert Figure 3 about here ------------------------------------------ 6 Although participants were instructed to focus on accuracy and not speed, a parallel analysis of response times found the same pattern of results (Supplementary Materials, section 1). Number line estimation task Data from one participant were lost due to computer failure. Thus, the analyses included responses from 80 participants. Due to a programming error, 5.8% of the natural number trials were lost. These lost trials were random and did not affect the results. We excluded outlier responses by discarding trials for which the RT was outside of the interval 1.5 – 15 sec (4.6% of the overall data). Our analyses focused on the second research question: Do people use referencing strategies to facilitate performance? Average absolute error and linearity. First, we analyzed average absolute error using a one-way ANOVA with type (natural numbers, one-digit radicals, perfect square radicals, and two-digit radicals) as the factor (Fig. 3). There was a main effect of type F(3, 316) = 14.13, p < .001, η2 = .118. Post hoc comparisons using Tukey’s procedure found that average absolute error was comparable for natural numbers and perfect squares (p = .836). Error for these two number types was lower than for the one-digit radicals and two-digit radicals (ps < .001), which were comparable to each other (p = .706).6 6 Although participants were instructed to focus on accuracy and not speed, a parallel analysis of response times found the same pattern of results (Supplementary Materials, section 1). 31 UNDERSTANDING IRRATIONAL NUMBERS Fig. 3. Average absolute error on the number line estimation task revealed that compared to natural numbers and perfect squares, one- and two-digit radicals were more difficult to process. Fig. 3. Average absolute error on the number line estimation task revealed that compared to natural numbers and perfect squares, one- and two-digit radicals were more difficult to process. rage absolute error on the number line estimation task revealed that compared to Fig. 3. Average absolute error on the number line estimation task revealed that compared to natural numbers and perfect squares, one- and two-digit radicals were more difficult to process. -------------------------------------------Insert Figure 4 about here ------------------------------------------ -------------------------------------------Insert Figure 4 about here -- UNDERSTANDING IRRATIONAL NUMBERS 32 How linear were participants’ estimates for natural numbers, perfect squares, one-digit radicals, and two-digit radicals? For each of the four types, best-fitting lines predicting the selected position from the correct position were plotted (Fig. 4). Estimates were highly linear across all four blocks. For natural numbers and perfect squares, the R2 values were both 1.00. For one-digit and two-digit radicals, the R2 values were .97 and 1.00, respectively. 33 UNDERSTANDING IRRATIONAL NUMBERS Fig.4. Number line estimation performance was highly linear on average. The dotted lines are the best-fitting regression lines. Fig.4. Number line estimation performance was highly linear on average. The dotted lines are the best-fitting regression lines. -------------------------------------------Insert Figure 5 about here ------------------------------------------ -------------------------------------------Insert Figure 5 about here --- UNDERSTANDING IRRATIONAL NUMBERS 34 To examine whether averaging performance concealed individual differences, we repeated them separately for each participant, estimating the best-fitting lines for each of the four number types (Fig. 5). Estimates for natural numbers and perfect squares were highly linear across participants. By contrast, there was greater variation in the linearity of participants’ estimates for one-digit radicals like √5 and two-digit radicals like √80. 35 UNDERSTANDING IRRATIONAL NUMBERS Fig. 5. Individual differences in the linearity of participants’ estimates were greatest for two-digit radicals Fig. 5. Individual differences in the linearity of participants’ estimates were greatest for two-digit Fig. 5. Individual differences in the linearity of participants’ estimates were greatest for two-digit radicals. -------------------------------------------Insert Table 3 about here ------------------------------------------- -------------------------------------------Insert Table 3 about here -- UNDERSTANDING IRRATIONAL NUMBERS 36 Estimation strategies. On the strategy questionnaire, participants self-reported the strategies they used for each block of the NLE task. After dropping illegible and incoherent responses, full data were available for 63 of 81 questionnaires. All 63 questionnaires were coded by the first author and a naïve volunteer. Interrater agreement, measured by Cohen’s kappa, averaged .74 across the eight prompts. Participants reported using multiple strategies, summarized in Table 3. Running head: UNDERSTANDING IRRATIONAL NUMBERS 37 Table 3. Strategies self-reported on the number line estimation strategy questionnaire and their frequency of use. ed on the number line estimation strategy questionnaire and their frequency of use. Table 3. Strategies self-reported on the number line estimation strategy questionnaire and their frequency of use. Number Type Strategy Stimulus Sample Response % Use Natural numbers landmark – 0, 5, or 10 4 “It had to be before the halfway mark.” 93.7 other 7 “About ¾ of the line…” 6.3 Perfect squares simplify & map √9 “√9 = 3 …” 95.2 other √81 “Guessed.” 4.8 One-digit radicals single perfect square landmark √8 “√9 = 3 near √9.” 26.2 double perfect square landmark √2 “√1 = 1, √4 = 2, between √1 and √4. So between 1-2.” 43.7 other – correct √2 “√2 is about 1.41… So it’s around 1.5.” 19.0 other – incorrect √8 “√8 is close to 8. So, estimate slightly to the left of 8.” 7.9 Two-digit radicals single perfect square landmark √60 “I knew √64 was 8, so a little to the left.” 25.4 double perfect square landmark √40 “I knew it was between 6 and 7 because √36 = 6 and √49 = 7. I put it slightly closer to where 6 was.” 60.3 other – correct √40 “√40 is 2√10. And √10 is about 3.5, so 2(3.5) is about 7.” 2.4 other – incorrect √60 “√60 is 2√15. So, estimate between 2 and 3.” 8.7 UNDERSTANDING IRRATIONAL NUMBERS 39 (single perfect square landmark, two perfect square landmarks, other – correct, other – incorrect) as a factor. We began with the first of two strategy prompts (“How did you decide where to place √2?”) in the one-digit radicals section of the questionnaire. There was a main effect of strategy (F(3, 58) = 10.75, p < .001, η2 = .357). Post hoc comparisons using Tukey’s procedure revealed that performance was comparable for the single perfect square landmark, two perfect square landmarks, and “other – correct” strategies (ps > .05); these strategies yielded more accurate estimates than “other – correct” strategies (ps < .001). We conducted a similar one-way ANOVA for the second prompt (“How did you decide where to place √8?”) and found a main effect of strategy (F(3, 58) = 11.52, p < .001, η2 = .373). Post hoc comparisons again found that utilizing either of the landmark strategies or an “other – correct” strategy produced equally accurate estimates (ps > .05). These estimates were more accurate than those produced by “other – incorrect” strategies (ps < .001). (single perfect square landmark, two perfect square landmarks, other – correct, other – incorrect) as a factor. We began with the first of two strategy prompts (“How did you decide where to place √2?”) in the one-digit radicals section of the questionnaire. There was a main effect of strategy (F(3, 58) = 10.75, p < .001, η2 = .357). Post hoc comparisons using Tukey’s procedure revealed that performance was comparable for the single perfect square landmark, two perfect square landmarks, and “other – correct” strategies (ps > .05); these strategies yielded more accurate estimates than “other – correct” strategies (ps < .001). We conducted a similar one-way ANOVA for the second prompt (“How did you decide where to place √8?”) and found a main effect of strategy (F(3, 58) = 11.52, p < .001, η2 = .373). Post hoc comparisons again found that utilizing either of the landmark strategies or an “other – correct” strategy produced equally accurate estimates (ps > .05). These estimates were more accurate than those produced by “other – incorrect” strategies (ps < .001). A parallel series of one-way ANOVAs was conducted for the two-digit radicals prompts. For the first prompt (“How did you decide where to place √40?”), a main effect of strategy was detected (F(3, 58) = 20.15, p < .001, η2 = .510). Running head: UNDERSTANDING IRRATIONAL NUMBERS 38 For natural numbers, the most frequent strategy was to reference the landmarks 0, 5, and 10. Next, estimates were adjusted slightly to the left or right. This strategy was reported by 93.7% of participants. For example, when estimating the position of the number 3, most participants found the midpoint (5) and then shifted their estimate slightly to the left. All additional strategies were categorized as other (6.3%). For perfect squares like √9, most participants chose to simplify the expression to 3 and map it to the natural number line – the simplify & map strategy (95.2%). The use of this strategy, which is related to the equivalence strategy participants used for the MC task, suggests that perfect squares enjoy privileged processing on the NLE task. All other strategies were labeled other (4.8%). For one-digit and two-digit radicals, participants continued to reference nearby landmarks during estimation. In this case, however, the landmarks were perfect squares. This finding informs the second research question concerning whether more concrete referents are used to understand the more abstract irrationals. Two main strategies were observed; we illustrate them using √7 as an example. The single perfect square landmark strategy was to reason that √7 < √9 and √9 = 3, so estimate slightly to the left of 3. The double perfect square landmark strategy was to bound the estimate from both sides. One might reason that √4 < √7 < √9 , √4 = 2, and √9 = 3. Thus, one should estimate between 2 and 3. Participants were more likely to use the double landmark strategy than the single perfect square landmark strategy for two-digit radicals like √70 (60.3%) than for one-digit radicals like √7 (43.7%). Correct, but rarely used strategies were categorized as “other – correct.” Vague, incorrect, or guess responses were categorized as “other – incorrect.” To determine whether strategy use improved accuracy when estimating the locations of one-digit radicals, a one-way ANOVA of average absolute error was conducted with strategy UNDERSTANDING IRRATIONAL NUMBERS 40 To summarize, participants who used the single or double perfect square landmark strategies were no more accurate than participants who used “other – correct” strategies. This was not because participants who used these strategies were at floor, as their estimates were more accurate than those of participants who used “other – incorrect” strategies. UNDERSTANDING IRRATIONAL NUMBERS Post hoc comparisons revealed that average absolute error was comparable when using a single perfect square landmark, two perfect square landmarks, and “other – correct” strategies (ps > .05). Error was worse when using “other – incorrect” strategies (ps < .001). For the second prompt (“How did you decide where to place √60?”), a main effect of strategy was detected again F(3, 58) = 6.75, p < .001, η2 = .259) and post hoc comparisons revealed that the perfect square and “other – correct” strategies were comparable to each other (ps > .05). Both of these strategies were superior to “other – incorrect” strategies (ps < .01). UNDERSTANDING IRRATIONAL NUMBERS -------------------------------------------Insert Table 4 about here ----------------------- -------------Insert Table 4 about here ------------------------------------------- Irrationals knowledge test All 81 participants completed a paper-and-pencil irrationals knowledge test composed of two sections – conceptual knowledge and procedural knowledge. These were in turn composed of five subsections – number concepts, density concepts, operation concepts, simple arithmetic, and complex arithmetic. Recall that Table 1 lists example items from each subsection. The full test is in the supplementary materials (section 2). Table 4 summarizes performance across all sections and subsections. 41 UNDERSTANDING IRRATIONAL NUMBERS 7 See Figure S2 in the Supplementary Materials for a plot of the two factors. UNDERSTANDING IRRATIONAL NUMBERS Table 4. Performance on all sections and subsections of the irrationals knowledge test. Section Subsection Mean (%) SD Conceptual Number concepts 75.1 20.4 Density concepts 49.0 26.4 Operation concepts 66.9 22.6 average 63.6 17.2 Procedural Simple arithmetic 61.0 27.0 Complex arithmetic 35.8 35.2 average 48.4 29.3 4. Performance on all sections and subsections of the irrationals knowledge test. -------------------------------------------Insert Table 5 about here --------------------- UNDERSTANDING IRRATIONAL NUMBERS 42 To determine the number of constructs being measured across these subsections, we conducted a principal components analysis using varimax rotation. The two procedural subsections loaded onto a single procedural factor (eigenvalue = 2.3, 46.0% of variance explained) and the three conceptual subsections loaded onto a single conceptual factor (eigenvalue = 1.1, 22.8% of variance explained).7 Hence, subsequent analyses focused on average scores on the conceptual section and the procedural section. To address the second research question, which concerns the use of referencing strategies during arithmetic problem-solving, we coded the strategies used on the arithmetic section of the irrationals knowledge test. Interrater agreement, measured by Cohen’s kappa, averaged .95. We focused on whether participants used inefficient or efficient strategies. Some participants used the inefficient prime factorization strategy exclusively (17%). It involves solving arithmetic problems by decomposing radicands into prime factors and then shifting pairs of common factors outside the radical sign. See Table 5 for an example. By contrast, 40% of participants used the perfect squares factorization strategy at least once. It involves directly identifying the largest perfect square factors of the radicand. This chunking strategy enabled participants to solve problems using fewer operations (Table 5). UNDERSTANDING IRRATIONAL NUMBERS 43 UNDERSTANDING IRRATIONAL NUMBERS 44 To evaluate whether arithmetic strategy use affected participants’ procedural scores on the irrationals knowledge test, a one-way ANOVA was conducted. The strategy factor had four levels: prime factorization, perfect squares factorization, both strategies, and other. The category “other” refers to participants who used incorrect strategies or guessed. There was a main effect of strategy on procedural scores (F(3, 77) = 29.71, p < .001, η2 = .537). Post hoc comparisons with Tukey’s procedure revealed that procedural scores were comparable between participants who used the prime factorization strategy and those who used the perfect squares factorization To evaluate whether arithmetic strategy use affected participants’ procedural scores on the irrationals knowledge test, a one-way ANOVA was conducted. The strategy factor had four levels: prime factorization, perfect squares factorization, both strategies, and other. The category “other” refers to participants who used incorrect strategies or guessed. There was a main effect of strategy on procedural scores (F(3, 77) = 29.71, p < .001, η2 = .537). Post hoc comparisons with Tukey’s procedure revealed that procedural scores were comparable between participants who used the prime factorization strategy and those who used the perfect squares factorization strategy (p = .260). The only significant score differences were between participants who used either of these strategies (or both) and participants who used “other” strategies (ps < .001). This indicates that participants who used the less efficient prime factorization strategy were not at floor, as they outperformed those who used “other” strategies (incorrect procedures). The perfect squares strategy is not associated with higher accuracy on complex arithmetic problems than the use of the prime factorization strategy. However, it may confer other advantages such as reducing processing time and working memory demand. We could not test these possibilities because we did not measure these dependent variables. UNDERSTANDING IRRATIONAL NUMBERS Table 5. Example of applying the two strategies to solve the complex arithmetic problem √48 + √75 −√27 from the irrationals knowledge test. Using the prime factorization strategy requires computing and maintaining more partial products than using the perfect squares factorization strategy. Strategy Step Partial Products Use Prime Factorization 0 1 2 3 4 5 6 7 8 9 10 11 12 √48 + √75 −√27 √48 = √2 × 24 √48 = √2 × 2 × 2 × 2 × 3 √48 = 2√2 × 2 × 3 √48 = 4√3 √75 = √3 × 25 √75 = √3 × 5 × 5 √75 = 5√3 √27 = √3 × 9 √27 = √3 × 3 × 3 √27 = 3√3 4√3 + 5√3 −3√3 = 6√3 17% Perfect Squares Factorization 0 1 2 3 4 5 6 7 √48 + √75 −√27 √48 = √16 × 3 √48 = 4√3 √75 = √25 × 3 √75 = 5√3 √27 = √9 × 3 √27 = 3√3 4√3 + 5√3 −3√3 = 6√3 40% Strategy Step Partial Products Use Predicting individual differences in knowledge of irrationals The third research question concerned whether individual differences in the magnitude representation and strategic processing of radical expressions predicts conceptual and procedural knowledge of irrationals. To answer this question, we extracted two sets of predictor variables from each participant, as described above. The first set consisted of the β weights for the three significant variables (distance, size, and type) when predicting response times on the magnitude comparison task. The β weights for the distance and size variables index the precision of magnitude representations and are common predictors of mathematical achievement in children -------------------------------------------Insert Table 6 about here ------------------------------------------- UNDERSTANDING IRRATIONAL NUMBERS 47 For the procedural knowledge measure, the magnitude comparison variables accounted for significant variance in step 1 (R2 = .140, F(3, 76) = 4.14, p = .009), and the NLE variables for significant additional variance in step 2 (∆R2 = .182, R2 = .322, F(2, 74) = 9.91, p < .001). In the final model, only the NLE variable two-digit radicals was significant (Table 6). These findings inform the second and third research questions. With respect to the second research question, participants’ ability to estimate the spatial locations of two-digit radicals (all irrational numbers) predicted conceptual and procedural scores. This indicates that strategically referencing perfect squares is associated with success on higher-level tests of mathematical knowledge. Concerning the third research question, neither the distance nor the size variables from the magnitude comparison task predicted knowledge of irrationals in the final models. This suggests that these variables, which index the precision of magnitude representations, are less important when predicting performance on unspeeded tasks measuring higher-level These findings inform the second and third research questions. With respect to the second research question, participants’ ability to estimate the spatial locations of two-digit radicals (all irrational numbers) predicted conceptual and procedural scores. This indicates that strategically referencing perfect squares is associated with success on higher-level tests of mathematical knowledge. Concerning the third research question, neither the distance nor the size variables from the magnitude comparison task predicted knowledge of irrationals in the final models. This suggests that these variables, which index the precision of magnitude representations, are less important when predicting performance on unspeeded tasks measuring higher-level mathematical knowledge. Further, the significance of the magnitude comparison variable type for predicting conceptual scores supports the role of the equivalence strategy. Understanding when judgments of irrational numbers can and cannot be simplified to judgments of natural numbers may depend on conceptual knowledge of number systems. UNDERSTANDING IRRATIONAL NUMBERS 45 (De Smedt et al., 2009). The β weight for the type variable indicates the additional time required to compare radical expressions versus natural numbers. In other words, it reflects the time needed to apply the equivalence strategy. The second set of predictor variables were the average absolute errors when estimating the positions of one-digit radicals and two-digit radicals on the NLE task. These variables reflect the efficiency of referencing neighboring perfect squares. The magnitude comparison variables were entered first and the NLE variables were entered second in separate hierarchical linear regressions predicting accuracy on the conceptual section and the procedural section. For the conceptual knowledge measure, the magnitude comparison variables added during the first step were nonsignificant (R2 = .074, F(3, 76) = 2.02, p = .119). Adding the NLE variables during the second step accounted for significant additional variance (∆R2 = .115, R2 = .188, F(2, 74) = 5.23, p = .007). In the final model, shown in Table 6, the magnitude comparison variable type and the NLE variable two-digit radicals were significant predictors. UNDERSTANDING IRRATIONAL NUMBERS 46 Table 6. Final models predicting conceptual and procedural knowledge of irrationals. Measure Predictor Std. β t p R2 Conceptual Knowledge Constant 11.87 .000 .188 MC – β Distance .039 .271 .788 MC – β Size .053 .370 .712 MC – β Type .235 2.21 .030 NLE 1D Radicals -.022 -.184 .855 NLE 2D Radicals -.355 -2.97 .004 Procedural Knowledge Constant 8.28 .000 .322 MC – β Distance .125 .945 .348 MC – β Size -.045 -.343 .732 MC – β Type .151 1.55 .125 NLE 1D Radicals -.104 -.952 .344 NLE 2D Radicals -.411 -3.76 .000 6. Final models predicting conceptual and procedural knowledge of irrationals. UNDERSTANDING IRRATIONAL NUMBERS 48 question by fitting two models to the magnitude comparison RT data. The first model, consistent with the equivalence strategy, defined the distance and size variables by the radicands’ magnitudes. The second model, consistent with the mental number line strategy, defined the distance and size variables by the holistic magnitudes of the radical expressions. The equivalence strategy model fit the data best. In addition, the type variable, which indicates whether the stimuli being compared are natural numbers or radical expressions, was a significant predictor above and beyond the distance and size variables. Taken together, these results suggest that people compare the magnitudes of radical expressions by referencing their natural number components (i.e., radicands). question by fitting two models to the magnitude comparison RT data. The first model, consistent with the equivalence strategy, defined the distance and size variables by the radicands’ magnitudes. The second model, consistent with the mental number line strategy, defined the distance and size variables by the holistic magnitudes of the radical expressions. The equivalence strategy model fit the data best. In addition, the type variable, which indicates whether the stimuli being compared are natural numbers or radical expressions, was a significant predictor above and beyond the distance and size variables. Taken together, these results suggest that people compare the magnitudes of radical expressions by referencing their natural number components (i.e., radicands). question by fitting two models to the magnitude comparison RT data. The first model, consistent with the equivalence strategy, defined the distance and size variables by the radicands’ question by fitting two models to the magnitude comparison RT data. The first model, consistent with the equivalence strategy, defined the distance and size variables by the radicands’ The second research question asked whether people reference perfect squares to make sense of irrational numbers. Examples of such referencing, besides the equivalence strategy, are evident on the NLE task. Strategy self-reports suggest that people reference nearby perfect squares to better estimate the locations of one-digit and two-digit irrationals – the single and double landmark strategies. Referencing strategies were also observed on the complex arithmetic subsection of the irrationals knowledge test. When simplifying radical expressions, 40% of participants factored radicands into perfect squares. This may be a more efficient strategy than the prime factorization strategy used by 17% of participants. Summary To study how people understand highly abstract mathematical concepts, we used irrational numbers as a test case. Specifically, we addressed three research questions that tested multiple hypotheses. The first question asked whether radical expressions are compared by referencing a mental number line or by using the equivalence strategy. We addressed this UNDERSTANDING IRRATIONAL NUMBERS The third research question asked whether individual differences in the speed and accuracy with which irrational numbers are processed on the simple MC and NLE tasks predicts individual differences in higher-level conceptual and procedural knowledge measures comparable to classroom mathematics tests. The type variable for the magnitude comparison task, which indicates whether the stimuli are natural numbers or radical expressions, was a significant predictor of conceptual knowledge. We interpret this as reflecting the importance of UNDERSTANDING IRRATIONAL NUMBERS 49 selectively accessing the natural number components of radical expressions (i.e., radicands and roots) when reasoning about irrationals. Accuracy with two-digit radicals on the NLE task was also a significant predictor. It explained both conceptual and procedural knowledge. This may reflect the cognitive value of referencing perfect squares when processing irrational numbers. Although magnitude representations are necessary for arithmetic competence, they are not as critical as for natural numbers, integers, and rational numbers. The role of magnitude representations appears to attenuate in importance as the abstraction of number systems increases. Hence, alternative mechanisms must be invoked to explain expertise with irrationals. Theoretical implications – a referential processing account Some theories of numerical and arithmetic development emphasize the recruitment, repurposing, and refinement of perceptuo-motor and visuospatial systems (Blair, Tsang, & Schwartz, 2013; Landy, Allen, & Zednik, 2014; Siegler, 2016). Based on our findings that natural numbers and perfect squares support an understanding of the irrationals on a range of tasks, we hypothesize that abstract mathematical concepts are understood by selectively recruiting more concrete representations that align with task demands and stimulus properties. We call these representations referents and the recruitment and alignment processes referencing or referential processing. Mathematical tasks like magnitude comparison, number line estimation, and arithmetic problem-solving can impose high computational demands on limited cognitive resources. Mathematical tasks like magnitude comparison, number line estimation, and arithmetic problem-solving can impose high computational demands on limited cognitive resources. Mathematical tasks like magnitude comparison, number line estimation, and arithmetic problem-solving can impose high computational demands on limited cognitive resources. Referential processing reduces these demands in task- and stimulus-specific ways. Magnitude comparison with radical expressions, for instance, can be performed using a “guess and check” algorithm to determine the greater or lesser value in number pairs like (√7 , √4 ). Due to the substantial computational demands of using this algorithm, people choose to use the more Referential processing reduces these demands in task- and stimulus-specific ways. Magnitude comparison with radical expressions, for instance, can be performed using a “guess and check” algorithm to determine the greater or lesser value in number pairs like (√7 , √4 ). Due to the substantial computational demands of using this algorithm, people choose to use the more UNDERSTANDING IRRATIONAL NUMBERS 50 efficient equivalence strategy instead. Radical expressions are interpreted as natural number referents like (7, 4) because the judgments are the same in both cases. On the NLE task, the computational demands of estimating the positions of one-digit and two-digit radicals are also high. They are circumvented by locating estimates with respect to neighboring perfect square referents – the single and double perfect square landmark strategies. Likewise, referential processing supports arithmetic problem-solving on the irrationals knowledge test by enabling expressions like √48 to be simplified efficiently via perfect squares factorization: √48 = √16 ∗3 = 4√3. √48 = √16 ∗3 = 4√3. This is less computationally demanding as it requires fewer steps and partial products than the prime factorization strategy: This is less computationally demanding as it requires fewer steps and partial products than the prime factorization strategy: √48 = √2 ∗24 = √2 ∗2 ∗12 = √2 ∗2 ∗2 ∗6 = √2 ∗2 ∗2 ∗2 ∗3 = 2√2 ∗2 ∗3 = 4√3 = 4√3 Critically, the referential processing account states that referents are task- and stimulus- specific. On the magnitude comparison task, for instance, the appropriate referents were natural numbers. In contrast, the appropriate referents on the NLE and arithmetic problem-solving tasks were perfect squares. To explain why particular concrete mathematical concepts are appropriate on some tasks but not others, it is useful to focus on the alignment between what a task demands and what a referent provides. An appropriate referent simplifies a task without altering its underlying structure. For example, the equivalence strategy aligns well with the magnitude comparison task because the comparisons (√𝑥, √𝑦) and (x, y) have the same response when x and y are non-negative. In this regard, the equivalence strategy’s focus on the natural number components of radical expressions constitutes a virtuous form of the whole number bias (Ni & UNDERSTANDING IRRATIONAL NUMBERS UNDERSTANDING IRRATIONAL NUMBERS 51 Zhou, 2005).8 Referential processing, then, can reduce computational demands and improve performance while preserving task structure. Zhou, 2005).8 Referential processing, then, can reduce computational demands and improve performance while preserving task structure. The extent to which referential processing enhances performance relative to default strategies depends on the task to be performed. On the MC task, referential processing improves how quickly irrational numbers are processed. During NLE, it improves accuracy compared to “other-incorrect” strategies. It is important to acknowledge that referential processing does not always improve accuracy. On the arithmetic problem-solving test, for example, the use of perfect square referents was not associated with higher accuracy compared to prime number referents. The benefits afforded in this context appear to be more modest and may be restricted to the speed domain, a prediction that the current study did not test. 8 We thank an anonymous reviewer for suggesting this connection between referential processing and the whole number bias. and the whole number bias. 8 We thank an anonymous reviewer for suggesting this connection between referent Referential processing across number systems and mathematical tasks Our referential processing account unifies a constellation of seemingly disparate phenomena in the mathematical cognition literature. The prominence of referents has been demonstrated using more open-ended prompts, in naturalistic studies, and in formal analyses of number naming systems. In production studies, where participants must generate numbers in a specified range like 1 – 100, the frequency with which numbers are produced follows a power law. Smaller numbers are produced more frequently than larger numbers. Critically, the exceptions are referents in the base-10 system like 10 and 100 or the larger culture like 12, 15, and 50. These numbers are produced more frequently than the power law predicts (Noma & Baird, 1975). Corpora studies reveal the same pattern; referents in the base-10 system like 10 and 9 Evidence exists against the referential processing account and in favor of continuous performance (whether logarithmic or linear) on the NLE task (Ashcraft & Moore, 2012; Opfer et al., 2011; Siegler & Booth, 2004; Siegler & Opfer, 2003; Opfer et al. 2016). UNDERSTANDING IRRATIONAL NUMBERS 52 100 are produced more frequently than expected given the otherwise power-law distribution of numbers (Dehaene & Mehler, 1992). In a corpus study and formal analysis of number words in the Dutch language, Pollman and Jansen (1996) found that referents in the base-10 system like the base raised to an integer power (e.g., 101 = 10) are among the “favourite numbers.” This finding was extended to Swedish by Ericsson, Biley, and Geary (2010). 100 are produced more frequently than expected given the otherwise power-law distribution of numbers (Dehaene & Mehler, 1992). In a corpus study and formal analysis of number words in the Dutch language, Pollman and Jansen (1996) found that referents in the base-10 system like the base raised to an integer power (e.g., 101 = 10) are among the “favourite numbers.” This finding was extended to Swedish by Ericsson, Biley, and Geary (2010). Studies using the MC and NLE tasks also support the use of referential processing across a range of number systems (Table 7). For natural numbers, referential processing has been observed when comparing one-digit numbers, with end-anchoring to zero (Banks, 1977; Pinhas & Tzelgov, 2012). Comparisons of multi-digit numbers are made with reference to intervening multiples of 10 (Brysbaert, 1995; Franklin, Jonides, & Smith, 2009). The question of whether referential processing is used when estimating the position of a natural number on a bounded number line (and if so, under which conditions) is a contentious one. We focus here on findings and models consistent with referential processing.9 Ebersbach at al. (2008) proposed that the largest number familiar to children serves as a referent during NLE. Moeller et al. (2009) proposed that before children have integrated the ones and tens places in their understanding of number, 10 serves as a referent when estimating numbers in the range 0 – 100. Landy et al. (2013) proposed that scale words like ‘million’ serve as referents when estimating across very large ranges like 1 thousand – 1 billion. Across these studies, variants of a bilinear model with a discontinuity at the proposed referent (the largest familiar number, 10, and 1 million, Studies using the MC and NLE tasks also support the use of referential processing across a range of number systems (Table 7). For natural numbers, referential processing has been observed when comparing one-digit numbers, with end-anchoring to zero (Banks, 1977; Pinhas & Tzelgov, 2012). UNDERSTANDING IRRATIONAL NUMBERS Comparisons of multi-digit numbers are made with reference to intervening multiples of 10 (Brysbaert, 1995; Franklin, Jonides, & Smith, 2009). The question of whether referential processing is used when estimating the position of a natural number on a bounded number line (and if so, under which conditions) is a contentious one. We focus here on findings and models consistent with referential processing.9 Ebersbach at al. (2008) proposed that the largest number familiar to children serves as a referent during NLE. Moeller et al. (2009) proposed that before children have integrated the ones and tens places in their understanding of number, 10 serves as a referent when estimating numbers in the range 0 – 100. Landy et al. (2013) proposed that scale words like ‘million’ serve as referents when estimating across very large ranges like 1 thousand – 1 billion. Across these studies, variants of a bilinear model with a discontinuity at the proposed referent (the largest familiar number, 10, and 1 million, 9 Evidence exists against the referential processing account and in favor of continuous performance (whether logarithmic or linear) on the NLE task (Ashcraft & Moore, 2012; Opfer et al., 2011; Siegler & Booth, 2004; Siegler & Opfer, 2003; Opfer et al. 2016). -------------------------------------------Insert Table 7 about here ----------------------- ----------------------Insert Table 7 about here ------------------------------------------- UNDERSTANDING IRRATIONAL NUMBERS 53 respectively) fit the data better than a linear or a logarithmic model. Finally, some have applied cyclical models to account for the NLE performance of children and adults (Barth & Paladino, 2011; Cohen & Blanc-Goldhammer, 2011; Slusser et al., 2013). For our purposes, these models can be characterized as positing referents at endpoints, midpoints, and quartiles. They also posit proportional estimation between referents in opposition to the linear estimation of the models just reviewed. Even natural number arithmetic can be supported by base-10 referents. For instance, one- digit addition problems such as 9 + 8 can be solved by decomposition to the referent 10: 9 + 8 = 9 + (1 + 7) = (9 + 1) + 7 = 10 + 7 = 17 (Baroody, Purpura, Eiland, Reid, & Paliwal, 2015). For multi-digit arithmetic, the referents are often multiples of 10, as when 49 + 38 is transformed to 50 + 37 (Torbeyns, de Smedt, Ghesquiere, & Verschaffel, 2009). UNDERSTANDING IRRATIONAL NUMBERS 54 Table 7. Across number systems and tasks, people use many kinds of referents flexibly. Naturals Integers Rationals Irrationals single-digit multi-digit fractions decimals Magnitude Comparison zero multiples of 10n zero unit fractions, decimals naturals naturalsa Number Line Estimation endpoints midpoints quartiles multiples of 10n, midpoints, quartiles zero unit fractions, decimals quartiles tenths perfect squaresb Arithmetic Computation 10 multiples of 10n zero ? ? perfect squaresc a Supported by the equivalence strategy on the MC task. 7. Across number systems and tasks, people use many kinds of referents flexibly. b Supported by the single and double perfect square landmark strategies on the NLE task. c Supported by the perfect squares factorization strategy on the irrationals knowledge test. UNDERSTANDING IRRATIONAL NUMBERS 55 Referential processing also extends to more abstract number systems. With integers, zero can be used as a referent when performing magnitude comparison, number line estimation, number bisection, and arithmetic computation (Gullick & Wolford, 2013; Tsang et al., 2015; Tsang & Schwartz, 2009; Varma & Schwartz, 2011). Fractions are often processed by referencing unit fractions like ½ when making magnitude comparison and number line estimation judgments (Fazio et al., 2016; Siegler & Thompson, 2014). When rational numbers are expressed as decimal proportions, their magnitudes can be referenced with respect to natural numbers (Varma & Karl, 2013). Finally, during number line estimation with decimals, quartiles like 0.25 and tenths like 0.10 are often referenced (Houseworth, 2016). UNDERSTANDING IRRATIONAL NUMBERS 56 factorization should be taught during formal instruction. Doing so may improve students’ adaptive expertise, or their ability to recognize and apply optimal strategies (Hatano & Oura, 2003). Third, it is worth noting that the correlation between magnitude knowledge and arithmetic expertise is weaker for irrationals than other number systems. Interventions that improve the magnitude representations of children result in greater arithmetic expertise for natural numbers, integers, and rational numbers (Fuchs et al., 2013; Siegler & Ramani, 2009; Tsang et al., 2015). Our individual differences analyses, however, did not find a relationship between the precision of magnitude representations as measured by the distance and size effects and conceptual and procedural knowledge of irrationals. This suggests that training magnitude representations may not produce similar learning gains for the irrationals. We tentatively suggest that a better approach for teaching more abstract mathematical concepts like the irrationals is to focus on strategies for relating them to referents according to stimulus properties and task demands. Instructional implications Our results have three potential implications for instructional design in mathematics. First, performance patterns on the MC and NLE tasks suggest that our sampled undergraduates represent the magnitudes of irrationals accurately. Nonetheless, performance on the NLE task reveals that some people place irrationals, denoted by one-digit expressions (e.g., √2) and two- digit expressions (e.g., √40), on the number line inaccurately. It might be possible to improve their performance by giving students explicit strategy instruction. They could be guided to estimate the values of irrationals by referencing perfect squares via the single and double perfect square landmark strategies. The second pedagogical implication concerns strategy differences during arithmetic problem-solving. Participants who used the prime factorization strategy required more steps and likely spent more time on task. Those who used the perfect squares factorization strategy, in contrast, could skip steps by directly accessing the perfect square factors under the radical signs. Not all learners discover such strategies on their own, suggesting that perfect squares UNDERSTANDING IRRATIONAL NUMBERS UNDERSTANDING IRRATIONAL NUMBERS 57 A second limitation is that much of the variation in conceptual and procedural knowledge of irrational numbers was unexplained. Unexplained variance in the conceptual knowledge measure might reflect differential understanding of the cardinalities of infinite sets such as the rationals, irrationals, and reals. This knowledge is crucial for defining and reasoning about these number systems. Unexplained variance in the procedural knowledge measure might reflect differential fluency in the multiplication and division skills necessary for factoring and simplifying radical expressions. Future studies should investigate these and other possible explanations. Limitations This study was constrained in several ways. First, the NLE task involved collecting strategy prompts after the entire task ended rather than after each trial in a block. This is potentially problematic because strategies can change on a trial-by-trial basis, as has been shown for fractions (Fazio et al., 2016). Future studies should collect strategies after every trial. This study was constrained in several ways. First, the NLE task involved collecting strategy prompts after the entire task ended rather than after each trial in a block. This is potentially problematic because strategies can change on a trial-by-trial basis, as has been shown for fractions (Fazio et al., 2016). Future studies should collect strategies after every trial. Relatedly, only two strategy prompts were presented for each block. It is possible that participants used different strategies for the unprompted items and that these strategies were not captured in the current study. These limitations could be addressed in a future study that focuses on the NLE task for irrational numbers and collects strategy prompts after every trial. This study was constrained in several ways. First, the NLE task involved collecting strategy prompts after the entire task ended rather than after each trial in a block. This is potentially problematic because strategies can change on a trial-by-trial basis, as has been shown for fractions (Fazio et al., 2016). Future studies should collect strategies after every trial. Relatedly, only two strategy prompts were presented for each block. It is possible that participants used different strategies for the unprompted items and that these strategies were not Conclusion Most numerical cognition research focuses on relatively concrete concepts like natural numbers, integers, and rational numbers. Our study of irrationals elucidates how the mind grasps abstract mathematical concepts incapable of being directly perceived or fully grounded in visuospatial referents. We suggest that the numerical magnitudes and symbolic procedures associated with irrationals are understood by referencing natural numbers and perfect squares. Such referential processing may explain performance across a wide range of tasks and number systems (Table 7), supporting an intuitive understanding of otherwise alien concepts. Thus far, we have only learned that experts use referential processing to understand irrationals. Our next step is to explore the development of referential processing in novices and how it supports the discovery, comprehension, and instruction of diverse mathematical content. Doing so will illuminate how the abstract becomes concrete. UNDERSTANDING IRRATIONAL NUMBERS 58 References Alibali, M. W., Knuth, E. J., Hattikudur, S., McNeil, N. M., & Stephens, A. C. (2007). 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Distance and size effects, defined by the radicand components and consistent with the equivalence strategy hypothesis, were found for natural number and radical expression magnitude comparisons. Shaded regions are 95% confidence envelopes. Fig. 2. Distributions of the β weights for the distance, size, type, perfect square, and type by perfect square interaction variables in linear regressions predicting each participant’s magnitude comparison RTs. Fig. 3. Average absolute error on the number line estimation task revealed that compared to natural numbers and perfect squares, one- and two-digit radicals were more difficult to process. Fig. 3. Average absolute error on the number line estimation task revealed that compared to natural numbers and perfect squares, one- and two-digit radicals were more difficult to process. Fig. 4. Number line estimation performance was highly linear on average. The dotted lines are the best-fitting regression lines. Fig. 4. Number line estimation performance was highly linear on average. The dotted lines are the best-fitting regression lines. Fig. 5. Individual differences in the linearity of participants’ estimates were greatest for two-digit radicals.
https://openalex.org/W4289287074	https://figshare.com/articles/journal_contribution/Ax-Lindemann-Weierstrass_with_derivatives_and_the_genus_0_Fuchsian_groups/14425937/1/files/27575531.pdf	English	null	Ax-Lindemann-Weierstrass with derivatives and the genus 0 Fuchsian groups	arXiv (Cornell University)	2,018	cc-by	23,949	To Keiji Nishioka on his retirement. ABSTRACT. We prove the Ax-Lindemann-Weierstrass theorem with derivatives for the uni- formizing functions of genus zero Fuchsian groups of the ﬁrst kind. Our proof relies on dif- ferential Galois theory, monodromy of linear differential equations, the study of algebraic and Liouvillian solutions, differential algebraic work of Nishioka towards the Painlev´e ir- reducibility of certain Schwarzian equations, and considerable machinery from the model theory of differentially closed ﬁelds. arXiv:1811.06583v3 [math.AG] 19 y y Our techniques allow for certain generalizations of the Ax-Lindemann-Weierstrass the- orem which have interesting consequences. In particular, we apply our results to give a complete proof of an assertion of Painlev´e (1895). We also answer certain cases of the Andr´e- Pink conjecture, namely in the case of orbits of commensurators of Fuchsian groups. arXiv:1811.06583v3 [math AX-LINDEMANN-WEIERSTRASS WITH DERIVATIVES AND THE GENUS 0 FUCHSIAN GROUPS 11.06583v3 [math.AG] 19 Sep 2020 G. Casale is partially supported by Math-AMSUD project “Complex geometry and foliations”. J. Freitag is partially supported by NSF grant DMS-1700095. J. Nagloo is partially supported by NSF grant DMS-1700336. 1We say that t1, . . . , tn are geodesically independent if ti is nonconstant for i = 1, . . . , n and there are no relations of the form ti = γtj for i ̸= j, i, j ∈{1, . . . , n} and γ is an element of the commensurator of Γ. 2010 Mathematics Subject Classiﬁcation. 11F03, 12H05, 03C60. p y pp y g g p y pp y g 1We say that t1, . . . , tn are geodesically independent if ti is nonconstant for i = 1, . . . , n and there are no relations of the form ti = γtj for i ̸= j, i, j ∈{1, . . . , n} and γ is an element of the commensurator of Γ. G. Casale is partially supported by Math-AMSUD project “Complex geometry and foliations”. J partially supported by NSF grant DMS-1700095. J. Nagloo is partially supported by NSF grant DMS 1 2010 Mathematics Subject Classiﬁcation. 11F03, 12H05, 03C60. 2010 Mathematics Subject Classiﬁcation. 11F03, 12H05, 03C60. G. Casale is partially supported by Math-AMSUD project “Complex geometry and foliations”. J. Freitag i partially supported by NSF grant DMS-1700095. J. Nagloo is partially supported by NSF grant DMS-1700336 1. INTRODUCTION In this paper our central work is to prove a series of functional transcendence results for the automorphic functions jΓ associated with a Fuchsian group Γ of genus 0. We will also refer to the automorphic function jΓ as a Hauptmodul or uniformizing function of Γ. Our general results are most easily expressed in the language of model theory and algebraic differential equations, but a special case of our functional transcendence results is what has come to be called the Ax-Lindemann-Weierstrass theorem with derivatives for jΓ: Theorem 1.1. Let C(V) be an algebraic function ﬁeld, where V ⊂An is an irreducible algebraic variety deﬁned over C. Let t1, . . . , tn ∈C(V) take values in the upper half complex plane H at some P ∈V and are geodesically independent 1. Then the 3n-functions jΓ(t1), j′ Γ(t1), j′′ Γ(t1) . . . , jΓ(tn), j′ Γ(tn), j′′ Γ(tn) (considered as functions on V(C) locally near P) are algebraically independent over C(V 1 2 G CASALE, J FREITAG, AND J NAGLOO G. CASALE, J. FREITAG, AND J. NAGLOO 2 One can also describe Theorem 1.1 in more geometric terms. Let W ⊂An(C) be an algebraic variety which has a nonempty intersection with Hn. Theorem 1.1 precisely char- acterizes those varieties W whose image under the automorphic function (and derivatives) applied to each coordinate: ¯jΓ : (t1, . . . , tn) 7→(jΓ(t1), j′ Γ(t1), j′′ Γ(t1) . . . , jΓ(tn), j′ Γ(tn), j′′ Γ(tn)) is contained in a proper algebraic subvariety of C3n. Intuitively, the function jΓ is highly transcendental, so the varieties obtained in this way should be restricted to a very special class. Indeed, Theorem 1.1 says that if ¯jΓ(W) is an algebraic variety, then W must have been deﬁned by instances of relation of the form ti = γtj where γ is an element of the commensurator of Γ, giving a very restrictive (countable) class of complex varieties coming from the image of ¯jΓ. As we will explain in additional detail below, our methods also allow for more general results, which are most naturally stated in the language of model theory. For instance, statements incorporating other transcendental functions on additional coordinates (such as Weierstrass ℘-functions and exponential functions on semi-abelian varieties) similar to Theorem 1.6 of [49] will follow from our general result. 1. INTRODUCTION However, following the Pila-Wilkie theorem, there has been an explosion of work. In [54], the Pila-Wilkie theorem was employed by Pila and Zannier to give a new proof of the Manin-Mumford conjecture. The strategy was immediately taken up by Masser and Zannier [30] to prove a a special case of Pink’s relative Manin-Mumford conjecture, while Pila [50] gave new proofs of results of a Manin- Mumford-Andr´e-Oort ﬂavor. The common line of reasoning in the results mentioned in the previous several para- graphs is to embed the problem in an o-minimal context by proving that the a certain ana- lytic function (restricted to an appropriate fundamental domain) is interpretable in Ran,exp, an o-minimal structure in which the deﬁnable sets are given by inequalities built from the algebraic functions, the exponential function, and real analytic functions restricted to bounded sets. Following this, variants of the Pila-Zannier strategy or deﬁnable versions of results from complex geometry [45] are used to detect and characterize algebraic relations. p g y g Our approach is completely different, and does not employ the theory of o-minimality at all. Rather, our proof relies on differential Galois theory, monodromy, the study of alge- braic and Liouvillian solutions to linear differential equations, differential algebraic work of Nishioka towards the Painlev´e irreducibility of certain Schwarzian equations, and con- siderable machinery from the model theory of differentially closed ﬁelds. y y y Recently there has been a surge in interest around functional transcendence statements of the type in Theorem 1.1, in part due to their connection with a class of problems from number theory called special points conjectures or problems of unlikely intersections; in [49] the Ax-Lindemann-Weierstrass theorem is central to the proof of the Andr´e-Oort conjec- ture for Cn. Each of the other functional transcendence results mentioned above can be applied in certain special points settings. For instance, in [10] Daw and Ren give applica- tions of the Ax-Schanuel conjecture proved in [34]. Our functional transcendence results are no exception - we apply them to certain cases of a special points conjecture called the Andr´e-Pink conjecture, following Orr [41, 42]. Numerous variations on the conjecture are possible (depending for instance, on the deﬁnition of Hecke-orbits one takes), but we will describe the speciﬁc setup next. 1. INTRODUCTION g Theorem 1.1 is a generalization of Theorem 1.6 of [49] and Theorem 1.1 [48], in which Pila established the special case with one group Γ = PSL2(Z) (in [49] without derivatives and later in [48] with derivatives). Theorem 1.1 also overlaps nontrivially with a number of recent results, which we detail next. Note that most of the following results do not in- volve the derivatives of the automorphic functions in question and are mainly concerned with arithmetic groups. Pila and Tsimerman [52] generalized Theorem 1.6 of [49] to the uniformizing functions associated with the moduli spaces of higher dimensional abelian varieties (their result specializes to Theorem 1.6 of [49] for purposes of comparing with Theorem 1.1). In a different direction, Pila and Tsimerman [51] generalized Theorem 1.6 of [49] to an Ax-Schanuel type statement for the j-function. In [66], Ullmo and Yafaev prove an Ax-Lindemann-Weierstrass result for the uniformizing functions of cocompact Shimura varieties (without derivatives), and so a statement of Theorem 1.1 without derivatives in the case that Γ is arithmetic and cocompact is a consequence of their work. Later, Klinger, Ullmo, and Yafaev [22] removed the assumption of cocompactness, and Gao [14] general- ized the result to mixed Shimura varieties. Finally, Mok, Pila, and Tsimerman [34] have established the (more general) Ax-Schanuel theorem with derivatives for the uniformizing function of a Shimura variety. The previous Ax-Lindemann-Weiestrass (ALW for short) results discussed above em- ploy various techniques from group theory, complex variables, and number theory, but each one also shares a common element in their approach: a tool called o-minimality orig- inating in model theory. The theory of o-minimality is a natural generalization of real algebraic geometry to include certain non-oscillatory transcendental functions. It was de- veloped starting in the 1980s by model theorists [71], but in the early 2000s, o-minimality was connected with various aspects of number theory in part through the work of Pila and Wilkie [53] and Peterzil and Starchenko [44, 47, 46]. The counting theorem of Pila- Wilkie has precursors coming from number theory before the connection to o-minimality was made. See, for instance, the work of Bombieri and Pila [6] and the related manuscript 3 AX-LINDEMANN-WEIERSTRASS WITH DERIVATIVES AND THE GENUS 0 FUCHSIAN GROUPS of Sarnak [61]. Diophantine properties of deﬁnable sets in o-minimal structures had also been previously investigated by Wilkie [74]. 1. INTRODUCTION Let V be a connected Shimura variety with (connected) Shimura datum (G, X+) such that V = Γ \ X+, for some congruence subgroup Γ ⊂G(Q) that stabilizes X+. The Andr´e- Pink conjecture predicts that when W is an algebraic subvariety of V and S is the orbit of the commensurator of Γ, Comm(Γ), on a point ¯a = (a1, . . . , an), if W ∩S is Zariski dense in W, then W is of a very restrictive form, which we will refer to as Γ-special, which we describe next. Let jΓ : X+ →V be a uniformisation map. When γ ∈Comm(Γ), it turns out that (jΓ(t), jΓ(γt)) are algebraically dependent and lie on an irreducible curve given by the vanishing of a polynomial in two variables which we will refer to as a Γ-special polynomial. The Γ-special varieties are intersections of zero sets of Γ-special polynomials and relations of the form xi = bi where bi is in the Comm(Γ)-orbit of ai. Orr [41, 42] proved various special cases of the conjecture (for instance, when W is an algebraic curve). In [12] Freitag and Scanlon used Pila’s ALW with derivatives theorem from [48] to prove the Andr´e-Pink conjecture when ¯a is assumed to be a transcendental point and Γ is commensurable with G. CASALE, J. FREITAG, AND J. NAGLOO 4 PSL2(Z). In this paper, we generalize that result to allow for an arbitrary Fuchsian group Γ. PSL2(Z). In this paper, we generalize that result to allow for an arbitrary Fuchsian group Γ. The central idea employed is a beautiful technique which has its origins in the work of Hrushovski [17] and Buium [7]. In order to understand intersections of algebraic varieties with an arithmetically deﬁned set of points (e.g. torsion points on an algebraic group, Hecke orbits, etc.), replace the arithmetic set with a more uniformly deﬁned algebraic ob- ject, the solution set of some algebraic differential differential or difference equation. j g q We replace our arithmetic objects (the orbits of the commensurators of some discrete groups, Γ) by the solution sets of certain differential equations satisﬁed by the uniformiz- ing functions jΓ. An inherent restriction of the technique is that it generally only works for diophantine problems in function ﬁelds, hence the assumption that ¯a is a tuple of transcen- dentals. 1. INTRODUCTION In pursuing our approach to the Andr´e-Pink conjecture, it becomes necessary to prove more far reaching functional transcendence results than the ALW theorem as stated above; our results are most naturally phrased in terms of the model theory of differential ﬁelds, one of the main tools we use to establish our results. One of the chief advantages of this approach is that it leads to an effective solution of our case of Andr´e-Pink, that is, we are able to give bounds on the degree of the Zariski closure of the intersection of Comm(Γi)- orbits with a variety V, which depend on algebro-geometric invariants of the variety V. So, for instance, if the variety V is a non-special curve (or a variety which does not contain a special curve), we can give a bound on the number of special points contained in the curve. At the relevant sections of our paper (e.g. 5) we will give equivalent formulations in algebro-geometric language of the model-theoretic properties we describe next. We prove that for any Fuchsian group Γ, the set deﬁned by the differential equation satisﬁed by the uniformizing function jΓ is strongly minimal and has geometrically trivial forking geome- try. This result generalizes work of [12] which covers the cases when Γ is commensurable with PSL2(Z). In particular, our work gives many new examples of geometrically trivial strongly minimal sets in differentially closed ﬁelds. This also establishes an interesting new connection between two important dividing lines on the logic and group theory: the differential equation satisﬁed by jΓ is ℵ0-categorical if and only if the group Γ is not arith- metic. Further, we characterize all instances of nonorthogonality between these sets (each such instance comes from commensurability of two groups Γ1 and Γ2). These results also have various interesting consequences related to determining the isomorphism invariants of differentially closed ﬁelds, which we will not explore further in this article. y p We should also mention that this work gives a complete proof of an assertion of Painlev´e [43, Page 519], concerning the irreducibility of the differential equations satisﬁed by jΓ for Γ a Fuchsian group. Irreducibility is closely related to the strong minimality of a differen- tial equation, a connection pointed out in detail by Nagloo and Pillay [36]. The original deﬁnition of irreducibility applies to nonlinear differential equations and was given by Painlev´e [43, pages 490-496]. 2. THE BASIC THEORY 2.1. Fuchsian groups and the associated Schwarzian equations. We direct the reader to [21] and [25] for the basics on Fuchsian groups and the corresponding automorphic functions. The appendices of [72] also give a very detailed introduction to the associated Schwarzian equations. q Let H be the upper half complex plane and let H := H ∪P1(R). Recall that SL2(R) and PSL (R) acts on H (and H) by linear fractional transformation: for a b ∈SL (R) and q Let H be the upper half complex plane and let H := H ∪P1(R). Recall that SL2(R) and b Let H be the upper half complex plane and let H := H ∪P1(R). Recall that SL2(R) and PSL2(R) acts on H (and H) by linear fractional transformation: for a b c d ∈SL2(R) and τ ∈H PSL2(R) acts on H (and H) by linear fractional transformation: for a b c d ∈SL2(R) and τ ∈H a b c d · τ = aτ + b cτ + d. This action yields all the orientation preserving isometries of H. This action yields all the orientation preserving isometries of H. Let Γ ⊂PSL2(R) be a Fuchsian group, that is, assume that Γ is a discrete subgroup of PSL2(R). A point τ ∈H is said to be a cusp if its stabilizer group Γτ = {g ∈Γ : g · τ = τ} has inﬁnite order. We also assume throughout that Γ is of ﬁrst kind (i.e., its limit set is P1(R)) and of genus zero2 (i.e., Γ \ H can be compactiﬁed to a compact Riemann surface of genus 0, cf. paragraph after Example 2.1). For any point τ ∈H, the group Γτ is ﬁnite and cyclic. A point τ ∈H is said to be elliptic of order ℓ≥2 if \|Γτ\| = ℓ. Our assumptions on Γ ensure that modulo Γ there are only ﬁnitely many orbits under Γ of elliptic points. If m1, . . . , mr denotes the orders of the elliptic points as well as of those of the cusps (which would be ∞’s), then Γ is said to have signature (0; m1, . . . , mr). The zero here reﬂects that Γ has genus 0. The group then has the following presentation Γ = g1, . . . , gr : gm1 1 = . . . 1. INTRODUCTION A deﬁnition (for functions) using more modern language was given by Umemura, for instance see [40, pages 754-755]. There have been claims (usu- ally non-speciﬁc) that Painlev´e’s deﬁnition is not completely rigorous. For instance, see the third paragraph of page 755 of [40] and page 772 of [69]. These claims seem to origi- nate with Umemura [68], however the only speciﬁc complaint with Painlev´e’s deﬁnitions 5 AX-LINDEMANN-WEIERSTRASS WITH DERIVATIVES AND THE GENUS 0 FUCHSIAN GROUPS which we ﬁnd there is related to some subtleties around algebraic and analytic groups (for instance, see pages three and eight). Similar points are made also in [67]. These com- plaints seem mainly to affect some proofs of results from [43], but not directly the deﬁnition of irreducibility. which we ﬁnd there is related to some subtleties around algebraic and analytic groups (for instance, see pages three and eight). Similar points are made also in [67]. These com- plaints seem mainly to affect some proofs of results from [43], but not directly the deﬁnition of irreducibility. y In [38] and [39], Nishioka proved a weak form of Painlev´e’s assertion; various tech- niques from Nihsioka’s paper have inspired our work. Acknowledgements. G.C and J.N take this opportunity to thank the organizers of the CIRM meeting ‘‘Algebra, Arithmetic and Combinatorics of Differential and Difference Equations” in May 2018, where this research collaboration started. We also thank the anonymous referees for their comments and suggestions. 2The methods of proof and results of the current article can be generalized with additional effort to the case of arbitrary genus. This will be tackled in a forthcoming work of the authors along with D. Bl´asquez-Sanz around Ax-Schanuel Theorems for Fuchsian functions. 2. THE BASIC THEORY = gmr r = g1 · · · gr = I When one or more of the mi’s are inﬁnity, one simply remove the relations containing the inﬁnite mi’s in the above presentation. 2The methods of proof and results of the current article can be generalized with additional effort to the case of arbitrary genus. This will be tackled in a forthcoming work of the authors along with D. Bl´asquez-Sanz around Ax-Schanuel Theorems for Fuchsian functions. G. CASALE, J. FREITAG, AND J. NAGLOO 6 Example 2.1. PSL2(Z) is a Fuchsian (triangle) group of type (0; 2, 3, ∞). Recall that tradi- tionally we might consider the following generators of SL2(Z): T = 1 1 0 1 , S = 0 −1 1 0 . Nonetheless, by setting g1 = −S, g2 = −T−1S and g3 = T one has that SL2(Z) = g1, g2, g3 : g2 1 = g3 2 = g1g2g3 = −I . PSL2(Z) is obtained from the above using the natural projection π : SL2(R) →PSL2(R). As is well known, Γ acts on the set CΓ of its cusps and the action of Γ on HΓ := H ∪CΓ, yields a compact Riemann surface Γ \ HΓ or equivalently a projective non-singular curve X(Γ), which is of genus zero. The group Γ is said to be cocompact if CΓ = ∅. In other words, if the quotient Γ \ H is already a compact space. By an automorphic function for Γ, we mean a meromorphic function f on H which is meromorphic at every cusp of Γ and which is invariant under the action of Γ: f(g · τ) = f(τ) for all g ∈Γ and τ ∈H. One has that the ﬁeld of automorphic functions A0(Γ) for Γ (or equivalently the ﬁeld of meromorphic functions of Γ \ HΓ) is isomorphic to the ﬁeld C(X(Γ)) of rational functions on X(Γ). By an Hauptmodul or uniformizer jΓ(t) for Γ we mean an automorphic function for Γ which generates A0(Γ) (and so C(jΓ) ≃C(X(Γ))). We will also write jΓ for the biholomorphism Γ \ HΓ →P1(C). Let us point out that the function jΓ is not unique. This follows from the existence of nontrivial automorphisms of the curve X(Γ). Moreover, it is well known that the function jΓ is unique once its values at three points have been speciﬁed. AX-LINDEMANN-WEIERSTRASS WITH DERIVATIVES AND THE GENUS 0 FUCHSIAN GROUP Γ = PSL2(Z) is an example of a triangle group. In the appendix the case of the Fuchsian triangle groups is explained in more details. We also direct the reader to [4] where more examples of uniformizers - beyond those attached to triangle groups - are studied. There is a long tradition of functional transcendence results around automorphic func- tions. For instance, a very weak form of our results was conjectured by Mahler, and an- swered by Nishioka: Fact 2.3 ([37]). The Hauptmodul jΓ satisﬁes no algebraic differential equation of order two or less over C(t, eut), for any u ∈C. The same is true for all Γ-automorphic functions. Using the Seidenberg’s embedding theorem and the composition rule of the Schwarzian derivative, we also have Lemma 2.4 (cf. [12]). Let K be an abstract differential ﬁeld extension of C(t) generated by y1, . . . , yn solutions of equation (⋆). Here C is a ﬁnitely generated subﬁeld of C. Then there are elements g1, . . . , gn ∈GL2(C) such that K ∼= C⟨t, jΓ(g1t), . . . , jΓ(gnt)⟩. Proof. By Seidenberg’s embedding theorem, we may assume that y1, . . . , yn are meromor- phic functions on some domain U contained in H. Since the jΓ is a non constant holo- morphic function from H to C, there are holomorphic functions ψi : U →H, such that yi(t) = jΓ(ψi(t)). Repeating the arguments in [12] - using the composition rule for S d dt (y) and the fact that jΓ(ψi(t)) is a solution of the equation (⋆) - we get that S d dt (ψi(t)) = 0. Hence ψi(t) = git for some gi ∈GL2(C). □ Remark 2.5. Notice that the g1, . . . , gn are not arbitrary elements of GL2(C). Indeed, since the yi(t)’s are meromorphic on U ⊂H, it must be that gi : U →H. Also, for each i, from the inverse g−1 i of gi we have well deﬁned solutions jΓ(g−1 i t) and jΓ(gjg−1 i t) of (⋆). Remark 2.5. Notice that the g1, . . . , gn are not arbitrary elements of GL2(C). Indeed, since the yi(t)’s are meromorphic on U ⊂H, it must be that gi : U →H. Also, for each i, from the inverse g−1 i of gi we have well deﬁned solutions jΓ(g−1 i t) and jΓ(gjg−1 i t) of (⋆). 2. THE BASIC THEORY j q p p The uniformizer jΓ also satisﬁes a third order ordinary differential equation of Schwarzian type: (⋆) S d dt (y) + (y′)2 · RjΓ(y) = 0 S d dt (y) + (y′)2 · RjΓ(y) = 0 (⋆) dt y y C(y) depends on the choice of jΓ. Moreover, the ‘shape’ of the function RjΓ, depends on knowing the fundamental half domain for the Γ-action on H: Let us assume that it is given by a polygon P with r vertices b1, . . . , br and whose sides are identiﬁed by pairs and having internal angles α1π, . . . , αrπ. Then C(y) depends on the choice of jΓ. Moreover, the ‘shape’ of the function RjΓ, depends on knowing the fundamental half domain for the Γ-action on H: Let us assume that it is given by a polygon P with r vertices b1, . . . , br and whose sides are identiﬁed by pairs and having internal angles α1π, . . . , αrπ. Then RjΓ(y) = 1 2 r ∑ i=1 1 −α2 i (y −ai)2 + r ∑ i=1 Ai y −ai where jΓ(bi) = ai and the Ai’s are real numbers that do not depend on jΓ and satisfy some very speciﬁc algebraic relations (cf. [72, page 142]). where jΓ(bi) = ai and the Ai’s are real numbers that do not depend on jΓ and satisfy some very speciﬁc algebraic relations (cf. [72, page 142]). Example 2.2. A well-known example is Γ = PSL2(Z) and jΓ is the classical j-function. In this case the equation is given with Rj(y) = y2 −1968y + 2654208 y2(y −1728)2 AX-LINDEMANN-WEIERSTRASS WITH DERIVATIVES AND THE GENUS 0 FUCHSIAN GROUPS 7 AX-LINDEMANN-WEIERSTRASS WITH DERIVATIVES AND THE GENUS 0 FUCHSIAN GROUP In this paper, depending on the context, we will freely alternate between thinking of solutions of the Schwarzian equation (⋆) as points in an abstract differential ﬁeld or as meromorphic functions of the form jΓ(gt). The latter form will always mean that g is an element of GL2(C) that maps (a subset of) H to H. 2.2. Arithmetic Fuchsian groups. We have already seen one important dividing line among those Γ, which we consider, namely whether or not Γ is cocompact. Another, perhaps even more important (for our work) property that Γ might possess is that of arithmeticity. We will begin by reviewing some key deﬁnitions. A standard reference for this subsection is [73]. Throughout Γ ⊂PSL2(R) is a Fuchsian group of ﬁrst kind of genus zero. Let F be a ﬁeld of characteristic zero and let A be a quaternion algebra over F: a cen- tral simple algebra of dimension 4 over F. Since the characteristic of F is zero, there are elements i and j in A and a, b ∈F∗such that i2 = a, j2 = b, ij = −ji, and A = F + Fi + Fj + Fij. As customary, we use the Hilbert symbol notation A = a,b F . For α = a0 + a1i + a2j + a3ij ∈A, we deﬁne its conjugation as α = a0 −a1i −a2j −a3ij ∈A. and A = F + Fi + Fj + Fij. As customary, we use the Hilbert symbol notation A = a,b F . For α = a0 + a1i + a2j + a3ij ∈A, we deﬁne its conjugation as α = a0 −a1i −a2j −a3ij ∈A. 8 G CASALE J FREITAG AND J NAGLOO G. CASALE, J. FREITAG, AND J. NAGLOO 8 Then, the reduced trace tr(α) is deﬁned to be α + α = 2a0 ∈F and the reduced norm n(α) is deﬁned to be αα = a2 0 −a2 1a −a2 2b + a2 3ab ∈F. Example 2.6. For example, the 2 × 2 matrices over F is given by M2(F) = 1,1 F and in this case the norm is simply the determinant. If F = R or a non-Archimedean local ﬁeld, then up to isomorphism, there are only two quaternion algebras: M2(F) or a division algebra. AX-LINDEMANN-WEIERSTRASS WITH DERIVATIVES AND THE GENUS 0 FUCHSIAN GROUP When F is a number ﬁeld and v a place of F, we say that A splits at v if the localization A ⊗F Fv is isomorphic to M2(Fv). Here Fv denote the completion of F with respect to v. If on the other hand A ⊗F Fv is isomorphic to a division algebra, we say A ramiﬁes at v. It is known that the number of ramiﬁed places is ﬁnite and the discriminant of A is deﬁned as the product of the ﬁnite ramiﬁed places. p p Assume now that F is a totally real number ﬁeld of degree k + 1 and we denote by OF its ring of integers. Assume further that A splits at exactly one inﬁnite place, that is, A ⊗Q R ≃M2(R) × Hk A ⊗Q R ≃M2(R) × Hk where H is Hamilton’s quaternion algebra −1,−1 R . Then, up to conjugation, there is a unique embedding ρ of A into M2(R). In particular for any α ∈A, one has that n(α) = det(ρ(α)). Let O be an order in A, namely a ﬁnitely generated OF-module that is also a ring with unity containing a basis for A, that is O ⊗OF F ≃A. Denote by O1 the norm-one group of O, that is O1 = {α ∈O : n(α) = 1}. Then the image ρ(O1) of O1 under ρ is a discrete subgroup of SL2(R). We denote by Γ(A, O) the projection in PSL2(R) of the group ρ(O1). Deﬁnition 2.7. The group Γ is said to be arithmetic if it is commensurable with a group of the form Γ(A, O). Perhaps the best known example of an arithmetic group is PSL2(Z). Recall that two groups Γ1 and Γ2 are commensurable, denoted by Γ1 ∼Γ2, if their intersection Γ1 ∩Γ2 has ﬁnite index in both Γ1 and Γ2. If Γ is arithmetic, then the quotient Γ \ H is called a Shimura curve. In this article, by abuse of terminology we will refer to Γ \ H as a Shimura curve of genus g if and only if Γ \HΓ is of genus g, and we are interested solely in the case where g = 0. As is well known, Shimura curves are generalizations of classical modular curves. We direct the reader to [3] and [65] where the Schwarzian equations for many examples of these curves are derived and studied. AX-LINDEMANN-WEIERSTRASS WITH DERIVATIVES AND THE GENUS 0 FUCHSIAN GROUP We now look at the connection between arithmeticity of Γ and existence of correspon- dences on P1(C) × P1(C) whose preimage under jΓ is also algebraic (cf. [33] and [62]). Let Comm(Γ) be the commensurator of Γ, namely Comm(Γ) = {g ∈PSL2(R) : gΓg−1 ∼Γ}. By a Comm(Γ)-correspondence on P1(C) × P1(C) we mean a subset Comm(Γ) = {g ∈PSL2(R) : gΓg−1 ∼Γ}. Comm(Γ) = {g ∈PSL2(R) : gΓg−1 ∼Γ}. Comm(Γ) = {g ∈PSL2(R) : gΓg ∼Γ}. By a Comm(Γ)-correspondence on P1(C) × P1(C) we mean a subset of the form X(ΓgΓ) = {jΓ(τ) × jΓ(g · τ) : τ ∈HΓ} By a Comm(Γ)-correspondence on P1(C) × P1(C) we mean a subset of the form X(ΓgΓ) = {jΓ(τ) × jΓ(g · τ) : τ ∈HΓ} By a Comm(Γ)-correspondence on P1(C) × P1(C) we mean a subset of the form X(ΓgΓ) = {jΓ(τ) × jΓ(g τ) : τ ∈HΓ} X(ΓgΓ) = {jΓ(τ) × jΓ(g · τ) : τ ∈HΓ} where g ∈Comm(Γ). It turns out that X(ΓgΓ) is an absolutely irreducible curve and that it depends only on the coset ΓgΓ and not on the choice of g (cf. [62] Chapter 7). We suppose where g ∈Comm(Γ). It turns out that X(ΓgΓ) is an absolutely irreducible curve and that it depends only on the coset ΓgΓ and not on the choice of g (cf. [62] Chapter 7). We suppose 9 AX-LINDEMANN-WEIERSTRASS WITH DERIVATIVES AND THE GENUS 0 FUCHSIAN GROUPS that X(ΓgΓ) is given by the equation Ψ ˜g(X, Y) = 0, so that Ψ ˜g(jΓ, jΓ(gt)) = 0. We write ˜g to highlight that the equation depends on ΓgΓ and not g. With this notation, for g1, g2 ∈ GL2(C) we more generally say that jΓ(g1t) and jΓ(g2t) are in Comm(Γ)-correspondence if Ψ ˜g(jΓ(g1t), jΓ(g2t)) = 0 for some ΓgΓ. One has the following result of Margulis: Fact 2.8 ([27]). The group Γ is arithmetic if and only if Γ has inﬁnite index in Comm(Γ) and as a result there are inﬁnitely many Comm(Γ)-correspondences Fact 2.8 ([27]). The group Γ is arithmetic if and only if Γ has inﬁnite index in Comm(Γ) and as a result there are inﬁnitely many Comm(Γ)-correspondences. The modular polynomials (also known as Hecke correspondences) are the classical ex- amples (when Γ = PSL2(Z)). Returning to the Schwarzian equations we see that arith- metic Fuchsian groups of genus 0 give examples of ODE’s with rich binary relations. AX-LINDEMANN-WEIERSTRASS WITH DERIVATIVES AND THE GENUS 0 FUCHSIAN GROUP 2.3. A touch of Model theory. We end this section by saying a few words about the con- cepts in model theory and differential algebra that will be required in the next sections. We will then be ready to state the main results in the paper. Throughout we work in a differentially closed ﬁeld of characteristic zero. Deﬁnition 2.9. A deﬁnable set Y is said to be strongly minimal if it is inﬁnite and every deﬁnable subset is ﬁnite or co-ﬁnite. Remark 2.10. Let Y be deﬁned by an ODE of the form y(n) = f(t, y, y′, . . . , y(n−1)), where f is rational over C(t) (this is of course the case for the sets deﬁned by the Schwarzian equations). Then Y is strongly minimal if and only if for any differential ﬁeld extension K of C and solution y ∈Y , tr.deg.KK ⟨y⟩= 0 or n. Strong minimality is fundamental to the model theoretic approach to differential algebra (cf. [36]). It is also closely related to the Painlev´e notion of irreducibility of the ODE with respect to classical functions [70]. It turns out that there is a very general classiﬁcation of strongly minimal sets in differentially closed ﬁelds about which we will say a few more words in Section 5.1. For now, we only mention the kind of strongly minimal set that is relevant for equation (⋆): Deﬁnition 2.11. Let Y be an F-deﬁnable strongly minimal set. Then Y is geometrically trivial if for any differential ﬁeld extension K of F, and for any distinct solutions y1, . . . , ym, if the collection consisting of y1, . . . , ym together with all their derivatives y(j) i is algebraically dependent over K then for some i < j, yi, yj together with their derivatives are algebraically dependent over K. So geometric triviality limits the complexity of the structure of the algebraic relations on the deﬁnable set. However, given such a set, for the results which we pursue, much greater precision is required. Throughout for simplicity, we will say that an ODE is strongly min- imal and geometrically trivial just in the case that its solution set is strongly minimal as a deﬁnable set. Our ﬁrst theorem is the following: Theorem 2.12. AX-LINDEMANN-WEIERSTRASS WITH DERIVATIVES AND THE GENUS 0 FUCHSIAN GROUP The Schwarzian equation (⋆) for the Hauptmodul jΓ of a genus 0 Fuchsian group Γ of ﬁrst kind is strongly minimal and geometrically trivial. We will give the proof in subsection 5.1. This result was previously only known for PSL2(Z) (the j-function see Example 2.2) as well as for arithmetic subgroups of PSL2(Z) G. CASALE, J. FREITAG, AND J. NAGLOO 10 (cf. [12]). Our proof, which handles all Schwarzian equations of genus zero Fuchsian functions at once, also is the ﬁrst which does not use o-minimality. The ﬁrst proof for PSL2(Z) (of [12]) relied on the main result of [48], where Pila employs the same strategy from [49], relying on o-minimality and counting of points of bounded height. Later, [2] also gave a proof of the special case of PSL2(Z) which relied on the Ax-Schanuel type results of [51], where again, an o-minimal strategy was employed. g gy p y It is worth mentioning that Painlev´e [43, Page 519] claimed that strong minimality (or irreducibility as he called it) would hold for the equations we consider. In [39], Nishioka proved a very weak form of that conjecture. Nevertheless, Nishioka’s paper contains tech- niques that inspired our own proof. We have also obtained a full description of the structure of the deﬁnable sets. One can think of these results as a weak form of the Ax-Lindemann-Weierstrass Theorem with derivatives for Γ.3 Theorem 2.13. Suppose that Γ is arithmetic and suppose that jΓ(g1t), ..., jΓ(gnt) are distinct solutions of the Schwarzian equation (⋆) that are pairwise not in Comm(Γ)-correspondence. Then the 3n functions jΓ(g1t), j′ Γ(g1t), j′′ Γ(g1t), . . . , jΓ(gnt), j′ Γ(gnt), j′′ Γ(gnt) are algebraically independent over C(t). Theorem 2.14. Suppose that Γ is non-arithmetic. Then there is a k ∈N such that if jΓ(g1t), ..., jΓ(gnt) are distinct solutions of the Schwarzian equation (⋆) satisfying tr.deg.C(t)C ⟨t, jΓ(g1t) . . . , jΓ(gnt)⟩= 3n, then for all other solutions jΓ(gt), except for at most n · k, tr.deg.C(t)C ⟨t, jΓ(g1t) . . . , jΓ(gnt), jΓ(gt)⟩= 3(n + 1). So, by the previous two theorems, we have that the set deﬁned by the Schwarzian equa- tion (⋆) is ℵ0-categorical if and only if the group Γ is non-arithmetic. 3The ALW statement we are pursuing allows for characterizing algebraic relations between functions which don’t formally satisfy the same differential equation, but we will use to Theorems 2.13 and 2.14 to prove our most general results, which imply the pertinent version of ALW. 4 4Later in the paper, it will be clear to model theorists that by “arise from” arithmetic Fuchsian groups, we mean ”are non-orthogonal to the differential equation (⋆) or one of its other ﬁbers”. An answer to the question is of interest in part because if there were a strong classiﬁcation of the geometrically trivial strongly minimal sets in differential ﬁelds, some of the strategy laid out in [18] for certain diophantine problems might be possible. 3The ALW statement we are pursuing allows for characterizing algebraic relations between functions which don’t formally satisfy the same differential equation, but we will use to Theorems 2.13 and 2.14 to prove our most general results, which imply the pertinent version of ALW. 4Later in the paper, it will be clear to model theorists that by “arise from” arithmetic Fuchsian groups, we mean ”are non-orthogonal to the differential equation (⋆) or one of its other ﬁbers”. An answer to the question is of interest in part because if there were a strong classiﬁcation of the geometrically trivial strongly minimal sets in differential ﬁelds, some of the strategy laid out in [18] for certain diophantine problems might be possible. AX-LINDEMANN-WEIERSTRASS WITH DERIVATIVES AND THE GENUS 0 FUCHSIAN GROUP It was a long-standing open problem in the model theory of differential ﬁelds (recently resolved by [12]) to ﬁnd a non-ℵ0-categorical geometrically trivial strongly minimal set; the non-existence of such sets was part of a strategy for certain diophantine problems suggested by Hrushovski [18, see page 292]. Theorem 2.13 gives many new examples of geometrically trivial non-ℵ0- categorical equations, and together with Theorem 2.14 also provides an interesting con- nection between categoricity and arithmetic groups. We view the following question as the next major challenge in the classiﬁcation of geometrically trivial strongly minimal sets in differentially closed ﬁelds: Question 2.15. In DCF0, are there non-ℵ0-categorical strongly minimal sets that do not arise from arithmetic Fuchsian groups?4 3The ALW statement we are pursuing allows for characterizing algebraic relations between functions which don’t formally satisfy the same differential equation, but we will use to Theorems 2.13 and 2.14 to prove our most general results, which imply the pertinent version of ALW. 4 4Later in the paper, it will be clear to model theorists that by “arise from” arithmetic Fuchsian groups, we mean ”are non-orthogonal to the differential equation (⋆) or one of its other ﬁbers”. An answer to the question is of interest in part because if there were a strong classiﬁcation of the geometrically trivial strongly minimal sets in differential ﬁelds, some of the strategy laid out in [18] for certain diophantine problems might be possible. AX-LINDEMANN-WEIERSTRASS WITH DERIVATIVES AND THE GENUS 0 FUCHSIAN GROUPS 11 Finally let us talk about the full Ax-Lindemann-Weierstrass Theorem with derivatives for Γ. We closely follow the description of the problem as in [48]. Let V ⊂An be an irre- ducible algebraic variety deﬁned over C such that V(C) ∩Hn ̸= ∅and V projects dom- inantly to each of its coordinates (each coordinate function is nonconstant). Let t1, . . . , tn be the functions on V induced by the canonical coordinate functions on An. We say that t1, . . . , tn are Γ-geodesically independent if there are no relations of the form ti = gtj where i ̸= j and g ∈Comm(Γ) acts by fractional linear transformations. where i ̸= j and g ∈Comm(Γ) acts by fractional linear transformations. Theorem 2.16. With the notation (and assumption V(C) ∩Hn ̸= ∅) as above, suppose that t1, . . . , tn are Γ-geodesically independent. 5If f is a solution of equation (⋆′) generating a differential ﬁeld extension of F of transcendence degree one or two, taking jR to be a realization of a non-forking extension of the type of f over F to the ﬁeld K = F(t) gives such a solution jR of (⋆′). AX-LINDEMANN-WEIERSTRASS WITH DERIVATIVES AND THE GENUS 0 FUCHSIAN GROUP Then the 3n functions jΓ(t1), j′ Γ(t1), j′′ Γ(t1) . . . , jΓ(tn), j′ Γ(tn), j′′ Γ(tn) (deﬁned locally) on V(C) are algebraically independent over C(V). We will prove Theorem 2.16 in section 6. Pila [48] had already proved the result for PSL2(Z) (see also [12] where the same is established for arithmetic subgroups of PSL2(Z)). 3. A CRITERION FOR STRONG MINIMALITY OF A GENERAL FUCHSIAN EQUATION We now aim to give a criterion that can be used to show that the Schwarzian equation (⋆) is strongly minimal. This criterion is applicable to Schwarzian equations in the general sense, namely to any equation of the form (⋆′) S d dt (y) + (y′)2 · R(y) = 0. S d dt (y) + (y′)2 · R(y) = 0. (⋆′) So here we do not assume the rational function R to necessarily correspond to some Haupt- modul. We only require that R is rational over C. By the Riccati equation attached to (⋆′) we mean the equation (⋆⋆) du dy + u2 + 1 2R(y) = 0. du dy + u2 + 1 2R(y) = 0. (⋆⋆) Condition 3.1. The Riccati equation (⋆⋆) has no solution in C(y)alg. Condition 3.1. The Riccati equation (⋆⋆) has no solution in C(y)alg. Theorem 3.2. Let (K, ∂) be any differential ﬁeld extension of C and let us assume that Condition 3.1 holds. If jR is a solution of the Schwarzian equation (⋆′) we have that tr.deg.KK ⟨jR⟩= 0 or 3. tr.deg.KK ⟨jR⟩= 0 or 3. tr.deg.KK ⟨jR⟩= 0 or 3. In other words, if Condition 3.1 holds, then equation (⋆′) is strongly minimal In other words, if Condition 3.1 holds, then equation (⋆′) is strongly minimal. Proof. For contradiction, assume that there is a ﬁnitely generated differential ﬁeld exten- sion F of C that witnesses non-strong minimality of the equation (⋆′) (i.e., an order 1 or 2 F-differential subvariety exists). Throughout, we write K = F(t) and let jR be a solution of the Schwarzian equation (⋆′) such that tr.deg.KK ⟨jR⟩= 1 or 2, respectively.5 5If f is a solution of equation (⋆′) generating a differential ﬁeld extension of F of transcendence degree one or two, taking jR to be a realization of a non-forking extension of the type of f over F to the ﬁeld K = F(t) gives such a solution jR of (⋆′). G. CASALE, J. FREITAG, AND J. NAGLOO 12 Furthermore, using Seidenberg’s embedding theorem we can assume that K is a subﬁeld of M (U) the ﬁeld of meromorphic functions on an open domain U ⊂C and that jR ∈ M (U). ( ) Let P ∈C[y] be a denominator of the rational function R(y). 3. A CRITERION FOR STRONG MINIMALITY OF A GENERAL FUCHSIAN EQUATION Let L = K[y, 1 P(y), y′, 1 y′ , y′′] be the polynomial ring equipped with the derivation • D = ∂+ y′ ∂ ∂y + y′′ ∂ ∂y′ + 3 2 y′′2 y′ −(y′)3R(y) ∂ ∂y′′ making L a universal (K, ∂)-algebra generated by a non constant solution of the Schwarzian equation. One also deﬁnes an action of psl2(C) by : making L a universal (K, ∂)-algebra generated by a non constant solution of the Schwarzian equation. One also deﬁnes an action of psl2(C) by : • X = ∂ • X = ∂ • X = ∂ • H = t∂−y′ ∂ ∂y′ −2y′′ ∂ ∂y′′ • Y = t2 2 ∂−ty′ ∂ ∂y′ −(2ty′′ + y′) ∂ ∂y′′ • H = t∂−y′ ∂ ∂y′ −2y′′ ∂ ∂y′′ y y • Y = t2 2 ∂−ty′ ∂ ∂y′ −(2ty′′ + y′) ∂ ∂y′′ It is easily veriﬁed that [X, H] = X, [H, Y] = Y, [X, Y] = H (the basis ˜X = X, ˜Y = 2Y and ˜H = 2H is a Chevalley basis, i.e., satisfying [ ˜X, ˜H] = 2 ˜X, [ ˜H, ˜Y] = 2 ˜Y, [ ˜X, ˜Y] = ˜H). Furthermore, the equalities [D, X] = 0, [D, H] = D, [D, Y] = tD can be easily veriﬁed. It is easily veriﬁed that [X, H] = X, [H, Y] = Y, [X, Y] = H (the basis ˜X = X, ˜Y = 2Y and ˜H = 2H is a Chevalley basis, i.e., satisfying [ ˜X, ˜H] = 2 ˜X, [ ˜H, ˜Y] = 2 ˜Y, [ ˜X, ˜Y] = ˜H). Furthermore, the equalities [D, X] = 0, [D, H] = D, [D, Y] = tD can be easily veriﬁed. It is easily veriﬁed that [X, H] = X, [H, Y] = Y, [X, Y] = H (the basis ˜X = X, ˜Y = 2Y and ˜H = 2H is a Chevalley basis, i.e., satisfying [ ˜X, ˜H] = 2 ˜X, [ ˜H, ˜Y] = 2 ˜Y, [ ˜X, ˜Y] = ˜H). Furthermore, the equalities [D, X] = 0, [D, H] = D, [D, Y] = tD can be easily veriﬁed. 3. A CRITERION FOR STRONG MINIMALITY OF A GENERAL FUCHSIAN EQUATION q [ ] [ ] [ ] When K = C(t), the algebraic group PSL2(C) acts on L by (3.1) h(t, y, y′, y′′) = h(t), y, y′ h′(t), y′′ (h′(t))2 −y′ h′′(t) (h′(t))3 (3.1) where h denotes the homography of the projective line associated to an element h of PSL2(C). For F ∈L, one deﬁnes (h)∗D · F = h ◦D ◦h−1(F) . Direct computations give that (h)∗D = 1 h′(t)D. This equality means that the set of meromorphic solutions of a Schwarzian equation is stable by the action of PSL2(C) by precomposition. The previously given action of psl2(C) is the inﬁnitesimal action of PSL2(C). When K ⊂M (U) then a ﬁxed element h ∈PSL2(C) maps L onto an isomorphic subﬁeld in M (h−1(U)) but the whole group does not act on L. Now let I ⊂L be the annihilator of the solution jR and Z be the zero locus of I ∩O(U)[y, y′, y′′] in U × C3, where O(U) is the ring of holomorphic functions on U. We have that Z is an analytic variety, afﬁne over U, and that its K-ﬁbers are algebraic varieties over K. We have the following lemma Lemma 3.3. The dimension of the subalgebra b of psl2(C) stabilizing I equals the dimension of Z over K. Lemma 3.3. The dimension of the subalgebra b of psl2(C) stabilizing I equals the dimension of Z over K. Proof. Let p ∈Z be a smooth point in the graph of (jR, j′ R, j′′ R). Then the evaluation of X, H, Y and D at p give a basis of Tp(U × C3). If v is in Tp(Z) ⊂Tp(U × C3) then there exists V ∈psl2 + CD whose value at p is v. Proof. Let p ∈Z be a smooth point in the graph of (jR, j′ R, j′′ R). Then the evaluation of X, H, Y and D at p give a basis of Tp(U × C3). If v is in Tp(Z) ⊂Tp(U × C3) then there exists V ∈psl2 + CD whose value at p is v. p We ﬁrst show that V · I ⊂I. To see this, notice that for P ∈I we have that D · (V · P) = V · (D · P) + f D · P, for some f ∈C(t). p We ﬁrst show that V · I ⊂I. To see this, notice that for P ∈I we have that D · (V · P) = V · (D · P) + f D · P, for some f ∈C(t). Here we use the equalities [D, X] = 0, [D, H] = D, [D, Y] = tD. Since by deﬁnition D · P ∈I, we have that the ideal J generated by I and V · I is stable by D. Moreover all elements of J vanish at p thus on the whole graph of (jR, j′ R, j′′ R). By maximality V · I ⊂I. (j jR jR) y y The stabilizer of I in CX + CH + CY + CD has the dimension of TpZ and thus that of Z. Because D is tangent to Z, the dimension of Z over K is the dimension of the stabilizer of I in psl □ Proof. Let p ∈Z be a smooth point in the graph of (jR, j′ R, j′′ R). Then the evaluation of X, H, Y and D at p give a basis of Tp(U × C3). If v is in Tp(Z) ⊂Tp(U × C3) then there exists V ∈psl2 + CD whose value at p is v. 3. A CRITERION FOR STRONG MINIMALITY OF A GENERAL FUCHSIAN EQUATION Here we use the equalities [D, X] = 0, [D, H] = D, [D, Y] = tD. Since by deﬁnition D · P ∈I, we have that the ideal J generated by I and V · I is stable by D. Moreover all elements of J vanish at p thus on the whole graph of (jR, j′ R, j′′ R). By maximality V · I ⊂I. (j jR jR) y y The stabilizer of I in CX + CH + CY + CD has the dimension of TpZ and thus that of Z. Because D is tangent to Z, the dimension of Z over K is the dimension of the stabilizer of I in psl2. □ AX-LINDEMANN-WEIERSTRASS WITH DERIVATIVES AND THE GENUS 0 FUCHSIAN GROUPS AX-LINDEMANN-WEIERSTRASS WITH DERIVATIVES AND THE GENUS 0 FUCHSIAN GROUPS 13 Our assumption tr.degKK ⟨jR⟩= 1 or 2 gives that the stabilizer, denoted by b, is a non- trivial proper subalgebra of psl2(C). Every such a proper subalgebra is contained in a 2-dimensional Lie subalgebra of psl2 = sl2. Furthermore, the group PSL2(C) acts on psl2 by the adjoint representation and under this action all Lie subagebras of psl2 of dimension 2 are conjugate to one another (cf. [20, Section 16]). j g Let g ∈PSL2(C) be an element conjugating a dimension two subalgebra containing b to the algebra generated by X and H. Then g acts as an homography on P1(C) and transforms K ⊂M (U) to Kg ⊂M (g−1(U)). ( ) (g ( )) The induced isomorphism of L to Lg = Kg[y, 1 P(y), y′, 1 y′ , y′′] sending y to y, y′ to y′g′(t) and y′′ to y′′g′(t)2 −y′g′′(t) preserves D up to multiplication by an element of K (see equa- tion 3.1) and induced the adjoint action on psl2. The transcendence degree of jR over K is the transcendence degree of jR ◦g over Kg but now we have ensured that the stabiliser is included in the Lie algebra generated by X and H. Let us forget that we change the ﬁeld and assume b is included in the triangular Borel subalgebra, that is in the Lie algebra generated by X and H. In L, we have that −y′′ y′2 vanishes when we apply the induced X and H. So the image of −y′′ y′2 in L/I belongs to the kernel of the action of b: namely, the algebraic closure of C[y, 1 P(y)] in L/I. Let z be this algebraic function. Direct computation shows that in L/I, −y′′ y′2 satisﬁes the following re-writing of equation (⋆′) (−y′′ y′2 )′ 1 y′ + 1 2(−y′′ y′2 )2 + R(y) = 0 meaning that z 2 is an algebraic solution of meaning that z 2 is an algebraic solution of du dy + u2 + 1 2R(y) = 0 du dy + u2 + 1 2R(y) = 0 in C(y)alg. This contradicts Condition 3.1. in C(y)alg. This contradicts Condition 3.1. □ The next section is devoted to proving that Condition 3.1 holds for Equation (⋆). 4. THE GENERAL PROOF OF STRONG MINIMALITY (4.2) Because the previous transformation only involves scaling by a Liouvillian element, the Liouvillian solutions of equation 4.2 are in bijective correspondence with the Liouvillian solutions to equation 4.1, and so without loss of generality, we may now assume that the order two equation in which we are interested is given in the following normal form: Because the previous transformation only involves scaling by a Liouvillian element, the Liouvillian solutions of equation 4.2 are in bijective correspondence with the Liouvillian solutions to equation 4.1, and so without loss of generality, we may now assume that the order two equation in which we are interested is given in the following normal form: z′′ = r(y)z (4) No solution of 4.3 are Liouvillian. The connection with Riccati equations is as follows. If we deﬁne u = z′ z where z is a solution to equation 4.3, then via direct computation we have that u′ + u2 −r(y) = 0. u′ + u2 −r(y) = 0. u′ + u2 −r(y) = 0. (4.3) where r(y) ∈C(y). Theorem 4.2. [24, page 5] With regard to the Liouvillian solutions of a second order linear differ- ential equation with coefﬁcients in C(y), there are four mutually exclusive options: The differential equation 4.3 has a solution of the form e R w where w ∈C(y). R (1) The differential equation 4.3 has a solution of the form e R w where w ∈C(y). R (1) The differential equation 4.3 has a solution of the form e R w where w ∈C(y). (2) The differential equation 4.3 has a solution of the form e R w where w ∈C(y)alg is an algebraic function of degree two over C(y). (1) The differential equation 4.3 has a solution of the form e R where w ∈C(y). (2) The differential equation 4.3 has a solution of the form e R w where w ∈C(y)alg is an algebraic function of degree two over C(y). (2) The differential equation 4.3 has a solution of the form e R w where w ∈C(y)alg is an algebraic function of degree two over C(y). (3) All of the solutions of 4.3 are algebraic over C(y). (4) No solution of 4.3 are Liouvillian. 4. THE GENERAL PROOF OF STRONG MINIMALITY 4. THE GENERAL PROOF OF STRONG MINIMALITY 4.1. Liouvillian solutions, algebraic solutions, and Picard-Vessiot theory. 4.1. Liouvillian solutions, algebraic solutions, and Picard-Vessiot theory. Deﬁnition 4.1. Fix a differential ﬁeld K extending C(y) such that the derivation on K ex- tends d dy. We say that K is Liouvillian if there is a tower of ﬁeld extensions C(y) ⊂K0 ⊂ K1 ⊂. . . ⊂Kn = K such that for each i = 1, . . . , n, Ki/Ki−1 is generated by an element ai such that one of the following holds: (1) a′ i ∈Ki−1. (2) a′ i ai ∈Ki−1. (3) ai ∈Kalg i−1. (2) a′ i ai ∈Ki−1. 14 G CASALE J FREITAG AND J NAGLOO G. CASALE, J. FREITAG, AND J. NAGLOO 14 If K is a ﬁeld of meromorphic functions, then in case 1, ai = R f for some f ∈Ki and in case 2, ai = e R f for some f ∈Ki. So, occasionally we will refer to these cases as integrals or exponentials of integrals. Consider the differential equation z′′ + pz′ + qz = 0 (4.1) where p, q are rational functions in C(y). The classiﬁcation of its Liouvillian solutions has been extensively studied, and in [24], an algorithmic solution to determining the Liouvil- lian solutions was given. Let z be a solution to equation 4.1, and let v = e 1 2 R pz. It follows by direct computation that v′′ + (b −1 4a2 −1 2a′)v = 0 v′′ + (b −1 4a2 −1 2a′)v = 0 v′′ + (b −1 4a2 −1 2a′)v = 0 (4.2) v′′ + (b −1 4a2 −1 2a′)v = 0 (4.4) (4.5) were to have an algebraic solution f ∈C(y)alg, then as in the previous subsection z = e R f is a Liouvillian solution of the linear equation were to have an algebraic solution f ∈C(y)alg, then as in the previous subsection z = e R f is a Liouvillian solution of the linear equation (4.6) d2z dy2 + 1 4 r ∑ i=1 1 −α2 i (y −ai)2 + r ∑ i=1 Ai/2 y −ai ! z = 0. (4.6) This equation is an example of the most general (normal) form of a Fuchsian equation of second order: This equation is an example of the most general (normal) form of a Fuchsian equation of second order: Deﬁnition 4.5. Consider the linear equation d2z dy2 + a1 dz dy + a2z = 0, where a1, a2 are rational functions in C(y). (1) A point p ∈C is called regular if the functions ai have no pole at p, otherwise p is called singular. To determine whether the point y = ∞is regular, one simply substitutes y = z−1 in the equation and veriﬁes whether z = 0 is regular for the new equation. q (2) A point p ∈C (resp. p = ∞) is called regular singular if it is singular and for each i = 1, 2, the limit limy→p(y −p)iai(y) (resp. limy→∞yiai(y)) exists and is ﬁnite. q (2) A point p ∈C (resp. p = ∞) is called regular singular if it is singular and for each i = 1, 2, the limit limy→p(y −p)iai(y) (resp. limy→∞yiai(y)) exists and is ﬁnite. 1 (3) The equation is called Fuchsian if all points of P1(C) are regular or regular singular. It is well-known (cf. [9, Chapter 7]) that if the equation is Fuchsian then the coefﬁcients a1 and a2 are of the form B ( ) ai(y) = Bi(y) ∏s j=1(z −βj)i j where Bi(y) is a polynomial of degree ≤i(s −1). j where Bi(y) is a polynomial of degree ≤i(s −1). Remark 4.6. We have already seen in the previous section how to obtain the normal form of the a second order linear equation (see equation 4.2) Remark 4.6. (4.4) Notice that z = ce R u for some constant c ∈C and in particular z1 = e R u is also a solution to 4.3. So using Theorem 4.2 we have the following lemma. Notice that z = ce R u for some constant c ∈C and in particular z1 = e R u is also a solution to 4.3. So using Theorem 4.2 we have the following lemma. Lemma 4.3. The Riccati equation 4.4 has an algebraic solution over C(y) if and only if the second order linear differential equation 4.3 has a Liouvillian solution. Lemma 4.3. The Riccati equation 4.4 has an algebraic solution over C(y) if and only if the second order linear differential equation 4.3 has a Liouvillian solution. Now, the veriﬁcation of Condition 3.1 follows from showing that equation 4.3 has no Liouvillian solutions. For this, we will need the following well-known result. Theorem 4.4. [24, page 8, case 4] Let G be the Picard-Vessiot group of 4.3. There are no Liouvil- lian solutions to equation 4.3 if and only if G = SL2(C). In the next subsection we will prove that, in the special case of a Fuchsian group Γ, the Picard-Vesiot group of the order two linear equation associated to the Riccati equation (⋆⋆) is SL2(C). AX-LINDEMANN-WEIERSTRASS WITH DERIVATIVES AND THE GENUS 0 FUCHSIAN GROUPS 15 4.2. Monodromy and the PV-group. At this point, let us recall that the the Schwarzian equation (⋆) we focus on is given with 4.2. Monodromy and the PV-group. At this point, let us recall that the the Schwarzian equation (⋆) we focus on is given with RjΓ(y) = 1 2 r ∑ i=1 1 −α2 i (y −ai)2 + r ∑ i=1 Ai y −ai , where the αi’s, Ai’s and ai’s are obtained from the fundamental domain for Γ-action on H. As discussed in the previous subsection, if the Riccati equation corresponding to (⋆) where the αi’s, Ai’s and ai’s are obtained from the fundamental domain for Γ-action on H. As discussed in the previous subsection, if the Riccati equation corresponding to (⋆) (4.5) du dy + u2 + 1 2RjΓ(y) = 0. (4.5) du dy + u2 + 1 2RjΓ(y) = 0. (4.4) We have already seen in the previous section how to obtain the normal form of the a second order linear equation (see equation 4.2) As it turns out, the problem of existence of Liouvillian solutions for Fuchsian equations of second order is a classical one. We direct the reader to [16] and [60] for some historical perspectives. We will only review parts of the theory that is relevant to this paper. Our focus will be the work of Poincar´e on the relationship between the monodromy group of the Fuchsian equation 4.6 and ‘its’ Fuchsian group Γ. It is this work - partly rediscovering Schwarz’s uniformization of P1(C) by the jΓ’s - that lead Poincar´e to introduce the theory of Fuchsian groups and functions, and to attack the problem of the uniformization of other Riemman surfaces. 6 G. CASALE, J. FREITAG, AND J. NAGLOO G. CASALE, J. FREITAG, AND J. NAGLOO 16 From now on, we assume that the equation From now on, we assume that the equation (4.7) d2z dy2 = r(y)z d2z dy2 = r(y)z (4.7) is Fuchsian and denote by S its set of singular points. For z ∈P1(C) \ S, let f1 and f2 be analytic solutions in a neighborhood of z. We also assume that f1 and f2 are a basis of solutions, i.e., that they are linearly independent over C. Given any γ ∈π1(P1(C) \ S; z), we can analytically continue f1 and f2 along γ and obtain new solutions ˜f1 and ˜f2 of 4.7. So there exists a matrix Mγ ∈GL2(C) such that ˜f1 ˜f2 = Mγ · f1 f2 ˜f1 ˜f2 = Mγ · f1 f2 The mapping ρ : π1(P1(C) \ S; z) →GL2(C), taking γ 7→Mγ is a group homomorphism called the monodromy representation. Its image M is called the monodromy group of equation 4.7. From the monodromy group, one can determine the Picard-Vessiot group of the equation: Fact 4.7. [9, Chapter 7] Let G be the Picard-Vessiot group of the Fuchsian equation 4.7. Then, (1) G ⊆SL2(C); (2) if M is its monodromy group, then G is the Zariski closure of M. (1) G ⊆SL2(C); ( ) ( ) (2) if M is its monodromy group, then G is the Zariski closure of M. (4.4) Note that in particular from (1), for the Fuchsian equation 4.6, the monodromy group M is a subgroup of SL2(C). We will now explain how in the case of equation 4.6, the monodromy group M is related to the Schwarzian equation. The following well-known fact - which can be easily veriﬁed - will be needed. Fact 4.8. Let t(y) = j−1 Γ (y) be a branch of the inverse of y = jΓ(t). Then t(y) satisﬁes the following equation S d dy(t) = RjΓ(y). s z1 = t ( dt dy) 1 2 z2 = 1 ( dt dy) 1 2 (4.8) S d dy(t) = RjΓ(y). (4.8) S d dy(t) = RjΓ(y). Furthermore, the functions z1 = t ( dt dy) 1 2 z2 = 1 ( dt dy) 1 2 (4.8) Furthermore, the functions z1 = t ( dt dy) 1 2 z2 = 1 ( dt dy) 1 2 z1 = t ( dt dy) 1 2 z2 = 1 ( dt dy) 1 2 y form a basis of solutions of the Fuchsian equation 4.6 form a basis of solutions of the Fuchsian equation 4.6 d2z dy2 + 1 2RjΓ(y)z = 0. d2z dy2 + 1 2RjΓ(y)z = 0. Notice that in particular z1(y) z2(y) = t(y). This allows us to deﬁne from M the projective monodromy of the equation 4.8. Namely, if Mγ = a b c d ∈SL2(C) is monodromy matrix (as above), then b Notice that in particular z1(y) z2(y) = t(y). This allows us to deﬁne from M the projective monodromy of the equation 4.8. Namely, if Mγ = a b c d ∈SL2(C) is monodromy matrix (as above), then t∗= at + b ct + d AX-LINDEMANN-WEIERSTRASS WITH DERIVATIVES AND THE GENUS 0 FUCHSIAN GROUPS 17 is again a solution of the equation 4.8. The collection of matrices ˆMγ : t 7→t∗is called the projective monodromy group ˆM of the equation 4.8. Of course ˆM is the image of M under the natural projection π : SL2(R) →PSL2(R). The following proposition is attributed to Poincar´e in various sources but we know of no reference for a proof of it and thus reproduce it here. Proposition 4.9. The projective monodromy group of the equation 4.8 is Γ. As a consequence, the monodromy group of the Fuchsian equation 4.6 is π−1(Γ). Proof. (4.4) Throughout t(y) = j−1 Γ (y) is a branch of the inverse of jΓ locally deﬁned on some small domain U and ˆM is the projective monodromy group. We have We have g ∈ˆM \ {I} ⇐⇒ gt(y) is another branch of the inverse of jΓ (deﬁned on some larger domain U′). ⇐⇒ y = jΓ(t(y)) = jΓ(gt(y)) ⇐⇒ g ∈Γ \ {I}. g ∈ˆM \ {I} ⇐⇒ gt(y) is another branch of the inverse of jΓ (deﬁned on some larger domain U′). ⇐⇒ y = jΓ(t(y)) = jΓ(gt(y)) ⇐⇒ g ∈Γ \ {I}. g ∈ˆM \ {I} ⇐⇒ gt(y) is another branch of the inverse of jΓ (deﬁned on some larger domain U′). ⇐⇒ y = jΓ(t(y)) = jΓ(gt(y)) ⇐⇒ g ∈Γ \ {I}. □ We have used here that jΓ is a globally deﬁned single-valued function. Proposition 4.10. There are no Liouvillian solutions of the Fuchsian equation 4.6. Consequently, Condition 3.1 holds for the Riccati equation 4.5. Proof. We have that π−1(Γ), the monodromy group of the Fuchsian equation 4.6, is Zariski dense in SL2(C). Hence by Fact 4.7, the Picard-Vessiot group is G = SL2(C). By Theorem 4.4 there are no Liouvillian solutions for the equation. □ Proof. We have that π−1(Γ), the monodromy group of the Fuchsian equation 4.6, is Zariski dense in SL2(C). Hence by Fact 4.7, the Picard-Vessiot group is G = SL2(C). By Theorem 4.4 there are no Liouvillian solutions for the equation. □ We thus obtain the ﬁrst part of Theorem 2.12; namely the Schwarzian equation (⋆) is strongly minimal. We thus obtain the ﬁrst part of Theorem 2.12; namely the Schwarzian equation (⋆) is strongly minimal. 6A complete proof, can be found in [56, Corollary 3.10] and in the arguments in the paragraphs leading up to Proposition 4.10 of [58]. A good guide/summary of the proof can also be found in [36, Section 2.1]. 5. GEOMETRIC TRIVIALITY AND ALGEBRAIC RELATIONS 5.1. The classiﬁcation of strongly minimal sets. In this section we will discuss some general model-theoretic results regarding strongly minimal sets in differentially closed ﬁelds. In particular, we will explain some consequences of the (unpublished6) work of Hrushovski and Sokolovi´c on the classiﬁcation of strongly minimal sets. We will, from these considerations, obtain geometric triviality of the Schwarzian equations satisﬁed by the uniformizing functions in the earlier sections. Let us denote by U the differentially closed ﬁeld of characteristic zero that we work in. We assume that U is saturated and that C (deﬁned by y′ = 0) is its ﬁeld of constants. Notice incidentally that C is itself a strongly minimal deﬁnable set. Indeed, it is the only deﬁnable strongly minimal subﬁeld of U. In what follows strongly minimal sets are understood to be deﬁned over some ﬁnitely gener- ated differential subﬁeld K of U. The zero set of any irreducible order one differential polynomial in a single variable (by irreducible, we will always mean as a polynomial) is also strongly minimal. Higher order G. CASALE, J. FREITAG, AND J. NAGLOO 18 linear differential equations are never strongly minimal (one can deﬁne linear subspaces using elements of a fundamental set of solutions). For higher order non-linear equations, it seems that it is in general difﬁcult to establish strong minimality. However, if the strong minimality of an equation is established, one can often employ a variety of model theoretic tools to establish even stronger results. g Other important examples of strongly minimal sets are given by the following Fact 5.1 ([8],[19]). Let A be an abelian variety deﬁned over U. We identify A with its set A(U) of U-points. Then Fact 5.1 ([8],[19]). Let A be an abelian variety deﬁned over U. We identify A with its set A(U) of U-points. Then (1) A has a (unique) smallest Zariski-dense deﬁnable subgroup, which we denote by A♯. ♯ A has a (unique) smallest Zariski-dense deﬁnable subgroup, which we denote by A♯. ♯ (2) If A is a simple abelian variety that does not descend to C, then A♯is strongly minimal. (2) If A is a simple abelian variety that does not descend to C, then A♯is strongly minimal. The subgroup A♯is called the Manin kernel of A (cf. [28]). 5. GEOMETRIC TRIVIALITY AND ALGEBRAIC RELATIONS The trichotomy theorem, gives a classiﬁcation of strongly minimal sets up to non-orthogonality, a notion we will explain following the statement of the theorem. Theorem 5.2 ([19],[56]). Let Y be a strongly minimal set. Then exactly one of the following holds: Theorem 5.2 ([19],[56]). Let Y be a strongly minimal set. Then exactly one of the following holds: (1) Y is nonorthogonal to the strongly minimal set C, ♯ 2) Y is nonorthogonal to A♯for some simple abelian variety A over U which does n descend to C, Y is nonorthogonal to A♯for some simple abelian variety A over U which does not descend to C, (3) Y is geometrically trivial. (3) Y is geometrically trivial. Deﬁnition 5.3. Let Y and Z be strongly minimal sets. Denote by π1 : Y × Z →Y and π2 : Y × Z →Z the projections to Y and Z respectively. We say that Y and Z are nonorthogonal if there is some inﬁnite deﬁnable relation R ⊂Y × Z such that π1\|R and π2\|R are ﬁnite-to-one functions. Deﬁnition 5.3. Let Y and Z be strongly minimal sets. Denote by π1 : Y × Z →Y and π2 : Y × Z →Z the projections to Y and Z respectively. We say that Y and Z are nonorthogonal if there is some inﬁnite deﬁnable relation R ⊂Y × Z such that π1\|R and π2\|R are ﬁnite-to-one functions. The sets Y and Z are deﬁned over some ﬁnitely generated differential subﬁeld K of U, and so for any differential ﬁeld F containing K, it makes sense to ask whether a relation R as above can be deﬁned over F. Deﬁnition 5.4. We say that Y is weakly orthogonal to Z over F if no such relation R can be deﬁned over F. Deﬁnition 5.4. We say that Y is weakly orthogonal to Z over F if no such relation R can be deﬁned over F. The following facts from the model theory of differential ﬁelds are well-known (see for instance, [29]). Fact 5.5. Let Y and Z be strongly minimal sets. (1) Nonorthogonality is an equivalence rela (1) Nonorthogonality is an equivalence relation on strongly minimal sets. (2) Nonorthogonality classes of strongly minimal differential equations reﬁne various basic invariants of the equations. For instance, if Y, Z are nonorthogonal then order(Y) = order(Z). 7We thank Dave Marker for a sketch of this proof. Nonorthogonality of Manin Kernels has been further classiﬁed in terms of isogeny classes of abelian varieties. Nonorthogonality of Manin Kernels has been further classiﬁed in terms of isogeny classes of abelian varieties. Fact 5.6. If A and B are two simple abelian varieties which do not descend to C, then A♯and B♯ are non-orthogonal if and only if A and B are isogenous. For relations R which witness nonorthogonality between trivial strongly minimal sets, there is an important and very general descent result: Fact 5.7. [55, Corollary 2.5.5] Two geometrically trivial strongly minimal sets are nonorthogonal if and only if they are non weakly orthogonal. That is, the relation R witnessing nonorthogonality of X and Y can be deﬁned over the differential ﬁeld generated by the parameters used in the equations deﬁning X and Y. Proposition 5.8. Let Y be a strongly minimal set of order > 1 and suppose that Y is deﬁned over C. Then Y is geometrically trivial. Proof. 7 First note that since order(Y) ̸= 1, Y is necessarily orthogonal to the constants C. So by Theorem 5.2, to show that Y is geometrically trivial, we only need to show that it is orthogonal to all Manin kernels. We argue by contradiction. g g y Suppose that Y is nonorthogonal to A♯for some simple abelian variety A over U which does not descend to C. Let (A, λ) be a principal polarization of A. We can use the fact that moduli spaces of principally polarized abelian varieties exist over any base ﬁeld (cf. [35, Chapter 7]). So let (V, ϕ) be a moduli space for (A, λ) over C. For some b in (V, ϕ), we have that (A, λ) = (Vb, ϕb). Using uniform deﬁnability of Manin kernels [36, Lemma 2.25], we have a formula φ(x) over C asserting that Y is non-orthogonal to V♯ x and such that φ(b) is true in U. If φ(c) holds, then, by Fact 5.6, it must be the case that Vb and Vc are isogenous. But there are only countably many abelian varieties isogenous to Vb. Hence the deﬁnable set {a ∈U : φ(a) is true in U} is countable and so must be ﬁnite. In other words c is algebraic over (and so in) C. But this contradicts the assumption that A does not descend to C. □ Corollary 5.9. For Γ a Fuchsian group, equation (⋆) deﬁnes a geometrically trivial strongly mini- mal set. 5. GEOMETRIC TRIVIALITY AND ALGEBRAIC RELATIONS Recall that the order of a deﬁnable set Y is given by order(Y) = sup{tr.deg.KK ⟨y⟩ y ∈Y}, where K is any countable differential ﬁeld over which Y is deﬁned. y } y ff ﬁ ﬁ (3) If Y and Z are nonorthogonal, then they fall into the same category of Theorem 5.2. ( ) f g y f g y f (4) Strongly minimal sets that fall in cases (2) and (3) of Theorem 5.2 are said to be locally modular (and in case (1) the sets are non-locally modular). AX-LINDEMANN-WEIERSTRASS WITH DERIVATIVES AND THE GENUS 0 FUCHSIAN GROUPS 19 (5) Orthogonality has a natural interpretation in terms of transcendence. Suppose that Y and Z are orthogonal strongly minimal sets deﬁned over K. Let a, b be solutions of Y, Z, respec- tively. Let F be any differential ﬁeld extending K. Then (5) Orthogonality has a natural interpretation in terms of transcendence. Suppose that Y and Z are orthogonal strongly minimal sets deﬁned over K. Let a, b be solutions of Y, Z, respec- tively. Let F be any differential ﬁeld extending K. Then (5) Orthogonality has a natural interpretation in terms of transcendence. Suppose that Y and Z are orthogonal strongly minimal sets deﬁned over K. Let a, b be solutions of Y, Z, respec- tively. Let F be any differential ﬁeld extending K. Then tr.deg.F(F⟨a, b⟩) = tr.deg.F(F⟨a⟩) + tr.deg.F(F⟨b⟩). Conversely, if the inequality does not hold for some a ∈Y and b ∈Z over F, then Y is not weakly orthogonal to Z over F. tr.deg.F(F⟨a, b⟩) = tr.deg.F(F⟨a⟩) + tr.deg.F(F⟨b⟩). tr.deg.F(F⟨a, b⟩) = tr.deg.F(F⟨a⟩) + tr.deg.F(F⟨b⟩). Conversely, if the inequality does not hold for some a ∈Y and b ∈Z over F, then Y is not weakly orthogonal to Z over F. Conversely, if the inequality does not hold for some a ∈Y and b ∈Z over F, then Y is not weakly orthogonal to Z over F. tr.deg.KK(j1, j′ 1, j′′ 1 , j2, j′ 2, j′′ 2 ) < 6 For i = 1, 2 consider K(yi, y′ i, y′′ i ), equipped with the derivation • Di = ∂+ y′ i ∂ ∂yi + y′′ i ∂ ∂y′ i + 3 2 y′′2 i y′ i −(y′ i)3RΓ(yi) ∂ ∂y′′ i One deﬁnes an action of psl2(C) by : One deﬁnes an action of psl2(C) by : • Xi = ∂ • Xi = ∂ i • Hi = t∂−y′ i ∂ ∂y′ i −2y′′ i ∂ ∂y′′ i • Yi = t2 2 ∂−ty′ i ∂ ∂y′ i −(2ty′′ i + y′ i) ∂ ∂y′′ i • Hi = t∂−y′ i ∂ ∂y′ i −2y′′ i ∂ ∂y′′ i • Y = t2 ∂ ty′ ∂ (2ty′′ + y′) ∂ • Hi = t∂−y′ i ∂ ∂y′ i −2y′′ i ∂ ∂y′′ i • Hi t∂ yi ∂y′ i 2yi ∂y′′ i • Yi = t2 2 ∂−ty′ i ∂ ∂y′ i −(2ty′′ i + y′ i) ∂ ∂y′′ i i i • Yi = t2 2 ∂−ty′ i ∂ ∂y′ i −(2ty′′ i + y′ i) ∂ ∂y′′ i Yi 2 ∂ tyi ∂y′ i (2tyi + yi) ∂y′′ i We have that [Xi, Hi] = Xi, [Hi, Yi] = Yi, [Xi, Yi] = Hi, and [Di, Xi] = 0, [Di, Hi] = Di, Di Yi] = tDi yi yi We have that [Xi, Hi] = Xi, [Hi, Yi] = Yi, [Xi, Yi] = Hi, and [Di, Xi] = 0, [Di, Hi] = Di, [Di, Yi] = tDi. yi yi We have that [Xi, Hi] = Xi, [Hi, Yi] = Yi, [Xi, Yi] = Hi, and [Di, Xi] = 0, [Di, Hi] = Di, [Di, Yi] = tDi. ( ) i i We have that [Xi, Hi] = Xi, [Hi, Yi] = Yi, [Xi, Yi] = Hi, and [Di, Xi] = 0, [Di, Hi] = Di, [Di, Yi] = tDi. As explained in the proof of Theorem 3.2, the above action of psl2(C) is the inﬁnitesimal action of PSL2(C) on C(t, yi, y′ i, y′′ i ). We “verticalize” this action by considering Xv i = Xi − Di, Hv i = Hi −tDi and Yv i = Yi −t2 2 Di. Now CXv i + CHv i + CYv i is a realization of psl2(C) acting K-linearly and commuting with Di. Nonorthogonality of Manin Kernels has been further classiﬁed in terms of isogeny classes of abelian varieties. We have hence established the entirety of Theorem 2.12. We have hence established the entirety of Theorem 2.12. G. CASALE, J. FREITAG, AND J. NAGLOO 20 Theorem 5.10. Let (K, ∂) be a differential extension of (C(t), ∂ ∂t) with no new constants. Let Γ be a Fuchsian group and j1, j2 be two solutions of the equation S d dt (y) + (y′)2 · RΓ(y) = 0. If tr.deg.KK(j1, j′ 1, j′′ 1 , j2, j′ 2, j′′ 2 ) < 6 tr.deg.KK(j1, j′ 1, j′′ 1 , j2, j′ 2, j′′ 2 ) < 6 tr.deg.KK(j1, j′ 1, j′′ 1 , j2, j′ 2, j′′ 2 ) < 6 then j1 or j2 is algebraic over K or there is a nonzero polynomial P(y1, y2) over C such that P(j1, j2) = 0. then j1 or j2 is algebraic over K or there is a nonzero polynomial P(y1, y2) over C such that P(j1, j2) = 0. The group PSL2(C) acts on pairs of solutions by precomposition. We will prove that the ideal of differential relations between (j1, j2) is stable under this action. Proof. From Theorem 3.2, it follows that if tr.deg.KK(j1, j′ 1, j′′ 1 , j2, j′ 2, j′′ 2 ) < 6 then it is either 0 or 3. But it follows from Fact 2.3 and Lemma 2.4 that if both j1 and j2 are not algebraic over K then tr.deg.KK(j1, j′ 1, j′′ 1 , j2, j′ 2, j′′ 2 ) = 3. By Fact 5.7, we can assume that K = C(t) and so throughout K = C(t) and ∂= ∂ ∂t. g ( ) ∂t We proceed as in the proof of Theorem 3.2. 5.2. Transcendence and orbits of the commensurator of Γ. Theorem 5.10. Let (K, ∂) be a differential extension of (C(t), ∂ ∂t) with no new constants. Let Γ be a Fuchsian group and j1, j2 be two solutions of the equation We claim that I is stable under the diagonal action of psl2. The algebra L/I is an algebraic extension of K(y1, y′ 1, y′′ 1) and of K(y2, y′ 2, y′′ 2), and as usual, D1, X1, H1, Y1, D2, X2, H2, Y2 and their ”verticalization” will also denote their unique extensions to L/I. Lemma 5.11. On L/I we have D1 = D(2) = D2. Lemma 5.11. On L/I we have D1 = D(2) = D2. Lemma 5.11. On L/I we have D1 = D(2) = D2. Proof. Restrict the derivation D(2) of L/I to its subalgebra L1. The deﬁnition of D(2) gives that this restriction is D1. Now, the extension to the algebraic extension L/I of L1 is unique then D(2) = D1. Same argument gives D(2) = D2. □ So we will just write this derivation as D. Lemma 5.12. There exists a ∈C such that, on L/I, X1 = X2, H1 = H2 + a(X2 −D) and Y1 = Y2 + a(H2 −tD) + a2 2 (X2 −D). Proof. Using Fact 5.7, we have that any algebraic relations between j1 and j2 (together with derivatives) can be deﬁned over C. Hence I is generated by I ∩C(y1, y′ 1, y′′ 1) ⊗C(y2, y′ 2, y′′ 2). Then, on L/I, both X1 and X2 coincide with ∂ ∂t, this proves X1 = X2. Then, on L/I, both X1 and X2 coincide with ∂ ∂t, this proves X1 = X2. ∂t The two triples Xv 1, Hv 1, Yv 1 and Xv 2, Hv 2, Yv 2 are two bases of the derivations of F = Frac(L/I) over K. Let A be the matrix with coefﬁcient in F such that (Xv 1, Hv 1, Yv 1 ) = (Xv 2, Hv 2, Yv 2 )A. From the bracket with D one gets 0 = [D, (Xv 1, Hv 1, Yv 1 )] = (Xv 2, Hv 2, Yv 2 )D(A). So the coefﬁcients of A are constant. Now the two triples are basis of two realisations of psl2(C) with the same structure constants, then A is an automorphism of the Lie algebra psl2(C). All automorphisms of psl2(C) are inner (see [59, Proposition 14.21]), thus there exists a g ∈PSL2(C) such that Ad(g) = A. This automorphism ﬁxes X1, hence there exists a ∈C such that g = 1 a 0 1 . AX-LINDEMANN-WEIERSTRASS WITH DERIVATIVES AND THE GENUS 0 FUCHSIAN GROUPS AX-LINDEMANN-WEIERSTRASS WITH DERIVATIVES AND THE GENUS 0 FUCHSIAN GROUPS 21 The ideal I is the kernel of the evaluation in (j1, j2) with values in a ﬁeld of mero- morphic function thus it is prime. From geometric triviality, the subﬁeld of constants of F = Frac(L/I) with respect to the derivation D(2) is C. We claim that I is stable under the diagonal action of psl2. tr.deg.KK(j1, j′ 1, j′′ 1 , j2, j′ 2, j′′ 2 ) < 6 g y g The ideal of the polynomial differential relations between j1 and j2 over K is an ideal in K[y1, y′ 1, . . . , y2, y′ 2, . . .]. Let J be the differential ideal generated by the third order dif- ferential equations satisﬁed by j1 and by j2 and K[y1, y′ 1, . . . , y2, y′ 2, . . .] →K(y1, y′ 1, y′′ 1) ⊗K K(y2, y′ 2, y′′ 2) be the quotient by J followed by localizations. (y y2 y2) q y y As j1 and j2 do not satisfy any lower order differential equations this ideal is the preim- age of an ideal I of L = K(y1, y′ 1, y′′ 1) ⊗K K(y2, y′ 2, y′′ 2) stable by (5.1) D(2) = ∂+ y′ 1 ∂ ∂y1 + y′ 2 ∂ ∂y2 + y′′ 1 ∂ ∂y′ 1 + y′′ 2 ∂ ∂y′ 2 + 3 2 y′′2 1 y′ 1 −(y′ 1)3RΓ(y1) ∂ ∂y′′ 1 + 3 2 y′′2 2 y′ 2 −(y′ 2)3RΓ(y2) ∂ ∂y′′ 2 . + y′ 2 ∂ ∂y2 + y′′ 1 ∂ ∂y′ 1 + y′′ 2 ∂ ∂y′ 2 + 3 2 y′′2 1 y′ 1 −(y′ 1)3RΓ(y1) ∂ ∂y′′ 1 + 3 2 y′′2 2 y′ 2 −(y′ 2)3RΓ(y2) ∂ ∂y′′ 2 . We claim that I is stable under the diagonal action of psl2. Then Now the two triples are basis of two realisations of psl2(C) with the same structure constants, then A is an automorphism of the Lie algebra psl2(C). All automorphisms of psl2(C) are inner (see [59, Proposition 14.21]), thus there exists a g ∈PSL2(C) such that Ad(g) = A. This automorphism ﬁxes X1, hence there exists a ∈C such that g = 1 a 0 1 . Then Then (Xv 1, Hv 1, Yv 1 ) = (Xv 2, Hv 2, Yv 2 )   1 a a2 2 0 1 a 0 0 1   □ This proves the lemma. In F, y2 is an algebraic function over C(t, y1, y′ 1, y′′ 1) satisfying X2(y2) = H2(y2) = Y2(y2) = 0. Hence, using Lemma 5.12, one easily computes that (5.2) X1(y2) = 0, (5.3) H1(y2) = −aD(y2), (5.4) Y1(y2) = (−at −a2 2 )D(y2) = (t + a 2)H1(y2). X1(y2) = 0, H1(y2) = −aD(y2), (5.2) X1(y2) = 0, (5.3) H1(y2) = −aD(y2), H1(y2) = −aD(y2), (5.3) (5.4) Y1(y2) = (−at −a2 2 )D(y2) = (t + a 2)H1(y2). (5.4) 22 G CASALE J FREITAG AND J NAGLOO G. CASALE, J. FREITAG, AND J. NAGLOO 22 We will prove that this system of partial differential equations over C(t, y1, y′ 1, y′′ 1) has an algebraic solution if and only if a = 0. For contradiction, assume not. We expand y2 as a Puiseux series in 1/z with z = y′′ 1 y′2 1 , that is we think of y2 as being an element of C(t, y1, y′ 1)alg 1 z : y2 = ∑ λ≤n Aλ(t, y1, y′ 1)zλ. In the coordinates t, y1, y′ 1, z, one has In the coordinates t, y1, y′ 1, z, one has ∂ • X1 = ∂ ∂t, • H1 = t ∂ ∂t −y′ 1 ∂ ∂y′ 1 , • Y1 = t2 2 ∂ ∂t −ty′ 1 ∂ ∂y′ 1 −1 y′ 1 ∂ ∂z, • D = ∂ ∂t −y′ 1 ∂ ∂y1 + z(y′ 1)2 ∂ ∂y′ 1 − 1 2z2y′ 1 + RΓ(y1)y′ 1 ∂ ∂z. y1 y1 • D = ∂ ∂t −y′ 1 ∂ ∂y1 + z(y′ 1)2 ∂ ∂y′ 1 − 1 2z2y′ 1 + RΓ(y1)y′ 1 ∂ ∂z. We claim that I is stable under the diagonal action of psl2. The induced continuous action of X1 on C(t, y1, y′ 1)alg 1 z gives The induced continuous action of X1 on C(t, y1, y′ 1)alg 1 z gives (5.5) X1(y2) = ∑ λ≤n ∂Aλ ∂t zλ, (5.5) The equations 5.2 and 5.5 give for all λ, ∂Aλ ∂t = 0. Then by direct computation one gets The equations 5.2 and 5.5 give for all λ, ∂Aλ ∂t = 0. Then by direct computation one gets The equations 5.2 and 5.5 give for all λ, ∂Aλ ∂t = 0. Then by direct computation one gets (5.6) H1(y2) = ∑ λ≤n −y′ 1 ∂Aλ ∂y′ 1 zλ, (5.6) H1(y2) = ∑ λ≤n −y′ 1 ∂Aλ ∂y′ 1 zλ, (5.6) (5.7) Y1(y2) = ∑ λ≤n −ty′ 1 ∂Aλ ∂y′ 1 (z)λ −λAλ 1 y′ 1 zλ−1, (5.7) (5.8) D(y2) = ∑ λ≤n RΓ(y1)y′ 1λAλzλ−1 + y′ 1 ∂Aλ ∂y1 zλ + (y′ 1)2 ∂Aλ ∂y′ 1 −1 2y′ 1λAλ zλ+1. (5.8) D(y2) = ∑ λ≤n RΓ(y1)y′ 1λAλzλ−1 + y′ 1 ∂Aλ ∂y1 zλ + (y′ 1)2 ∂Aλ ∂y′ 1 −1 2y′ 1λAλ zλ+1. (5.8) Lemma 5.13. If y2 is an algebraic solution of 5.2, 5.3, 5.4 then H1(y2) = 0. Lemma 5.13. If y2 is an algebraic solution of 5.2, 5.3, 5.4 then H1(y2) = 0. Proof. If a = 0 there is nothing to prove. Assume it is not. We have already seen that ∂Aλ ∂t = 0. Let q ∈Q be such that Aq ̸= 0. One can assume that q is maximal among the elements q′ ∈q + Z such that Aq′ ̸= 0. From 5.4, one gets −ty′ 1 ∂Aq ∂y′ 1 = (t + a 2) −y′ 1 ∂Aq ∂y′ 1 and then ∂Aq ∂y′ 1 = 0. Now 5.3 gives (y′ 1)2 ∂Aq ∂y′ 1 −1 2y′ 1qAq = 0, this implies q = 0 so that n = 0 and the range of λ is −N. g The equation 5.4 can be written as: ∀k ∈N, ∂A A g The equation 5.4 can be written as: ∀k ∈N, (5.4 (k)) a 2 ∂A−k−1 ∂y′ 1 = k A−k y′ 1 g The equation 5.4 can be written as: ∀k ∈N, (5.4 (k)) a 2 ∂A−k−1 ∂y′ 1 = k A−k y′ 1 (5.4 (k)) Lemma 5.15. For g1 /∈Comm(Γ), jΓ(t) is algebraically independent from jΓ(gt) over C. Proof. Let g /∈Comm(Γ). For a contradiction, assume ﬁrst that P is an algebraic relation over C holding between jΓ(t) and jΓ(gt). Then for all a ∈H, we have that P(jΓ(a), jΓ(ga)) = 0. For γ ∈Γ, consider the point bγ = γ · a. Letting a = bγ, we have that P(jΓ(bγ), jΓ(gbγ)) = 0. But, since jΓ(bγ) = jΓ(a), we have that P(jΓ(a), jΓ(gbγ)) = 0. Now, by the Γ-invariance of jΓ, we have that for any γ1 ∈Γ, P(jΓ(γ1a), jΓ(γ1gγa)) = 0. But jΓ(γ1a) = jΓ(a), we have that P(jΓ(a), jΓ(γ1gγa)) = 0 (5.4 (k)) Let k0 be the maximal integer such that for all strictly positive k smaller than k0, A−k = 0. The equality 5.4 (0) gives that A−1 does not depend on y′ 1. Then 5.4 (1) is an equality between a derivative of an algebraic function in y′ 1 and and rational function with a simple pole at 0. This implies that the latter is identically zero: k0 is greater than 2. S AND THE GENUS 0 FUCHSIAN = 0 and 5.4 (k0) is ∂A−k0−1 ∂y′ 1 simple pole, A−k0 = 0 whi how that D(y2) = 0. But t This contradicts the assump Y2. These three derivation ormulas for these derivation ch that tr.deg.CN ≥1, tr.de N over C is 1. This proves ee that P generates I as a D .10 also holds for all gener minimal (and so geometrica chsian groups are involved corresponding Riccati equ AX-LINDEMANN-WEIERSTRASS WITH DERIVATIVES AND THE GENUS 0 FUCHSIAN GROUPS AX-LINDEMANN-WEIERSTRASS WITH DERIVATIVES AND THE GENUS 0 FUCHSIAN GROUPS 23 Now if k0 is ﬁnite then 5.4 (k0 −1) is ∂A−k0 ∂y′ 1 = 0 and 5.4 (k0) is ∂A−k0−1 ∂y′ 1 = A−k0 y′ 1 . As a derivative of an algebraic function can not have simple pole, A−k0 = 0 which contradicts the existence of k0. Then 5.6 proves the lemma. If a ̸= 0 Lemma 5.13 and the equation 5.3 show that D(y2) = 0. But the subﬁeld of constants of D in F is C and y2 is not contant. This contradicts the assumption on a and one gets a = 0. g Now, on F, X1 = X2, H1 = H2 and Y1 = Y2. These three derivations are linearly independent and their kernel is denoted by N. Formulas for these derivations give y1 ∈N and y2 ∈N. y The sequence of extensions C ⊂N ⊂F is such that tr.deg.CN ≥1, tr.deg.NF ≥3 and tr.deg.CF = 4 then the transcendence degree of N over C is 1. This proves that I contains some nonzero P ∈C[y1, y2]. It is not difﬁcult to see that P generates I as a D-ideal. □ Remark 5.14. It is not hard to see that Theorem 5.10 also holds for all general Schwarzian equations (⋆′) provided that they are strongly minimal (and so geometrically trivial). In- deed the above proof did not use the fact that Fuchsian groups are involved. In particular, Theorem 5.10 holds if Condition 3.1 is true of the corresponding Riccati equations. It now remains to understand the kind of polynomials P ∈C[y1, y2] that can occur. Notice that if P(jΓ(g1t), jΓ(g2t)) = 0 gives an algebraic relation between two solutions jΓ(g1t) and jΓ(g2t), then there trivially is an algebraic relation between jΓ(t) and jΓ(g2g−1 1 t), namely P(jΓ(t), jΓ(g2g−1 1 t)) = 0. So it sufﬁces to characterize interalgebraicity with jΓ(t). Lemma 5.15. For g1 /∈Comm(Γ), jΓ(t) is algebraically independent from jΓ(gt) over C. emma 5.15. For g1 /∈Comm(Γ), jΓ(t) is algebraically independent from jΓ(gt) over C. are algebraically independent over K. Proof. For contradiction, assume that the 3n functions Proof. For contradiction, assume that the 3n functions jΓ(g1t), j′ Γ(g1t), j′′ Γ(g1t), . . . , jΓ(gnt), j′ Γ(gnt), j′′ Γ(gnt) are algebraically dependent over K. Deﬁne the ﬁeld ˜K as ˜K = K jΓ(g2t), j′ Γ(g2t), j′′ Γ(g2t) . . . , jΓ(gnt), j′ Γ(gnt), j′′ Γ(gnt) = K ⟨jΓ(g2t), . . . , jΓ(gnt)⟩. By strong minimality of equation (⋆), it must be that jΓ(g1t) ∈ ˜Kalg and by geometric triviality of (⋆), we have that jΓ(g1t) ∈K ⟨jΓ(git)⟩alg for some i = 2, . . . , n. Using Theorem 5.10 we get that jΓ(g1t) ∈C(jΓ(git))alg d jΓ(g1t) ∈K ⟨jΓ(git)⟩alg for some i = 2, . . . , n. Using Theorem 5.10 we get that jΓ(g1t) ∈C(jΓ(git))alg jΓ(g1t) ∈C(jΓ(git))alg and so jΓ(t) ∈C(jΓ(gig−1 1 t))alg Now using Lemma 5.15, it must be the case that g = gig−1 1 ∈Comm(Γ). So for the Γ-special polynomial Ψ ˜g, we get Ψ ˜g(jΓ(t), jΓ(gig−1 1 t)) = 0 and hence jΓ(t) ∈C(jΓ(gig 1 1 t))alg Now using Lemma 5.15, it must be the case that g = gig−1 1 ∈Comm(Γ). So for the Γ-special polynomial Ψ ˜g, we get Ψ (j (t) j (g g−1t)) 0 Now using Lemma 5.15, it must be the case that g = gig−1 1 ∈Comm(Γ). So for the Γ-special polynomial Ψ ˜g, we get Now using Lemma 5.15, it must be the case that g = gig−1 1 ∈Comm(Γ). So for the Γ-special polynomial Ψ ˜g, we get Ψ ˜g(jΓ(t), jΓ(gig−1 1 t)) = 0 Ψ ˜g(jΓ(t), jΓ(gig−1 1 t)) = 0 Ψ ˜g(jΓ(g1t), jΓ(git)) = 0. P(jΓ(a), jΓ(γ1gγa)) = 0 f or all γ1, γ ∈Γ. However, jΓ is precisely Γ-invariant, and for g /∈Comm(Γ), there are inﬁnitely many left coset representatives of Γ among the double coset ΓgΓ. Then there are inﬁnitely many distinct points y for which P(jΓ(a), y) = 0 holds, contradicting the fact that P = 0 gives an algebraic relation. □ Lemma 5.16. [62, Section 7.2] For g ∈Comm(Γ), jΓ(t) is algebraically dependent with jΓ(gt) over C. Deﬁnition 5.17. By Lemma 5.16, when g ∈Comm(Γ), there is an irreducible polynomial Ψ ˜g(x, y) ∈C[x, y] such that Ψ ˜g(jΓ(t), jΓ(gt)) = 0. We call Ψ ˜g a Γ-special polynomial, and the zero set of such a polynomial a Γ-special curve. G. CASALE, J. FREITAG, AND J. NAGLOO 24 Now from Theorems 2.12 and 5.10 and Lemmas 5.15 and 5.16, one gets the weak form of the Ax-Lindemann-Weierstrass Theorems 2.13 and 2.14. Theorem 5.18. Let K be a differential extension of (C(t), ∂ ∂t) and jΓ(g1t), ..., jΓ(gnt) be dis- tinct solutions of the Schwarzian equation (⋆) that are not algebraic over K nor pairwise related by Γ-special polynomials. Then the 3n functions jΓ(g1t), j′ Γ(g1t), j′′ Γ(g1t), . . . , jΓ(gnt), j′ Γ(gnt), j′′ Γ(gnt) jΓ(g1t), j′ Γ(g1t), j′′ Γ(g1t), . . . , jΓ(gnt), j′ Γ(gnt), j′′ Γ(gnt) are algebraically independent over K. and hence g This contradicts our assumption that jΓ(g1t) and jΓ(git) are not related by any Γ-special polynomials. □ 6. ORTHOGONALITY AND THE AX-LINDEMANN-WEIERSTRASS THEOREM In the previous sections, we have understood the structure of the solution set of S d dt (y) + (y′)2 · RjΓ(y) = 0. Deﬁne χΓ, d dt (y) = S d dt (y) + (y′)2 · RjΓ(y). (6.1) (6.1) In this section, we consider equations of the form χΓ, d dt (y) = a for a an element in some differential ﬁeld extension of Q, and produce a similar analysis. In this section, we consider equations of the form χΓ, d dt (y) = a for a an element in some differential ﬁeld extension of Q, and produce a similar analysis. dt differential ﬁeld extension of Q, and produce a similar analysis. AX-LINDEMANN-WEIERSTRASS WITH DERIVATIVES AND THE GENUS 0 FUCHSIAN GROUPS 25 6.1. Strong minimality and algebraic relations on other ﬁbers. First, we prove the so- lution set of the equation χΓ, d dt (y) = a is strongly minimal and characterize the algebraic relations between solutions. Essentially, the analysis from [12, Section 5.1] adapts to this case, but for the sake of completeness, we will provide a brief explanation here. p p p Let a ∈K be an element in some differential ﬁeld extension of Q. By Seidenberg’s em- bedding theorem, we can, without loss of generality, assume a = a(t) is given by a mero- morphic function over some domain U, and the derivation is given by d dt. After sufﬁcently shrinking the domain, there is some meromorphic function ˜a(t) satisfying S d dt (˜a) = a such that χΓ, d dt (jΓ(˜a(t))) = a(t). The following Lemma follows by the Schwarzian chain rule and is nearly identical to [12, Lemma 5.1]: Lemma 6.1. Let K be a differentially closed d dt-ﬁeld containing a. There exists ∂∈K d dt such that χΓ,∂(y) = 0. Proof. The equation S d dt (˜a) = a, with unknown ˜a, can be considered as a differential equa- tion over C⟨a⟩. By Seidenberg’s theorem this ﬁeld can be assumed to be a ﬁeld of mero- morphic functions on some domain U ⊂C and by the usual Cauchy theorem, one can build a solution, holomorphic on some domain U′ ⊂U. By the differential Nullstellensatz there exists ˜a ∈K a solution of S d dt (˜a) = a. and hence Then ∂= 1 ˜a′ d dt. □ Theorem 6.2. The sets deﬁned by χΓ, d dt (y) = a are strongly minimal and geometrically trivial. If a1, . . . , an satisfy χΓ, d dt (ai) = a and are dependent, then there exist i, j ≤n and a Γ-special polynomial P such that P(ai, aj) = 0. The proof of Theorem 6.2 is quite similar to that of [12] Proposition 5.2, but we include it here for completeness. Proof. We ﬁrst explain why χΓ, d dt (y) = a is strongly minimal; it sufﬁces to show that over some differentially closed ﬁeld which contains the coefﬁcients of the equation, that every differentially constructible set is ﬁnite or coﬁnite. Using properties of differentially closed ﬁeld, one can ﬁnd in K ˜a as above. By Lemma 6.1, K is a ∂-differential ﬁeld and the sets χΓ, d dt (y) = a and χΓ,∂(y) = 0 coincide. Now strong minimality follows by Theorem 3.2 and the fact that d dt-differentially constructible sets are ∂-differentially constructible (over K). By Lemma 6.1, K is a ∂-differential ﬁeld and the sets χΓ, d dt (y) = a and χΓ,∂(y) = 0 coincide. Now strong minimality follows by Theorem 3.2 and the fact that d dt-differentially constructible sets are ∂-differentially constructible (over K). Algebraic dependencies among elements of the set χΓ, d dt (y) = a give algebraic depen- dencies among elements of the set χΓ,∂(y) = 0, and thus by Theorem 5.18 must be given by Γ-special polynomials. □ Algebraic dependencies among elements of the set χΓ, d dt (y) = a give algebraic depen- dencies among elements of the set χΓ,∂(y) = 0, and thus by Theorem 5.18 must be given by Γ-special polynomials. □ The ﬁnal piece of our analysis of the ﬁbers of χ shows that there are no algebraic relations between different ﬁbers. 26 G CASALE J FREITAG AND J NAGLOO G. CASALE, J. FREITAG, AND J. NAGLOO 26 Theorem 6.3. For a ̸= b, the strongly minimal sets deﬁned by χΓ, d dt (y) = a and by χΓ, d dt (y) = b are orthogonal. Theorem 6.3. For a ̸= b, the strongly minimal sets deﬁned by χΓ, d dt (y) = a and by χΓ, d dt (y) = b are orthogonal. and hence Theorem 6.3 is more general than [12] Theorem 5.4, but the proof there cannot be adapted to the case of non-arithmetic fuchsian groups. Proof. Throughout, we respectively use M (U) and D(p, r) for the ﬁeld of meromorphic functions on a domain U ⊂C, and the open complex disk centered at p ∈C with ra- dius r. As both χ−1 Γ, d dt (a) and χ−1 Γ, d dt (b) are strongly minimal and geometrically trivial, if d d χ−1 Γ, d dt (a) ̸⊥χ−1 Γ, d dt (b), then there is a ﬁnite-to-ﬁnite correspondence between the sets, de- ﬁned over Q⟨a, b⟩. Using Seidenberg’s embedding theorem, we regard a, b as meromor- phic functions on a domain U ⊂C. Recall that ˜a denotes a meromorphic function such that S d dt (˜a) = a. The function ˜b is deﬁned similarly. χ−1 Γ, d dt (a) ̸⊥χ−1 Γ, d dt (b), then there is a ﬁnite-to-ﬁnite correspondence between the sets, de- ﬁned over Q⟨a, b⟩. Using Seidenberg’s embedding theorem, we regard a, b as meromor- phic functions on a domain U ⊂C. Recall that ˜a denotes a meromorphic function such that S d dt (˜a) = a. The function ˜b is deﬁned similarly. dt Using the holomorphic inverse function theorem, we claim that without loss of general- ity, it is enough to prove the result for the case a = 0. Indeed, since jΓ(˜a(t)) is interalgebraic with jΓ(g˜b(t)) for some g ∈GL2(C), we have that jΓ(t) is interalgebraic with jΓ(g˜b(˜a−1(t)) (since ˜b is deﬁned up to composition with linear fractional transformations, we can assume that there is a common regular point for ˜a and ˜b and work locally around this point). Let- ting ˜c = ˜b ◦˜a−1 and c = S d dt (˜c), we see that χ−1 Γ, d dt (0) ̸⊥χ−1 Γ, d dt (c) and by geometric triviality this occurs over Q⟨c⟩. ⟨⟩ So we assume that a = 0. Let p be a regular point for ˜b(t) and let D1 = D(p, ǫ) be a disc of regular points of ˜b(t). Also let γ be a linear fractional transformation sending D2 = D(p, 1 2ǫ) to H. 2 Since χ−1 Γ, d dt (0) ̸⊥χ−1 Γ, d dt (b), we have that for some g ∈GL2(C), the solution jΓ(g˜b(t)) is algebraic over Q⟨b, jΓ(γt)⟩⊂M (D1)(jΓ ◦γ, j′ Γ ◦γ, j′′ Γ ◦γ) ⊂M (D2). and hence But notice that for any domain U such that D2 ⊆U ⊆D1, if jΓ(g˜b(t)) is algebraic over M (U), then jΓ(γt) will also be algebraic over M (U). This follows from the fact that M (D1) ⊆M (U), and jΓ(g˜b(t)) is interalgebraic with jΓ(γt) over Q ⟨b⟩⊂M (D1). But jΓ(t) cannot be extended algebraically on a neighborhood of H, hence U = D2. Since χ−1 Γ, d dt (0) ̸⊥χ−1 Γ, d dt (b), we have that for some g ∈GL2(C), the solution jΓ(g˜b(t)) is algebraic over Q⟨b, jΓ(γt)⟩⊂M (D1)(jΓ ◦γ, j′ Γ ◦γ, j′′ Γ ◦γ) ⊂M (D2). But notice that for any domain U such that D2 ⊆U ⊆D1, if jΓ(g˜b(t)) is algebraic over M (U), then jΓ(γt) will also be algebraic over M (U). This follows from the fact that M (D1) ⊆M (U), and jΓ(g˜b(t)) is interalgebraic with jΓ(γt) over Q ⟨b⟩⊂M (D1). But jΓ(t) cannot be extended algebraically on a neighborhood of H, hence U = D2. The disc D2 is thus the maximal among domains U such that jΓ(g˜b(t)) is algebraic over M (U). But such a domain satisﬁes g˜b(D2) = H, that is the image of D2 by the reg- ular holomorphic map ˜b is the disc g−1H. A corollary of Schwarz’s lemma gives that biholomorphisms from a disc to a disc are restrictions of homographies. Hence ˜b is an homography h ∈PSL2(C) and so b = 0. □ 8Fix a subset of the coordinates such that there is an algebraic dependence as described above. Then there is some minimal such set. Picking i, j to be any two coordinates of this minimal set, the subset is the collection of coordinates in the remainder of the minimal set. We can ﬁnally turn to the proof of the Ax-Lindemann-Weierstrass Theorem 2.16. We can ﬁnally turn to the proof of the Ax-Lindemann-Weierstrass Theorem 2.16 Proof of Theorem 2.16. Recall that V ⊂An and for each i = 1, . . . , n, the variety V is as- sumed to project dominantly onto A1 under projection to the ith coordinate. Thus, the ith coordinate function is nonconstant, and it is possible to equip the ﬁeld generated by the ith coordinate functions with various differential structures, which will be essential to the technique in our proof. AX-LINDEMANN-WEIERSTRASS WITH DERIVATIVES AND THE GENUS 0 FUCHSIAN GROUPS 27 Lemma 6.4. There is a derivation δ on C(V) such that for each of the coordinate functions ti for i = 1, . . . , n, δ(ti) ̸= 0. Lemma 6.4. There is a derivation δ on C(V) such that for each of the coordinate functions ti for i = 1, . . . , n, δ(ti) ̸= 0. Proof. Let z1, . . . , zk be a transcendence basis of C(V) over C and α1, . . . , αk be Q-linearly independent complex numbers. As C(V) is an algebraic extension of C(z1, . . . , zk) the derivation δ = ∑i αizi ∂ ∂zi extends a derivation of C(V) and the ﬁeld of constants in C(V) is an algebraic extension of the ﬁeld of constant in C(z1, . . . , zk). The latter is C. As the projection of V on the ith coordinate is dominant, δ(ti) ̸= 0. □ The transcendence degree over C(V) of the 3n functions The transcendence degree over C(V) of the 3n functions jΓ(t1), j′ Γ(t1), j′′ Γ(t1) . . . , jΓ(tn), j′ Γ(tn), j′′ Γ(tn) jΓ(t1), j′ Γ(t1), j′′ Γ(t1) . . . , jΓ(tn), j′ Γ(tn), j′′ Γ(tn) is identical to that of the 3n functions is identical to that of the 3n functions jΓ(t1), δ(jΓ(t1)), δ2(jΓ(t1)), . . . , jΓ(tn), δ(jΓ(tn)), δ2(jΓ(tn).) Now, for any ti, since jΓ(ti) is not an algebraic function, it follows by strong minimality that jΓ(ti) is a generic solution to a δ-differential equation of the form χΓ,δ(y) = ai with ai = Sδ(ti) ∈C(V). We can ﬁnally turn to the proof of the Ax-Lindemann-Weierstrass Theorem 2.16. If the 3n functions are not algebraically independent, then there exist i, j such that the functions If the 3n functions are not algebraically independent, then there exist i, j such that the functions 2 2 j(ti), δ(j(ti)), δ2(j(ti)), j(tj), δ(j(tj)), δ2(j(tj)) are algebraically dependent over K, the δ-ﬁeld extension of C(V) generated by j(tk) for those k in some subset of {1, . . . , n} \ {i, j}. Moreover one can choose8 K such that j(ti) and j(tj) are not algebraic over K. j But then by strong minimality of the equations χΓ,δ(y) = ai and χΓ,δ(y) = aj (Theorem 6.2), there is a ﬁnite-to-ﬁnite correspondence between χΓ,δ(y) = ai and χΓ,δ(y) = aj deﬁned over K. By Theorem 6.3, it must be that ai = aj and ti and tj are Γ-geodesically dependent. A contradiction. □ 6.2. Orthogonality and commutators. In this section, we analyze the algebraic relations between solutions of S d dt (y) + (y′)2 · RjΓ1(y) = 0 (6.2) S d dt (y) + (y′)2 · RjΓ2(y) = 0 (6.3) S d dt (y) + (y′)2 · RjΓ1(y) = 0 S d dt (y) + (y′)2 · RjΓ2(y) = 0 (6.2) (6.3) when Γ1 is not necessarily commensurable with Γ2. If Γ1 is commensurable with Γ2, then it is well known that jΓ1 is interalgebraic with jΓ2 over C. Moreover this is not the whole story: we say that Γ1 is commensurable with Γ2 in the wide sense if Γ1 is commensurable to some conjugate of Γ2. When such is the case and Γ1 is commensurable with g−1Γ2g then again one has that jΓ1 is interalgebraic with jΓ2 ◦g over C. Notice that if Γ1 is commensurable with Γ2 in the wide sense, then Comm(Γ1) is conju- gate to Comm(Γ2). G. CASALE, J. FREITAG, AND J. NAGLOO 28 Theorem 6.5. Suppose that Γ1 is not commensurable with Γ2 in the wide sense. Then the sets deﬁned by equations 6.2 and 6.3 are orthogonal. In particular, for any differential ﬁeld K tr.deg.KK jΓ1(t1), j′ Γ1(t1), j′′ Γ1(t1), jΓ2(t2), j′ Γ2(t2), j′′ Γ2(t2) = tr.deg.KK jΓ1(t1), j′ Γ1(t1), j′′ Γ1(t1) + tr.deg.KK jΓ2(t2), j′ Γ2(t2), j′′ Γ2(t2) . Proof. Let XΓ1 and XΓ2 be the set deﬁned by equations 6.2 and 6.3 respectively. Assume for contradiction that XΓ1 ̸⊥XΓ2. Since XΓ1 and XΓ2 are trivial strongly minimal sets, we have that nonorthogonality is witnessed over C (i.e., the sets are non weakly orthogonal). So for any solution y1 ∈XΓ1 there is a solution y2 ∈XΓ2 such that y1 ∈C ⟨y2⟩alg. By invoking Fact 2.3, we have that jΓ1(t) ∈C ⟨jΓ2(gt)⟩alg for some g ∈GL2(C). Let us write P(jΓ1(t), jΓ2(gt), j′ Γ2(gt), j′′ Γ2(gt), t) = 0 for this algebraic relation over C. For any γ1 ∈Γ1, using the fact that jΓ1(γ1t) = jΓ1(t), we have that for this algebraic relation over C. For any γ1 ∈Γ1, using the fact that jΓ1(γ1t) = jΓ1(t), we have that P(jΓ1(t), jΓ2(gγ1t), j′ Γ2(gγ1t), j′′ Γ2(gγ1t), γ1t) = 0. So this implies that for any γ1 ∈Γ1, we get that jΓ1(t) ∈C ⟨jΓ2(gγ1t)⟩alg. In particular C ⟨jΓ2(gt)⟩alg = C ⟨jΓ2(gγ1t)⟩alg for all γ1 ∈Γ1. By Theorem 5.18, it must be the case that gγ1g−1 ∈Comm(Γ2) for all γ1 ∈Γ1, that is it must be that gΓ1g−1 ⊆Comm(Γ2). So this implies that for any γ1 ∈Γ1, we get that jΓ1(t) ∈C ⟨jΓ2(gγ1t)⟩alg. In particular C ⟨jΓ2(gt)⟩alg = C ⟨jΓ2(gγ1t)⟩alg for all γ1 ∈Γ1. By Theorem 5.18, it must be the case that gγ1g−1 ∈Comm(Γ2) for all γ1 ∈Γ1, that is it must be that gΓ1g−1 ⊆Comm(Γ2). Now, to get our contradiction, we consider three cases (without loss of generality) (1) Assume Γ1 is arithmetic and Γ2 is nonarithmetic. In this case, χΓ1 is not ℵ0-categorical while χΓ2 is ℵ0-categorical (this follows from Theorem 5.18). This case could also be handled in a more elementary manner similar to our technique in the third case. y q (2) Assume that both Γ1, Γ2 are arithmetic groups. We have, by the above arguments, that gΓ1g−1 is contained in Comm(Γ2). We will be done if we show that gΓ1g−1 and Γ2 are commensurable in the strict sense. This follows by arguments of [32, see page 4], where the following fact is shown: for any two arithmetic Fuchsian groups G1 and G2, if G1 is contained in the commensurator of G2 then G1 and G2 are commensurable in the strict sense. (3) Assume that both Γ1 and Γ2 are non-arithmetic. By the above argument, we have that gΓ1g−1 ≤Comm(Γ2) for some g ∈GL2(C). By a symmetric argument, we have some h ∈GL2(C) such that hΓ2h−1 ≤Comm(Γ1). Replacing one of Γi with a suitable conjugate, we may assume that Γ1 ≤Comm(Γ2) and Γ2 ≤Comm(Γ1). From this, we will show that Γ1 and Γ2 are commensurable. By Margulis’ Theo- rem, Γi is ﬁnite index in Comm(Γi). We need only show that Γ2 is ﬁnite index in Comm(Γ1). We have that Γ1 is contained in Comm(Γ2), Γ1 contains only ﬁnitely many left coset representatives of Γ2. Since Γ1 is ﬁnite index in its own commensurator, the conclusion follows. (3) Assume that both Γ1 and Γ2 are non-arithmetic. By the above argument, we have that gΓ1g−1 ≤Comm(Γ2) for some g ∈GL2(C). By a symmetric argument, we have some h ∈GL2(C) such that hΓ2h−1 ≤Comm(Γ1). Remark 6.6. The following stronger result should hold: The sets deﬁned by χΓ1, d dt (y) = a1 and by χΓ2, d dt (y) = a2 are orthogonal if Γ1 is not commensurable with Γ2 in the wide sense. However, we have not been able to prove it yet. 7. EFFECTIVE FINITENESS RESULTS AROUND THE ANDR´E-PINK CONJECTURE The Andr´e-Pink conjecture predicts that when W is an algebraic subvariety of a Shimura variety and S is a Hecke orbit, if W ∩S is Zariski dense in W, then W is weakly special. For details, deﬁnitions, and proofs of certain special cases of the conjecture see [41, 42, 13]. p p j In the setting of the present paper the conjecture concerns the intersection of an algebraic variety W ⊂An with the image, under jΓ applied to each coordinate, of the orbit under Comm(Γ)n of some point in ¯a ∈H. ( ) p Given a Fuchsian group Γ and a point a ∈C, we denote, by IsoΓ(a), the collection of points b ∈C such that P(a, b) = 0 for some Γ-special polynomial P. Equivalently, for some (all) ˜a, ˜b ∈H such that jΓ(˜a) = a and jΓ(˜b) = b, there is γ ∈Comm(Γ) such that γ˜a = ˜b. j ( ) j ( ) ( ) Given a Fuchsian group Γ and a point ¯a = (a1, . . . , an) ∈An(C), let IsoΓ(¯a) denote the product of the orbits of the points a1, . . . , an under Γ-special polynomials, that is IsoΓ(¯a) = n ∏ i=1 IsoΓ(ai). We call a polynomial p(x1, . . . , xn) (Γ)-(a1, . . . , an)-special if We call a polynomial p(x1, . . . , xn) (Γ)-(a1, . . . , an)-special if (1) p( ¯x) = xi −bi where bi ∈IsoΓ(a), or (1) p( ¯x) = xi −bi where bi ∈IsoΓ(a), or ( ) p( ) i i i ( ) (2) For some i, j, IsoΓ(ai) = IsoΓ(aj), and p( ¯x) is a Comm(Γ)-special polynomial in xi, xj. (2) For some i, j, IsoΓ(ai) = IsoΓ(aj), and p( ¯x) is a Comm(Γ)-special polynomial in xi, xj. An irreducible subvariety of Cn will be called (Γ)-(a1, . . . , an)-special if it is given by a ﬁnite conjunction of (Γ)-(a1, . . . , an)-special polynomials. If an irreducible variety V is (Γ)- (a1, . . . , an)-special, then it follows that V has a Zariski dense set of points from IsoΓ(¯a). Our ﬁrst result of this section shows that the converse holds, at least when ¯a is a tuple of transcendental numbers (perhaps with algebraic relations between them). Theorem 7.1. AX-LINDEMANN-WEIERSTRASS WITH DERIVATIVES AND THE GENUS 0 FUCHSIAN GROUPS 29 P(jΓ1(t), jΓ2(gγ1t), j′ Γ2(gγ1t), j′′ Γ2(gγ1t), γ1t) = 0. Replacing one of Γi with a suitable conjugate, we may assume that Γ1 ≤Comm(Γ2) and Γ2 ≤Comm(Γ1). From this, we will show that Γ1 and Γ2 are commensurable. By Margulis’ Theo- rem, Γi is ﬁnite index in Comm(Γi). We need only show that Γ2 is ﬁnite index in Comm(Γ1). We have that Γ1 is contained in Comm(Γ2), Γ1 contains only ﬁnitely many left coset representatives of Γ2. Since Γ1 is ﬁnite index in its own commensurator, the conclusion follows. □ Remark 6.6. The following stronger result should hold: The sets deﬁned by χΓ1, d dt (y) = a1 and by χΓ2, d dt (y) = a2 are orthogonal if Γ1 is not commensurable with Γ2 in the wide sense. However, we have not been able to prove it yet. Remark 6.6. The following stronger result should hold: The sets deﬁned by χΓ1, d dt (y) = a1 and by χΓ2, d dt (y) = a2 are orthogonal if Γ1 is not commensurable with Γ2 in the wide sense. However, we have not been able to prove it yet. AX-LINDEMANN-WEIERSTRASS WITH DERIVATIVES AND THE GENUS 0 FUCHSIAN GROUPS 7. EFFECTIVE FINITENESS RESULTS AROUND THE ANDR´E-PINK CONJECTURE Fix a complex algebraic variety V ⊂An(C), a genus zero Fuchsian group Γ of the ﬁrst kind, and a point ¯a = (a1, . . . , an) ∈An(C) such that for all but at most one i ∈{1, . . . , n}, ai /∈Qalg. Then V ∩IsoΓ(¯a) Zar is a ﬁnite union of (Γ)-(a1, . . . , an)-special varieties. Proof. The (perhaps reducible) variety V ∩IsoΓ(¯a) Zar consists of ﬁnitely many components W1, . . . , Wk, and so we need only show that the varieties Wi are (Γ)-(a1, . . . , an)-special. Working component by component, it sufﬁces to show that for an arbitrary irreducible variety V, if IsoΓ(¯a) is Zariski dense in V, then V is (Γ)-(a1, . . . , an)-special. y p Without loss of generality, assume that all of the coordinates of ¯a, except perhaps a1, are transcendental over Q. We also assume a1 ∈Qalg without loss of generality - otherwise just ignore arguments about this coordinate in the proof. j g g Embed Q(a2, . . . , an) into the ﬁeld of meromorphic functions on some connected subset of H such that a2, . . . , an are non-constant. Let ˜a2, . . . , ˜an be as in the proof of Theorem 6.2 - that is, jΓi(˜ai) = ai for i = 2, . . . , n. In the differential closure K of the ﬁeld generated by the ai over Q we have, by Theorem 6.2, that {x ∈K \| χΓ(x) = χΓ(ai)} = IsoΓ(ai), so χΓ(ai) = χΓ(aj) if and only if IsoΓ(ai) = IsoΓ(aj). so χΓ(ai) = χΓ(aj) if and only if IsoΓ(ai) = IsoΓ(aj). G. CASALE, J. FREITAG, AND J. NAGLOO 30 Consider the collection of i ∈{1, . . . , n} such that V projects dominantly onto the coordi- nate corresponding to xi. Then if IsoΓ(¯a) is dense in V, and we let b2, . . . , bn be a collection of generic solutions of χΓ(bi) = χΓ(ai) and let b1 be a generic constant, we have that the tuple ¯b is dependent over C, but as b2, . . . , bn satisfy equations which are strongly mini- mal and trivial, it must be that two of the coordinates are nonorthogonal. 7. EFFECTIVE FINITENESS RESULTS AROUND THE ANDR´E-PINK CONJECTURE But now we are done, since all instances of nonorthogonality are given by Theorem 6.2, since none of the coordinates 2, . . . , n can be nonorthogonal to b1, a constant. □ Remark 7.2. The assumption in Theorem 7.1 that all but at most one of the elements in the tuple ¯a are transcendental is an inherent restriction of the method we employ, which is similar to the technique employed in various applications of differential algebra to dio- phantine problems. We replace an arithmetic (discrete) object by the solution to a system of differential equations, then reduce the general case to an analytic statement using a strong version of Seidenberg’s embedding theorem. Generally speaking, the technique works when the discrete set satisﬁes some interesting differential equation, which one is able to understand. But the only derivation on Qalg is the trivial one, and so such a coordinate can not . For other instances of applications of this general idea, see [15, 17, 57, 7]. It would be interesting to see if the methods here might be combined with methods solv- ing other special cases of the conjecture (e.g. [41]) to remove the transcendence restrictions of Theorem 7.1. Remark 7.3. The technique by which we prove Theorem 7.1 has natural limitations de- scribed in Remark 7.2, but it also has an interesting natural advantage over other tech- niques. Because we replace an arithmetic object, whose deﬁnition is very non-uniform, with a differential algebraic variety, results from differential algebraic geometry can be used to give effective bounds the degree of the Zariski-closure of the solutions set. A general purpose Bezout-type theorem for algebraic differential equations (generaliz- ing a theorem of Hrushovski and Pillay) was established in [11]. In what follows, τℓAn denotes the ℓth-prolongation space of An and for a differential ﬁeld K, we deﬁne (X, S \ T)♯(K) = {a ∈X(K) : (a, a′, . . . , a(ℓ)) ∈S \ T(K)}. Theorem 7.4. Let X be a closed subvariety of An, with dim(X) = m, and let S, T be closed subvarieties (not necessarily irreducible) of τℓAn for some ℓ∈N. Then the degree of the Zariski closure of (X, S \ T)♯(C) is at most deg(X)ℓ2mℓdeg(S)2mℓ−1. In particular, if (X, S \ T)♯(C) is a ﬁnite set, this expression bounds the number of points in that set. 7. EFFECTIVE FINITENESS RESULTS AROUND THE ANDR´E-PINK CONJECTURE Next, we aim to put our differential relations in a form such that we may apply Theorem 7.4. Recall our Schwarzian differential equation: S d dt (y) + (y′)2 · RjΓ(y) = 0 (⋆) S d dt (y) + (y′)2 · RjΓ(y) = 0 (⋆) dt (y) y 2 y ( dt) jΓ C(y) depends on the choice of jΓ. For the purposes of this section, all that matters is the degree of the rational function RjΓ (the coefﬁcients, which are complex numbers, will not AX-LINDEMANN-WEIERSTRASS WITH DERIVATIVES AND THE GENUS 0 FUCHSIAN GROUPS 31 be important in stating or proving our results). If the Γ-action on H has a fundamental half domain given by a r-sided polygon P (note that this is the case for any Fuchsian group of the ﬁrst kind as r is equal to the number of generators of Γ [21]), then RjΓ(y) = 1 2 r ∑ i=1 1 −α2 i (y −ai)2 + r ∑ i=1 Ai y −ai , RjΓ(y) = 1 2 r ∑ i=1 1 −α2 i (y −ai)2 + r ∑ i=1 Ai y −ai , where the coefﬁcients are complex numbers depending on speciﬁc characteristics of the domain. The crucial point for our results is that the degree of RjΓ (by which we mean the maximum of the degree of the numerator and the denominator) is given by 2r where r is the number of generators of Γ. Clearing the denominator of the rational function and the Schwarzian in equation (⋆), we obtain: (Q(⋆)) 0 = (y′′′y′ −3 2(y′′)2) r ∏ i=1 (y −αi)2 + (y′)4  1 2 r ∑ i=1  (1 −αi) ∏ j∈[r], j̸=i (y −ai)2  + r ∑ i=1  Ai(y −ai) ∏ j∈[r],j̸=i (y −aj)2     As a polynomial, the previous equation has degree 2r + 2. As a polynomial, the previous equation has degree 2r + 2. Theorem 7.5. Fix a complex algebraic variety V ⊂An(C), a genus zero Fuchsian group Γ of the ﬁrst kind, and a point ¯a = (a1, . . . , an) ∈An(C) such that for all i ∈{1, . . . , n}, ai /∈Qalg. Then V ∩IsoΓ(¯a) Zar is a ﬁnite union of (Γ)-(a1, . . . 7. EFFECTIVE FINITENESS RESULTS AROUND THE ANDR´E-PINK CONJECTURE , an)-special varieties, and the sum of the degrees of the varieties in this union is at most ((2r + 2)n · deg(V))23n−1. Proof. We need only put the equations appearing in Theorem 7.1 in a form suitable to apply Theorem 7.4. We can write the Schwarzian differential equations as ∇−1 3 (S) on each coordinate, where S is the locus of (Q(⋆)) in τ3(A1). On each coordinate, this equation has degree 2r, so the intersection of these relations with V is a variety in τ3(An) of degree at most (2r + 2)n deg(V). Now the degree bound follows from Theorem 7.4 with X = An, l = 3, and V as given above. □ Remark 7.6. 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Saint Gervais, Uniformisation des surfaces de Riemann: retour sur un th´eoreme centenaire, ENS Editions, Lyon, 2010. [61] P. Sarnak, Torsion points on varieties and homology of abelian covers, unpublished manuscript, 1988. G. CASALE, J. FREITAG, AND J. NAGLOO 34 [62] G. APPENDIX A. STRONG MINIMALITY FOR THE SPECIAL CASE OF TRIANGLE GROU In this appendix, we discuss an alternate method of proving strong minimality of the Schwarzian equation in the special case of triangle groups. As before, we assume that Γ is a Fuchsian group of ﬁrst kind and of genus zero. The group Γ is said to be a Fuchsian triangle group of type (k, l, m) if its signature is (0; k, l, m) (see Section 2). We will without loss of generality always assume that 2 ≤k ≤l ≤m ≤∞. We write Γ(k,l,m) for the Fuchsian triangle group of type (k, l, m). The fundamental domain in H of Γ(k,l,m) is the union of a hyperbolic triangle with angles π k , π l and π m at the vertices vk, vl and vm respectively, together with its image via hyperbolic reﬂection of one side connecting the vertices. Notice that since k, l, m relates to the angle of an hyperbolic triangle, if Γ(k,l,m) is a triangle group then 1 k + 1 l + 1 m < 1. 1 k + 1 l + 1 m < 1. Also, the vertices vk, vl and vm are the ﬁxed points of the generators g1, g2 and g3 respec- tively. Deﬁnition A.1. The function j(k,l,m) will denote the (unique) Hauptmodul Γ(k,l,m) \HΓ(k,l,m) → P1(C) sending vk, vl, vm to 1, 0, ∞respectively. AX-LINDEMANN-WEIERSTRASS WITH DERIVATIVES AND THE GENUS 0 FUCHSIAN GROUPS 35 With this deﬁnition (cf. [1, Chapter 5]) we have that j(k,l,m) satisﬁes the Schwarzian equation (⋆) with With this deﬁnition (cf. [1, Chapter 5]) we have that j(k,l,m) satisﬁes the Schwarzian equation (⋆) with Rj(k,l,m)(y) = 1 −l−2 y2 + 1 −k−2 (y −1)2 + k−2 + l−2 −m−2 −1 y(y −1) . (A.1) Notice that with Deﬁnition A.1, the Hauptmodul j(2,3,∞) for PSL2(Z) is not the classical j-funtion. Rather, one has that j = 1728j(2,3,∞) (see Example 2.2). Notice that with Deﬁnition A.1, the Hauptmodul j(2,3,∞) for PSL2(Z) is not the classical j-funtion. Rather, one has that j = 1728j(2,3,∞) (see Example 2.2). ( , , ) Finally let us mention that there is a full classiﬁcation, up to PSL2(R)-conjugation, of the arithmetic triangle groups Fact A.2. Up to PSL2(R)-conjugation, there are ﬁnitely many arithmetic triangle groups; 76 cocompact and 9 non-cocompact [63]. Among these, there are 19 distinct commensurability classes represented [64]. JAMES FREITAG, UNIVERSITY OF ILLINOIS CHICAGO, DEPARTMENT OF MATHEMATICS, STATISTICS, AND COMPUTER SCIENCE, 851 S. MORGAN STREET, CHICAGO, IL, USA, 60607-7045. E-mail address: freitag@math.uic.edu APPENDIX A. STRONG MINIMALITY FOR THE SPECIAL CASE OF TRIANGLE GROU In the special case of triangle groups, proving that the Riccati equation 4.4 has no alge- braic solutions (and thus establishing the strong minimality of the associated order three nonlinear Schwarzian differential equations) can be accomplished without any appeal to Picard-Vesiot theory but instead by using classical work around the hypergeometric equa- tion. Already, in [39, see page 601], Nishioka shows that equation 4.5 has no algebraic solutions in the case the Γ is a cocompact triangle group (which corresponds to the case that none of k, l, m are ∞). Hence Condition 3.1 and thus Theorem 3.2 holds in the case of cocompact triangle groups. We will, via a very similar argument, show the same result holds in the case that Γ is not cocompact. To emphasize, these results are a special case of our general result on Fuchsian groups, but we feel their inclusion is worthwhile in part because the method, which deals more directly with the order two linear equation 4.1 and Riccati equation 4.5, might generalize to Schwarzian equations of the form of equation (⋆’) which do not necessarily come from a group action of Γ on H. This restriction appears to be more inherent in our main approach of the previous section. L λ = 1 l (A.2) µ = 1 k (A.3) ν = 1 m (A.4) (A.2) (A.3) (A.4) m where the integers 2 ≤k ≤l ≤m ≤∞are as above. We have already seen λ + µ + ν < 1. Now let α, β and γ be any complex numbers such that, λ = 1 −γ, µ = γ −α −β, and ν = α −β. Now, we know that the second order equation 4.1 corresponding to equation (⋆) with ra- tional function A.1 (equation (5) of [39]) is reducible if and only if one of α, β, γ −α, γ −β is an integer. Since [39] covers the cocompact case, we can assume without loss of generality that m = ∞, equivalently ν = 0. Thus, in the above notation, α = β. Now, α = 1 −1 l −1 k 2 . α = 1 −1 l −1 k 2 . 6 G CASALE J FREITAG AND J NAGLOO G. CASALE, J. FREITAG, AND J. NAGLOO 36 In this case, by the triangle requirement, 1 l + 1 k < 1, so α is never an integer. JOEL NAGLOO, DEPARTMENT OF MATHEMATICS AND COMPUTER SCIENCE, BRONX COMMUNITY COL- LEGE CUNY, BRONX, NY 10453, USA. E-mail address: joel.nagloo@bcc.cuny.edu GUY CASALE, UNIV RENNES, CNRS, IRMAR-UMR 6625, F-35000 RENNES, FRANCE E-mail address: guy.casale@univ-rennes1.fr APPENDIX A. STRONG MINIMALITY FOR THE SPECIAL CASE OF TRIANGLE GROU Further we have Further, we have γ −α = 1 −1 l + 1 k 2 . γ −α = 1 −1 l + 1 k 2 . This quantity is never an integer, since 1 l + 1 k < 1. Thus, in the non-cocompact case, we have that the corresponding equation 4.1 is always irreducible, which, by the correspon- dence explained in 4 implies that there are no rational solutions to equation 4.4 in this case. Now, under the assumption of irreducibility of equation 4.1, we have that there is an algebraic (but irrational) solution of 4.4 if and only if two of λ −1 2, µ −1 2, ν −1 2 are integers [31, pages 96-100]. This is impossible for any triangle group as at most one of these is an integers as long as λ + µ + ν < 1. g g µ Thus, we have shown, in a more direct way, that Condition 3.1 and thus Theorem 3.2 also holds in the case of non-cocompact triangle groups. Remark A.3. At ﬁrst glance the above arguments only seem to show that the differential equations for the unformizers j(k,l,m) are strongly minimal. However, all other uniformizers are rational functions (over C) of the j(k,l,m)’s. From this, strong minimality follows for the other equations as well. GUY CASALE, UNIV RENNES, CNRS, IRMAR-UMR 6625, F-35000 RENNES, FRANCE E-mail address: guy.casale@univ-rennes1.fr GUY CASALE, UNIV RENNES, CNRS, IRMAR-UMR 6625, F-35000 RENNES, FRANCE E-mail address: guy.casale@univ-rennes1.fr
https://openalex.org/W1983565785	https://journals.plos.org/ploscompbiol/article/file?id=10.1371/journal.pcbi.1000623&type=printable	English	null	A Stochastic Model for Microtubule Motors Describes the In Vivo Cytoplasmic Transport of Human Adenovirus	PLOS computational biology/PLoS computational biology	2,009	cc-by	7,229	Introduction The small number of motor proteins involved in microtubule transport implies a system where the fluctuations in the behavior of motors and the randomness of molecular reactions are essential characteristics [13] suggesting a stochastic modeling of the governing processes. Here we propose a stochastic representation of the main events involved in motor transport, namely stepping along microtubules and binding and unbinding of molecular motors to the cargo. The function of eukaryotic cells relies on the transport of macromolecules and organelles throughout the cytoplasm. Path- ogenic viruses can exploit a cell’s cytoplasmic transport mecha- nisms [1,2] in order to reach their site of replication. Cytoplasmic transport involves three types of molecular motors. Kinesin and dynein motors use microtubule tracks to move cargo throughout the cytoplasm, while myosin motors interact with actin filaments to move their cargoes [3,4]. Microtubule based transport is usually bidirectional and its mechanism can be explained by the exclusive binding of dynein and kinesin motors to the cargo, motor cooperation and regulation, or a stochastic tug-of-war [5–8]. Exclusive binding of motors has not been reported in cells, while in systems with cooperating motors, additional factors such as on/off switches or coordinators between motors have been postulated for bidirectional transport of large cargo, such as vesicles [7]. The mechanism of bidirectional motor transport by non-coordinated motors of opposite polarity has been the basis of tug-of-war models [7,9]. The proposed model has six parameters, namely the binding, unbinding and stepping rates of plus-end and minus-end motors (herein presumed to be dynein and kinesin, respectively). The step sizes of the motors were set to 28/+8 nm for dynein/kinesin as suggested by the results of single molecule experiments [14,15]. We note that we do not impose any geometrical information on the motors and their binding sites on the virus capsid. The motor protein binding sites on the adenovirus capsid are not known even though a recent cryo-EM image of the structure of the human adenovirus type 2 temperature sensitive mutant revealed the organization of the surface of the virus capsid [16]. In this work we propose a stochastic model for motor transport on microtubules and we systematically identify its parameters using virus trajectories obtained by in vivo imaging (Fig. 1). Trajectories are obtained by live cell microscopy of fluorescently labelled human adenovirus type 2 (Ad2) using confocal microsco- py. Abstract Cytoplasmic transport of organelles, nucleic acids and proteins on microtubules is usually bidirectional with dynein and kinesin motors mediating the delivery of cargoes in the cytoplasm. Here we combine live cell microscopy, single virus tracking and trajectory segmentation to systematically identify the parameters of a stochastic computational model of cargo transport by molecular motors on microtubules. The model parameters are identified using an evolutionary optimization algorithm to minimize the Kullback-Leibler divergence between the in silico and the in vivo run length and velocity distributions of the viruses on microtubules. The present stochastic model suggests that bidirectional transport of human adenoviruses can be explained without explicit motor coordination. The model enables the prediction of the number of motors active on the viral cargo during microtubule-dependent motions as well as the number of motor binding sites, with the protein hexon as the binding site for the motors. Editor: Herbert M. Sauro, University of Washington, United States of America Received June 22, 2009; Accepted November 19, 2009; Published December 24, 2009 Received June 22, 2009; Accepted November 19, 2009; Published December 24, 2009 Gazzola et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which pe ribution, and reproduction in any medium, provided the original author and source are credited. Copyright: 2009 Gazzola et al. This is an open-access article distributed under the terms of the Creative Commons Attr unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the SNSF and the ETH Zurich Research Foundation, and SNSF, Novartis and Kanton Zurich. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. * E-mail: ufgreber@zool.uzh.ch (UFG); petros@ethz.ch (PK) * E-mail: ufgreber@zool.uzh.ch (UFG); petros@ethz.ch (PK) PLoS Computational Biology \| www.ploscompbiol.org Mattia Gazzola1, Christoph J. Burckhardt2, Basil Bayati1, Martin Engelke2, Urs F. Greber2, Petros Koumoutsakos1 1 Chair of Computational Science, ETH Zurich, Zurich, Switzerland, 2 Institute of Zoology, University of Zurich, Zurich, Switzerland December 2009 \| Volume 5 \| Issue 12 \| e1000623 Author Summary The stochastic model reproduces directed motion length and velocity distributions of human adenovirus, and predicts the optimal number of either common or separate binding sites for dynein and kinesin motors on the capsid. (A) Dynein (D, blue) and kinesin (K, orange) bind to, and unbind from the capsid and transport it along a microtubule (green). Equations [1]–[6] describe the dynamics of the model: [3]/[4] dynein binding/unbinding and decrease/increase of the number (r) of the available motor binding sites on the virus capsid with da/dd binding/ unbinding rates, [5]/[6] kinesin binding/unbinding and decrease/ increase of the number(r) of the available motor binding sites on the virus capsid with ka/kd binding/unbinding rates, and [1]/[2] dynein/ kinesin motor stepping with dm/km stepping rates. Cost function (B) and parameter values (C) (blue = dm, green = km, red = da, cyan = ka, yellow = dd, magenta = kd) versus number of evaluations during the optimization of the 14 common binding sites model. Probability distribution of directed motion length (D) and velocity (E) for the in vivo and in silico (black/red) trajectories. (F) Plot of the cost function versus the number of motor binding sites for the common (red) and the separate binding sites model (blue, grey, dark blue, black colours). The separate binding sites have a total number of 8 (blue), 10 (grey), 12 (dark blue) and 14 (black) binding sites for dynein plus kinesin motors. The central dot in each curve represents 50% dynein and 50% kinesin occupancy (e.g. black curve: 7+7). The remaining dots denote permutations with decreasing/increasing dynein binding sites (e.g. 6 dynein + 8 kinesin on the right and 8 dynein +6 kinesin on the left of the central dot of the black curve). (G, H) examples of segmented in silico 1D trajectories for the 14 common (G) or 7+7 separate (H) binding sites models. The distance (mm) traveled along the 1D microtubule is plotted against the time in seconds, and the directed motions are depicted in red. doi:10.1371/journal.pcbi.1000623.g002 Figure 1. Imaging, tracking and trajectory segmentations of single adenoviruses. (A) HeLa cells were infected with fluorescent adenovirus type 2 for 30 min, and imaged by spinning disc confocal fluorescence microscopy [42]. Virus tracks (black lines) recorded by a single particle tracking algorithm [10] using the nucleus (red square) as a reference point are displayed over a phase contrast image of the infected cell. Author Summary It has also been reported for drosophila lipid droplets, that multiple processive motors do not move cargoes faster [24]. It is likely that yet unknown mechanisms account for the high velocities measured in in vivo biological systems. These mechanisms may involve motors which are able to increase their Figure 2. A stochastic model for microtubule-dependent movements of human adenovirus. The stochastic model reproduces directed motion length and velocity distributions of human adenovirus, and predicts the optimal number of either common or separate binding sites for dynein and kinesin motors on the capsid. (A) Dynein (D, blue) and kinesin (K, orange) bind to, and unbind from the capsid and transport it along a microtubule (green). Equations [1]–[6] describe the dynamics of the model: [3]/[4] dynein binding/unbinding and decrease/increase of the number (r) of the available motor binding sites on the virus capsid with da/dd binding/ unbinding rates, [5]/[6] kinesin binding/unbinding and decrease/ increase of the number(r) of the available motor binding sites on the virus capsid with ka/kd binding/unbinding rates, and [1]/[2] dynein/ kinesin motor stepping with dm/km stepping rates. Cost function (B) and parameter values (C) (blue = dm, green = km, red = da, cyan = ka, yellow = dd, magenta = kd) versus number of evaluations during the optimization of the 14 common binding sites model. Probability distribution of directed motion length (D) and velocity (E) for the in vivo and in silico (black/red) trajectories. (F) Plot of the cost function versus the number of motor binding sites for the common (red) and the separate binding sites model (blue, grey, dark blue, black colours). The separate binding sites have a total number of 8 (blue), 10 (grey), 12 (dark blue) and 14 (black) binding sites for dynein plus kinesin motors. The central dot in each curve represents 50% dynein and 50% kinesin occupancy (e.g. black curve: 7+7). The remaining dots denote permutations with decreasing/increasing dynein binding sites (e.g. 6 dynein + 8 kinesin on the right and 8 dynein +6 kinesin on the left of the central dot of the black curve). (G, H) examples of segmented in silico 1D trajectories for the 14 common (G) or 7+7 separate (H) binding sites models. The distance (mm) traveled along the 1D microtubule is plotted against the time in seconds, and the directed motions are depicted in red. doi:10.1371/journal.pcbi.1000623.g002 Figure 2. A stochastic model for microtubule-dependent movements of human adenovirus. Introduction The central dot in each curve represents 50% dynein and 50% kinesin occupancy (e.g. black curve: 7+7). The remaining dots denote permutations with decreasing/increasing dynein binding sites (e.g. 6 dynein + 8 kinesin on the right and 8 dynein +6 kinesin on the left of the central dot of the black curve). (G, H) examples of segmented in silico 1D trajectories for the 14 common (G) or 7+7 separate (H) binding sites models. The distance (mm) traveled along the 1D microtubule is plotted against the time in seconds, and the directed motions are depicted in red. doi:10.1371/journal.pcbi.1000623.g002 Introduction Motility information extracted through single virus tracking [10], and trajectory segmentation [11] are implemented in order to study the properties of virus transport by employing a systems identification process [12] for a stochastic model of cargo transport on microtubules. The six model parameters are inferred through a system identification process using the velocity and displacement distributions of segmented trajectories as the cost function of our optimization. An evolutionary algorithm, capable of handling noisy cost functions, is used to obtain the rates that minimize the distance between the velocity and displacement distributions of the in silico and in vivo trajectories. The velocity distribution in virus trajectories has led to several suggestions regarding the cooperation or lack thereof between molecular motors. High velocities, in the order of a few microns 1 December 2009 \| Volume 5 \| Issue 12 \| e1000623 December 2009 \| Volume 5 \| Issue 12 \| e1000623 A Stochastic Model for Adenovirus Transport Figure 2. A stochastic model for microtubule-dependent movements of human adenovirus. The stochastic model reproduces directed motion length and velocity distributions of human adenovirus, and predicts the optimal number of either common or separate binding sites for dynein and kinesin motors on the capsid. (A) Dynein (D, blue) and kinesin (K, orange) bind to, and unbind from the capsid and transport it along a microtubule (green). Equations [1]–[6] describe the dynamics of the model: [3]/[4] dynein binding/unbinding and decrease/increase of the number (r) of the available motor binding sites on the virus capsid with da/dd binding/ unbinding rates, [5]/[6] kinesin binding/unbinding and decrease/ increase of the number(r) of the available motor binding sites on the virus capsid with ka/kd binding/unbinding rates, and [1]/[2] dynein/ kinesin motor stepping with dm/km stepping rates. Cost function (B) and parameter values (C) (blue = dm, green = km, red = da, cyan = ka, yellow = dd, magenta = kd) versus number of evaluations during the optimization of the 14 common binding sites model. Probability distribution of directed motion length (D) and velocity (E) for the in vivo and in silico (black/red) trajectories. (F) Plot of the cost function versus the number of motor binding sites for the common (red) and the separate binding sites model (blue, grey, dark blue, black colours). The separate binding sites have a total number of 8 (blue), 10 (grey), 12 (dark blue) and 14 (black) binding sites for dynein plus kinesin motors. Author Summary Molecular motors, due to their transportation function, are essential to the cell, but they are often hijacked by viruses to reach their replication site. Imaging of virus trajectories provides information about the patterns of virus transport in the cytoplasm, leading to improved understanding of the underlying mechanisms. In turn improved understand- ing may suggest actions that can be taken to interfere with the transport of pathogens in the cell. In this work we use in vivo imaging of virus trajectories to develop a computational model of virus transport in the cell. The model parameters are identified by an optimization procedure to minimize the discrepancy between in vivo and in silico trajectories. The model explains the in vivo trajectories as the result of a stochastic interaction between motors. Furthermore it enables predictions on the number of motors and binding sites on pathogens, quantities that are difficult to obtain experimentally. Beyond the understanding of mechanisms involved in pathogen transport, the present paper introduces a systematic parameter identification algorithm for stochas- tic models using in vivo imaging. The discrete and noisy characteristics of biological systems have led to increased attention in stochastic models and this work provides a methodology for their systematic development. per second, were observed for intracellular viruses (Fig. 2E) [17]. Similar high speeds have been observed for vesicles moving along microtubules such as peroxisomes [18] and endosomes [19]. These velocities are above the maximum velocities measured for single motors without load (3 mm/s for dynein, [14]; 0.4 mm/s for kinesin-1, [20]; 3 mm/s for kinesin-1, [21]; 0.8 mm/s for kinesin-1, [22]; 0.8 mm/s kinesin-1 and 0.5 mm/s kinesin-2, [23] in in vitro experiments. It has also been reported for drosophila lipid droplets, that multiple processive motors do not move cargoes faster [24]. It is likely that yet unknown mechanisms account for the high velocities measured in in vivo biological systems. These mechanisms may involve motors which are able to increase their per second, were observed for intracellular viruses (Fig. 2E) [17]. Similar high speeds have been observed for vesicles moving along microtubules such as peroxisomes [18] and endosomes [19]. These velocities are above the maximum velocities measured for single motors without load (3 mm/s for dynein, [14]; 0.4 mm/s for kinesin-1, [20]; 3 mm/s for kinesin-1, [21]; 0.8 mm/s for kinesin-1, [22]; 0.8 mm/s kinesin-1 and 0.5 mm/s kinesin-2, [23] in in vitro experiments. A Stochastic Model for Adenovirus Transport parameters and at the same time provides a sensitivity analysis of the model. The standard deviations of the six principal axes are shown to converge (Fig. 3), thus yielding a minimum (Text S1). velocities up to few microns per second or motors are able to act additively to achieve higher speeds. Both assumptions have not been experimentally validated or discarded in in vivo experiments. Additive behaviour of motors is an underlying assumption in our model (Fig. 2A). The additive behaviour is inherent to the Stochastic Simulation Algorithm [25] used herein to simulate the model, since the time step to the next event depends on the total propensity (numbers and event rates). The proposed stochastic model does not impose any explicit coordination between motor proteins, e.g. a switching mechanism that selects a certain motor protein type to be active. After the convergence of the optimization process (Fig. 3) we found that the directed motion length and velocity distributions of the in silico trajectories, under the optimal set of parameters, matched with high accuracy the experimental data (Fig. 2D, E). The maximum number of motors attached to the viral cargo is limited by the number of binding sites on the virus. The present model enables predictions on the number of motor binding sites on the viral capsid, a quantity that is difficult to determine experimentally but important for understanding the mechanisms of transport. We first estimated the number (between 2 and 20) of motor binding sites on the virus by an optimization procedure (Fig. 2F). In models with 6–16 binding sites, the cost function values were almost constant around the minimum value obtained for 14 binding sites (Text S1). For less than 6 motor binding sites, the optimization process did not converge to the experimentally observed directed motion length and velocity distributions. Above 16 binding sites, an unbalanced configuration of motors was feasible only at low binding and unbinding rates, and yielded largely unidirectional trajectories due to infrequent motor binding to the virus. We concluded that 14 common binding sites for dynein and kinesin correspond optimally to the experimental data. We emphasize that our model does not aim at a mechanistic description at the motor level. Forces are known to affect motor properties, but it is not clear how they are distributed among multiple motors [26]. A Stochastic Model for Adenovirus Transport Furthermore while it is possible to obtain data relating forces for certain motors in vitro, there is no such data for in vivo experiments. In the present model the forces between molecular motors and cargo are implicitly taken into account through the binding/unbinding/stepping rates of the stochastic models. Results The model contains no a-priori assumptions on the existence of either a tug- of-war or coordination between molecular motors. In turn, the model parameters are systematically identified with a derando- mized evolution strategy that minimizes the difference between the length and velocity distributions of directed motions (fast microtubule dependent runs [11]) produced by the model and those of fluorescently labelled human adenovirus type 2 (Ad2) as measured by confocal microscopy at 25 Hz temporal resolution. The two-dimensional virus trajectories are extracted by a single particle tracking algorithm [10] (Fig. 1A, B). Directed motions along microtubules are classified by trajectory segmentation [11] and the distance travelled along the microtubule is determined as a function of time (1D trajectory shown in Fig. 1C). The same analysis is applied to trajectories obtained by the simulation of our model using the Stochastic Simulation Algorithm (SSA) [27]. These trajectories are also subsequently segmented to classify directed motions [11]. In turn an optimization algorithm is employed to identify the parameters of the stochastic model [28]. Since it is not possible to differentiate between common and separate binding sites, we additionally investigated the possibility that the experimental data support separate binding sites for the different motors. We optimized a model where dynein and kinesin have distinct binding sites, namely 4+4, 5+5, 6+6, 7+7 binding sites, and various permutations thereof (Fig. 2F, Text S1), and found that an equal number of motor binding sites was optimal in all cases. This is consistent with the observation that center and periphery directed length and velocity distributions were almost symmetric (Fig. 2D, E). We note that the optimal number of binding sites, i.e., 14, is the same for the models with common and separate binding sites (Fig. 2F, black curve). Molecular motors carrying cargo on microtubules operate as individuals or as an ensemble. We found that, on average, during virus directed motions, 1.5660.56 dynein or kinesin (for minus- end and plus-end directed motions, respectively) motors, and 0.1560.22 motors of opposite polarity were bound to the virus (Fig. 4A). The probability of binding more than four motors to one Here the six model parameters (binding, unbinding and stepping for both kinesin and dynein, Fig. 2A) were identified by minimizing the Symmetric Kullback-Leibler divergence between the in silico and in vivo length and velocity distributions using an Evolution Strategy with Covariance Matrix Adaptation (CMA-ES) [29] (Fig. 2B,C). Results The simulation of the stochastic model produces cargo trajectories that depend on the parameter settings. The model contains no a-priori assumptions on the existence of either a tug- of-war or coordination between molecular motors. In turn, the model parameters are systematically identified with a derando- mized evolution strategy that minimizes the difference between the length and velocity distributions of directed motions (fast microtubule dependent runs [11]) produced by the model and those of fluorescently labelled human adenovirus type 2 (Ad2) as measured by confocal microscopy at 25 Hz temporal resolution. The two-dimensional virus trajectories are extracted by a single particle tracking algorithm [10] (Fig. 1A, B). Directed motions along microtubules are classified by trajectory segmentation [11] and the distance travelled along the microtubule is determined as a function of time (1D trajectory shown in Fig. 1C). The same analysis is applied to trajectories obtained by the simulation of our model using the Stochastic Simulation Algorithm (SSA) [27]. These trajectories are also subsequently segmented to classify directed motions [11]. In turn an optimization algorithm is employed to identify the parameters of the stochastic model [28]. Here the six model parameters (binding, unbinding and stepping for both kinesin and dynein, Fig. 2A) were identified by minimizing the Symmetric Kullback-Leibler divergence between the in silico and in vivo length and velocity distributions using an Evolution Strategy with Covariance Matrix Adaptation (CMA-ES) [29] (Fig. 2B,C). The proposed de-randomized optimization algorithm is an essential aspect of our method. CMA-ES samples the six-dimensional multivariate normal distribution involving the parameters of this problem at each iteration and it encodes relations between the parameters of the model and the objective that is being optimized without requiring explicitly the gradients of the cost function [29]. The CMA-ES is a method capable of optimizing noisy cost functions (such as those from the present stochastic model) and its efficiency, reliability and robustness were demonstrated over a number of benchmark problems [30,31]. The CMA-ES is particularly suitable to this optimization problem as it is know to perform best [29] in problems that are low dimensional (here six parameters), inherently noisy (here a stochastic model), multimodal and computationally expensive (for each parameter set thousands of trajectories are generated and segmented to collect reliable statistics). The algorithm identifies an optimal set of The simulation of the stochastic model produces cargo trajectories that depend on the parameter settings. Author Summary (B) Two-dimensional projection of a single virus trajectory with directed motion segments in red. (C) Distance travelled along the trajectory shown in Fig. 1B plotted as a function of time. Reduction to 1D is justified, since in cultured cells microtubules are largely flat and straight over distances of many micrometers [43]. Negative/positive values indicate displacements towards the cell centre/periphery. doi:10.1371/journal.pcbi.1000623.g001 Figure 1. Imaging, tracking and trajectory segmentations of single adenoviruses. (A) HeLa cells were infected with fluorescent adenovirus type 2 for 30 min, and imaged by spinning disc confocal fluorescence microscopy [42]. Virus tracks (black lines) recorded by a single particle tracking algorithm [10] using the nucleus (red square) as a reference point are displayed over a phase contrast image of the infected cell. (B) Two-dimensional projection of a single virus trajectory with directed motion segments in red. (C) Distance travelled along the trajectory shown in Fig. 1B plotted as a function of time. Reduction to 1D is justified, since in cultured cells microtubules are largely flat and straight over distances of many micrometers [43]. Negative/positive values indicate displacements towards the cell centre/periphery. doi:10.1371/journal.pcbi.1000623.g001 December 2009 \| Volume 5 \| Issue 12 \| e1000623 2 PLoS Computational Biology \| www.ploscompbiol.org A Stochastic Model for Adenovirus Transport PLoS Computational Biology \| www.ploscompbiol.org Results By considering the size (90 nm in diameter) and icosahedral geometry of the virus and the cylindrical microtu- bule (25 nm in diameter), we can postulate that the maximum number of microtubule motor-capsid interactions occurs along the edge of a capsid facet, in this case on hexon (Fig. 5A, B). This arrangement implies that 9 hexon trimers are aligned with the microtubule, giving a maximum of 27 hexon binding sites for the motors. This is above the value of 14 binding sites predicted from the simulations. If we assume, however, that the motor protein binding sites are located at the interface of two trimeric hexons, one microtubule filament could cover 1–15 sites (Fig. 5B, red lines), which is within the predicted range of 6–16. In addition to hexon, 6 to 8 binding sites were available for pIX, and less than 5 for penton base which detaches to a significant extent from the incoming virions before reaching the cytosol [34]. We analyzed trajectories of pIX-deficient adeno- viruses to distinguish between hexon and pIX binding sites for motor proteins [35]. The directed motion length and velocity distributions of pIX-deleted adenovirus were similar to those from wild-type viruses without significant deviations or asym- metries, indicating that pIX may not provide a binding site for microtubule dependent motors during cytoplasmic transport (Fig. 5C, D). Therefore, we predict that hexon harbours the binding sites for dynein and kinesin motors. Figure 4. A low number of dynein and kinesin motors mediate directed motions of adenovirus. (A) In silico probabilities of the number of bound dynein (black) and kinesin (red) motors during periphery directed motions. Similar results were observed for center directed motions with dynein prevalence. Phase diagrams of the length (B) of directed motions versus the average number of bound dynein (black) and kinesin (red). Positive (negative) lengths correspond to periphery (center) directed motions. The results were obtained with the optimized model with 14 common binding sites. doi:10.1371/journal.pcbi.1000623.g004 virus particle was below 1023, and most often only one type of motor was bound (Fig. 4A, B). These data are in agreement with low number of motors estimated on vesicular cargo in squid axoplasm by cryo-EM [7]. For other organelles, the estimates for motor numbers range from a few to dozens based on immunological detections in chemically fixed cells. Results In order to quantify the correlation between the number of bound motors and the directed motion length, the Sample Pearson Product Moment correlation coefficient (with a range of 0 to 1, where 1 is maximal correlation) between motor numbers and directed motion length was computed to be 0.51 for dynein and 0.49 for kinesin for minus-end and plus-end directed motions, respectively. This implies a weak correlation between the number of bound motors and the directed motion length, showing that long runs do not necessarily require many motors, as two or three already account for lengths in the order of micrometers (Fig. 4B). This result is consistent with the recent in vitro observation that two motors are sufficient to move a cargo over several micrometers [32]. Figure 5. Hexon not protein IX is the predicted to be motor binding site on the adenovirus capsid. (A) Schematic model of the icosahedral adenovirus capsid with the major capsid protein hexon (blue hexagonal structures representing trimers), and pIX (yellow lines representing trimers). An icosahedral facette is enlarged in (B), where hexon-hexon trimer interfaces are depicted in red and in light green the microtubule orientation that maximizes the overlap with one facet. Note that the fourth trimer of pIX is covered by the red lines depicting hexon-hexon interfaces. (C, D) Directed motion length and velocity distributions for protein IX deficient adenovirus recorded in HeLa cells 30 to 90 min post infection. doi:10.1371/journal.pcbi.1000623.g005 The velocities, derived from optimized stepping rates, for single dynein and kinesin motors were 866 nm/s and 833 nm/s, respectively, consistent with values reported for dynein and conventional kinesin-1 or kinesin-2 [21–23,33]. Although kinesins are currently not known to be involved in cytoplasmic transport of adenovirus [1], the model makes a clear prediction for a plus end directed motor in cytoplasmic transport of adenovirus. Our findings indicate that microtubule-based motility of adenovirus requires a low number of bound motors compared to the number of binding sites on the capsid. This allows configurations where only one motor type is bound, and thereby produce directed motions. Low numbers of motors allow fast switches between directions and therefore, bidirectional motion. Importantly, the binding and unbinding rates were much smaller than the stepping rates, which is key for directed motion runs (Fig. 2C). Small perturbations of binding and unbinding rates greatly affect the model dynamics (Text S1). PLoS Computational Biology \| www.ploscompbiol.org Results The proposed de-randomized optimization algorithm is an essential aspect of our method. CMA-ES samples the six-dimensional multivariate normal distribution involving the parameters of this problem at each iteration and it encodes relations between the parameters of the model and the objective that is being optimized without requiring explicitly the gradients of the cost function [29]. The CMA-ES is a method capable of optimizing noisy cost functions (such as those from the present stochastic model) and its efficiency, reliability and robustness were demonstrated over a number of benchmark problems [30,31]. The CMA-ES is particularly suitable to this optimization problem as it is know to perform best [29] in problems that are low dimensional (here six parameters), inherently noisy (here a stochastic model), multimodal and computationally expensive (for each parameter set thousands of trajectories are generated and segmented to collect reliable statistics). The algorithm identifies an optimal set of Figure 3. Convergence of the standard deviation of the principal axes. Convergence is shown for the six axes of the distribution which CMA-ES samples from. The evolution of the standard deviations during the optimization procedure is shown for the non- competing binding sites model with 14 receptors. doi:10.1371/journal.pcbi.1000623.g003 Figure 3. Convergence of the standard deviation of the principal axes. Convergence is shown for the six axes of the distribution which CMA-ES samples from. The evolution of the standard deviations during the optimization procedure is shown for the non- competing binding sites model with 14 receptors. doi:10.1371/journal.pcbi.1000623.g003 PLoS Computational Biology \| www.ploscompbiol.org December 2009 \| Volume 5 \| Issue 12 \| e1000623 3 A Stochastic Model for Adenovirus Transport Figure 4. A low number of dynein and kinesin motors mediate directed motions of adenovirus. (A) In silico probabilities of the number of bound dynein (black) and kinesin (red) motors during periphery directed motions. Similar results were observed for center directed motions with dynein prevalence. Phase diagrams of the length (B) of directed motions versus the average number of bound dynein (black) and kinesin (red). Positive (negative) lengths correspond to periphery (center) directed motions. The results were obtained with the optimized model with 14 common binding sites. doi:10.1371/journal.pcbi.1000623.g004 present on cytosolic viruses that have undergone stepwise disassembly [34]. Cytosolic particles contain the major protein hexon, a large fraction of the pentameric penton base at the icosahedral vertex, and protein IX (pIX), which stabilizes hexon. Materials and Methods HeLa cells were grown to 30% confluency on 18 mm glass cover slips (Menzel Glaser) and kept in Hanks buffered salt solution containing 0.5% BSA (Sigma) and 1 mg/ml ascorbic acid (Sigma). Adenovirus serotype 2 and protein IX deficient adenoviruses were grown, isolated, and labeled with atto565 (Atto-tec, Germany) as described by Nakano and Greber in [41] and Suomalainen et al. in [17]. In our work kinetic rates of a stochastic model are determined via an evolutionary optimization approach using experimental data. The identified model enables a number of predictions. First, it shows that one to four motors are active on virus particles during microtubule-dependent motions, although the number of motor binding sites is estimated to be 6–16. The observation that the cost function value is constant within this range suggests that the virus may align with the microtubule in different orientations (Fig. 5B) and still preserve its motility. This range is consistent with the maximum of 15 hexon trimer-trimer interfaces along the edge of a capsid facet. The low number of motors involved in directed motions supports an emerging concept from wet lab experiments and in silico simulations, that key events of cell functions are in many cases executed by only a few polypeptides [36]. HeLa cells were infected with Ad-atto565 and imaged between 30 and 90 minutes post infection at 25 Hz. Flat regions of the cell were chosen for imaging in order to minimize the cytoplasmic volume above the imaging plane. The center of the cell was detected by differential interference contrast imaging to assign directionality to the virus motions. Images were recorded using a spinning disc confocal micro- scope (Olympus IX81) fitted with an UplanApo100x objective of N.A. 1.35 on a back-illuminated monochrome Cascade 512 EM-CCD camera (Photometrics) containing a 5126512 pixel chip (with 16616 micrometer large pixels). For the computational methods see Text S1. Second, if equal numbers of opposite motors are attached, the cargo oscillates and eventually stops, or remains confined to small areas. This may be an important mechanism for fine-tuning the subcellular velocity to achieve localized delivery of the cargo. We anticipate that viral transport is tuned by the binding and unbinding rates of motors to microtubules or the cargo, rather than by additional regulatory factors. Acknowledgments We thank Diego Rossinelli for his support during the code parallelization and Jort Vellinga for Figure 4A. Results The susceptibility of motor based cargo transport to these parameters has been reported in other theoretical studies [26] and hints to a possible mechanism to regulate the run length of the motors [32]. Figure 5. Hexon not protein IX is the predicted to be motor binding site on the adenovirus capsid. (A) Schematic model of the icosahedral adenovirus capsid with the major capsid protein hexon (blue hexagonal structures representing trimers), and pIX (yellow lines representing trimers). An icosahedral facette is enlarged in (B), where hexon-hexon trimer interfaces are depicted in red and in light green the microtubule orientation that maximizes the overlap with one facet. Note that the fourth trimer of pIX is covered by the red lines depicting hexon-hexon interfaces. (C, D) Directed motion length and velocity distributions for protein IX deficient adenovirus recorded in HeLa cells 30 to 90 min post infection. doi:10.1371/journal.pcbi.1000623.g005 The present results enabled an assessment on the virus binding sites for motor proteins. The outer surface of adenovirus particles is composed of five polypeptides, three of which are still December 2009 \| Volume 5 \| Issue 12 \| e1000623 4 A Stochastic Model for Adenovirus Transport Supporting Information Text S1 Supplementary information includes details on the computational methods used. In particular it describes the trajectory segmentation process, the models studied, the stochastic simulation algorithm and the definition of the cost function used in the optimization procedure. Found at: doi:10.1371/journal.pcbi.1000623.s001 (1.05 MB PDF) Author Contributions Conceived and designed the experiments: MG CJB BB UFG PK. Performed the experiments: CJB ME. Analyzed the data: MG CJB BB. Conceived and designed the experiments: MG CJB BB UFG PK. Performed the experiments: CJB ME. Analyzed the data: MG CJB BB. We close by noting that besides the results on motor transport on microtubules, the algorithm taken here is in line with reverse engineering and systems identification approaches [28,38–40] Contributed reagents/materials/analysis tools: PK. Wrote the paper: MG CJB BB UFG PK. Contributed reagents/materials/analysis tools: PK. Wrote the paper: MG CJB BB UFG PK. Contributed reagents/materials/analysis tools: PK. Wrote the paper: MG CJB BB UFG PK. Discussion which are gaining significance as discovery and model validation tools in systems biology. The CMA-ES algorithm is capable of handling noisy and multimodal cost functions that are inherently associated with stochastic models. The CMA-ES optimization algorithm along with image analysis of in vivo systems can be a robust process to help identify parameters of stochastic models employed in several areas of systems biology. In this study, we use in vivo imaging to identify a stochastic model of cargo transport by molecular motors on microtu- bules. The model parameters were systematically identified using live imaging data of virus trajectories and a de- randomized optimization algorithm to minimize the Kull- back-Leibler divergence between the length and velocity distributions of adenovirus directed motions on microtubules with the in silico trajectories produced by the model. The model accounts for directed motions at mm/s speeds, processive stepping over hundreds of nanometres, and periods of stationary behaviour. The results show that the stochastic model can result in bidirectional support without an explicit coordination mechanism. Materials and Methods Such tuning could be cell- type specific [17], and could control the number of engaged motors and motor configuration, and also provide specific segregation or guidance cues for traffic. In support of this, it has been suggested that the microtubule binding protein Tau can fine- tune the distance that the cargo travels by reducing microtubule binding of kinesin in distal parts of neuronal axons [7,37]. In addition, motor properties can be tuned by post-translational modifications, such as phosphorylation of dynein or kinesin binding partners, which could affect their enzymatic functions and hence their stepping rates [7]. 2. Brandenburg B, Zhuang XW (2007) Virus trafficking-learning from single-virus tracking. Nature Reviews Microbiology 5: 97–208. 12. Astrom KJ, Eykhoff P (1971) System Identification – Survey. Automatica 7: 123–162. 1. Greber UF, Way M (2006) A superhighway to virus infection. Cell 124: 741–754. 3. Schliwa M, Woehlke G (2003) Molecular motors. Nature 422: 759–765. 14. Gennerich A, Carter AP, Reck-Peterson SL, Vale RD (2007) Force-induced bidirectional stepping of cytoplasmic dynein. Cell 131: 952–965. 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https://openalex.org/W4311304169	https://tmfv.com.ua/journal/article/download/1880/1530	English	null	Assessment of Training Loads of Highly Qualified Basketball Players	Teorìâ ta metodika fìzičnogo vihovannâ	2,022	cc-by	4,509	© Koryahin, V., 2022. Abstract h The purpose of the study is: 1) development and unification of a system of training loads monitoring by the nature of the physiological impact on the basketball players’ body; 2) study of the strategy of preparing a highly qualified basketball team for important international competitions, namely: to find out the nature of the distribution of means of different physiological, as well as technical-tactical and game orientation at different stages of preparation; 3) test basketball players before and after training. t Materials and methods. A team of highly qualified basketball players (masters of sports and masters of sports of international class) in the number of 15 people participated in the research. The assessment of physical load by the nature of the physiological impact on the body of basketball players during 18 microcycles using 4 – 1 system, that is, 4 working days and 1 day off, was carried out. Athletes were tested before and after training.h ft Results. The dynamics of the means of training by the nature of the physiological impact on the athletes’ body have been established. The nature of the distribution of means at different stages of preparation has been established. The test results before and after training are shown. ht Conclusions. The research results showed that a certain accounting system based on assessments of the nature of the tasks to be solved and the degree of compliance with the specifics of their game activities has developed in basketball. At the same time, research have indicated the need for accounting and analyzing of the training loads by the nature of physiological impact on the body of athletes.i Keywords: basketball, training loads, highly qualified basketball players. ТМФВ ТОВ ОВС Viktor Koryahin1ABCDE 1Lviv Polytechnic National University Authors’ Contribution: A – Study design; B – Data collection; C – Statistical analysis; D – Manuscript Preparation; E – Funds Collection Authors’ Contribution: A – Study design; B – Data collection; C – Statistical analysis; D – Manuscript Preparation; E – Funds Collection Corresponding Author: Viktor Koryahin, E-mail: koryahinv@meta.ua Accepted for Publication: Oсtober 25, 2022 Published: November 30, 2022 Corresponding Author: Viktor Koryahin, E-mail: koryahinv@meta.ua Accepted for Publication: Oсtober 25, 2022 Published: November 30, 2022 DOI: 10.17309/tmfv.2022.3s.19 ISSN 1993-7989 (print) ISSN 1993-7997 (online) ISSN-L 1993-7989 Introduction stages of training; 4) development and unification of the sys tem of control over the level of training of basketball players.hii In the theory and practice of basketball, the issues of planning and managing the training process are resolved, as a rule, based on the experience and intuition of coach es (Kondrashin & Koryahin, 1978; Koryahin, 2018; Anas tasiadis, 2006).f The first step towards creating a unified system of ac counting and analysis of the training loads can be the clas sification of exercises used as training tools according to certain characteristics (Altberg, 1971; Poplavsky, 2004; Semashko, 1976).h Effective management of the training of basketball play ers is possible only when solving the following tasks (Kon drashin & Koryahin, 1978; Koryahin, 1998): 1) creation of a unified system of accounting and analysis of the train ing loads; 2) determination of the most effective means and methods of training; 3) optimization of the construction of the training process, i.e. finding ways of the most rational distribution of means and methods of training at different The systems developed earlier were built on the basis of assessments of the nature of the technical-tactical tasks being solved and the system of their compliance with the specifics of the game activity of basketball players (Raiola, Altavilla, Tafuri, & Lipoma, 2016; Ben Abdelkrim, Castagna, El Fazaa, & El Ati, 2010; Gonzalo-Skok, Sánchez-Sabaté, Izquierdo- Lupón, & Sáez de Villarreal, 2019). Another approach to systematization is based on accounting of the physiological impact of loads on the body of athletes (Hoare, 2000; Mont gomery, Pyne, & Minahan, 2010; Ciuti, Marcello, Macis, Onnis, Solinas, & Lai, 2009). S137 S137 SN 1993-7997. ISSN-L 1993-7989. Physical Education Theory and Methodology. Vol. 22, Num. 3 Supplement ISSN 1993-7989. eISSN 1993-7997. ISSN-L 1993-7989. Physical Education Theory and Methodology. Vol. 22, N the amount of 15 people took part in the research during the period of preparation for important international competitions. During the training period, a 4–1 microcycle was used, that is, 4 working days and 1 day off. A total of 18 microcycles were performed. During the training period, basketball players were tested in order to determine the level of their physical and technical preparedness before and after training. Introduction The following tests were used: 6 m and 20 m run, jumping height, shots from the spot (the number of hits out of 40 shots and the time of operation were determined), free throws out of 30 (number of hits), running across the court from one front line to another 3 times for 40 seconds after 1 minute of rest (number of meters covered), Cooper test (Koryahin, 1998; Koryahin, Hrebinka, Prystynskyi, & Prystynska, 2022). When establishing the gradations of training loads in basketball, two fundamental approaches can be used (Kon drashin & Koryahin, 1978): y 1. Systematization of exercises, taking into account the characteristics of physical loads. y 1. Systematization of exercises, taking into account the characteristics of physical loads. p y 2. Gradation of loads on the base of accounting of physi ological changes in the body of athletes that occur when performing exercises. g However, in basketball, such a determination of the pa rameters of physical exertion is not always possible and a greater priority should be given to the accounting of the physiological reactions of the body that occur during physi cal loads. Based on the established ratios between the indi cators of physical load and physical shifts in the body, all exercises used in the training of highly qualified basketball players were divided into 4 types of training loads: y 1. Aerobic exercises. 1. Aerobic exercises. Results The results on the distribution of means of different physiological orientation are presented in Fig. 1 As can be seen from Fig. 1, the volume of means of aero bic impact gradually increased from stage to stage, but de creased noticeably during the period of the team’s participa tion in international tournaments (8–9, 13–16 microcycles, see Fig. 2). At the same time, the volume of aerobic-anaero bic exercises increased significantly. With the main interna Statistical analysis 2. Exercises of mixed aerobic-anaerobic orientation. To maintain a laboratory journal, to perform operations on sorting the results obtained and their storage, and to cal culate statistical data, the MS Excel program was used. The choice of mathematical statistics methods was an adequate to the goals and objectives of our study and included the use of the following methods: the arithmetic mean of the primary data, the error of the average value, the standard deviation and the reliability of the difference between the data obtained. 3. Exercises of anaerobic-alactate orientation. 4. Exercises of anaerobic glycolytic orientation. It should be noted that the determination of the im mediate training effect of special exercises and their system atization by the nature of the physiological impact do not yet determine how rationally it will be built as a whole. The main question in this problem is: which and in what volume the exercises should be used? Therefore, it is important to find out what is the strategy of training in basketball, what means are given more attention, what are the dynamics of these means at different stages of training? Clarifying these issues allows us to summarize previous experience and sub sequently make adjustments to the training process. Participants and research organization A team of highly qualified basketball players (masters of sports and masters of sports of international class) in Fig. 1. Dynamics of the volume of training means of different physiological orientation, which were used during the preparation of a highly qualified basketball team for important international competitions: Ordinate axis – the volume of training load, min; Abscissa axis – microcycles, number; a – volume of training load, mainly aerobic orientation; b – aerobic-anaerobic orientation; c – anaerobic alactate and anabolic orientation; d – anaerobic glycolytic orientation. Fig. 1. Dynamics of the volume of training means of different physiological orientation, which were used during the preparation of a highly qualified basketball team for important international competitions: Ordinate axis – the volume of training load, min; Abscissa axis – microcycles, number; a – volume of training load, mainly aerobic orientation; b – aerobic-anaerobic orientation; c – anaerobic alactate and anabolic orientation; d – anaerobic glycolytic orientation. S138 Koryahin, V. (2022). Assessment of Training Loads of Highly Qualified Basketball Players Table 1. Results of control tests of basketball players before and after the preparation for important international competitions (n = 15) tional competitions approaching, the volume of aerobic and mixed aerobic-anaerobic means decreased. At this stage, the volume of speed-power means (alactate anaerobic impact) increased and for the first time during the entire training pe riod, exercises aimed at developing the speed endurance of basketball players (anaerobic-glycolytic effects) were used.h Table 1. Results of control tests of basketball players before and after the preparation for important international competitions (n = 15) Indicators Examination Difference reliability 1st 2nd Running at 6 m, sec. 1.61±0.67 0.107 1.36±0.067 0.107 >0.99 Running at 20 m, sec. Participants and research organization 3.55±0,079 0.125 3.06±0.168 0.267 >0.99 Jumping height, cm 48.8±4.25 6.75 49.0±4.44 7.05 <0.95 Throws out of 40 number of hits time of work, sec. 20.5±2.7 4.29 4.21±0.1 0.159 24.0±2.12 3.37 4.04±0.173 0.276 >0.95 <0.95 Free throws out of 30, number of hits 20.1±2,51 3.99 23.5±1.73 2.76 >0.95 Running 3×40 sec., m 563±16.6 26.4 572±17.0 27.0 <0.95 Cooper Test, m 2939±87 138 3057±96 153 >0.95 y g y yf The results of the distribution of means of technical, tactical, technical- tactical and game training are presented in Fig. 2. Fig. 2. Dynamics of distribution of means of technical, technical-tactical, tactical and game training at different stages of preparation of basketball players for important international competitions: Ordinate axis – amount of time, min. Abscissa axis – microcycles, number. Columns in Fig. 1, 2, 3 – participation in international tournaments. Technical training Technical–tactical training Tactical training Game training 1 2 3 1 2 3 400 300 200 100 0 0 2 4 6 8 10 12 14 16 18 400 300 200 100 0 0 2 4 6 8 10 12 14 16 18 0 2 4 6 8 10 12 14 16 18 0 2 4 6 8 10 12 14 16 18 1 2 3 1 2 3 400 300 200 100 0 400 300 200 100 0 Technical training Technical–tactical training 1 2 3 1 2 3 400 300 200 100 0 0 2 4 6 8 10 12 14 16 18 400 300 200 100 0 0 2 4 6 8 10 12 14 16 18 Technical training 1 2 3 400 300 200 100 0 0 2 4 6 8 10 12 14 16 18 Tactical training Game training 0 2 4 6 8 10 12 14 16 18 0 2 4 6 8 10 12 14 16 18 1 2 3 1 2 3 400 300 200 100 0 400 300 200 100 0 ball, “scoring” the ball into the basket from top to bottom, etc.), helps to maintain the level of jumping ability. However, strength training cannot be ignored. As shown by a study by Jim Hanley, 1973, qualified athletes have fewer opportunities to increase results in jumping ability. The most effective for the strength developing, strength increasing and jumping ability in qualified athletes are exercises with large and maxi mum weights, which includes jumping on a bench, lifting a barbell, half-squats and deep squats. Participants and research organization Our experiment with qualified basketball players confirms this. Physical train ing, which was allotted 31.9% of the time, included crosses, fartlek, gymnastic exercises, acceleration, jumping with one or both legs pushing up and moving forward, stairs, over barriers, etc. Exercises with weights were not used, which affected the results in the jumping test. Fig. 2. Dynamics of distribution of means of technical, As can be seen from Fig. 2, there is a certain pattern in the distribution of means aimed at improving technical, tactical and game training. The volume of technical training gradually decreases as official responsible international com petitions approach, the volume of tactical and game training increases.ht f Studies have shown that it is necessary to carry out the gradation of loads based not only on traditional assessments of the nature of the technical-tactical tasks being solved, but also on the basis of accounting of the physiological impact of loads on the body of athletes. A significant contribution here can be the establishment of an urgent training effect of special exercises of basketball players and, on this basis, the creation of a unified system of accounting of the training loads, their systematization by the nature of the physiologi cal changes that they cause in the body of basketball players. The results of testing basketball players before and after the preparation for important international competitions are shown in Table 1. As can be seen from the table, basketball players in creased their performance in all tests, with the exception of jumping height. It should be noted that in such tests as the time of work in throws from spots, running 3×40 sec onds, the differences are not reliable (p < 0.95), but, in any case, there is no decrease in indicators. There are practi cally no differences in the average values of jumping height indicators (1st examination – 48.8 cm, 2nd examination – 49.0 cm). Therefore, coaches should not ignore the thesis that qualified basketball players can develop jumping ability without using exercises with weights. This, of course, does not mean that you do not need to use jumping exercises in training. In addition, jumping exercises develop “explosive” strength, coordination of movements, specific skills (for ex ample, during a jump shot, fighting for the rebound of the Discussion The question of the importance of planning and manag ing the training process was considered by many scientists and coaches at different stages of basketball development (Semashko, 1976; Yukhno, & Preobrazhensky, 1967; Sha blinsky, 1989; Koryahin, 2018).h The results of our research coincide with a number of scientific studies (Ben Abdelkrim, Castagna, El Fazaa, & El Ati, 2010; Poplavskyi, 2004; Ferreira, Volossovitch, & Sam S139 S139 SN 1993-7997. ISSN-L 1993-7989. Physical Education Theory and Methodology. Vol. 22, Num. 3 Supplement SSN 1993-7989. eISSN 1993-7997. ISSN-L 1993-7989. Physical Education Theory and Methodology. Vol. 22, Num. paio, 2014) on the need to develop a system for planning and managing the training load, creating a unified system for accounting and analysis of training load.h Koryahin, V. (2018). Effectiveness of basketball players training process. Journal of Physical Education and Sport, 16(2), 163, 1029-1030h Anastasiadis, M. (2006). The basketball training. Athens: IN. Elvekalt EPE. The results of our studies showed a certain accounting system has developed in basketball based on assessments of the nature of the tasks to be solved and the degree of compliance with the specifics of their game activities (Su shko & Mustafa, 2015; Mitova & Ivchenko, 2014). At the same time, studies have indicated the need of accounting and analysis of training loads by the nature of the physiologi cal impact on athletes’ bodies (Koryahin, Blavt, Doroshenko, Prystynskyi & Stadnyk, 2020; Anastasiadis, 2006). Koryahin, V. (1998). Training of the highly skilled basketball players. Textbook for the correct physical education. Lviv: Publishing house «Krai», 192. Altberg, O. (1971). Study of the process of training and competitive loads intensification for basketball players of the highest ranks. Author’s abstract. Dis. cand. Tartu. Poplavsky, L. Y. (2004). Basketball. Kyiv:Publishing House “Olympic literature”, 446. y y y y Based on the study results, it can be stated that in order to effectively manage the training process, a certain accounting system is needed. Suchlike at this stage of scientific research is the gradation of special exercises of basketball players ac cording to 4 groups, namely: exercises of aerobic, mixed aer obic-anaerobic, anaerobic alactate and anaerobic glycolytic orientation (Koryahin, Hrebinka, Prystynskyi, & Prystynska, 2022; Koryahin, Blavt, Prystynskyi, & Stadnyk, 2021).h Semashko, N. (1976). Basketball. Moscow: Physical culture and sport. 264 Raiola, G., Altavilla, G., Tafuri, D., & Lipoma, M. (2016). Analysis of learning a basketball shot. Journal of Physical Education and Sport, 16(1), 1, 3-7. Discussion https://doi.org/10.7752/jpes.2016.01001 Ben Abdelkrim, N., Castagna, C., El Fazaa, S., & El Ati, J. (2010). The effect of players’ standard and tactical strategy on game demands in men’s basketball. Journal Strength and Conditioning Research, 24, 2652-62. https://doi.org/10.1519/jsc.0b013e3181e2e0a3 y y y y y The proposed system of accounting for training loads in basketball opens up new opportunities for improving the management system of training processes. Further research should be aimed at finding the nature of the interaction of urgent training effects of special exercises of basketball play ers and on this basis improve the system of accounting for loads by the nature of the physiological impact of the exer cises used in the training of basketball players. Gonzalo-Skok, O., Sánchez-Sabaté, J., Izquierdo-Lupón, L., & Sáez de Villarreal, E. (2019). Influence of force-vector and force application plyometric training in young elite basketball players. European Journal of Sport Science, 19(3), 305-314. https://doi.org/10.1080/17461391.2018.1502357 Conclusions Studies have shown that the dynamics of the volume of training means of different physiological orientation dur ing the preparation of a highly qualified team for important international competitions is uneven, which affects the re sults of control examinations in special tests (see Table 1). Therefore, the distribution of training means at different stages of training should take into account not only techni cal, technical-tactical, tactical and game training, but also without fail, taking into account the nature of the physiologi cal impact of exercises on the body of athletes. Koryahin, V., Hrebinka, H., Prystynskyi, V., & Prystynska, T. (2022). Methodology for Determining the SpeedPower Capabilities of Basketball Players. Physical Education Theory and Methodology, 22(1), 14-18. https://doi.org/10.17309/tmfv.2022.1.02 Hanley, B. J. (1973). Strength training for jumping ability. Scholastic Coach, 43(3). Yukhno, M., & Preobrazhensky, I. (1967). Control exercises for special physical and technical training for basketball players of «A» class teams, national teams of the republics and the USSR. Methodical development. Moscow. 14. The author declares no conflict of interest. The author declares no conflict of interest. Ferreira, A. P, Volossovitch, A., & Sampaio, J. (2014). Towards the game critical moments in basketball: a grounded theory approach. International Journal of Performance Analysis in Sport, 14(2), 428-442. https://doi.org/10.1080/24748668.2014.11868732 Conclusions Hoare, D.G. (2000). Predicting success in junior elite basketball players–the contribution of anthropometic and physiological attributes. Journal Sci Med Sport, 3(4): 391- 405. https://doi.org/10.1016/s1440-2440(00)80006-7h 1. Studies have shown that effective management of the training process of highly qualified basketball players is pos sible not only on the basis of assessing the nature of the tech nical and tactical tasks to be solved and the degree of their compliance with the game activity of basketball players, but also, which is very important, by accounting the physiologi cal impact of loads on the body of athletes.h 1. Studies have shown that effective management of the training process of highly qualified basketball players is pos sible not only on the basis of assessing the nature of the tech nical and tactical tasks to be solved and the degree of their compliance with the game activity of basketball players, but also, which is very important, by accounting the physiologi cal impact of loads on the body of athletes.h Montgomery, P. G., Pyne, D. B., & Minahan, C. L. (2010). The physical and physiological demands of basketball training and competition. International Journal of Sports Physiology and Performance, 5, 75-86. https://doi.org/10.1123/ijspp.5.1.75 p y 2. The proposed gradation of the loads of highly quali fied basketball players based on the accounting of physi ological changes caused by the performance of exercises, allows effectively manage the training process. Ciuti, S., Marcello, S., Macis, A., Onnis, E., Solinas, R., & Lai, S. (2009). Improved aerobic power by detraining in basketball players mainly trained for strength. Journal Sports Medicine, Training and Rehabilitation, 6(4), 325- 335. https://doi.org/10.1080/15438629609512063 f y g g p 3. Studies have shown that the dynamics of the volume of training means of different physiological orientation dur ing the preparation of a highly qualified team for important international competitions is uneven, which affects the re sults of control examinations in special tests (see Table 1). Therefore, the distribution of training means at different stages of training should take into account not only techni cal, technical-tactical, tactical and game training, but also without fail, taking into account the nature of the physiologi cal impact of exercises on the body of athletes. f y g g p 3. Kondrashin, V., & Koryahin, V. (1978). Training of the basketball players of high grade. Kyiv: Publishing house “Zdorov’ya”, 93. Conflict of interest Shablinsky, V. (1989). Basketball. What do I know about it? Kyiv: Publishing house “Molod’”, 141. Реферат. Стаття: 5 с., 1 табл., 2 рис., 22 джерела. Мета дослідження: 1. Розробка і уніфікація системи контролю за тренувальними навантаженнями за характером фізіологічного впливу на організм баскетболістів. 2. Дослідити стратегію підготовки баскетбольної команди високої кваліфікації до відповідальних міжнародних змагань, а саме: з’ясувати характер розподілу засобів різної фізіологічної, а також техніко-тактичної і ігрової спрямованості на різних етапах підготовки. Матеріали і методи. В дослідженнях брала участь команда баскетболістів високої кваліфікації (майстри спорту і майстри спорту міжнародного класу) у кількості 15 осіб у період підготовки до відповідальних міжнародних змагань. У період підготовки використовувався мікроцикл 4-1, тобто 4 робочих дні і 1 вихідний. Усього було проведено 18 мікро циклів. У період підготовки проводилося тестування баскетболістів з метою визначення рівня їх фізичної і технічної під готовленості до початку і після закінчення підготовки. Використовувались такі тести: біг на 6 м і 20 м; висота вистрибу вання; кидки з точок (визначалась кількість попадань з 40 кидків і час роботи); штрафні кидки з 30 (кількість попадань); біг майданчиком від одної лицьової лінії до іншої 3 рази по 40 секунд через 1 хвилину відпочинку (кількість подоланих метрів); тест Купера (Koryahin, 1998; Koryahin, Hrebinka, Prystynskyi, & Prystynska, 2022). у y y y y y y y Результати. Проведені дослідження показали, що ефективне керування тренувальним процесом баскетболістів висо кої кваліфікації можливе не тільки на основі оцінок характеру вирішуваних техніко-тактичних і ігрових задач, але й при обліку характеру фізіологічного впливу навантажень на організм спортсменів. Результати тестування баскетболістів до початку проведення й після закінчення показали, що в цілому система підготовки позитивно відобразилась на результа тах у всіх тестах, окрім висоти вистрибування (перше обстеження 48,8±4.25 см, друге обстеження – 49,0±4.44 см, Р<0.95), часу роботи у кидковому тесті (перше обстеження – 4,21±0.1 сек., друге обстеження – 4,04±0.173 сек., Р<0.95), у тесті з бігу 3×40 сек. (перше обстеження – 563±16.6 сек., друге обстеження – 572,04±17.0 сек., Р<0.95). Це свідчить про необхідність більш ефективного, науково обґрунтованого розподілу засобів на різних етапах підготовки. Висновки. Дослідження показали, що ефективне керування тренувальним процесом баскетболістів високої кваліфі кації можливе не тільки на основі оцінок характеру вирішуваних технічних, тактичних задач й ступеня їх відповідності ігровій активності баскетболістів, але й, що є досить важливим, обліку фізіологічного впливу навантажень на організм спортсменів. Запропонована градація навантажень баскетболістів високої кваліфікації на основі обліку фізіологічних змін, що викликані виконанням вправ, дозволяє ефективно керувати тренувальним процесом. Динаміка об’ємів тре нувальних засобів різної фізичної спрямованості у період підготовки висококваліфікованої команди до відповідальних міжнародних змагань, носить нерівномірний характер, що відображається на результатах контрольних випробувань у спеціальних тестах. References Kondrashin, V., & Koryahin, V. (1978). Training of the basketball players of high grade. Kyiv: Publishing house “Zdorov’ya”, 93. Kondrashin, V., & Koryahin, V. (1978). Training of the basketball players of high grade. Kyiv: Publishing house “Zdorov’ya”, 93. S140 S140 Koryahin, V. (2022). Assessment of Training Loads of Highly Qualified Basketball Players Sushko, R., & Mustafa, A. (2015). Evaluation of technical and tactical activities of basketball players with taking into account the differences of the periods of play activity. Kyiv: Publishing house “Olympic literature”, 177.h Koryahin, V., Blavt, O., Prystynskyi, V., & Stadnyk, V. (2021). Research of training loads of basketball players. Physical education theory and methodology, 21(2), 107-112. https://doi.org/10.17309/tmfv.2021.2.02 Mitova, O., & Ivchenko, O. (2014). The current state of control over the level of integrated training of basketball players at the stage of preliminary basic training. Sports Herald, Prydniprovya, 3, 72-76. Mitova, O., & Ivchenko, O. (2014). The current state of control over the level of integrated training of basketball players at the stage of preliminary basic training. Sports Herald, Prydniprovya, 3, 72-76. Koryahin, V., Blavt, O., Doroshenko, E., Prystynskyi, V., & Stadnyk, V. (2020). Training effect of special basketball exercises. Physical education theory and methodology, 20(3), 137-141. https://doi.org/10.17309/tmfv.2020.3.02 Koryahin, V., Blavt, O., Prystynskyi, V., & Stadnyk, V. (2021). Research of training loads of basketball players. Physical education theory and methodology, 21(2), 107-112. https://doi.org/10.17309/tmfv.2021.2.02 Віктор Корягін1АВCDE 1Національний університет «Львівська політехніка» 1Національний університет «Львівська політехніка» Реферат. Стаття: 5 с., 1 табл., 2 рис., 22 джерела. Koryahin, V., Blavt, O., Doroshenko, E., Prystynskyi, V., & Stadnyk, V. (2020). Training effect of special basketball exercises. Physical education theory and methodology, 20(3), 137-141. https://doi.org/10.17309/tmfv.2020.3.02 Koryahin, V., Blavt, O., Prystynskyi, V., & Stadnyk, V. (2021). Research of training loads of basketball players. Physical education theory and methodology, 21(2), 107-112. https://doi.org/10.17309/tmfv.2021.2.02 Information about the authors: Koryahin V.: viktor.m.koryahin@lpnu.ua; https://orcid.org/0000-0003-1472-4846; Department of Physical Education, Lviv Polytechnic National University, Bandera St, 12, Lviv, 79013, Ukraine. Cite this article as: Koryahin, V. (2022). Assessment of Training Loads of Highly Qualified Basketball Players. Physical Education Theory and Methodology, 22(3s), S137-S141. https://doi.org/10.17309/tmfv.2022.3s.19 Received: 02.09.2022. Accepted: 25.10.2022. Published: 30.11.2022 This work is licensed under a Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0). Реферат. Стаття: 5 с., 1 табл., 2 рис., 22 джерела. Тому розподіл засобів тренування на різних етапах підготовки повинен здійснюватися із врахуван ням не тільки технічної, техніко-тактичної і ігрової підготовки, але й у обов’язковому порядку з урахуванням характеру фізіологічного впливу вправ на організм спортсменів. б б б б ф ф у р р р Ключові слова: баскетбол, тренувальні навантаження, баскетболісти високої кваліфікації. Information about the authors: Koryahin V.: viktor.m.koryahin@lpnu.ua; https://orcid.org/0000-0003-1472-4846; Department of Physical Education, Lviv Polytechnic National University, Bandera St, 12, Lviv, 79013, Ukraine. Cite this article as: Koryahin, V. (2022). Assessment of Training Loads of Highly Qualified Basketball Players. Physical Education Theory and Methodology, 22(3s), S137-S141. https://doi.org/10.17309/tmfv.2022.3s.19 Received: 02.09.2022. Accepted: 25.10.2022. Published: 30.11.2022 This work is licensed under a Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0). Information about the authors: d under a Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0). This work is licensed under a Creative Commons Attribution 4.0 International License (http://creativecommons S141
https://openalex.org/W2121651169	https://europepmc.org/articles/pmc3564776?pdf=render	English	null	Deep transcriptome-sequencing and proteome analysis of the hydrothermal vent annelid Alvinella pompejana identifies the CvP-bias as a robust measure of eukaryotic thermostability	Biology direct	2,013	cc-by	13,577	* Correspondence: gaspar.jekely@tuebingen.mpg.de; fulvia.bono@tuebingen. mpg.de 1Max-Planck-Institute for Developmental Biology, Spemannstr. 35, Tübingen D-72076, Germany Full list of author information is available at the end of the article Holder et al. Biology Direct 2013, 8:2 http://www.biology-direct.com/content/8/1/2 Holder et al. Biology Direct 2013, 8:2 http://www.biology-direct.com/content/8/1/2 Deep transcriptome-sequencing and proteome analysis of the hydrothermal vent annelid Alvinella pompejana identifies the CvP-bias as a robust measure of eukaryotic thermostability Deep transcriptome-sequencing and proteome analysis of the hydrothermal vent annelid Alvinella pompejana identifies the CvP-bias as a robust measure of eukaryotic thermostability Thomas Holder1, Claire Basquin3, Judith Ebert3, Nadine Randel1, Didier Jollivet2, Elena Conti3, Gáspár Jékely1* and Fulvia Bono1* Abstract Background: Alvinella pompejana is an annelid worm that inhabits deep-sea hydrothermal vent sites in the Pacific Ocean. Living at a depth of approximately 2500 meters, these worms experience extreme environmental conditions, including high temperature and pressure as well as high levels of sulfide and heavy metals. A. pompejana is one of the most thermotolerant metazoans, making this animal a subject of great interest for studies of eukaryotic thermoadaptation. Results: In order to complement existing EST resources we performed deep sequencing of the A. pompejana transcriptome. We identified several thousand novel protein-coding transcripts, nearly doubling the sequence data for this annelid. We then performed an extensive survey of previously established prokaryotic thermoadaptation measures to search for global signals of thermoadaptation in A. pompejana in comparison with mesophilic eukaryotes. In an orthologous set of 457 proteins, we found that the best indicator of thermoadaptation was the difference in frequency of charged versus polar residues (CvP-bias), which was highest in A. pompejana. CvP-bias robustly distinguished prokaryotic thermophiles from prokaryotic mesophiles, as well as the thermophilic fungus Chaetomium thermophilum from mesophilic eukaryotes. Experimental values for thermophilic proteins supported higher CvP-bias as a measure of thermal stability when compared to their mesophilic orthologs. Proteome-wide mean CvP-bias also correlated with the body temperatures of homeothermic birds and mammals. Conclusions: Our work extends the transcriptome resources for A. pompejana and identifies the CvP-bias as a robust and widely applicable measure of eukaryotic thermoadaptation. Reviewer: This article was reviewed by Sándor Pongor, L. Aravind and Anthony M. Poole. © 2013 Holder et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Holder et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. RESEARCH Open Access Open Access Background temperatures of approximately 68°C, compared to tem- peratures of approximately 20°C in the surrounding water [3]. Given the steep temperature gradient inside the tubes and the difficulty of carrying out such in situ measure- ments, the maximum body temperature A. pompejana can tolerate is unclear [1]. Direct temperature preference and tolerance experiments using a high-pressure aquar- ium with a thermal gradient have not yet been carried out on adult A. pompejana. However, such experiments have shown that a North-Pacific relative of A. pompejana, Paralvinella sulfincola, prefers temperatures between 40°C and 50°C and tolerates temperatures up to 55°C [4]. The Alvinella pompejana is one of the most heat tolerant of all animals known to date [1]. This annelid worm inhabits deep-sea hydrothermal vent chimney walls in self-made glycoprotein tubes [2], where it is exposed to extreme en- vironmental conditions (high pressure, high temperature, low pH, anoxia, heavy metals). In situ measurements in- side occupied tubes, near the animals’ tails, revealed Page 2 of 16 Holder et al. Biology Direct 2013, 8:2 http://www.biology-direct.com/content/8/1/2 habitat of A. pompejana is similar to that of P. sulfincola, and it is likely that adult A. pompejana have a similar ther- mal preference. Given its high temperature tolerance, there has been considerable interest in studying the mechanisms of thermoadaptation in A. pompejana, and in establishing sequence-resources for this organism [5]. stability for A. pompejana proteins [14], or measured parameters (e.g. optimal temperatures for enzyme acti- vity) that only allowed an indirect assessment of thermal stability. For example, the extracellular giant hemoglobin of A. pompejana showed higher oxygen affinity than that of other annelids, and exhibited other functional proper- ties related to the vent environment [15], but its macro- molecular assembly was unstable at 50°C, and it was not more thermostable than earthworm hemoglobin [14]. A thermoadapted metazoan proteome could greatly benefit structural biology research. The advantage of using thermostable proteins for structural studies has been well documented. In general, proteins from ther- mophiles are more stable and less flexible than their mesophilic counterparts. Consequently, these proteins are more amenable for expression, purification and crystallization experiments, and have often been used to solve the structure of large macromolecular complexes. Well-known examples include the ribosome, whose complete atomic structure was first determined from the thermophilic eubacterium Thermus thermophilus [6], and the exosome, first purified and crystallized from the archaebacterium Sulfolobus solfataricus [7]. Background Among the eukaryotes, the fungus Chaetomium thermophilum has re- cently been shown to have a thermoadapted proteome, and this facilitated the structural study of nuclear pore components [8]. Among the metazoans, A. pompejana po- tentially represents a promising source of thermostable proteins and complexes. Given the small number of biochemical studies and the uncertainties about the thermotolerance of the ani- mals, the general degree of thermoadaptation of the A. pompejana proteome is still unclear. Sequence analysis of a large number of proteins could reveal general features of thermoadaptation. There have been many attempts to correlate protein thermal stability with sequence or struc- ture derived features [16]. When comparing sequence composition of thermophiles and mesophiles, the most apparent difference is an enrichment of charged residues in combination with a decrease in the number of polar residues in the thermophilic proteins. Both the (E + K)/(Q + H) ratio and the CvP-bias (difference in the frequency between charged and polar residues) can discriminate hyperthermophiles from mesophiles [17] as well as baro- philes [18]. Another study identified a universal set of residues, I,V,Y,W,R,E,L (IVYWREL measure), enriched in thermophiles [19]. More complex discrimination func- tions have also been proposed, such as the function employed by THERMORANK [20]. This method uses a linear combination of 10 sequence-based features to rank a set of input sequences by relative thermostability. Ano- ther method, the Tm-Index tool, uses dipeptide compo- sition to predict a melting point index for a single protein sequence [21]. These measures have not yet been systematically tested on the Alvinella proteome. Although A. pompejana was first described more than 30 years ago, biochemical data have only been published for a small subset of its proteins. Melting temperature (Tm) values, the most direct measure of thermal stability, have only been published for cuticular and interstitial collagen [9,10]. These proteins have melting tempera- tures of 45–46°C, 17 degrees higher than that of col- lagens from shallow seawater annelids [9,10]. The activity of A. pompejana and human recombinant DNA polymer- ase η (Pol η) following high temperature incubation has also been tested. A. pompejana Pol η maintained high activity following incubation at temperatures up to 49°C, whereas human Pol η maintained high activity only until 43°C. At 52°C the activity of Pol η from both species dropped below 20% [11]. A. Background large orthologous dataset with a taxon sampling that included other annelids, as well as the thermostable fungus, Chaetomium thermophilum [8]. To investigate the extent of thermoadaptation of A. pompejana we then performed a systematic survey of the available sequence-based thermo- stability measures previously established for prokaryotes on its proteome. Testing the THERMORANK, IVYWREL, Tm-Index, (E + K)/(Q + H), and loop-length gave conflict- ing results. Our analyses identified the CvP-bias as the best measure to discriminate A. pompejana from mesophilic eukaryotes. The CvP-bias also discriminated the thermo- philic fungus C. thermophilum from mesophilic eukaryotes. The correlation of the CvP-bias with thermostability was also supported by experimentally-determined thermostabi- lity data. into a reference transcriptome dataset. Large-scale EST sequencing projects have also been carried out by the Joint Genome Institute (JGI) and Genoscope [5], yielding a total of 218,458 publicly available ESTs (as of Feb 2011). We also assembled these sequences with our resource, creating a combined MPI + JGI + Genoscope dataset. To estimate the number of novel sequences in our resource (MPI dataset, Additional file 1) we compared it with the already available ESTs (JGI + Genoscope dataset, Additional file 2), as well as the combined dataset (MPI + JGI + Genoscope dataset, Additional file 3). First, we com- pared the length distribution of contigs and singletons in the three assemblies (Figure 1A). The final combined assem- bly nearly doubled the available transcriptome resources for A. pompejana, with 60,475 contigs longer than 500 bp, compared to 34,860 in the JGI + Genoscope dataset. Due to the small size of the 454 and Illumina reads, the MPI and the MPI + JGI + Genoscope datasets were dominated by short contigs (Figure 1A). However, they also contained a larger number of long contigs (>1,500 bp) than the JGI + Genoscope dataset (7,269 in MPI and 9,639 in MPI + JGI + Genoscope compared to 3,634 in JGI + Genoscope). Background pompejana superoxide dismu- tase (SOD) was also shown to have enhanced chemical stability relative to its human counterpart by guanidine de- naturation, a measure thought to correlate with thermal stability. In this study the structure of A. pompejana SOD was also solved at high resolution and for the first time in complex with H2O2 [12]. The A. pompejana splicing factor U2AF65 in complex with RNA has also been shown to have a slightly increased thermal stability (using RNA- binding as a readout) relative to the human protein (6°C higher) [13]. Recently, a large A. pompejana cDNA resource, pre- pared from three different tissues and whole animals was published [5]. Analysis of this dataset in comparison with various metazoan homologs showed that A. pompe- jana protein sequences have the highest proportion of charged amino acids. This bias was interpreted as a sign of protein thermostability [5]. Another EST resource, generated by the Joint Genome Institute (JGI, http:// www.jgi.doe.gov/), is also publicly available. These A. pompejana EST resources could form the basis for re- constituting and determining structures of metazoan proteins and multiprotein complexes. However, in order for structural biologists to identify all components of large multiprotein complexes, where the lack of a single component will impede reconstitution, more extensive sequence coverage is essential. To increase the available dataset, we first carried out deep sequencing of the A. pompejana transcriptome. Using this resource and the published sequences, we established a The aforementioned studies showed increased thermo- stability of some A. pompejana proteins. In contrast, other studies either did not reveal higher thermal Page 3 of 16 Holder et al. Biology Direct 2013, 8:2 http://www.biology-direct.com/content/8/1/2 Holder et al. Biology Direct 2013, 8:2 http://www.biology-direct.com/content/8/1/2 Holder et al. Biology Direct 2013, 8:2 http://www.biology-direct.com/content/8/1/2 large orthologous dataset with a taxon sampling that included other annelids, as well as the thermostable fungus, Chaetomium thermophilum [8]. To investigate the extent of thermoadaptation of A. pompejana we then performed a systematic survey of the available sequence-based thermo- stability measures previously established for prokaryotes on its proteome. Testing the THERMORANK, IVYWREL, Tm-Index, (E + K)/(Q + H), and loop-length gave conflict- ing results. Our analyses identified the CvP-bias as the best measure to discriminate A. pompejana from mesophilic eukaryotes. The CvP-bias also discriminated the thermo- philic fungus C. thermophilum from mesophilic eukaryotes. The correlation of the CvP-bias with thermostability was also supported by experimentally-determined thermostabi- lity data. Results Deep sequencing of the A. pompejana transcriptome To gain insights into the mechanisms of thermoadaptation of A. pompejana, we first performed deep sequencing of its transcriptome. We used a combination of the Sanger, Roche/454 and Illumina technologies. With the 454 tech- nology we obtained 2,717,445 reads of an average length of 220 bp. Using Illumina paired-end sequencing (~300 bp fragments) we obtained 87 million reads of an average length of 76 bp. These resources, and an additional 10,063 novel Sanger ESTs we generated, were assembled Annotation of predicted A. pompejana proteins Next we searched the nucleotide datasets for potential open reading frames and analyzed the predicted protein sequences (Additional file 4, 5 and 6). We identified B Length of predicted proteins (aa) 0 2,000 4,000 6,000 8,000 60-80 100-120 140-160 180-200 220-240 260-280 300-320 340-360 380-400 420-440 460-480 >500 Number of predicted proteins MPI MPI+JGI+Genoscope JGI+Genoscope equences in three A. pompejana datasets. (A) Histograms representing PI JGI G ( d) d JGI G ( ) bli 0 5,000 10,000 15,000 20,000 200-300 400-500 600-700 800-900 1000-1100 1200-1300 1400-1500 >1500 MPI MPI+JGI+Genoscope JGI+Genoscope Length of contigs (bp) Number of contigs A B Length of predicted proteins (aa) 0 2,000 4,000 6,000 8,000 60-80 100-120 140-160 180-200 220-240 260-280 300-320 340-360 380-400 420-440 460-480 >500 Number of predicted proteins MPI MPI+JGI+Genoscope JGI+Genoscope Figure 1 Size distribution of cDNA contigs and predicted protein sequences in three A. pompejana datasets. (A) Histograms representing the number of contigs in the different size-ranges for the MPI (black), MPI + JGI + Genoscope (red) and JGI + Genoscope (cyan) assemblies. (B) Histograms representing the number of predicted proteins in the different size-ranges for the MPI (black), MPI + JGI + Genoscope (red) and JGI + Genoscope (cyan) datasets. 0 5,000 10,000 15,000 20,000 200-300 400-500 600-700 800-900 1000-1100 1200-1300 1400-1500 >1500 MPI MPI+JGI+Genoscope JGI+Genoscope Length of contigs (bp) Number of contigs A A B MPI MPI+JGI+Genoscope JGI+Genoscope Number of predicted proteins Number of contigs Length of predicted proteins (aa) Figure 1 Size distribution of cDNA contigs and predicted protein sequences in three A. pompejana datasets. (A) Histograms representing the number of contigs in the different size-ranges for the MPI (black), MPI + JGI + Genoscope (red) and JGI + Genoscope (cyan) assemblies. (B) Histograms representing the number of predicted proteins in the different size-ranges for the MPI (black), MPI + JGI + Genoscope (red) and JGI + Genoscope (cyan) datasets. Page 4 of 16 Holder et al. Biology Direct 2013, 8:2 http://www.biology-direct.com/content/8/1/2 datasets, including the A. pompejana datasets. We then counted the number of C. teleta proteins that had a sig- nificant hit (e-value 1e-5) in all animals, but not outside animals, or all eukaryotes. These two subsets of proteins (general animal and general eukaryotic) are also expected to be present in A. pompejana. We therefore counted how many of these two subsets had a BLAST hit in the A. pompejana datasets (Table 2). Annotation of predicted A. pompejana proteins We found that the com- bined A. pompejana resource is about 74-99% complete, depending on the subset of proteins examined. Animal- specific proteins were highly covered, between 74-94%, depending on whether we considered all sequences or only full-length sequences. Proteins present in all eukar- yotes had even higher percent coverage (87-99%). This is probably due to the higher expression levels of genes with general eukaryotic cellular functions. When we con- sidered only full-length A. pompejana proteins, the MPI + JGI + Genoscope dataset was 74% complete for animal- specific proteins, compared to the 55% completion of the JGI + Genoscope dataset. These searches show that the A. pompejana resource is about 74-99% complete at the level of eukaryotic paralogous families and protein domains. Overall, our sequencing efforts greatly extended the known A. pompejana transcriptome and proteome. many novel full-length and partial sequences (Figure 1B and Table 1), and also extended the length of already known partial proteins. We then compared the A. pompejana predicted proteins in the MPI + JGI + Genoscope dataset to those already available in the JGI + Genoscope dataset. We found 13,301 sequences (>100 aa) in the MPI + JGI + Genoscope dataset with no identical BLAST hit (fraction identical <90%) to the JGI + Genoscope dataset. We then performed BLASTP searches with these sequences in the predicted proteome of the annelid Capitella teleta, the closest relatives of A. pompejana with complete genome information (phylogen- etic tree in Additional file 7). We found that 3,897 C. teleta sequences had one or more significant hits (e-value 1e-5) among the novel A. pompejana proteins. These represent newly identified, conserved A. pompejana proteins. In addition, our data also extended the length of many truncated protein sequences in the JGI + Genoscope dataset. In a BLASTP comparison of the predicted protein datasets, 2,776 query sequences from the MPI + JGI + Genoscope dataset were at least 40 amino acids longer than their corre- sponding fragments in the JGI + Genoscope dataset. Next, we annotated the combined predicted A. pompe- jana proteome using BLASTP (Additional file 8). We defined the sequences based on their first hit in the SwissProt database, and further annotated them with the first hit in the C. teleta, human, Danio rerio and Drosophila melanogaster proteomes. Annotation of predicted A. pompejana proteins We also identified 775 sequences with no BLASTP hit in 26 eukaryotic genomes (including 18 animals) but significant hits in prokaryotes (in the UniRef90 database). These were annotated as potential contaminants or genes that originated by recent lateral gene transfer. A future A. pompejana genome sequencing project could distinguish between these two possibilities. th start and stop codon), partial (with stop codon), and total number of predicted protein sequences in the three datasets clustered at tity. Generation of an orthologous set of protein sequences Generation of an orthologous set of protein sequences As a prerequisite for a thorough assessment of the thermo- adaptation of the A. pompejana proteome we generated a large set of orthologous protein sequences from A. pompe- jana and nine other eukaryotic species. Importantly, our orthologous set also included three other lophotrochozoan species, the annelids C. teleta and Helobdella robusta and the mollusk Lottia gigantea. To be able to identify general features of thermoadaptation in eukaryotes, we also included the thermotolerant fungus, Chaetomium thermo- philum, and a mesophilic yeast, Saccharomyces cerevisiae in the orthologous set. In order to estimate the completion of the A. pompe- jana datasets, we performed extensive BLASTP searches in the proteome of 15 animal and 8 additional eukaryotic species. We used C. teleta protein sequences as query and performed BLASTP searches in the 23 eukaryotic The orthologous set was generated using an all-against- all BLASTP search and the reciprocal best-hit approach, Table 1 Size of A. pompejana protein datasets Table 1 Size of A. pompejana protein datasets Dataset % identity #full-length #partial with stop #total MPI (New data) 100 6 272 15 886 28 169 98 5 778 14 893 26 992 90 5 667 14 502 26 433 JGI + Genoscope (Existing data) 100 6 233 15 539 23 962 98 5 360 13 365 19 890 90 5 008 12 341 18 155 MPI + JGI + Genoscope (Combined data) 100 10 778 26 068 42 665 98 9 359 23 131 38 185 90 8 722 21 288 35 235 Number of full-length (with start and stop codon), partial (with stop codon), and total number of predicted protein sequences in the three datasets clustered at 100%, 98% and 90% identity. Table 1 Size of A. pompejana protein datasets Number of full-length (with start and stop codon), partial (with stop codon), and total number of predicted protein sequences in the three datasets clustered at 100%, 98% and 90% identity. Page 5 of 16 Page 5 of 16 Holder et al. Biology Direct 2013, 8:2 http://www.biology-direct.com/content/8/1/2 Table 2 Estimated completion of the A. pompejana proteome Table 2 Estimated completion of the A. pompejana proteome C. teleta A. Sequence based thermostability ranking Protein sequence composition of thermophilic prokaryotes differs significantly from mesophilic protein sequences. The general trend of an increased number of charged residues and a decreased number of polar residues was reported sev- eral times [16,17,22]. We next performed sequence-based thermostability ranking calculations using a variety of other computa- tional methods. All measures were used to rank the whole orthologous set of 457 proteins. We found that THERMORANK [20], “Tm Predictor” [21], the IVY- WREL measure [19] and the (E + K)/(Q + H) ratio [23], all failed to rank A. pompejana or C. thermophilum as the most thermostable species (Figure 3A-D). In the THERMORANK analysis, for example, A. pompejana ranks behind H. robusta and L. gigantea, also highlighting the im- portance of broad taxon-sampling when performing these comparisons. Additionally, the length of the protein sequences in the trimmed multiple alignments (an indica- tion of the length of surface loops) [24] did not identify A. pompejana proteins as thermostable, and ranked the two fungi as the species with the most compact proteins It was previously reported that A. pompejana proteins were enriched in charged amino acids [5]. This was inter- preted as a sign for enhanced thermostability. Our set of orthologous sequences only partly supports this observa- tion. Compared to the protein sequences from Drosophila melanogaster, Danio rerio and Homo sapiens, A. pompe- jana is strongly enriched in Lys, and slightly enriched in Asp, but not in Glu or Arg. However, the enrichment in Lys and Asp is shared with H. robusta and L. gigantea, two other lophotrochozoan species that do not live at high temperatures. These two species also have fewer Ala than A. pompejana, a sequence feature that has previously been proposed to be associated with thermostability [20]. Figure 2 Amino acid compositions in the orthologous protein datasets of the analysed species. Percent of amino acid content calculated from the orthologous set of 457 protein sequences in the 10 species: Alvinella pompejana (Apo), Capitella teleta (Cap), Helobdella robusta (Hel), Lottia gigantea (Lot), Homo sapiens (Hum), Drosophila melanogaster (Dro), Danio rerio (Dan), Caenorhabditis elegans (Cae), Saccharomyces cerevisiae (Sac), Chaetomium thermophilum (Cha). Figure 2 Amino acid compositions in the orthologous protein datasets of the analysed species. Generation of an orthologous set of protein sequences pompejana JGI + Genoscope (Existing data) MPI (New data) MPI + JGI + Genoscope (Combined data) JGI + Genoscope full length (Existing data) MPI full length (New data) MPI + JGI + Genoscope full length (Combined data) Hits to all 14 animals 1164 (100%) 932 (80%) 1017 (87%) 1074 (92%) 639 (55%) 708 (61%) 859 (74%) Hits to all 22 eukaryotes 3958 (100%) 3862 (98%) 3849 (97%) 3922 (99%) 3036 (77%) 3179 (80%) 3454 (87%) Number of BLAST hits in the different A. pompejana datasets using two subsets of the Capitella proteome: sequences that only give hits in all of the 14 animals included, but not outside metazoa (hits to all 14 animals only), and sequences that have hits in all 14 animals included and 8 other non-metazoan eukaryotes. Total number of hits is given as well as the percentage of a hit relative to the total number of Capitella queries (100%). Number of BLAST hits in the different A. pompejana datasets using two subsets of the Capitella proteome: sequences that only give hits in all of the 14 animals included, but not outside metazoa (hits to all 14 animals only), and sequences that have hits in all 14 animals included and 8 other non-metazoan eukaryotes. Total number of hits is given as well as the percentage of a hit relative to the total number of Capitella queries (100%). and contained 457 members (Additional file 9). We used this dataset to search for signals of thermoadaptation using a broad range of sequence-based methods that have been proposed in the thermostability literature. The fungus C. thermophilum is strongly enriched in Ala, Gly, Pro, and Arg. This reflects the high GC-content of this species (see below). However, the enrichment in Arg is compensated by a reduction in Lys content, resulting in no global enrichment in charged residues in C. thermophilum. We conclude that the number of charged residues alone does not distinguish A. pompejana and C. thermophilum from mesophilic species (Figure 2). Sequence based thermostability ranking Percent of amino acid content calculated from the orthologous set of 457 protein sequences in the 10 species: Alvinella pompejana (Apo), Capitella teleta (Cap), Helobdella robusta (Hel), Lottia gigantea (Lot), Homo sapiens (Hum), Drosophila melanogaster (Dro), Danio rerio (Dan), Caenorhabditis elegans (Cae), Saccharomyces cerevisiae (Sac), Chaetomium thermophilum (Cha). Holder et al. Biology Direct 2013, 8:2 http://www.biology-direct.com/content/8/1/2 Page 6 of 16 Lot Alv Sac Cap Dan Cae Dro Cha Hel Alv Cha Alv Cha Alv Cha Alv Cha Alv Cha Alv Cha Alv Cha Alv A B C D E F G H 0 100 200 300 400 0 Number of Sequences Cumulative ranks Intrinsic disorder Cha I Figure 3 Cumulative ranking of the orthologous protein set from the analyzed species based on various thermostability measures. (A-E) Cumulative ranks for the orthologus set based on (A) THERMORANK, (B) IVYWREL, (C) Tm-index, (D) (E + K)/(Q + H), (E) length of trimmed sequence in alignment. (F) Average hydrophobicity versus charged residue content for the orthologous set. (G-I) Cumulative ranks for the orthologus set based on (G) Serine content, (H) CvP-bias, and (I) intrinsic disorder (IUPred score [25]). The species are as in Figure 2. Alv Cha A Alv Cha C Alv Cha B C A B Alv Cha D D F E Alv Cha E Lot Alv Sac Cap Dan Cae Dro Cha Hel F G Alv Cha G I Alv Cha H I Alv 0 100 200 300 400 0 Number of Sequences Cumulative ranks Intrinsic disorder Cha I H Intrinsic disorder Intrinsic disorder Figure 3 Cumulative ranking of the orthologous protein set from the analyzed species based on various thermostability measures. (A-E) Cumulative ranks for the orthologus set based on (A) THERMORANK, (B) IVYWREL, (C) Tm-index, (D) (E + K)/(Q + H), (E) length of trimmed sequence in alignment. (F) Average hydrophobicity versus charged residue content for the orthologous set. (G-I) Cumulative ranks for the orthologus set based on (G) Serine content, (H) CvP-bias, and (I) intrinsic disorder (IUPred score [25]). The species are as in Figure 2. (Figure 3E). The average hydrophobicity versus charged residues, a measure that showed A. pompejana as an outlier [5], clustered A. pompejana with mesophilic L. gigantea (Figure 3F). A reduction in intrinsic protein disorder may also correlate with thermoadaptation. However, when we performed protein disorder predictions [25] on the ortholo- gous set, A. pompejana ranked as average. C. Close correlation of the CvP value with experimentally determined thermal stabilities Out of all the sequence-based thermostability measures used, only the CvP-bias ranked both A. pompejana and C. thermophilum highest. To test how well this measure correlates with thermal stability, we calculated the CvP- bias for proteins with experimentally determined protein stabilities (Table 4). Despite limited sample size, the CvP-bias values correlated well with experimentally determined stabilities for A. pompejana Pol η [11], collagen [9,10] and U2AF65 [13], and for C. thermophi- lum Nup170, Nup192 [8], and xylanase [30], when com- pared to mesophilic proteins. We also calculated the GC-content for the nucleotide sequences corresponding to the protein sequences in our trimmed orthologous set (Table 3). We found that C. thermophilum had the highest GC-content, A. pompejana had a GC-content below the average of the dataset, and L. gigantea, H. robusta and S. cerevisiae had the lowest GC- content values. We plotted the ratio of GC-rich codons (GARP residues) against the ratio of AT-rich codons (FYMINK residues; Figure 5A) [29] across the ortologous set and found a very strong correlation, indicating that GC-content strongly influences amino acid composition. We also tested how individual amino acids are influenced by GC-content and found that the frequency of GARP residues as well as of Trp, Ile and Lys correlates with GC- content (Additional file 10). The thermostability measures we applied, however, do not correlate with GC-content To further test the reliability of the CvP-bias measure, we searched for A. pompejana proteins with the lowest CvP-bias ranking among metazoans. We identified 26 proteins where A. pompejana ranked lowest, including the exosome component Rrp4. We expressed and puri- fied recombinant Rrp4 proteins from A. pompejana, False positive rate True positive rate 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 False positive rate True positive rate 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 False positive rate True positive rate 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 Dataset 1 False positive rate True positive rate 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 IVYWREL 0.991 Tm-index 0.754 (E+K)/(Q+H) 0.966 CvP 0.996 Serine (neg.) 0.925 THERMORANK 0.961 IUPred (short, neg.) 0.933 Thermus vs. Sequence based thermostability ranking thermophilum ranked highest (Figure 3I), probably due to the high content of Pro, Ala, Gly and Arg, residues that strongly contribute to structural disorder [25]. We found only two thermostability measures that weakly discriminated A. pompejana and C. thermophilum from the other species. Low serine content [26] ranks A. pompejana and C. thermophilum first and second in cumulative rank- ings, although C. thermophilum receives very similar scores to human (Figure 3G). The CvP-bias [17] was the only measure that ranked A. pompejana and C. thermophilum as the two most thermostable species (Figure 3H). (Figure 3E). The average hydrophobicity versus charged residues, a measure that showed A. pompejana as an outlier [5], clustered A. pompejana with mesophilic L. gigantea (Figure 3F). A reduction in intrinsic protein disorder may also correlate with thermoadaptation. However, when we performed protein disorder predictions [25] on the ortholo- gous set, A. pompejana ranked as average. C. thermophilum ranked highest (Figure 3I), probably due to the high content of Pro, Ala, Gly and Arg, residues that strongly contribute to structural disorder [25]. (Figure 3E). The average hydrophobicity versus charged residues, a measure that showed A. pompejana as an outlier [5], clustered A. pompejana with mesophilic L. gigantea (Figure 3F). A reduction in intrinsic protein disorder may also correlate with thermoadaptation. However, when we performed protein disorder predictions [25] on the ortholo- gous set, A. pompejana ranked as average. C. thermophilum ranked highest (Figure 3I), probably due to the high content of Pro, Ala, Gly and Arg, residues that strongly contribute to structural disorder [25]. We validated all the measures used on prokaryotic thermophile-mesophile test datasets. All measures robustly Page 7 of 16 Holder et al. Biology Direct 2013, 8:2 http://www.biology-direct.com/content/8/1/2 (Figure 5B-F), indicating that the observed ranking is not influenced by GC-content. discriminated a self-compiled Thermus thermophilus (a thermophilic bacterium) vs. Deinococcus radiodurans (an extremophilic but not thermophilic bacterium) data- set. Other published datasets were also tested, and most measures distinguished thermophiles from mesophiles [20,27,28] (Figure 4). Close correlation of the CvP value with experimentally determined thermal stabilities Deinococcus Dataset 3 Dataset 4 A B C D E False positive rate True positive rate 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 IVYWREL ex (E+K)/(Q+H) CvP Ser r THERMORANK Dataset 2 IVYWREL 0.898 Tm-index 0.605 (E+K)/(Q+H) 0.965 CvP 0.925 Serine (neg.) 0.728 THERMORANK 0.984 IUPred (short, neg.) 0.593 IVYWREL 0.860 Tm-index 0.566 (E+K)/(Q+H) 0.728 CvP 0.880 Serine (neg.) 0.777 THERMORANK 0.731 IUPred (short, neg.) 0.650 IVYWREL 0.956 Tm-index 0.579 (E+K)/(Q+H) 0.945 CvP 0.959 Serine (neg.) 0.776 THERMORANK 0.959 IUPred (short, neg.) 0.758 IVYWREL 0.909 Tm-index 0.533 (E+K)/(Q+H) 0.649 CvP 0.890 Serine (neg.) 0.894 THERMORANK 0.504 IUPred (short, neg.) 0.588 Figure 4 Thermostability measures in prokaryotic thermophile-mesophile ortologous protein datasets. (A-E) Receiver operating characteristic (ROC) curves for (hyper-)thermophile-mesophile homologous protein pairs from (A) one self-compiled dataset (Thermus thermophilus vs. Deinococcus radiodurans) and (B-E) four published datasets were tested with the different thermostability measures to discriminate thermophiles from mesophiles. Dataset 1 (B) is from ref. [20], dataset 2 (C) is from ref. [27], dataset 3 and 4 (D, E) are from ref. [28] (containing hyperthermophiles versus mesophiles and thermophiles versus mesophiles respectively). True positive rate 0.0 0.0 0.2 0.4 0.6 0.8 1.0 Dataset 1 False positive rate True positive rate 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 IVYWREL 0.991 Tm-index 0.754 (E+K)/(Q+H) 0.966 CvP 0.996 Serine (neg.) 0.925 THERMORANK 0.961 IUPred (short, neg.) 0.933 Thermus vs. Deinococcus A B C False positive rate True positive rate 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 IVYWREL 0.898 Tm-index 0.605 (E+K)/(Q+H) 0.965 CvP 0.925 Serine (neg.) 0.728 THERMORANK 0.984 IUPred (short, neg.) 0.593 False positive rate True positive rate 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 C Dataset 2 IVYWREL 0.860 Tm-index 0.566 (E+K)/(Q+H) 0.728 CvP 0.880 Serine (neg.) 0.777 THERMORANK 0.731 IUPred (short, neg.) 0.650 IVYWREL 0.991 Tm-index 0.754 (E+K)/(Q+H) 0.966 CvP 0.996 Serine (neg.) 0.925 THERMORANK 0.961 IUPred (short, neg.) 0.933 Thermus vs. -content (total and at different codon positions) in the orthologous set and GC-content enrichment, relative to the mean of each speci ch position. l and at different codon positions) in the orthologous set and GC-content enrichment, relative to the mean of each species studied at Close correlation of the CvP value with experimentally determined thermal stabilities Deinococcus A False positive rate True positive rate 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 Dataset 1 False positive rate True positive rate 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 B IVYWREL 0.898 Tm-index 0.605 (E+K)/(Q+H) 0.965 CvP 0.925 Serine (neg.) 0.728 THERMORANK 0.984 IUPred (short, neg.) 0.593 A B C D E False positive rate True positive rate 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 Dataset 3 D IVYWREL 0.956 Tm-index 0.579 (E+K)/(Q+H) 0.945 CvP 0.959 Serine (neg.) 0.776 THERMORANK 0.959 IUPred (short, neg.) 0.758 False positive rate True positive rate 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 Dataset 4 E IVYWREL 0.909 Tm-index 0.533 (E+K)/(Q+H) 0.649 CvP 0.890 Serine (neg.) 0.894 THERMORANK 0.504 IUPred (short, neg.) 0.588 Figure 4 Thermostability measures in prokaryotic thermophile-mesophile ortologous protein datasets. (A-E) Receiver operating characteristic (ROC) curves for (hyper-)thermophile-mesophile homologous protein pairs from (A) one self-compiled dataset (Thermus thermophilus vs. Deinococcus radiodurans) and (B-E) four published datasets were tested with the different thermostability measures to discriminate thermophiles from mesophiles. Dataset 1 (B) is from ref. [20], dataset 2 (C) is from ref. [27], dataset 3 and 4 (D, E) are from ref. [28] (containing hyperthermophiles versus mesophiles and thermophiles versus mesophiles respectively). Holder et al. Biology Direct 2013, 8:2 http://www.biology-direct.com/content/8/1/2 Page 8 of 16 Table 3 GC-content of nucleotide sequences corresponding to the trimmed orthologous protein set Species GC (%) GC enrichment relative to mean GC total (%) #1 #2 #3 #1 #2 #3 H. sapiens 56.23 38.06 54.67 1.05 1.02 1.09 49.65 S. cerevisiae 46.54 35.51 38.25 0.87 0.95 0.76 40.1 C. elegans 54.83 37.63 42.69 1.03 1.01 0.85 45.05 C. teleta 54.46 37.32 55.46 1.02 1 1.11 49.08 H. robusta 49.2 35.62 46.4 0.92 0.96 0.93 43.74 D. melanogaster 56.98 37.57 69.85 1.07 1.01 1.39 54.8 A. pompejana 52.2 36.42 41.08 0.98 0.98 0.82 43.23 C. thermophilum 59.75 41.02 70.83 1.12 1.1 1.41 57.2 D. rerio 55.62 37.68 57.52 1.04 1.01 1.15 50.27 L. Close correlation of the CvP value with experimentally determined thermal stabilities gigantea 47.59 35.77 24.82 0.89 0.96 0.49 36.06 mean 53.34 37.26 50.16 46.919 GC-content (total and at different codon positions) in the orthologous set and GC-content enrichment, relative to the mean of each species studied at each position Table 3 GC-content of nucleotide sequences corresponding to the trimmed orthologous protein set Species GC (%) GC enrichment relative to mean GC total (% t of nucleotide sequences corresponding to the trimmed orthologous protein set Table 3 GC-content of nucleotide sequences corresponding to the trimmed orthologous protein set Figure 5 Influence of GC-content on amino acid composition and thermostability measures. (A) Plot of the frequency of GC-rich codons (GARP) against the frequency of AT-rich codons (FYMINK) in the species of the orthologous set. (B) Average IVYWREL, (C) (E + K)/(Q + H), (D) average hydrophobicity, (E) CvP-bias, (F) and IUPred values plotted as a function of GC3-content (GC-content at the third codon position) in the species of the orthologous set. The regression lines with 95% confidence intervals and correlation coefficients with p-values are shown. Figure 5 Influence of GC-content on amino acid composition and thermostability measures. (A) Plot of the frequency of GC-rich codons (GARP) against the frequency of AT-rich codons (FYMINK) in the species of the orthologous set. (B) Average IVYWREL, (C) (E + K)/(Q + H), (D) average hydrophobicity, (E) CvP-bias, (F) and IUPred values plotted as a function of GC3-content (GC-content at the third codon position) in the species of the orthologous set. The regression lines with 95% confidence intervals and correlation coefficients with p-values are shown. Holder et al. Biology Direct 2013, 8:2 http://www.biology-direct.com/content/8/1/2 Page 9 of 16 Table 4 Correlation of CvP-bias differences with experimentally determined Tm value differences for various orthologous protein pairs Protein Thermophilic (A. pompejana or C. thermophilum) Mesophilic (human, S. cerevisiae or T. reesei) Difference Rrp4 (A. pompejana vs. human) CvP-bias 0.408 6.122 5.714 Tm 38°C 43.1°C 5.1 Pol η (A. pompejana vs. human) CvP-bias 5.71 3.67 −2.04 Tm 49°C 43°C −6 Collagen (A. pompejana vs. human) CvP-bias 8.696 5.804 −2.892 Tm 45-46°C 38-42°C −3 to −8 U2AF65 (A. pompejana vs. human) CvP-bias 11.90 5.68 −6.22 Tm 47°C 43°C −4 Nup170 (C. thermophilum vs. S. cerevisiae) CvP-bias −1.13 −4.86 −3.73 Tm 57°C 36°C −21 Nup192 (C. thermophilum vs. S. cerevisiae) CvP-bias 3.47 −3.33 −6.8 Tm 57°C 36°C −21 Xylanase (C. thermophilum vs. Close correlation of the CvP value with experimentally determined thermal stabilities Trichoderma reesei) CvP-bias −20.276 −22.523 −2.24 Tm 60°C 50°C −10 Validation of CvP-bias with experimentally determined thermal stabilities for orthologous protein pairs from refs. [8,30]. Table 4 Correlation of CvP-bias differences with experimentally determined Tm value differences for various orthologous protein pairs h h l h l Table 4 Correlation of CvP-bias differences with experimentally determined Tm value differences for various orthologous protein pairs CvP-bias differences with experimentally determined Tm value differences for various d thermal stabilities for orthologous protein pairs from refs. [8,30]. show such a trend. These results suggest that the CvP- bias, in comparison to mesophilic orthologs, is a good predictor of the thermal stability of eukaryotic proteins. human, and yeast and performed thermal denaturation experiments. As predicted, we found that human Rrp4 was more thermoresistant than the A. pompejana pro- tein (Figure 6A and Table 4). Close correlation of the CvP value with the body temperatures of homeothermic vertebrates Overall, the difference in the thermal stability values between the thermophilic and mesophilic orthologous pairs and the difference in the CvP-bias showed a con- sistent trend. The proteins that showed higher thermal stability than their ortholog consistently showed a higher CvP-bias (Figure 6B and Table 4). Three other measures (Tm-Index, IVYWREL, and (E + K)/(Q + H)) did not The thermal stability experiments performed to date in- dicate that A. pompejana proteins have approximately 4–8°C higher thermal denaturation values than their mesophilic counterparts. Nevertheless the CvP-bias was able to discriminate A. pompejana from mesophilic 0 10,000 20,000 30,000 40,000 50,000 60,000 70,000 20 25 30 35 40 45 50 55 60 65 70 75 Fluorescence Unit Temperature°C Tm Apo Spo Hum Sce 38°C 38.2°C 43.1°C 46.9°C 80 A B Figure 6 Experimental validation of the CvP-bias as a predictor of thermal stability. (A) Thermal denaturation curves for A. pompejana (Apo), human (Hum), Saccharomyces cerevisiae (Sce) and Schisosaccharomyces pombe (Spo) recombinant Rrp4 proteins. (B) Correlation between CvP-bias difference and Tm difference in orthologous protein pairs with experimentally determined Tm values from A. pompejana and C. thermophilum. Shading indicates the areas where the values are expected, if the predictors work. Related to Table 4. B or of thermal stability. (A) Thermal denaturation curves for A. pompejana h b (S ) bi t R 4 t i (B) C l ti b t B 0 10,000 20,000 30,000 40,000 50,000 60,000 70,000 20 25 30 35 40 45 50 55 60 65 70 75 Fluorescence Unit Temperature°C Tm Apo Spo Hum Sce 38°C 38.2°C 43.1°C 46.9°C 80 A A B Temperature°C Figure 6 Experimental validation of the CvP-bias as a predictor of thermal stability. (A) Thermal denaturation curves for A. pompejana (Apo), human (Hum), Saccharomyces cerevisiae (Sce) and Schisosaccharomyces pombe (Spo) recombinant Rrp4 proteins. (B) Correlation between CvP-bias difference and Tm difference in orthologous protein pairs with experimentally determined Tm values from A. pompejana and C. thermophilum. Shading indicates the areas where the values are expected, if the predictors work. Related to Table 4. Page 10 of 16 Holder et al. Biology Direct 2013, 8:2 http://www.biology-direct.com/content/8/1/2 will clarify this. In our orthologous set the proportion of Ala residues in A. pompejana is not higher than in most metazoans. The thermophilic C. thermophilum is highly enriched in Ala, but this is probably due to the high GC- content of this species. Validation of CvP-bias with body temperatures of homeothermic vertebrates. The number of sequences as well as the mean values of the indicated measures are shown for 10 vertebrate species. Pearson’s correlation coefficients of thermostability measures to body temperature are shown. Close correlation of the CvP value with the body temperatures of homeothermic vertebrates animals, indicating that it is a sensitive measure of ther- moadaptation. Different homeothermic vertebrates can also show up to 5°C difference in body temperature, with dolphins and whales having a body temperature of 36°C and birds 41°C. To test whether the CvP-bias also corre- lated with body temperatures among the homeothermic vertebrates we analysed the mean CvP-bias of the pro- teomes of 10 mammalian and bird species. We found a significant correlation between body temperature of a species and the mean CvP-bias of its proteins (Pearson’s r = 0.71, p = 0.02). In contrast, the Tm-Index, IVYWREL, and intrinsic disorder did not show significant correl- ation (Table 5). Our approach compared A. pompejana to a broad se- lection of taxa, including other annelids, as well as very distantly related eukaryotes (fungi). This global compari- son indicated that the CvP-bias may be a robust measure of thermostability across eukaryotes. CvP-bias ranked both A. pompejana and the thermophilic fungus C. thermophi- lum with the highest score among 10 eukaryotic species. Importantly, these ranking results cannot be explained by differences in GC-content, given that GC-content and CvP-bias are not correlated. Discussion We also tested the CvP-bias against the limited bio- chemical data available, and found a good correlation between thermal stability and CvP-bias, when comparing orthologous protein pairs. For a thorough validation or for the development of other, more sensitive measures, more biochemical and genomic data will be needed. Taking advantage of an extended sequence dataset, we searched for global signals of thermoadaptation in the proteome of the hydrothermal vent annelid A. pompejana. We used a broad phylogenetic framework, comparing orthologous eukaryotic proteins across vast evolutionary distances. For a detailed understanding of thermoadapta- tion, one would ideally focus on several closely related thermophilic and mesophilic species. However, there are no similar large-scale sequence resources available from other alvinellids, and such analyses are also confounded by the uncertain evolutionary history of thermoadapta- tion within the group. A recent analysis compared A. pompejana to a closely related mesophilic species, Para- lvinella grasslei [29]. This study revealed a higher propor- tion of Ala residues in A. pompejana than in P. grasslei. Given the possibility that P. grasslei may only recently have become a mesophile, evolving from a thermophilic common ancestor with A. pompejana, the direction of change and the role of Ala in thermoadaptation in alvi- nellids are unclear. Only an increased taxon sampling and further analyses within and outside the alvinellids One surprising finding was that the CvP-bias could also predict whether an A. pompejana protein would be less thermostable than its human ortholog, as shown for Rrp4. This suggests that not all A. pompejana protein are ther- moadapted. The thermoadaptation of a subset of proteins with certain functions (e.g. collagen, a major component of the cuticle), together with the up-regulation of heat-shock proteins [5], may be sufficient to enable A. pompejana to cope with higher temperatures. idation of CvP-bias with body temperatures of homeothermic vertebrates. The number of sequences as well as the mean values own for 10 vertebrate species. Pearson’s correlation coefficients of thermostability measures to body temperature are shown. Alvinella pompejana samples Transcript sequences were translated with ESTScan [33] and trimmed to the longest stop codon-free protein fragment. If a 5’ stop-codon was present, the sequence was scanned to the next methionine and that was con- sidered as the protein start. Fragments shorter than 60 amino acids were discarded. The sequence assemblies can be queried by BLAST at http://jekely-lab.tuebingen. mpg.de/blast/. Samples were collected from the hydrothermal vent chimney sites Julie and Parigo at 13N°/EPR (East Pacific Rise) during the cruises HOT 1996 and PHARE 2002 in the Pacific Ocean using the telemanipulated arms of the submersible Nautile and the ROV Victor. The samples were brought back to the surface into an insulated bas- ket and snap-frozen in liquid nitrogen and stored at −80°C until RNA extraction. Amino acid composition of protein sequences and measures derived from amino acid composition were calculated with custom scripts in Python. The CvP bias was calculated as (C - P)/length × 100, where C and P represents the number of charged (EDKR) and polar residues (STNQ) respectively. Conclusions Our deep-sequencing efforts greatly enhance the existing transcriptome data for A. pompejana, nearly doubling the number of full-length cDNAs. This extended resource will be valuable for further comparative genomic studies of Table 5 Correlation of CvP-bias with the body temperature of homeothermic vertebrates Species Number of sequences CvP-bias Tm-Index IUPred IVYWRE ble 5 Correlation of CvP-bias with the body temperature of homeothermic vertebrates Table 5 Correlation of CvP-bias with the body temperature of homeothermic vertebrates Species Number of sequences CvP-bias Tm-Index IUPred IVYWREL Body temperature °C HUMAN 50 006 0.93 0.9 0.31 36.87 37°C MOUSE 36 368 0.97 0.93 0.29 37.16 37°C TURTR (bottle-nosed dolphin) 307 0.99 0.79 0.28 38.8 36.8°C RAT 30 926 1.02 0.97 0.28 37.17 37°C BOVIN 23 358 1.34 0.96 0.27 37.6 37°C CHICK 19 671 1.42 0.94 0.28 37.3 41°C CANFA (dog) 24 002 1.43 0.96 0.28 37.5 37°C PIG 24 692 1.61 0.98 0.28 37.43 37°C MELGA (turkey) 15 899 1.72 0.94 0.27 37.66 41°C TAEGU (zebra finch) 16 717 2.32 0.97 0.27 37.72 41°C Pearson’s r 0.71 0.23 −0.44 0.02 Validation of CvP-bias with body temperatures of homeothermic vertebrates. The number of sequences as well as the mean values of the indicated measures are shown for 10 vertebrate species Pearson’s correlation coefficients of thermostability measures to body temperature are shown Page 11 of 16 Holder et al. Biology Direct 2013, 8:2 http://www.biology-direct.com/content/8/1/2 sequences, and passed to the CAP3 assembler with de- fault parameters [32]. To assess the amount of newly obtained transcripts from our own sequencing effort, our sequencing data and the JGI + Genoscope ESTs (218,458) were also assembled separately with CAP3. Contigs and singletons were joined for each assembly to compose the final set of transcript sequences. metazoans and extremophiles. The correlation of the CvP- bias with thermal stability may be used to identify the most thermoadapted proteins from A. pompejana and other thermophilic eukaryotes, potentially facilitating pro- tein structure determination studies. A. pompejana transcriptome sequencing, assembly and analysis For the sequencing of the A. pompejana transcriptome we used a combination of techniques. We generated a custom, normalized, full-length cDNA library (with m7Gppp affinity purification to limit bacterial RNA contamination; Invitro- gen), cloned into the pENTR222.1 vector from two adult worms. After plating, we sequenced 10,063 randomly picked clones using the Sanger technology (ABI 3730) and the M13-FP primer. The programs Phred and Cross-match were used for base calling and vector trimming. For the multi-species BLAST comparisons Capitella tel- eta, Helobdella robusta, Lottia gigantea, and Daphnia pulex protein sequences were downloaded from JGI (http://www.jgi.doe.gov/, Filtered Models). Protein se- quences for Caenorhabditis elegans, Drosophila melanoga- ster, Anopheles gambiae, Strongylocentrotus purpuratus, Branchiostoma floridae, Ciona intestinalis, Danio rerio, Gallus gallus, Homo sapiens, Hydra magnipapillata, Nematostella vectensis, Trichoplax adhaerens, Monosiga brevicollis, Neurospora crassa, Schizosaccharomyces pombe, Dictyostelium discoideum, Naegleria gruberi, Chlamydomo- nas reinhardtii, Paramecium tetraurelia, and Arabidopsis thaliana were downloaded from the NCBI RefSeq database (http://www.ncbi.nlm.nih.gov/RefSeq/). We also performed 454 sequencing (GS FLX, Roche/454) on the PCR-amplified cDNA library, following concaten- ation and fragmentation. After adaptor trimming (pDONR), quality (0.05), and length filtering (50 bp cutoff) with the software package CLC Genomics Workbench 4.5.1, we obtained 2,717,445 reads of an average length of 220 bp. For Illumina sequencing we used total RNA isolated from a third animal, a frozen A. pompejana male using the RNaesy Kit (Qiagen), following the breaking up of the tissue (pieces of 20–30 mg). We performed paired- end sequencing following reverse transcription of total RNA using the Smart cDNA Construction Kit (Clontech), m7cap-primed second strand synthesis, co-ligation and nebulization of cDNA, gel fractionation (approximately 350 bp) and adapter ligation. Samples were run on a HiSeq 2000 sequencer obtaining 91,670,518 paired reads. After adaptor trimming (pDONR222, Illumina PCR Pri- mer, Illumina Paired End PCR Primer 2.0), quality (0.05) and length (30 bp cutoff) filtering, using the CLC Genom- ics Workbench 4.5.1, we obtained 87,799,426 high-quality reads of an average length of 76 bp. Thermorank analysis Thermorank analysis For local usage, the THERMORANK tool was re- implemented according to ref. [20], using the Python programming language. Tripeptide residue accessible surface area values were used as described in ref. [38]. The 8 protein sequences in each orthologous set were ranked and the cumulative rank sums over the 457 sets calculated to assess the overall trend of ranking. Ranking calculations for all measures were performed using the R software environment (http://www.r-project.org/). I recommend the following points to the attention of the authors: At the first sight, the title and abstract does not make it clear whether or not the CvP index is defined here, or is it already known. I suggest to clarify this, for instance by adding a restrictive adjective, something like “…iden- tifies the CvP-bias as a (reliable, robust) measure of eukaryotic thermostability”. CvP-bias was described in several articles including PMID: 16494505. Since CvP- bias is part of the main message, these papers could be cited in the introduction. Cloning, expression and purification of A. pompejana proteins A. pompejana Rrp4 was cloned and expressed as a His- tagged fusion together with Rrp4 orthologues from Schiso- saccharomyces pombe, Homo sapiens and Saccharomyces cerevisiae. DNA was transformed in BL21Gold pLysS (Stratagene), grown overnight at 18°C and induced with 0.5 mM IPTG. Cells were resuspended and lysed by sonic- ation in a buffer containing Tris pH 7.5, 500 mM NaCl, 20 mM Imidazole, 5 mM beta-mercaptoethanol and 10% gly- cerol. All proteins were purified using affinity chromatog- raphy on Talon resin (Clontech) (elution with 250 mM imidazole), followed by size exclusion chromatography on a GF200 column. We have now changed the title to “..identifies the CvP- bias as a robust measure…”. We also changed one sen- tence in the abstract to clearly state that we looked at already known measures “We then performed an exten- sive survey of previously established prokaryotic thermo- adaptation measures” We also included a reference to PMID: 16494505 (ref. 18). Reviewers’ comments Reviewers’ comments Thermophile-mesophile benchmark datasets Thermophile-mesophile benchmark datasets Protein sequences for Thermus thermophilus and Deinococ- cus radiodurans were downloaded from UniProtKB by fil- tering on taxonomy ids 262724 and 1299. The Li et al. [20] dataset (Dataset 1, THERMORANK) was downloaded from http://www.abl.ku.edu/thermorank/suppl.html. The Monta- nucci et al. [27] dataset (Dataset 2) was downloaded from http://lipid.biocomp.unibo.it/~ludovica/thermo-meso- MUT/ and only the first pair was considered from each cluster. The Taylor and Vaisman datasets (Datasets 3 and 4) were obtained from the supplementary material of ref. [28]. The authors present transcriptome sequencing studies on the Alvinella pompejana worm that lives in high temperature environments. A. pompejana is an attractive eukaryotic model organism both for studying thermoa- daptation and also because thermophylic proteins hold promise for structural studies. The paper presents a sig- nificant advance in the sequencing of this organism, a large number of potential ORFs were discovered which warrants publication in itself. The manuscript also pre- sents a variety of data coming from and evaluated by different techniques. The authors also conducted bio- physical tests that showed that not all A. pompejana proteins are thermotolerant, indicating that the thermo- tolerance of this species may not be as outstanding as previously thought. Orthologous sets d l d d We downloaded the Swissprot and TrEMBL sequences for Homo sapiens, Drosophila melanogaster, Caenorhabdi- tis elegans, Danio rerio, and Saccharomyces cerevisiae from UniprotKB. For Capitella teleta, Helobdella robusta and Lottia gigantea protein sequences were downloaded from JGI (http://www.jgi.doe.gov/, Filtered Models). The pre- dicted Chaetomium thermophilum proteins were down- loaded from http://ct.bork.embl.de. Each proteome was clustered by 98% sequence identity and arbitrary length difference, using CD-HIT [34]. A total of 45 pairwise BLASTP searches were performed, best hits that were consistent in all searches were considered as an ortholo- gous set, yielding 457 sets. For each orthologous set, a multiple sequence alignment was created using MUSCLE [35]. Because many of the protein sequences were not full length, the alignments were trimmed from both ends to the first column without a gap symbol, so that each All quality trimmed and vector screened 454 and Illu- mina reads were assembled using Velvet version 1.1. and Oases [31]. The k-mer length used was 25. The resulting contigs and singletons were joined with all published A. pompejana ESTs from NCBI dbEST, and with our EST Page 12 of 16 Holder et al. Biology Direct 2013, 8:2 http://www.biology-direct.com/content/8/1/2 nm, respectively. To obtain the temperature midpoint for the protein unfolding transition (Tm), a Boltzmann model was used to fit the fluorescence data [39]. protein is represented with the same fragment. The sequences from the trimmed alignments were used for all further analyses (Additional file 9). The tree, based on the recently published phylogeny of annelids [36] and the con- sensus animal phylogeny [37], in Additional file 7 repre- sents the relationships of the species in the orthologous set. Reviewer’s reports Reviewer 1: Sándor Pongor, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy Thermal shift assay A key point made by this study is that the earlier analysis of Alvinella sequences might not have necessarily identified the actual basis for thermostability of proteins in this organism. In particular, the reported features are shared with mesophilic lophotrochozoans, suggesting that they might not be genuine discriminants of thermophily. In this regard, the extended proteome generated from se- quence data obtained by the authors is a useful resource. Further, the study shows that the metrics for thermophily that were found to be successful in discriminating pro- karyotic thermophily cannot be uncritically applied to eukaryotes. While the data is currently very limited, are there any pos- sible explanations for the CvP being more successful as a discriminant than other measures in eukaryotes? One possible explanation is that some of the other mea- sures are over-fitted to a training dataset (e.g. the Tm- Index uses dipeptide composition). The CvP-bias is a simple measure with few parameters combining infor- mation from two classes of amino acids, the changes of which have previously been linked to thermoadaptation. CvP-bias also performs best on the prokaryotic datasets. A larger number of Alvinella and Chaetomium protein structures and their comparison to mesophilic ortolo- gues will help to clarify the role of charged and polar residues in thermoadaptation. The authors might want to consider a few points: Reviewer 3: Anthony M. Poole, School of Biological Sciences, University of Canterbury, Christchurch, New Zealand While they used length of the trimmed sequence in the alignment as a possible discriminant, it might be better to directly measure intrinsic disorder or sequence entropy and use them as potential discriminants. This is of interest because in general eukaryotes have much great amount of low complexity sequence in their proteins than prokar- yotes (especially low complexity sequence enriched in charge or polar residues or both). While other factors affect the amount of low complexity in eukaryotes does thermophily have a negative effect on it. Using transcriptomics data, this paper examines whether there is a signal of thermoadaptation in proteins from the thermotolerant annelid worm, Alvinella pompejana. As well as significantly increasing the available sequence data for this species, which is a worthy effort in itself, the authors examine whether there is a compositional signa- ture for thermoadaptation in proteins from this species and from the thermotolerant yeast Chaetomium thermo- philum. Thermal shift assay Even though the technical parts are well separated from the main text of the paper, at times the manuscript is still difficult to read, simply because many techniques are used in the project. This could be helped by empha- sizing the main messages clarifying the details. For in- stance, ROC curves presented in Figure 4 could be complemented by a tabular comparison of AUC values. The panels of Figure 4 are too tiny and crowded in my Solutions containing 5 μl of 2 mg/ml protein with 35x of Sypro Orange (Invitrogen) and 45 μl of buffer screen were added to the wells of a 96-well PCR plate (Eppendorf). The plate was sealed and heated in a real-time PCR system (Eppendorf) from 20°C to 80°C in increments of 0.2°C. Fluorescence changes were monitored simultaneously. The wavelengths for excitation and emission were 470 and 550 Page 13 of 16 Holder et al. Biology Direct 2013, 8:2 http://www.biology-direct.com/content/8/1/2 Page 13 of 16 higher body temperatures (Gallus/Zebra Finch ~41 C). Would they show a higher rank in the CvP measure than other species in the current figure? opinion. Also, more details in the Figure legends would improve readability. We have included the AUC values in Figure 4 and give more details in the figure legends. We thank the reviewer for this insightful comment. In- deed, among the homeothermic metazoans temperature differences can be quite large (36 to 41 C), and this could be reflected in their proteomes. Although a full analysis in homeothermic metazoans is beyond the scope of this paper, we determined the average CvP values of the pro- teomes of several homeothermic vertebrates and found a strong correlation between body temperature and the CvP values, as shown in Table 5. This question can be addressed in more detail in the future when more bird genomes and genomes from vertebrates with low body temperature (e.g. dolphins, whales) become available. Reviwer 2: L. Aravind, National Center for Biotech- nology Information, National Library of Medicine, National Institutes 702 of Health, Bethesda, USA Since its discovery, A. pompejana has been of great interest in regard to the question of how a metazoan might tolerate environmental extremes such as those it faces. Holder et al. use deep transcriptome sequence combined with bioinformatics analysis to attempt to ex- plain the unusual thermotolerance of Alvinella pompe- jana. Competing interests Th h h Competing interests The authors have no competing interests. Thermal shift assay In contrast to a comparison of two prokaryote species (here there is a large literature on thermotoler- ant proteins), one a thermophile, and the other not, the authors find only a relatively faint indication of thermo- tolerance for these two eukaryotes. Of the nine ther- mostability measures presented, only one discriminates the two thermotolerant eukaryotes, whereas all were sufficient for discriminating between Thermus thermo- philus (thermophilic) and Deinococcus radiodurans (dessication/radioresistant but not thermophilic). We have performed calculations of intrinsic protein dis- order and included a new panel in Figure 3 showing the results. Using this measure (IUPred), Chaetomium ranks the highest. This is likely due to the high GC content of this species. Alvinella ranks average, indicating that in- trinsic disorder is not a general discriminator of thermo- philic and mesophilic eukaryotes. Most homeothermic metazoans tend to maintain higher body temperatures than the rest. In their analysis of CvP Homo, a homeothermic metazoan is ranked next after the thermophilic species. However, a species with a much lower preferred temperature is also close. Is there any significance to this? Would it be possible to use some bird species in the comparison as they have much This result is interesting in that it indicates that ther- moadaptation of A. pompejana (and C. thermophilum) Holder et al. Biology Direct 2013, 8:2 http://www.biology-direct.com/content/8/1/2 Holder et al. Biology Direct 2013, 8:2 http://www.biology-direct.com/content/8/1/2 Page 14 of 16 Holder et al. Biology Direct 2013, 8:2 http://www.biology-direct.com/content/8/1/2 Additional files Additional file 1: Transcriptome dataset 1 MPI transcriptome dataset generated by the clustering of all new A. pompejana sequence data in this study. The file was compressed with tar -jcvf archive_name.tar.bz2 file_to_compress, use tar –jxvf to uncompress it. Contains 74762 sequences. Additional file 2: Transcriptome dataset 2 JGI + Genoscope transcriptome dataset generated by the clustering of all publicly available A. pompejana ESTs (as of Feb 2011). The file was compressed with tar -jcvf archive_name.tar.bz2 file_to_compress, use tar –jxvf to uncompress it. Contains 39155 sequences. In the case of the two eukaryotes, the degree to which proteins may be subject to high temperature is debated, particularly for A. pompejana. It is a pity that the authors did not include the thermotolerant red alga Cyanidioschyzon merolae, which was isolated from hot springs (45°C) for which there is also sequence data (Matsuzaki et al. 2004 Nature 428:653). Including this species would be fascinating from the point of view of the general question of understanding thermoadapta- tion at the protein level in eukaryotes, but is not likely to change the primary conclusions drawn about A. pompejana. Additional file 3: Transcriptome dataset 3 MPI + JGI + Genoscope transcriptome dataset generated by the clustering of all publicly available A. pompejana ESTs (as of Feb 2011) and all new A. pompejana sequence data in this study. The file was split into two and compressed with tar -jcvf archive_name.tar.bz2 file_to_compress, use tar –jxvf to uncompress it. Contains 97285 sequences. Additional file 3: Transcriptome dataset 3 MPI + JGI + Genoscope transcriptome dataset generated by the clustering of all publicly available A. pompejana ESTs (as of Feb 2011) and all new A. pompejana sequence data in this study. The file was split into two and compressed with tar -jcvf archive_name.tar.bz2 file_to_compress, use tar –jxvf to uncompress it. Contains 97285 sequences. Additional file 4: Predicted protein dataset 1 MPI predicted protein dataset generated by ORF prediction on all new clustered A. pompejana sequence data in this study. Potential full-length protein sequences are indicated as ‘closed = (1/1)’. The file was compressed with tar -jcvf archive_name.tar.bz2 file_to_compress, use tar –jxvf to uncompress it. Contains 29013 sequences. Additional file 5: Predicted protein dataset 2 JGI + Genoscope predicted protein dataset generated by ORF prediction on the clustered transcripts derived from all publicly available A. pompejana ESTs (as of Feb 2011). Potential full-length protein sequences are indicated as ‘closed (1/1)’. Additional files melano- gaster, 41.80; S. cerevisiae, 46.39; C. elegans, 47.42; C. thermophilum, 49.24; A. pompejana, 52.89; H. sapiens, 60.63. Since we do not see a trend and Alvinella and Chaetomium rank average, we did not include this ana- lysis in the paper. Given that the signal for proteins is so subtle, it would be interesting to look at non-coding RNAs - I suspect at least some will be present in the raw read data. This could be worthwhile in that GC-content of rRNA cor- relates with optimal growth temperature, whereas the GC content of genomic DNA, protein-coding genes or 3rd codon position of such genes does not (Hurst & Merchant 2001 Proc Roy Soc Lond B 268:493). If the signal in proteins is weak, as the present authors show, perhaps an analysis of RNA can help shed light on whether there is a molecular signature of thermoadap- tation. This would be helpful in establishing whether the subtle difference observed here is an indicator that, of the various attributes that contribute to thermoa- daptation, CvP is the most sensitive, and thus the first to show a signal under moderate thermoadaptation. ll h ’ h l k d Additional file 7: Phylogenetic tree of the 10 species used in the orthologous set. The tree is based on the recently published phylogeny of annelids [36] and the consensus animal phylogeny [37]. Additional file 7: Phylogenetic tree of the 10 species used in the orthologous set. The tree is based on the recently published phylogeny of annelids [36] and the consensus animal phylogeny [37]. Additional file 8: Annotated predicted A. pompejana proteins 42665 predicted A. pompejana protein sequences, defined by their first BLASTP hit in the SwissProt database and annotated with the first BLASTP hits in the Capitella teleta, Homo sapiens, Danio rerio and Drosophila melanogaster proteomes. The file was compressed with tar -jcvf archive_name.tar.bz2 file_to_compress, use tar –jxvf to uncompress it. Contains 42665 sequences. Additional file 9: Orthologous set. Trimmed multiple sequence alignments of the orthologous set of 457 proteins from the 10 species used in the study. Additional file 10: Frequency of individual amino acids as a function of GC-content. The frequency of amino acids is plotted as a function of GC3-content (GC-content at the third codon position) in the Additional file 10: Frequency of individual amino acids as a function of GC-content. Additional files is not reflected in strong changes in the composition of individual proteins themselves. Of the nine measures employed here (Figures 2 & 3), only one (CvP) suggests a signature of thermoadapation in the two thermotoler- ant eukaryote species. It is worth pointing out that the two bacterial cases examined show much greater differ- ences in their optimal growth temperature, whereas, as discussed in the text, this may not be particularly high for A. pompejana. is not reflected in strong changes in the composition of individual proteins themselves. Of the nine measures employed here (Figures 2 & 3), only one (CvP) suggests a signature of thermoadapation in the two thermotoler- ant eukaryote species. It is worth pointing out that the two bacterial cases examined show much greater differ- ences in their optimal growth temperature, whereas, as discussed in the text, this may not be particularly high for A. pompejana. Additional file 1: Transcriptome dataset 1 MPI transcriptome dataset generated by the clustering of all new A. pompejana sequence data in this study. The file was compressed with tar -jcvf archive_name.tar.bz2 file_to_compress, use tar –jxvf to uncompress it. Contains 74762 sequences. Additional file 2: Transcriptome dataset 2 JGI + Genoscope transcriptome dataset generated by the clustering of all publicly available A. pompejana ESTs (as of Feb 2011). The file was compressed with tar -jcvf archive_name.tar.bz2 file_to_compress, use tar –jxvf to uncompress it. Contains 39155 sequences. Additional file 3: Transcriptome dataset 3 MPI + JGI + Genoscope transcriptome dataset generated by the clustering of all publicly available A. pompejana ESTs (as of Feb 2011) and all new A. pompejana sequence data in this study. The file was split into two and compressed with tar -jcvf archive_name.tar.bz2 file_to_compress, use tar –jxvf to uncompress it. Contains 97285 sequences. Additional file 4: Predicted protein dataset 1 MPI predicted protein dataset generated by ORF prediction on all new clustered A. pompejana sequence data in this study. Potential full-length protein sequences are indicated as ‘closed = (1/1)’. The file was compressed with tar -jcvf archive_name.tar.bz2 file_to_compress, use tar –jxvf to uncompress it. Contains 29013 sequences. Additional file 1: Transcriptome dataset 1 MPI transcriptome dataset generated by the clustering of all new A. pompejana sequence data in this study. The file was compressed with tar -jcvf archive_name.tar.bz2 file_to_compress, use tar –jxvf to uncompress it. Contains 74762 sequences. Additional files The frequency of amino acids is plotted as a function of GC3-content (GC-content at the third codon position) in the species of the orthologous set. The regression lines with 90% confidence intervals are shown. Additional file 10: Frequency of individual amino acids as a function of GC-content. The frequency of amino acids is plotted as a function of GC3-content (GC-content at the third codon position) in the species of the orthologous set. The regression lines with 90% confidence intervals are shown. Following the reviewer’s suggestion we have looked at 28S rRNA and obtained GC-content from the trimmed alignment (1200 nucleotides) for six species: D. melano- gaster, 41.80; S. cerevisiae, 46.39; C. elegans, 47.42; C. thermophilum, 49.24; A. pompejana, 52.89; H. sapiens, 60.63. Since we do not see a trend and Alvinella and Chaetomium rank average, we did not include this ana- lysis in the paper. Additional files The file was compressed with tar -jcvf archive_name.tar.bz2 file_to_compress, use tar –jxvf to uncompress it. Contains 36328 sequences. Additional file 5: Predicted protein dataset 2 JGI + Genoscope predicted protein dataset generated by ORF prediction on the clustered transcripts derived from all publicly available A. pompejana ESTs (as of Feb 2011). Potential full-length protein sequences are indicated as ‘closed (1/1)’. The file was compressed with tar -jcvf archive_name.tar.bz2 file_to_compress, use tar –jxvf to uncompress it. Contains 36328 sequences. We thank the reviewer for this suggestion. We had a look at the Cyanidioschyzon merolae proteome, but could only identify 16 proteins that give reciprocal best blast hits to our orthologous set. Given that this red alga is phylogenetically very far from yeasts + metazoans, we could not include it in the comparisons. However, it would certainly be worth comparing Cyanidioschyzon merolae to mesophilic red algae in the future, once more sequence data will become available. Additional file 6: Predicted protein dataset 3 MPI + JGI + Genoscope predicted protein dataset generated by ORF prediction on the clustered transcripts derived from all publicly available A. pompejana ESTs (as of April 2011) and all new A. pompejana sequence data in this study. Potential full-length protein sequences are indicated as ‘closed = (1/1)’. The file was compressed with tar -jcvf archive_name.tar.bz2 file_to_compress, use tar –jxvf to uncompress it. Contains 42665 sequences. q Given that the signal for proteins is so subtle, it would be interesting to look at non-coding RNAs - I suspect at least some will be present in the raw read data. This could be worthwhile in that GC-content of rRNA cor- relates with optimal growth temperature, whereas the GC content of genomic DNA, protein-coding genes or 3rd codon position of such genes does not (Hurst & Merchant 2001 Proc Roy Soc Lond B 268:493). If the signal in proteins is weak, as the present authors show, perhaps an analysis of RNA can help shed light on whether there is a molecular signature of thermoadap- tation. This would be helpful in establishing whether the subtle difference observed here is an indicator that, of the various attributes that contribute to thermoa- daptation, CvP is the most sensitive, and thus the first to show a signal under moderate thermoadaptation. Following the reviewer’s suggestion we have looked at 28S rRNA and obtained GC-content from the trimmed alignment (1200 nucleotides) for six species: D. References 1. Chevaldonne P, Fisher C, Childress J, Desbruyeres D, Jollivet D, Zal F, 1. 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Author details 1 1Max-Planck-Institute for Developmental Biology, Spemannstr. 35, Tübingen D-72076, Germany. 2Equipe GAME, UMR 7144 CNRS-UPMC, Station Biologique, Place Georges Teissier, Roscoff 29680, France. 1Max-Planck-Institute for Developmental Biology, Spemannstr. 35, Tübingen D-72076, Germany. 2Equipe GAME, UMR 7144 CNRS-UPMC, Station Biologique, Place Georges Teissier, Roscoff 29680, France. 20. Li Y, Middaugh CR, Fang J: A novel scoring function for discriminating hyperthermophilic and mesophilic proteins with application to predicting relative thermostability of protein mutants. BMC Bioinformatics 2010, 11:62. %U http://www.ncbi.nlm.nih.gov/pubmed/20109199. 3Max-Planck-Institute of Biochemistry, Department of Structural Cell Biology, Am Klopferspitz 18, Martinsried D-82152, Germany. 21. Ku T, Lu P, Chan C, Wang T, Lai S, Lyu P, Hsiao N: Predicting melting temperature directly from protein sequences. 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Chothia C: The nature of the accessible and buried surfaces in proteins. J Mol Biol 1976, 105:1–12. %U http://www.ncbi.nlm.nih.gov/pubmed/ 994183. 39. Ericsson UB, Hallberg BM, Detitta GT, Dekker N, Nordlund P: Thermofluor- based high-throughput stability optimization of proteins for structural studies. Anal Biochem 2006, 357:289–298. doi:10.1186/1745-6150-8-2 Cite this article as: Holder et al.: Deep transcriptome-sequencing and proteome analysis of the hydrothermal vent annelid Alvinella pompejana identifies the CvP-bias as a robust measure of eukaryotic thermostability. Biology Direct 2013 8:2. 36. Struck TH, Paul C, Hill N, Hartmann S, Hosel C, Kube M, Lieb B, Meyer A, Tiedemann R, Purschke G, Bleidorn C: Phylogenomic analyses unravel annelid evolution. Nature 2011, 471:95–98. 37. Telford MJ, Copley RR: Improving animal phylogenies with genomic data. Trends Genet 2011, 27:186–195. 38. Chothia C: The nature of the accessible and buried surfaces in proteins. 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